Prelims.indd i 5/15/2012 10:20:01 AM Textbook of Microbiology and Immunology Prelims.indd i 5/15/2012 10:20:01 AM “This page intentionally left blank" Prelims.indd ii 5/15/2012 10:20:02 AM Textbook of Microbiology and Immunology 2nd Edition Subhash Chandra Parija MBBS, MD, PhD, DSc, FRCPath FAMS, FICPath, FICAI, FABMS, FISCD, FIAVP, FIATP, FIMSA Professor and Head Department of Microbiology Jawaharlal Institute of Postgraduate Medical Education and Research Puducherry, India ELSEVIER A division of Reed Elsevier India Private Limited Prelims.indd iii 5/15/2012 10:20:02 AM Textbook of Microbiology and Immunology, 2/e Parija ELSEVIER A division of Reed Elsevier India Private Limited Mosby, Saunders, Churchill Livingstone, Butterworth-Heinemann and Hanley & Belfus are the Health Science imprints of Elsevier. © 2012 Elsevier First Edition 2009 Second Edition 2012 All rights are reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise without the prior permission of the publisher. ISBN: 978-81-312-2810-4 Medical knowledge is constantly changing. As new information becomes available, changes in treatment, procedures, equipment and the use of drugs become necessary. The author, editors, contributors and the publisher have, as far as it is possible, taken care to ensure that the information given in this text is accurate and up-to-date. However, readers are strongly advised to confirm that the information, especially with regard to drug dose/usage, complies with current legislation and standards of practice. Please consult full prescribing information before issuing prescriptions for any product mentioned in this publication. Published by Elsevier, a division of Reed Elsevier India Private Limited. Registered Office: 305, Rohit House, 3 Tolstoy Marg, New Delhi-110 001. Corporate Office: 14th Floor, Building No. 10B, DLF Cyber City, Phase II, Gurgaon–122 002, Haryana, India. Sr. Commissioning Editor: Shukti Mukherjee Bhattacharya Managing Editor (Development): Shabina Nasim Development Editor: Shravan Kumar Copy Editor: Shrayosee Dutta Manager Publishing Operations: Sunil Kumar Manager Production: NC Pant Typeset by Mukesh Technologies Pvt. Ltd., Pudhucherry, India. Printed and bound at EIH Unit Ltd. Press, Manesar. Prelims.indd iv 5/15/2012 10:20:03 AM To my father Late Shri Managovinda Parija and mother Late Smt Nishamani Parija Prelims.indd v 5/15/2012 10:20:03 AM “This page intentionally left blank" Prelims.indd vi 5/15/2012 10:20:03 AM Preface to the Second Edition Like the first edition, the second edition of Textbook of Microbiology and Immunology consists of six sections. Section I and II deal with general micro biology and immunology, respectively. Section III, IV, and V deal with bacteriology, virology, and mycology, respectively. Lastly, Section VI deals with applied microbiology and includes epidemiology and control of community infections, hospital infections, antimicrobial chemotherapy, water analysis, and immunization. Medical microbiology is a rapidly changing and evolving field. The threat of emerging and reemerging pathogens and the changing epidemiology of known pathogens have made it imperative that we keep abreast of the changes and developments in the field if we are to efficiently deal with them. The second edition of the Textbook of Microbiology and Immunology has been revised thoroughly and revamped to include the latest information in the field of medical microbiology. Special emphasis has been placed on molecular methods of diagnosis, which have revolutionized the diagnosis of infectious diseases and have made it possible to detect hitherto unknown and uncultivable pathogens from clinical specimens. The problem of antimicrobial resistance with respect to emerging mechanisms, changing epidemiology, and newer ways of detection has been dealt with in detail. This text aims at not only providing basic information about microbiology and immunology, but also deals with the clinical applications of microbiology in the study of infectious diseases. In each chapter, key points are highlighted. Clinical case studies have been included so that students can evaluate their understanding of the various microbes. Photographs and pertinent line diagrams have been included to aid the learning process. The book has been trimmed down so as to include the necessary information without overburdening the students. This textbook aims at providing comprehensive information about microbiology and it’s applications for medical students, paramedical students and workers in the field of infectious diseases. I sincerely hope the book serves this purpose and also helps in creating interest towards the subject among students. Readers’ views and suggestions for further improving the book in the coming years are welcome. Suggestions may kindly be e-mailed at subhashparija@yahoo.co.in. Subhash Chandra Parija Prelims.indd vii 5/15/2012 10:20:03 AM Prelims.indd viii 5/15/2012 10:20:03 AM Preface to the First Edition The intent of the book is to provide an up-to-date information on microbial diseases which are emerging as an important health problem world wide. This book has been written to provide a comprehensive coverage of basic and clinical microbiology, including immunology, bacteriology, virology, and mycology, in a clear and succinct manner. The book also intends to provide an accurate presentation of clinically relevant information to the learners of medical microbiology. Textbook of Microbiology and Immunology consists of six sections. Section I and II deals with general micro biology and immunology, respectively. Section III, IV, and V deals with bacteriology, virology, and mycology, respectively. Lastly, Section VI deals with applied microbiology and includes epidemiology and control of community infections, hospital infections, antimicrobial chemotherapy, water analysis, and immunization. Emphasis, throughout the text, is made on the clinical applications of microbiology to study infectious diseases. Cultivation and identification of each organism along with pathogenesis of diseases, clinical manifestations, diagnostic laboratory tests, treatment, and prevention and control of resulting infections are thoroughly updated to include most recent advances in the field. Details are summarized in the tabular format. Clinical cases are provided in most of the chapters. The book is profusely illustrated with line diagrams and photomicrographs both black & white and color. I believe this book will be a useful source of comprehensive information for students mainly the undergraduate students of medicine, allied sciences, and others who are interested in medical microbiology. I welcome reader’s views and suggestions for further improvement of the book in the future edition. Suggestions may kindly be e-mailed at subhashparija@yahoo.co.in or at infoindia@elsevier.com. Subhash Chandra Parija Prelims.indd ix 5/15/2012 10:20:03 AM Acknowledgements I am grateful for the valuable professional help and support provided by the staff at Elsevier, New Delhi, namely, Mr Sumeet Rohatgi, Mr Vidhu Goel, Ms Shabina Nasim, and Mr Shravan Kumar during a period of last more than one year. It has been really a wonderful and learning experience while working in particular with Ms Shabina Nasim and Mr Shravan Kumar; their professional contributions are immense for the development of manuscript to the present book format. I gratefully acknowledge all my colleagues, friends, and students for their valuable advice, constructive criticism, and assistance in preparation of the manuscript. I owe special debt of profound gratitude to my mother late Smt Nishamani Parija and father late Shri Managovinda Parija without whose encouragement the book would not have been possible. I am indeed grateful to my wife Smt Jyothirmayee Parija for all her support throughout the period of preparation of the manuscript of the book. It is my pleasure to thank my niece Er Kukumina Parija, son-in-law Er Subhasis Ray, nephew Er Shri Rajkumar Parija, daughterin-law Ms Smriti Parija, and daughters, Ms Dr Madhuri Parija, son-in-law Dr Ajay Halder, Ms Er Mayuri Parija and son-in-law Er Shailesh Nandan for their untiring secretarial help towards the preparation of the manuscript. Subhash Chandra Parija Prelims.indd x 5/15/2012 10:20:03 AM Contents Preface to the Second Edition Preface to the First Edition Acknowledgements Color Photos vii ix x CP1 6. Laboratory Identification of Bacteria and Taxonomy   SECTION I  GENERAL MICROBIOLOGY 1. History of Microbiology 3 7. Bacterial Genetics        Introduction Historical Background Microorganisms as a Cause of Disease Study of Viruses Phenomenon of Immunity Chemotherapeutic Agents 2. Morphology and Physiology of Bacteria        Introduction Size of Bacteria Microscopy Study of Bacteria Structure and Functions of Bacterial Cell Envelope Growth and Multiplication of Bacteria Bacterial Nutrition 3. Sterilization and Disinfection     Introduction Definition of Frequently Used Terms Sterilization Disinfection 4. Culture Media    Introduction Ingredients of Culture Media Types of Culture Media 5. Culture Methods    Prelims.indd xi Introduction Methods of Culture Anaerobic Culture 3 3 3 6 7 8 9 9 9 9 12 14 21 23 24 24 24 24 29 34 34 34 34 38 38 38 39 Introduction Identification of Bacteria Bacterial Taxonomy       Introduction Chromosomal Substances Mutations Extrachromosomal DNA Substances Transfer of DNA Within Bacterial Cells Transfer of DNA Between Bacterial Cells Recombination 8. Genetic Engineering and Molecular Methods         Introduction DNA: An Amazing Molecule Genetic Engineering Nucleic Acid Probes Polymerase Chain Reaction Recombinant DNA Technology Genetically Modified Organisms Gene Therapy 9. Antimicrobial Agents: Therapy and Resistance       Introduction Mechanisms of Action of Antimicrobial Drugs Resistance to Antimicrobial Drugs Basis of Resistance Antibiotic Sensitivity Testing Antibacterial Assays in Body Fluids 41 41 41 45 47 47 47 48 49 50 51 54 55 55 55 55 57 57 59 60 60 61 61 61 64 65 68 71 10. Microbial Pathogenesis 72 Introduction Types of Microorganisms Infection 72 72 72    5/15/2012 10:20:03 AM xii CONTENTS   Stages of Pathogenesis of Infections Stages of an Infectious Disease 73 82 17. Immune Response   SECTION II IMMUNOLOGY   11. Immunity   Introduction Types of Immunity 85 85 85  90             Introduction Determinants of Antigenicity Antigenic Specificity Species Specificity Isospecificity Autospecificity Organ Specificity Heterophile Specificity Haptens Superantigens 13. Antibodies    Introduction Immunoglobulins Abnormal Immunoglobulins 14. Antigen–Antibody Reactions     Introduction General Features of Antigen–Antibody Reactions Stages of Antigen–Antibody Reactions Types of Antigen–Antibody Reactions 90 90 91 92 92 92 92 92 93 93 94 94 94 100 101 101 101 102 102         Introduction The Complement System Activation of Complement Regulation of Complement System Biological Effects of Complement Deficiency of Complement Biosynthesis of Complement Quantitation of Complement 16. Structure and Function of Immune System      Prelims.indd xii Introduction Lymphoid Tissues and Organs Lymphatic Circulatory System Cells of the Lymphoreticular System Major Histocompatibility Complex 116 116 116 116 119 120 121 121 121 134 134 138 142 142 143 Introduction Primary Immunodeficiencies Secondary Immunodeficiencies 143 143 147 19. Hypersensitivity            149 Introduction Type I (Anaphylactic) Hypersensitivity Type II (Cytotoxic) Hypersensitivity Type III (Immune-Complex) Hypersensitivity Type IV Delayed (Cell-Mediated) Hypersensitivity Type V (Stimulatory Type) Hypersensitivity 20. Autoimmunity Introduction Tolerance Pathogenesis of Autoimmunity Animal Models of Autoimmunity Autoimmune Diseases 149 149 152 153 154 155 156 156 156 156 158 158 21. Immunology of Transplantation and Malignancy 161 Introduction Transplant Immunology Tumor Immunology 161 161 164    15. Complement System Introduction Humoral Immunity Cell-Mediated Immunity Transfer Factor Immunological Tolerance 18. Immunodeficiency  12. Antigen 134 22. Immunohematology       Introduction ABO Blood Group System Rh Blood Group System Blood Transfusion Hemolytic Disease of Newborn (Erythroblastosis Fetalis) ABO Hemolytic Diseases SECTION III 167 167 167 168 168 169 170 BACTERIOLOGY 122 122 122 124 124 131 23. Staphylococcus     Introduction Staphylococcus Staphylococcus aureus Coagulase-Negative Staphylococci 173 173 173 173 181 5/15/2012 10:20:03 AM CONTENTS    Micrococcus Planococcus Stomatococcus 182 182 182     24. Streptococcus and Enterococcus        Introduction Streptococcus Streptococcus pyogenes Streptococcus agalactiae Other Hemolytic Streptococci Viridans Streptococci Enterococcus 183 183 183 184 191 191 192 192       32. Salmonella  25. Pneumococcus   Introduction Streptococcus pneumoniae 26. Neisseria     Introduction Neisseria gonorrhoeae Neisseria meningitidis Other Neisseria Species 194 194 194 201 201 201 207 211        Introduction Corynebacterium diphtheriae Other Pathogenic Corynebacterium Species Other Coryneform Genera 213 213 213 220 221      Introduction Bacillus anthracis Anthracoid Bacilli 29. Clostridium       Introduction Clostridium Clostridium perfringens Clostridium tetani Clostridium botulinum Clostridium difficile 30. Nonsporing Anaerobes    Introduction Anaerobic Cocci Anaerobic Bacilli 31. Coliforms    Prelims.indd xiii Introduction Escherichia Escherichia coli 222 222 222 229 231 231 231 232 237 242 245 247 247 247 247 251 251 252 252 Introduction Shigella 34. Yersinia    Introduction Yersinia pestis Yersinia enterocolitica Yersinia pseudotuberculosis 35. Vibrio, Aeromonas, and Plesiomonas  28. Bacillus Introduction Salmonella Salmonella Gastroenteritis Salmonella Bacteremia 33. Shigella  27. Corynebacterium Edwardsiella Citrobacter Klebsiella Enterobacter Hafnia Serratia Proteus Morganella Providencia Erwinia        Introduction Vibrio cholerae Noncholera Vibrios Vibrio mimicus Halophilic Vibrios Other Vibrio Species Aeromonas Plesiomonas 36. Campylobacter and Helicobacter      Introduction Campylobacter Helicobacter Helicobacter pylori Other Helicobacter Species 37. Pseudomonas, Burkholderia, and Moraxella       Introduction Pseudomonas Pseudomonas aeruginosa Other Pseudomonas Species Burkholderia Moraxella xiii 261 261 261 263 264 264 265 267 268 268 269 269 269 280 280 281 281 281 286 286 286 291 292 294 294 294 302 302 302 303 304 304 305 305 305 308 308 311 313 313 313 313 318 318 319 5/15/2012 10:20:03 AM xiv CONTENTS 38. Haemophilus, Pasteurella, and Actinobacillus        Introduction Haemophilus Haemophilus influenzae Other Haemophilus Species Pasteurella Actinobacillus HACEK Group of Bacteria 45. Mycoplasma and Ureaplasma 321 321 321 321 326 327 327 328      46. Actinomycetes  39. Bordetella and Francisella 330         Introduction Bordetella Bordetella pertussis Bordetella parapertussis Bordetella bronchiseptica Francisella tularensis 40. Brucella   Introduction Brucella 41. Mycobacterium tuberculosis   Introduction Mycobacterium tuberculosis 330 330 330 334 335 335 338 338 338 345 345 346       358 Introduction Photochromogens Scotochromogens Nonphotochromogens Rapid Growers 358 358 359 359 360      43. Mycobacterium leprae and Mycobacterium lepraemurium          Introduction Mycobacterium leprae Mycobacterium lepraemurium         362 362 362 370            Prelims.indd xiv Introduction Treponema Treponema pallidum Nonvenereal Treponematosis Nonpathogenic Treponemes Borrelia Borrelia recurrentis Borrelia vincenti Borrelia burgdorferi Leptospira Leptospira interrogans Complex Introduction Listeria monocytogenes Erysipelothrix rhusiopathiae Alcaligenes faecalis Chromobacterium violaceum Flavobacterium meningosepticum Calymmatobacterium Streptobacillus and Spirillum Streptobacillus moniliformis Spirillum minus Legionella Legionella pneumophila Bartonella Capnocytophaga Gardnerella vaginalis 48. Rickettsia, Orientia, Ehrlichia, and Coxiella   44. Treponema, Borrelia, and Leptospira Introduction Actinomyces Nocardia Rhodococcus Gordonia and Tsukamurella Thermophilic Actinomyces Tropheryma whippelii Dermatophilus Oerskovia 47. Miscellaneous Bacteria  42. Nontuberculous Mycobacteria Introduction Mycoplasma pneumoniae Genital Mycoplasma Species Ureaplasma urealyticum Atypical Pneumonia 371  371 371 371 377 378 378 378 381 381 381 382            Introduction Genus Rickettsia Typhus Fever Group Rickettsia prowazekii Rickettsia typhi Spotted Fever Group Rickettsia rickettsiae Other Rickettsial Species in the Spotted Fever Group Rickettsia akari Genus Orientia Orientia tsutsugamushi Genus Ehrlichia Genus Coxiella Coxiella burnetii 386 386 386 391 391 392 393 393 393 395 397 398 398 398 398 398 399 399 399 400 400 400 400 400 401 401 401 401 402 404 406 406 407 407 407 408 408 410 411 411 413 413 413 413 414 415 416 5/15/2012 10:20:03 AM CONTENTS 49. Chlamydia and Chlamydophila 418 Introduction Chlamydia Chlamydia trachomatis Chlamydophila Chlamydophila pneumoniae Chlamydophila psittaci 418 418 419 423 424 424       56. Papovaviruses    57. Herpesviruses   SECTION IV VIROLOGY   50. General Properties of Viruses      Introduction Morphology of Viruses Replication of Viruses Viral Genetics Nomenclature and Taxonomy of Viruses 51. Pathogenesis of Viral Infections       Introduction Stages of Viral Infections Viral Pathogenesis at the Cellular Level Host Response to Viral Infections Clinical Manifestations of Viral Diseases Epidemiology 52. Antiviral Agents    Introduction Mechanism of Action of Antiviral Drugs Classification of Antiviral Drugs 429 429 429 433 435 437 440 440 440 442 445 446 447 449 449 449 449      453 Introduction Methods of Laboratory Diagnosis 453 453   54. Bacteriophages      Introduction Morphology Life Cycle Uses of Bacteriophages Bacteriophage Typing 460 460 460 460 462 462              Prelims.indd xv Introduction Variola (Smallpox) Virus Vaccinia Virus Monkeypox Buffalopox Cowpox Orf Molluscum Contagiosum Tanapox Yabapox 463 463 463 466 467 467 467 467 468 468 468 Introduction Adenovirus Adeno-Associated Viruses 59. Parvoviruses   Introduction Parvovirus B19 60. Picornaviruses        Introduction Enteroviruses Poliovirus Coxsackieviruses Echoviruses Other Enteroviruses Rhinoviruses Hepatitis A Virus 61. Orthomyxoviruses   Introduction Influenza Viruses 62. Paramyxoviruses  55. Poxviruses Introduction Herpes Simplex Virus Herpesvirus Simiae: B Virus Varicella Zoster Virus Epstein–Barr Virus Cytomegalovirus Human Herpesvirus 6 Human Herpesvirus 7 Human Herpesvirus 8 58. Adenoviruses  53. Laboratory Diagnosis of Viral Diseases Introduction Human Papillomaviruses Polyomaviruses        Introduction Measles Virus Parainfluenza Virus Mumps Virus Respiratory Syncytial Virus Nipah Virus Hendra Virus Human Metapneumovirus 63. Reoviruses   Introduction Orbiviruses xv 469 469 469 471 473 473 473 479 479 481 485 487 488 488 489 489 489 492 493 493 493 496 496 496 496 501 502 503 503 504 505 505 505 514 514 514 517 519 521 522 522 522 524 524 524 5/15/2012 10:20:03 AM xvi CONTENTS    Coltiviruses Orthoreoviruses Rotavirus 64. Rhabdoviruses    Introduction Rabies Virus Rabies-Related Viruses 525 525 526 529 529 529 534    SECTION V          Introduction Important Properties of Arboviruses Roboviruses Togaviruses Flaviviruses Bunyaviridae Viruses Reoviridae Rhabdoviridae Ungrouped Arboviruses 536 536 536 537 537 539 544 546 546 546        66. Hepatitis Viruses        Introduction Hepatitis A Virus Hepatitis B Virus Hepatitis C Virus Hepatitis D Virus Hepatitis E Virus Hepatitis G Virus 67. Retroviruses      Introduction Retroviruses Human T-Lymphotropic Viruses Endogenous Retroviruses Other Oncogenic Viruses 68. Human Immunodeficiency Virus   Introduction HIV Virus 547 547 547 550 555 557 558 559 560 560 560 563 565 565 566 566 566      Introduction Slow Diseases Caused by Prions Slow Diseases Caused by Conventional Viruses in Humans Slow Diseases Caused by Conventional Viruses in Animals 70. Miscellaneous Viruses    Prelims.indd xvi Introduction Rubella Virus Norwalk Virus 578 578 578 Introduction Superficial Mycoses Cutaneous Mycoses Subcutaneous Mycosis 73. Systemic Mycoses 597 597 597 598 600 603 74. Opportunistic Fungal Infections 608                Introduction Candidiasis Candida albicans Aspergillosis Aspergillus Species Zygomycosis Pneumocystosis Pneumocystis jiroveci Penicilliosis Penicillium marneffei Pseudoallescheria boydii Infection Fusarium solani Infection SECTION VI 582 75. Normal Microbial Flora 583 583 585 593 593 593 594 595 596 603 603 604 604 605 606  582 583 593 Introduction Coccidioidomycosis Paracoccidioidomycosis Histoplasmosis Blastomycosis Cryptococcosis   69. Slow Viruses and Prions Introduction Classification of Fungi Reproduction of Fungi Pathogenesis of Fungal Infection Laboratory Diagnosis Antifungal Drugs 72. Superficial, Cutaneous, and Subcutaneous Mycoses  586 586 588 MYCOLOGY 71. Introduction to Mycology  65. Arboviruses Viral Hemorrhagic Fever Arenavirus Coronaviruses     608 608 608 610 610 612 612 613 614 614 615 615 APPLIED MICROBIOLOGY Introduction Functions of Resident Flora Factors Determining the Colonization by Microbes Normal Flora at Various Sites of the Body 619 619 619 619 620 5/15/2012 10:20:03 AM CONTENTS 76. Bacteriology of Water, Milk, and Air     Introduction Bacteriology of Water Bacteriology of Milk Bacteriology of Air 623 78. Biomedical Waste Management 623 623 626 628    Introduction Types of Biomedical Waste Waste Treatment and Disposal 79. Immunoprophylaxis 77. Nosocomial Infections      Prelims.indd xvii Introduction Factors Affecting Hospital-Acquired Infection Epidemiology of Hospital-Acquired Infection Diagnosis of Hospital-Acquired Infections Prevention and Control of Hospital-Acquired Infections 629  629   629 629 632 632    Index Introduction Active Immunization Immunization Schedule Passive Immunization Combined Active and Passive Immunization Individual Immunization xvii 634 634 634 634 637 637 637 638 638 639 639 641 5/15/2012 10:20:03 AM 1 History of Microbiology Introduction Medical microbiology is a branch of microbiology that deals with the study of microorganisms including bacteria, viruses, fungi, and parasites of medical importance that are capable of causing diseases in humans. It also includes the study of microbial pathogenesis, disease pathology, immunology, and epidemiology of diseases. Medical microbiology is among the most widely studied branches of Microbiology. It has given mankind a chance to fight the organisms that, at one point of time, were pure nemesis to us. This has also provided an in-depth knowledge and in-detail understanding of the nature of pathogens that cause disease in humans. This field of microbiology has been the precursor to the wide gamut of immunological innovations in the field of medical science. This field not only has helped to develop vaccines against many invading organisms, it has also, in a more holistic way, given mankind a second shot at life. Deadly and debilitating diseases like smallpox, polio, rabies, plague, etc. have been either eradicated or have become treatable now because of the efforts of scientists and researchers in the field of medical microbiology. Microbes are the most significant life forms sharing this planet with humans because of their pervasive presence. Depending on their food sources, microbes may have either beneficial roles in maintaining life or undesirable roles in causing human, animal, and plant diseases. These microbes cause frequent and often severe diseases, such as AIDS, cholera, tuberculosis, rabies, malaria, etc. The ubiquitous presence of microbes in large numbers have given rise to the many mutants, which in part are responsible for emerging diseases such as AIDS, Ebola hemorrhagic fever, and multidrug-resistant tuberculosis (MDRTB). Historical Background Microbial diseases have undoubtedly played a major role in historical events, such as the decline of the Roman Empire and the conquest of the New World. In 1347, plague or Black Death struck Europe with a brutal force. By 1351, about 4 years later, the plague had killed one-third of the population (about 25 million people). Over the next 80 years, the disease has struck repeatedly, eventually wiping out 75% of the European population. Some historians believe that this disaster changed European culture and prepared the way for the Renaissance. This is just an example from many such epidemics, which while being devastating in their scope spared not even the high and mighty of the times. Apart from the bubonic plague, measles (now thankfully extinct) and smallpox too played their roles as epidemic diseases causing high mortality and morbidity. The first recorded epidemic of smallpox was in the year 1350 BC in Egypt. The disease was unknown in the population of the New World until the Portuguese and Spanish explorers made their appearance. Smallpox then traveled across America, devastating the previously unexposed population. It was already known at that time that the disease spreads through the skin lesions and scabs, and that survivors of the infection were immune to reinfection on further exposure. Though adopted much later in America and Europe, the practice of inoculation or variolation, whereby people were intentionally exposed to smallpox to make them immune, was already being practiced in India, China, and Africa for centuries. Microorganisms as a Cause of Disease Among various causes, the causes suggested for the occurrence of disease were the effect of supernatural phenomena like planetary alignments and effect of bad bodily humors; the faulty environment was also implicated. Even before microorganisms were seen, some investigators suspected their existence and responsibility for disease. Among others, the Roman philosopher Lucretius (about 98–55 BC) and the physician Girolamo Fracastoro (1478–1553 AD) suggested that disease was caused by invisible living creatures. Fracastoro was much more than an author of the popular poem on syphilis. In his book “De contagione, contagiosis morbis et curatione (On Contagion, Contagious Diseases, and their Treatment),” published in 1546, he proposed the revolutionary theory that infectious diseases are transmitted from person to person by minute invisible particles. He further suggested that infections spread from person to person by minute invisible seeds, or seminaria, that are self-replicating and act on the humors of the body to cause disease. His theories were ahead of their time, and it took about 200 years for the microscope to be invented and his theories to be proved. 4 GENERAL MICROBIOLOGY Chapter 43 Section III Chapter 1 Section I Antony van Leeuwenhoek: The Microscopist The first person to observe and describe microorganisms accurately was an amateur microscopist Antony van Leeuwenhoek (1632–1723) of Delft, Holland. Leeuwenhoek earned his living as a draper and haberdasher (a dealer in men’s clothing and accessories), but spent much of his spare time constructing simple microscopes composed of double convex glass lenses held between two silver plates. His microscopes could magnify around 50–300 times. It is believed that he may have illuminated his liquid specimens by placing them between two pieces of glass and shining light on them at 45-degree angle to the specimen plane. This would have provided a form of dark-field illumination and made bacteria clearly visible. In 1673, Leeuwenhoek sent detailed letters describing his discoveries to the Royal Society of London. It is clear from his descriptions that he saw both bacteria and protozoa. But he did not evaluate these organisms as agents of disease. Theory of Spontaneous Generation There was a considerable controversy surrounding the origin of microbial pathogens. Some proposed that microorganisms originated from nonliving things by spontaneous generation even though larger organisms did not (theory of spontaneous generation). They pointed out that boiled extracts of hay or meat would give rise to microorganisms after sometime. Needham (1713–1781) on the basis of his experiments proposed that all organic matter contained a vital force that could confer the property of life to nonliving matter. Louis Pasteur: Father of Microbiology Louis Pasteur, French Microbiologist, is known as the father of medical microbiology for his immense contributions to the field of medical microbiology. He first coined the term “microbiology” for the study of organisms of microscopic size. Many of his important contributions are discussed below. ◗ Germ theory of disease Many other scientists have contributed to the theory of spontaneous generation with their experiments, but it was Louis Pasteur (1822–1895) who settled it once for all. Pasteur first filtered air through cotton and found that objects resembling plant spores had been trapped. If a piece of cotton was placed in a sterile medium after air had been filtered through it, microbial growth appeared. Next he placed nutrient solutions in flasks, heated their necks in a flame, and drew them out into a variety of curves, while keeping the ends of the necks open to the atmosphere. Pasteur then boiled the solutions for a few minutes and allowed them to cool. No growth took place even though the contents of the flasks were exposed to the air. Pasteur pointed out that no growth occurred because dust and germs had been trapped on the walls of the curved necks. If the necks were broken, growth commenced immediately. By this Pasteur proved that all life even microbes arose only from their like and not de novo ( germ theory of disease). Pasteur had not only resolved the controversy by 1861 but also had shown how to keep solutions sterile. Support for the germ theory of disease began to accumulate in the early nineteenth century. Agostino Bassi (1773–1856) first showed that a microorganism could cause disease when he demonstrated in 1835 that the silkworm disease was due to a fungal infection. He also suggested that many diseases were due to microbial infections. In 1845, MJ Berkeley proved that the great potato blight of Ireland was caused by a fungus. ◗ Pasteurization Pasteur for the first time demonstrated that he could kill many microorganisms in wine by heating and then rapidly cooling the wine, a process now called pasteurization. While developing methods for culturing microorganisms in special liquid broths, Pasteur discovered that some microorganisms require air, specifically oxygen, while others are active only in the absence of oxygen. He called these organisms as aerobic and anaerobic organisms, respectively. ◗ Vaccination In 1877, Pasteur studied anthrax, a disease mainly of cattle and sheep. He developed a vaccine using a weakened strain of the anthrax bacillus, Bacillus anthracis. He attenuated the culture of anthrax bacillus by incubation at high temperature of 42–43°C and inoculated the attenuated bacilli in the animals. He demonstrated that animals receiving inoculation of such attenuated strains developed specific protection against anthrax. The success of this concept of immunization was demonstrated by a public experiment on a farm at Pouilly-le-Fort in the year 1881. In that public demonstration, he vaccinated sheeps, goats, and cows with anthrax bacillus attenuated strains, but equal numbers of these animals were nonvaccinated. All the vaccinated as well as nonvaccinated animals were subsequently challenged with a virulent anthrax bacillus culture, after which only the vaccinated animals survived whereas nonvaccinated group of animals died of anthrax. In 1885, he also developed the first vaccine against rabies in humans that saved millions of human life worldwide. Pasteur coined the term “vaccine” to commemorate Edward Jenner who used such preparations for protection against smallpox. The Pasteur Institute, Paris and subsequently similar institutions were established in many countries of the world for the preparation of vaccines and for the study of infectious diseases. ◗ Control of silkworm disease Following his successes with the study of fermentation, Pasteur was asked by the French government to investigate the cause of pébrine disease of silkworms that was disrupting the silk industry. After several years of work, he showed that the disease was due to a protozoan parasite. The disease was controlled by raising caterpillars from eggs produced by healthy moths. HISTORY OF MICROBIOLOGY Joseph Lister: The Pioneer of Antiseptics Koch’s postulates Koch’s postulates (criteria) were useful to prove the claim that a microorganism isolated from a disease was indeed causally related to it. A microorganism was accepted as the causative agent of infectious disease, only when it satisfied all the following criteria (Fig. 1-1): 1. The microorganism must be present in every case of the disease but absent from healthy host. 2. The suspected microorganism must be isolated and grown in a pure culture from lesions of the disease. Chapter 43 Isolation of microorganism from diseased animal Growth medium Identification of microorganism by microscopy Isolated microorganism injected into healthy animal The disease is reproduced in healthy animal Microorganisms are isolated from infected animal Identical microorganisms are identified FIG. 1-1. Koch’s postulates. Section III The first direct demonstration of the role of bacteria in causing disease came from the study of anthrax by the German physician Robert Koch (1843–1910). Koch used the criteria proposed by his former teacher, Jacob Henle (1809–1885), to establish the relationship between B. anthracis and anthrax, and he published ◗ Chapter 1 Robert Koch: The Founder of Koch’s Postulates his findings in 1876 briefly describing the scientific method he followed. In this experiment, Koch injected healthy mice with a material from diseased animals, and the mice became ill. After transferring anthrax by inoculation through a series of 20 mice, he incubated a piece of spleen containing the anthrax bacillus in beef serum. The bacilli grew, reproduced, and produced spores. When the isolated bacilli or spores were injected into mice, anthrax developed. During Koch’s studies on bacterial diseases, it became necessary to isolate suspected bacterial pathogens. His criteria for proving the causal relationship between a microorganism and a specific disease are known as Koch’s postulates. Section I Indirect evidence that microorganisms are the agents of human disease came from the work of an English surgeon Joseph Lister (1827–1912) on the prevention of wound infections. Lister, impressed with Pasteur’s studies on the involvement of microorganisms in fermentation and putrefaction, developed a system of antiseptic surgery designed to prevent microorganisms from entering wounds. Instruments were heat sterilized and phenol was used on surgical dressings and at times sprayed over the surgical area. The approach was remarkably successful and transformed surgery after Lister published his findings in 1867. It also provided strong indirect evidence for the role of microorganisms in disease because phenol, which killed bacteria, also prevented wound infections. 5 6 Chapter 1 Section I 3. The isolated organism, in pure culture, when inoculated in suitable laboratory animals should produce a similar disease. 4. The same microorganism must be isolated again in pure culture from the lesions produced in experimental animals. The specific antibodies to the bacterium should be demonstrable in the serum of patient suffering from the disease. This was an additional criterion that was introduced subsequently. Most of the human bacterial pathogens satisfy Koch’s postulates except for those of Mycobacterium leprae and Treponema pallidum, the causative agent of leprosy and syphilis, respectively. Both these bacteria are yet to be grown in cell-free culture media. Section III ◗ Chapter 43 GENERAL MICROBIOLOGY Discovery of important bacterial agents causing human diseases Scientist Bacteria Year Hansen Mycobacterium leprae 1874 Koch Bacillus anthracis 1876 Neisser Neisseria gonorrhoeae 1879 Ogston Staphylococcus aureus 1880 Loeffler Corynebacterium diphtheriae 1884 Fraenkel Streptococcus pneumoniae 1886 Weichselbaum Neisseria meningitidis 1887 Bruce Brucella melitensis 1887 Kitasato Clostridium tetani 1889 Yersin Yersinia pestis 1890 Solid medium for culture of bacteria Koch pioneered the use of agar as a base for culture media. He developed the pour plate method and was the first to use solid culture media for culture of bacteria. This development made possible the isolation of pure cultures that contained only one type of bacterium and directly stimulated progress in all areas of bacteriology. Koch also developed media suitable for growing bacteria isolated from the body. Because of their similarity to body fluids, meat extracts and protein digests were used as nutrient sources. The result was the development of nutrient broth and nutrient agar media that are still in wide use today. By 1882, Koch had used these techniques to isolate the bacillus that caused tuberculosis in humans. Koch also discovered that cholera was caused by Vibrio cholerae. He invented the hot air oven and steam sterilizer, and also introduced methods to find out the efficacy of antiseptics. There followed a golden age of about 30–40 years in which most of the major bacterial pathogens were isolated. ◗ TABLE 1-1 Koch’s phenomenon Koch’s phenomenon is a hypersensitivity reaction against tuberculosis bacilli demonstrated in guinea pigs. This was first demonstrated by Koch, who showed that guinea pigs already infected with tubercle bacillus, on challenge with tubercle bacillus or its protein, developed an exaggerated inflammatory response. Self-Experimentation Studies To study diseases in a more elaborate and controlled fashion, there were a few dedicated researchers who went to the extremes of self-experimentation. The discovery that hookworm infestation spread by fecal–oral route was first demonstrated by Arthur Loos in 1898. This was known during his attempts at studying Strongyloides stercoralis by swallowing its larvae and accidentally swallowing a fecal inoculum with hookworm eggs instead! These attempts did not always have happy and productive endings as is illustrated by the case of Daniel Carrion (1858–1895), a medical student in Lima, Peru. He managed to prove that the same organism (later identified to be Bartonella bacilliformis) caused a chronic skin lesion called verruga peruana and another serious disease called Oroya fever. This he did by inoculating himself with material from the warts of the skin lesion. He did develop Oroya fever as he had hypothesized, but the experiment costed him his life when he succumbed to the disease. In the subsequent 50 years, numerous microorganisms were identified as the causative agents of important human diseases and their discovery elucidated (Table 1-1). Study of Viruses In 1892, Dmitri Ivanovsky, a Russian scientist working in St. Petersburg, demonstrated that the sap of leaves infected with tobacco mosaic disease retains its infectious properties even after filtration through Chamberland filter candles. This was an important observation, because it provided an operational definition of viruses and also an experimental technique by which an agent could be considered as a virus. Beijerinck, a Dutch soil microbiologist, showed that the filtered sap could be diluted and then regain its strength after replication in living and growing tissue of the plant. The agent could reproduce itself (which meant that it was not a toxin) but only in living tissues, not in the cell-free sap of the plant. This explained the failure to culture the pathogen outside its host. All these observations contributed immensely to the discovery of an organism smaller than bacteria (a filterable agent) that is not observable in the light microscope and is able to reproduce itself only in living cells or tissues. Beijerinck called this agent a contagium vivum fluidum, or a contagious living liquid. The concept of contagium vivum fluidum or a contagious living liquid began a 25-year debate about the nature of viruses; whether they were liquids or particles? This conflict was laid to rest when d’Herelle developed the plaque assay in 1917 and subsequent development of electron microscopy by HISTORY OF MICROBIOLOGY The earliest written reference to the phenomenon of immunity can be traced back to Thucydides, the great historian of the Peloponnesian War. Describing a plague in Athens, in 430 BC, he wrote that only those who had recovered from the plague could nurse the sick because they would not contract the disease a second time. The earliest known smallpox inoculation took place in China, perhaps as early as the fifth century AD. The Chinese method was reported to the Royal Society by an English merchant, John Lister, during early 1900s. A Jesuit priest, Father d’Entrecolles, provided details of the method, which he said was to collect scabs from the pustules and blow a powder made from them into an infant’s nose. The scabs or a thread coated with the pus could be stored, but the operation was usually done face-to-face with a sick patient. The same method was used in Japan beginning in 1747. In precolonial India, a tika or dot would be made on a child, usually on the sole of the The experimental work of Emil von Behring and Shibasaburo Kitasato in 1890 gave the first insight into the mechanism of immunity. They demonstrated that serum contained elements that protected against infections thus laying the foundation for the identification of humoral immunity. In recognition of this work, von Behring received the Nobel Prize in Medicine in 1901. In 1884, even before the discovery that a serum component could transfer immunity, Elie Metchnikoff demonstrated that cells also contribute to the immune state of an animal. He observed that certain white blood cells, which he termed phagocytes, were able to ingest (phagocytose) microorganisms and other foreign material. Noting that these phagocytic cells were more active in animals that had been immunized, Metchnikoff hypothesized that cells, rather than serum components, were the major effector of immunity. The active phagocytic cells identified by Metchnikoff were most likely blood monocytes and neutrophils. Specificity of the Antibody Molecule One of the greatest enigmas facing early immunologists was the specificity of the antibody molecule for foreign material or antigen. Following theories were proposed to explain this mechanism of specificity: 1. The selective theory: The earliest conception of the selective theory dates to Paul Ehrlich in 1900. In the 1930s and 1940s, the selective theory was challenged by various instructional theories, in which antigen played a central role in determining the specificity of the antibody molecule. 2. The instructional theory: According to the instructional theories, a particular antigen would serve as a template around which the antibody would fold. This concept was first postulated by Friedrich Breinl and Felix Haurowitz in the 1930s and redefined in the 1940s in terms of protein folding by Linus Pauling. Chapter 43 Phenomenon of Immunity Mechanisms of Immunity Section III Twort and d’Herelle (1915) independently observed a lytic phenomenon in bacterial cultures, which they attributed to viruses. d’Herelle named these viruses as bacteriophages. He developed the use of limiting dilutions with the plaque assay to titer the virus preparation. He suggested that the appearance of plaques in the plaque assay show the viruses to be particulate, or corpuscular. d’Herelle also demonstrated that the attachment (adsorption) of the virus to the host cell is the first step in the pathogenesis of a virus infection. The attachment of a virus occurred only when bacteria sensitive to the virus were mixed with it, demonstrating the host range specificity of a virus at the adsorption step. He described the process of cell lysis and subsequently the release of infectious virus particles. He developed many other techniques that are still used in virology. d’Herelle was in many ways one of the founders of the principles of modern virology. Chapter 1 d’Herelle and Twort: Founders of the Principles of Modern Virology foot, by traditional tikadars who were invited into home (this professional niche was later blacklisted by colonial-era medical practitioners). The method was significantly improved by the English physician Edward Jenner in 1798. Jenner was intrigued by the fact that milkmaids who had contracted the mild disease cowpox were subsequently immune to smallpox, a disfiguring and often fatal disease. He believed that introducing fluid from a cowpox pustule into people (i.e., inoculating them) might protect them from smallpox. To test this idea, he inoculated an 8-year-old boy with fluid from a cowpox pustule and later intentionally infected the child with smallpox. As predicted, the child did not develop smallpox. Pasteur followed this up with development of vaccines for chicken cholera, anthrax, and rabies. Although Pasteur proved that vaccination worked, but he did not understand how. Section I Ruska (1934), when the first electron micrographs of tobacco mosaic virus (TMV) were taken in 1939. The viruses were accepted as particles. Loeffler and Frosch (1898) described and isolated the first filterable agent from animals, the foot-and-mouth disease virus of cattle. Walter Reed and his team in Cuba (1902) recognized the first human filterable virus, yellow fever virus. Landsteiner and Popper (1909) demonstrated that poliomyelitis was caused by a filterable virus and also successfully transmitted the infection to monkeys. Goodpasture (1930) used chick embryos for cultivation of viruses. The term virus (taken from the Latin for slimy liquid or poison) was at that time used interchangeably for any infectious agent and so was applied to TMV and then further to all agents of the class. 7 Chapter 1 Section I 8 GENERAL MICROBIOLOGY 3. The clonal selection theory: The instructional theories were formally disproved in the 1960s, during which information was beginning to appear regarding the structure of DNA, RNA, and protein. These information offered new insights into the vexing problem of how an individual could make antibodies against almost anything. In the 1950s, selective theories resurfaced as a result of new experimental data and through the pioneering contributions of Niels Jerne, David Talmadge, and F Macfarlane Burnet, who refined into a theory that came to be known as the clonal selection theory. Chapter 43 Section III Chemotherapeutic Agents Until the 1930s, there had been no chemical treatment available to fight bacterial infections in general. Prevention was the main means of protecting patients, and an obsession with the threat of germs and the moral responsibility to avoid infection was deeply instilled in Western cultures. At the same time, there were repeated hopes for a wonder drug. Louis Pasteur’s pupil Paul Vuillemin coined the term “antibiosis” in 1889 to denote a process by which life could be used to destroy life. Paul Ehrlich was an exceptionally gifted histological chemist and invented the precursor technique to Gram-staining of bacteria. He demonstrated that dyes react specifically with various components of blood cells and the cells of other tissues. He began to test the dyes for therapeutic properties to determine whether they could kill the pathogenic microbes. He developed Salvarsan, an arsenical compound in 1909. The compound known as “magic bullet” was capable of destroying T. pallidum, the causative agent of syphilis. This treatment proved effective against syphilis. This work was of epochal importance, stimulating research that led to the development of sulfa drugs, penicillin, and other antibiotics. He, therefore, is known as the father of chemotherapy. Antibiotics The word “antibiotic” did not follow immediately, but the drug pyocyanase, a weakly effective antibiotic, was marketed from TABLE 1-2 Year the late nineteenth century into the 1930s. Early in the 1920s, there was an excitement about the potential of the newly identified phage viruses to kill bacteria. The discovery of an antibacterial factor in the exudates of the fungus Penicillium by Sir Alexander Fleming at St. Mary’s Hospital in 1928 was therefore not totally unexpected. He accidentally discovered that a substance produced by the fungus destroyed the pyogenic bacteria, staphylococci. This initiated the beginning of the antibiotics era. Other similar antibiotics were discovered in rapid succession. The sulfonamide drugs discovered subsequently offered cures for a wide range of bacterial infections. Introduction of antibiotics in medicine raised a lot of expectations among both doctors and patients. Certain terrifying infectious diseases (e.g., rheumatic fever, syphilis, pneumonia, tuberculosis) and unpleasant skin conditions (e.g., carbuncles) became easily treatable, and their disappearance appeared to be certain. Surgeons could risk more dangerous operations and the use of drugs that compromised immune systems. Patients who had once turned to many kinds of alternative medicine, or refused treatment, now entrusted themselves to antibiotics. Medical uses of antibiotics on human patients were harder to limit—even though, from the 1940s, the fear that public enthusiasm would promote the selection of resistant strains did lead—to some constraints. In Britain, the Penicillin Act of 1948 was explicitly intended to, for the first time, limit through prescription the public’s access to a drug that was not a poison. Nonetheless, during the 1950s, a penicillin-resistant strain of staphylococcus aureus termed 80/81 infected hospitals and maternity wards across the world. Newborn babies in hospital crèches were infected and postoperative infections proved common. By the late 1990s, although many variants of older drugs had been produced, new families of antibiotics were, however, not being discovered. However, a sustained effort is now being made to develop more effective antibiotics to treat a wide range of infections. The field of medical microbiology has been enriched by contributions of many eminent microbiologists both in the past and in the recent times. The work of many of them has been recognized worldwide by the award of Nobel Prizes (Table 1-2). List of recent Nobel Prize winners Name/Names of scientists Research done 1993 Kary Mullis Polymerase chain reaction 1996 Peter C Doherty and Rolf M Zinkernagel Cell-mediated immune defenses 1997 Stanley B Prusiner Prion discovery 2005 Barry J Marshall, J Robin Warren Discovery of Helicobacter pylori and its role in gastritis and peptic ulcer disease 2008 Herald Zur Hausen Discovery of Human Papilloma viruses causing cervical cancer 2008 Francoise Barre Sinoussi, Luc Montagnier Discovery of Human immunodeficiency virus 2011 Ralph M. Steinman Discovery of dendritic cell and its role in adaptive immune response 2011 Bruce A. Beutler, Jules A. Hoffmann Discoveries concerning activation of innate immunity 432 Morphology and Physiology of Bacteria Mycobacterium Leprae Introduction All living beings can be classified into three kingdoms: Plant, Animal, and Protista. Microorganisms are a heterogeneous group of several distinct living structures of microscopic size, classified under the kingdom Protista. The kingdom Protista includes unicellular organisms, such as bacteria, fungi, protozoa, and algae. Based on the differences in cellular organization and biochemistry, the kingdom Protista has been divided into three groups: prokaryotes, eukaryotes, and the most recently described archaebacteria. 1. Prokaryotes: Bacteria and blue green algae are prokaryotes. Bacteria are unicellular free living organisms having both DNA and RNA. They are capable of performing all essential processes of life, e.g., growth, reproduction, and metabolism. They do not show any true branching except Actinomycetales, the higher bacteria. Bacteria lack chlorophyll unlike blue green algae, which contain chlorophyll. 2. Eukaryotes: Fungi, algae other than blue green, protozoa, and slime moulds are eukaryotes. 3. Archaebacteria: These are more closely related to eukaryotes than prokaryotes. They however do not include any human pathogens. Differences between prokaryotes and eukaryotes have been summarized in Table 2-1. Size of Bacteria Bacteria are microscopic and very small in size. The size of bacteria is measured in units of length called microns. A micron (micrometer, ␮m) is the unit of measurement used in bacteriology. 1 micron (␮m) ⫽ 1/1000 millimeter (mm). 1 nanometer (nm) ⫽ 1/1000 micron (␮m). 1 Angstrom unit (A⬚) ⫽ 1/10 nm (nanometer). Bacteria of medical importance measure 2–5 ␮m (length) ⫻ 0.2–1.5 ␮m (width). Microscopy Microscopy is an important component of diagnostic microbiology. Bacteria being very small cannot be visualized by the naked eye, because the limit of resolution with the unaided eye is about 200 microns. So, the study of bacteria requires the use of microscopes. A microscope is an instrument that uses one or more lenses to produce a magnified image of an object that is invisible to the unaided eye. Types of Microscopy The following types of microscopy are used for the examination of microorganisms including bacteria: ◗ Light microscopy Light microscopy, as the name suggests, uses natural or artificial transmitted light as the source of light. Resolving power of microscope is an important component of light microscopy. It is the ability of the lens system to distinguish two closely placed objects as distinct and separate entities. It is dependent on the wavelength of light used to illuminate the object and on the numerical aperture of the microscope. It is about half of the wavelength of light being used. For example, the smallest particle which can be resolved by yellow light with a wavelength of 0.4 ␮m is about 0.2 ␮m. Proper use of condenser that focuses light on the plane of the object facilitates optimization of the resolving power of the microscope. Resolving power of the microscope is enhanced further by adjusting the medium through which light passes between the object and objective lens. The use of immersion oil, whose refractive index is similar to that of the glass, improves the resolution of the microscope. The numerical aperture of the microscope is defined as the light gathering power of the microscope. Different types of light microscopy include (a) bright-field microscopy, (b) dark-ground microscopy, (c) phasecontrast microscopy, and (d) interference microscopy. 1. Bright-field microscopy: Bright-field microscopy (always referred to as ordinary light microscopy) is the most common form of light microscopy that uses a compound light microscope. A compound light microscope primarily consists of a compound lens system that contains a number of objective lenses, such as lenses of low power (⫻10), high power (⫻40), and oil immersion (⫻100). It also contains a fixed ocular (eye piece) lens, usually of ⫻10 or ⫻5. Final magnification of an object is the multiplication of lens power of the objective with that of the eye piece (Fig. 2-1). The bright-field microscopy has many uses. Chapter 2 Section I 10 GENERAL MICROBIOLOGY TABLE 2-1 Structure Eye Differences between prokaryotic and eukaryotic cells Prokaryotes Eukaryotes Ocular lens Nucleus Nuclear membrane Absent Present Nucleolus Absent Present Chromosome One, circular More than one, linear Location Free in the cytoplasm, Contained in attached to mesosomes membrane bound structure Replication Binary fission Mitotic division Extrachromosomal DNA Plasmid Inside the mitochondria Objective lens Specimen Condenser lens Section III Cytoplasm Cytoplasmic organelles like mitochondria, Golgi apparatus, and endoplasmic reticulum Absent Present Cytoplasmic streaming Absent Present Lysosomes Absent Present Ribosomes—protein production site 70S (50S ⫹ 30S), free in cytoplasm or bound to cell membrane 80S (60S ⫹ 40S), attached to rough endoplasmic reticulum Source of light FIG. 2-1. Principle of compound light microscope. Eye Ocular lens Chapter 43 Chemical composition Cell wall Present Absent, except for fungi that have chitinous cell wall Sterols Absent Present Muramic acid Present Absent Energy production site Electron transport chain located in the cell membrane Within membrane bound mitochondria ■ ■ ■ It may be used to examine either wet films or “hanging drop” for demonstration of the motility of flagellated bacteria (e.g., Escherichia coli, Pseudomonas aeruginosa, etc.) and protozoa (e.g., Trichomonas vaginalis, Giardia intestinalis, etc.). The wet preparation is also useful for demonstration of microorganisms in urine or feces, and also for detection of fungi in the skin. It is useful for demonstration of the structural details. It is also useful for measuring approximate size of the bacteria, fungi, and protozoa in stained preparations. 2. Dark-ground microscopy: The dark-ground microscopy makes use of dark-ground microscope, a special type of compound light microscope. The dark-field condenser with a central circular stop, which illuminates the object with a cone of light, is the most essential part of the dark-ground microscope. This microscope uses reflected light instead of transmitted light used in the ordinary light microscope (Fig. 2-2). It prevents light from falling directly on the objective lens. Light rays falling on the object are reflected or scattered onto the objective lens with the result that the microorganisms Objective lens Only light reflected by specimen enters the objective lens Specimen Condenser lens Opaque disc Source of light FIG. 2-2. Principle of dark-ground microscopy. appear brightly stained against a dark background. The darkground microscopy has following uses: ■ ■ It is useful for demonstration of very thin bacteria (such as, spirochetes) not visible under ordinary illumination, since the reflection of the light makes them appear larger. This is a frequently used method for rapid demonstration of Treponema pallidum in clinical specimens. It is also useful for demonstration of motility of flagellated bacteria and protozoa. 3. Phase-contrast microscopy: Phase-contrast microscopy makes use of a specific optical system that converts differences MORPHOLOGY AND PHYSIOLOGY OF BACTERIA Eye Observer Fluorescent light Light wave splitting mirror Diffraction plate Section I Ocular lens Undiffracted light unaltered by specimen 11 Exciter filter Refracted or diffracted light by specimen Light source Excitation light Specimen Chapter 2 Objective lens Excitation light Condenser lens Annular diaphragm Fluorochrome stained microorganisms present in the specimen Source of light FIG. 2-3. Principle of phase-contrast microscopy. ■ It is immensely useful for examination of living microorganisms particularly protozoa (e.g., T. vaginalis, Entamoeba histolytica, etc.) It is useful for examining the internal structures of a living cell by improving the contrast and differentiating structures within the cell that differs in their thickness and refractive index. 4. Interference microscopy: This is another specialized application of light microscopy used for demonstrating cell organelles. It is also useful for quantitative measurement of the chemical constituents of the cells, such as proteins, lipids, and nucleic acids. ◗ Fluorescence microscopy Fluorescence microscopy is based on the principle that the specimens stained with fluorescent dye when exposed to ultraviolet light result in emission of longer wavelength of light (i.e., visible light) (Fig. 2-4). The bacteria stained with fluorescent dye appear as a brightly glowing object against a dark background. Fluorescence microscopy needs a fluorescence microscope fitted with an ultraviolet light source. Auramine O, acridine orange, and rhodamine are fluorescent dyes used to visualize bacteria. The resolving power of a fluorescence microscope is increased due to the short wavelength of ultraviolet light. Auramine O, acridine orange, and rhodamine are fluorescent dyes used to visualize bacteria. Fluorescence microscopy is widely used in diagnostic microbiology in the following ways: ■ ◗ It is used for direct demonstration of antigen of a pathogen in clinical specimens by direct fluorescent antibody test (e.g., direct detection of Neisseria gonorrhoeae, Corynebacterium diphtheriae, etc. directly in clinical specimens). It is also used for the estimation of antibodies in the serum by indirect fluorescent antibody test (IFA) (e.g., IFA in leptospirosis, syphilis, brucellosis, etc.). Electron microscopy Electron microscopy utilizes a beam of electrons instead of a beam of light used in the light microscopy. The electron beam is focused by electromagnets, analogous to the lenses used in the light microscopy. The object to be examined is kept on the path of the beam that scatters the electrons and produces an image which is focused on a screen (Fig. 2-5). The resolving power of the electron microscope is extremely high, theoretically 100,000 times than that of a light microscope. This is because the electron microscope uses electrons whose wavelength is approximately 0.005 nm as compared to 5000 nm wavelength of the visible light. As mentioned earlier, the resolving power is half of the wavelength. In practice, the resolving power of the electron microscope, however, is about 0.1 nm. There have been many developments in electron microscopy that include (a) shadow casting, (b) scanning electron microscopy, (c) immunoelectron microscopy, and (d) freeze-etching, etc. ■ Shadow casting is an important technique that is carried out by depositing a thin layer of platinum or other metal on the microorganism to be examined. This platinum-coated organism, on bombardment with electron beams, scatters the electron and produces an image that is focused on a fluorescent screen. Chapter 43 ■ ■ Principle of fluorescence microscopy. Section III FIG. 2-4. in phase in an organism into differences in intensity of light thereby producing light and dark contrast in the image (Fig. 2-3). The optical system includes a special condenser and objective lens which can be fitted to an ordinary light microscope to convert it into a phase-contrast microscope. The phase-contrast microscopy has following uses: GENERAL MICROBIOLOGY Section I 12 V A C U U M Cathode ray tube for viewing Chapter 2 Cathode ray tube for photography Scanning circuit VACUUM FIG. 2-5. Section III Electron gun Condenser lenses Scanning coil Primary electrons Photo multiplier Chapter 43 TABLE 2-2 ■ ■ ■ Detector Secondary electrons Specimen Specimen holder Principle of electron microscopy. Scanning electron microscopy is another development that provides a three-dimensional image of the object as well as high resolution. Immunoelectron microscopy is a method to enhance sensitivity and specificity by reacting the specimen with specific antiviral antibody that results in clumping of viral particles. In this method also, antibody may be conjugated with gold to visualize and determine the location of specific antigenic determinants in a specimen. Freeze-etching is the method by which live organisms can be visualized unlike in traditional methods of electron microscopy in which living cells cannot be examined. This method is useful for the study of cellular ultrastructure of the microorganisms in the living state. This method is based on rapid cooling of specimens by deep-freezing in liquid gas and the subsequent formation of carbon platinum replica of the specimen. Electron microscope is widely used for: ■ ■ Rapid detection of viruses directly in clinical specimens. This is especially useful for detection of noncultivable viruses. Ultrastructural study of various microorganisms. Differences between electron microscope and light microscope are summarized in Table 2-2. ◗ Newer microscopic methods These include the following: ■ ■ Confocal microscopy: This is useful to obtain high resolution images and for three dimensional reconstruction of biological models. Scanning probe microscopy: This measures surface features by moving a sharp probe over the object’s surface. There are two types of scanning probe microscope: (a) scanning tunneling microscope and (b) atomic force microscope. Differences between electron microscope and light microscope Characteristics Light microscope Electron microscope Source Visible light Electron beam Medium of transmission Air High vacuum Nature of lens Glass Electromagnet Focusing mechanism Lens position is adjusted mechanically Current to the magnetic lens is adjusted Changing the magnification Switch the objective lens Adjust the current to the magnetic lens Source of contrast Differential light absorption Scattering of electrons Specimen mount Glass slide Metal grid Best resolution 0.2 ␮m 0.5 nm Highest practical magnification 1000–1500 Over 100,000 Affordability Cheaper Expensive Study of Bacteria Bacteria can be studied either in unstained (wet mount) or stained preparation. ■ ■ The wet mount preparation is useful for demonstration of motility of bacteria by light microscopy or demonstration of spirochetes by dark-ground microscopy. Stained preparations are used to demonstrate structural details of the bacteria by producing color contrast. Staining Methods The common staining techniques used in diagnostic microbiology are discussed below: ◗ Simple stains Methylene blue and basic fuchsin are the simple stains that provide color contrast but impart the same color to all the bacteria in a smear. ◗ Negative staining Bacteria are mixed with dyes, such as Indian ink that produces a uniform dark-colored background against which the unstained organisms stand out in contrast. This is used for demonstration of bacterial capsule that are usually not stained by simple stains. India Ink method for demonstration of the fungus Cryptococcus neoformans is a common example. ◗ Impregnation stains Cells and structures that are too thin to be visualized by the light microscope can be rendered visible by impregnation of silver on their surface. Silver impregnation method is a common method used for staining spirochetes, such as T. pallidum, Leptospira, Borrelia, etc. MORPHOLOGY AND PHYSIOLOGY OF BACTERIA ◗ Differential stains Key Points Gram staining is an essential procedure that is used in the identification of bacteria. The stain differentiates bacteria into two broad groups: ■ There are certain groups of bacteria, such as Mycobacterium tuberculosis and Mycobacterium leprae, that cannot be considered typical Gram-negative or Gram-positive bacteria. This is because these bacteria either do not take up the Gram stain or they have a different type of envelope. These mycobacteria possess a waxy envelope containing complex glycolipids that make them impervious to the Gram stain. Gram stain also provides useful information on the structure of bacterial cell envelope. Acid-fast stain: The acid-fast stain was discovered by Ehrlich, who found that after staining with aniline dyes, tubercle bacilli resist decolorization with acid. The method, subsequently, was modified by Ziehl and Neelsen, hence is widely known as Ziehl– Neelsen (ZN) stain. M. tuberculosis, M. leprae, Nocardia, Actinomyces, and Oocysts of intestinal coccidian parasites (such as Cryptosporidium, Cyclospora, Isospora, etc.). Albert’s stain: Albert’s stain is used for staining the volutin granules of C. diphtheriae. These granules have an affinity for basic dyes and are called metachromatic granules. These granules are stained bluish-black against green protoplasm on staining by Albert’s stain. Shape of Bacteria Depending on their shape, bacteria are classified into several types (Fig. 2-6): 1. Cocci: The cocci (kokkos, berry) are oval or spherical cells. These may be arranged in pairs (e.g., pneumococci, meningococci, and gonococci), tetrads (micrococci), chains (e.g., streptococci), and clusters (e.g., staphylococci). 2. Bacilli: The bacilli (bacillus, rod) are rod shaped. These bacilli may show either of the following arrangement: (a) Coccobacilli: Length of the bacteria is approximately the same as its width, e.g., Brucella. (b) Streptobacilli: These are arranged in chains, e.g., Streptobacillus. (c) Comma shaped: They exhibit curved appearance, e.g., Vibrio. (d) Spirilla: They exhibit rigid spiral forms, e.g., Spirillum. 3. Spirochetes: Spirochetes (spira, coil; chaite, hair) are slender, flexuous spiral forms, e.g., Treponema. 4. Actinomycetes: Actinomycetes (actin, ray; mykes, fungus) are branching filamentous bacteria resembling fungi. They possess a rigid cell wall. Coccus Coccobacillus Bacillus Spirochete Fusiform bacillus Vibrio Key Points Acid fast staining method consists of following methods: ■ ■ ■ Fixed smears are first stained by a strong carbol fuchsin with the application of heat. Heating facilitates entry of phenolic carbol fuchsin stain into the bacteria. It is then decolorized with 5–20% (depending on the bacteria to be stained) sulfuric acid. It is then counterstained with a contrasting dye, such as methylene blue. The acid-fast bacilli (AFB) retain the red color of carbol fuchsin and appear bright red in stained smears. Pus cells and epithelial cells present in the smear, on other hand, take up the blue color of the counterstain and appear blue. Spirillum FIG. 2-6. Different morphological types of the bacteria. Chapter 43 Gram-positive bacteria are those that resist decolorization and retain the primary dye-iodine complex, appearing violet. They have a relatively thick amorphous wall and more acidic protoplasm which are believed to retain the basic violet dye and iodine complex within the cell. Gram-negative bacteria are decolorized by organic solvents and take counterstain, appearing red. The decolorizing agent, such as acetone or ethanol, used during staining disrupts this membran-ous envelope, and the dye and iodine complex is washed out of Gram-negative bacteria. ■ Section III ■ ■ Chapter 2 (i) Primary staining with basic dyes, such as methyl violet, crystal violet, etc. (ii) Application of mordant in the form of dilute solution of iodine. (iii) Decolorization with ethanol, acetone, or aniline. (iv) Counterstaining with acidic dyes, such as carbol fuchsin, safranine, or neutral red. Acid fastness is due to the (a) high content of lipids, fatty acids, components of mycolic acid, and (b) higher alcohols found in the cell wall of the Mycobacterium. Acid fastness also depends on integrity of the cell wall. The ZN smear is best used for staining: Section I Differential stains impart different colors to different bacteria or different bacterial structures. The commonly used differential stains include Gram stain, acid-fast stain, and Albert’s stain. Gram stain: Gram stain was devised by Christian Gram, a Danish microbiologist, in 1884, as a convenient method for classifying bacteria. The Gram staining method essentially consists of four steps: 13 Chapter 2 Section I 14 GENERAL MICROBIOLOGY Structure and Functions of Bacterial Cell Envelope The outer layer or cell envelope provides a structural and physiological barrier between the protoplasm (inside) of the cell and the external environment. The cell envelope protects bacteria against osmotic lysis and gives bacteria rigidity and shape. The cell envelope primarily consists of two components: a cell wall and cytoplasmic or plasma membrane. It encloses the protoplasm, which consists of (i) cytoplasm, (ii) cytoplasmic inclusions (mesosomes, ribosomes, inclusion granules, vacuoles), and (iii) a single circular DNA (Fig. 2-7). cell wall consists primarily of teichoic and teichuronic acids. These two components account for up to 50% of the dry weight of the wall and 10% of the dry weight of the total cell. 1. Teichoic acids: Teichoic acids are polymers of polyribitol phosphate or polyglycerol phosphate containing ribitol and glycerol. These polymers may have sugar or amino acid substitutes, either as side chain or within the chain of polymer. Teichoic acids are of two types—wall teichoic acid (WTA) and lipoteichoic acids (LTA). They are connected to the peptidoglycan by a covalent bond with the six hydroxyl of N-acetylmuramic acid in the WTA and to plasma membrane lipids in LTA. Key Points Chapter 43 Section III Cell Wall Prokaryotic cells almost always are bounded by a fairly rigid and chemically complex structure present between the cell membrane and capsule/slime layer called the cell wall. Peptidoglycan is the main component of the cell wall and is responsible for the shape and strength of the cell. It is a disaccharide and contains two sugar derivatives—N-acetylglucosamine and N-acetylmuramic acid—joined together by short peptide chains. N-acetylmuramic acid carries a tetrapeptide side chain consisting of D- and L-amino acids (D-glutamic acid and L-alanine) with mesodiaminopimelic acid (Gram-negative bacteria) or L-lysine (Gram-positive bacteria). Tetrapeptide side chains are interconnected by pentaglycine bridges. Most Gram-negative cell walls lack interpeptide bridge. Cell wall provides shape to the cell and protects bacteria from changes inosmotic pressure, which within the bacteria cell measures 5–20 atmospheres. Bacterial cells can be classified into Gram-positive or Gram-negative based on the structural differences between Gram-positive and Gram-negative cell walls. The cell walls of the Gram-positive bacteria have simpler chemical structures compared to Gram-negative bacteria. ◗ Gram-positive cell wall The Gram-positive cell wall is thick (15–80 nm) and more homogenous than that of the thin (2 nm) Gram-negative cell wall. The Gram-positive cell wall contains large amount of peptidoglycan present in several layers that constitutes about 40–80% of dry weight of the cell wall (Fig. 2-8). The Gram-positive Single circular supercoiled chromosome Ribosome Plasmid Plasma membrane FIG. 2-7. Flagellum Cell wall Schematic diagram of structure of a bacteria. Teichoic acids have many functions: ■ ■ ■ ■ ■ ■ They constitute major surface antigens of those Grampositive species that possess them. In Streptococcus pneumoniae, the teichoic acids bear the antigenic determinants called Forssman antigen. In Streptococcus pyogenes, LTA is associated with the M protein that protrudes from the cell membrane through the peptidoglycan layer. The long M protein molecules together with the LTA form microfibrils that facilitate the attachment of S. pyogenes to animal cells; They are also used as antigen for serological classification of bacteria; They serve as substrates for many autolytic enzymes; They bind magnesium ion and may play a role in supply of this ion to the cell; They play a role in normal functioning of the cell wall and provide an external permeability barrier to Gram-positive bacteria; and Membrane teichoic acid serves to anchor the underlying cell membrane. 2. Teichuronic acid: Teichuronic acid consists of repeat units of sugar acids (such as N-acetylmannuronic or D-glucuronic acid). They are synthesized in place of teichoic acids when phosphate supply to the cell is limited. Gram-positive cell wall also contains neutral sugars (such as mannose, arabinose, rhamnose, and glucosamine) and acidic sugars (such as glucuronic acid and mannuronic acid), which occur as subunits of polysaccharides in the cell wall. ◗ Gram-negative cell wall The Gram-negative cell wall is much more complex than the Gram-positive cell wall. Peptidoglycan content in the Gram-negative cell wall is significantly less than the Grampositive cell wall. Only 1–2 layers of peptidoglycan (2–8 nm) are present just outside the cell membrane. The Gram-negative cell wall outside the peptidoglycan layer contains three main components—(a) lipoprotein layer, (b) outer membrane, and (c) lipopolysaccharides (Fig. 2-9). Lipoprotein layer: The lipoprotein layer is mainly composed of Braun’s lipoprotein. Braun’s lipoprotein is a small lipoprotein that is covalently joined to the underlying peptidoglycan and embedded in the outer membrane by its hydrophobic end. MORPHOLOGY AND PHYSIOLOGY OF BACTERIA 15 Peptidoglycan Cell wall Teichoic acid Structural and enzymatic proteins FIG. 2-8. Chapter 2 Cytoplasmic membrane Section I Lipoteichoic acid Schematic diagram of the cell wall of the Gram-positive bacteria. Lipopolysaccharide Outer membrane Section III Porin proteins (Pore) Lipoprotein Nutrient-binding protein Peptidoglycan Carrier protein Cytoplasmic membrane FIG. 2-9. Schematic diagram of the cell wall of the Gram-negative bacteria. The lipoprotein stabilizes the outer membrane of the Gramnegative cell wall. Outer membrane: The outer membrane is a bilayered structure; its inner part resembles in composition with that of the cell membrane, while its outer part contains a distinctive component called lipopolysaccharide. The outer membrane and plasma membrane appear to be in direct contact at many sites in the Gram-negative wall. The outer membrane has a variety of proteins as follows: (a) Porins: The outer membrane has special channels consisting of protein molecules called porins. These porins have many functions: ■ ■ ■ They permit the passive diffusion of low-molecular weight hydrophilic compounds, such as sugars, amino acids, and certain ions; They exclude hydrophobic molecules; and They serve to protect the cell. (b) Outer membrane proteins (OMPs): These include the following: ■ ■ ■ Omp C, D, F, and PhoE&LamB are the four major proteins of the outer membrane that are responsible for most of the transmembrane diffusion of maltose and maltodextrins. Tsx, the receptor for T6 bacteriophage, is responsible for the transmembrane diffusion of nucleosides and some amino acids. Omp A protein anchors the outer membrane to the peptidoglycan layer. It is also the sex pilus receptor in F-mediated bacterial conjugation. The outer membrane also contains proteins that are involved in the transport of specific molecules, such as vitamin B12 and iron-siderophore complexes; it also contains a limited number of minor proteins, such as enzymes, phospholipases, and proteases. Chapter 43 Periplasmic space 16 GENERAL MICROBIOLOGY Section I TABLE 2-3 Chapter 2 O-polysaccharide Core Core Lipid A Smooth form LPS FIG. 2-10. Lipid A Rough form LPS (or lipo-oligosaccharide) Structure of a lipopolysaccharide. Differences between Gram-positive and Gram-negative bacteria cell wall Characters Gram-positive cell wall Gram-negative cell wall Thickness 15–80 nm 2 nm Lipid content 2–5% 15–20% Teichoic acid Present Absent Variety of amino acid Few Several Aromatic amino acid Absent Present Action as endotoxin No Yes Sulfur-containing amino acid Absent Present Treatment with lysozyme Protoplast Spheroplast Differences between Gram-positive and Gram-negative cell walls are summarized in Table 2-3. ■ Chapter 43 Section III Periplasmic space Lipopolysaccharides: Lipopolysaccharides (LPS) are complex molecules present in the outer membrane of the Gram-negative bacteria. Structurally, the LPS consist of three main components— lipid A, the core oligosaccharide, and the O polysaccharide or O-antigen (Fig. 2-10). ■ ■ Lipid A: This consists of phosphorylated glucosamine disaccharide units, to which a number of long-chain fatty acids are attached. This also consists of hydroxymyristic acid, a unique fatty acid, which is associated with endotoxic activity of the LPS. There is a little variation in the structure of the lipid A among different species of the Gram-negative bacteria. However, it remains the same within the bacteria of the same species. Core oligosaccharide: The core oligosaccharide includes two characteristic sugars—ketodeoxyoctanoic acid (KDO) and a heptose—both joined together by lipid A. This is genus specific and similar in all Gram-negative bacteria. Lipooligosaccharides (LOS) are smaller glycolipids. They have relatively short, multiantennary (i.e., branched) glycans present in bacteria (e.g., Neisseria meningitidis, N. gonorrhoeae, Haemophilus influenzae, and Haemophilus ducreyi) that colonize mucosal surfaces. They exhibit extensive antigenic and structural diversity even within a single strain. LOS is an important virulence factor. Epitopes on LOS have a terminal N-acetyllactosamine (Gal(␤)l-4-GlcNAc) residue, which is immunochemically similar to the precursor of the human erythrocyte i antigen. Sialylation of the N-acetyllactosamine residue in vivo provides the organism with the environmental advantages of molecular mimicry of a host antigen and the biologic masking thought to be provided by sialic acids. O polysaccharide or O-antigen: It is the portion extending outwards from the core. It has several peculiar sugars and varies in composition between bacterial strains, conferring species-specific antigen specificity. It is exposed to host– immune system. Gram-negative bacteria may thwart host defenses by rapidly changing the nature of their O side chains to avoid detection. Periplasmic space is a distinct space between cell membrane and outer membrane (innermost layer of Gram-negative cell wall) in Gram-negative bacteria. This space is filled with a loose layer of peptidoglycan matrix. The periplasmic space of Gramnegative bacteria contains many proteins that participate in nutrient acquisition, and many hydrolytic enzymes, betalactamases binding proteins, and enzymes that participate in the peptidoglycan synthesis. Polymers of D-glucose, called membrane-derived oligosaccharides, appear to play a role in osmoregulation. The periplasmic space is less distinct in Grampositive cell walls. ◗ Cell wall of acid-fast bacilli The cell wall of acid-fast bacilli, such as M. tuberculosis, contains large amounts of waxes known as mycolic acids. The cell wall is composed of peptidoglycan and an outer asymmetric lipid bilayer. The inner lipid bilayer contains mycolic acids linked to an arabinoglycan protein and the outer layer contains other extractable lipids. This hydrophobic structure renders these bacteria resistant to many harsh chemicals including detergents and strong acids. During staining, if dye is introduced into these cells by brief heating or treatment with detergents, they resist decolorization by sulfuric acid or acid alcohol, and are therefore called acid-fast organisms. ◗ Atypical forms of bacteria Atypical forms of bacteria include (i) cell wall deficient forms, (ii) pleomorphic bacteria, and (iii) involution forms. Many agents, such as antibiotics, lysozyme, and bacteriophages interfere or inhibit the synthesis of bacterial cell wall components, resulting in the formation of defective bacteria. 1. Cell wall deficient forms: The cell wall could be removed by hydrolysis with lysozyme or by blocking peptidoglycan synthesis with an antibiotic such as penicillin. These defective bacteria are believed to play a role in the persistence of pyelonephritis and other chronic infections. Cell wall deficient forms MORPHOLOGY AND PHYSIOLOGY OF BACTERIA ■ ■ ■ Demonstration of cell wall The cell walls can be demonstrated by (a) differential staining procedure, (b) electron microscopy, (c) plasmolysis, (d) microdissection, (e) mechanical rupture of the cell, and (f) serological test by exposure to specific antibodies. Cell Membrane Cell membrane or plasma membrane is a thin (5–10 nm) semipermeable membrane that acts as an osmotic barrier. It lies beneath the cell wall separating it from the cell cytoplasm. Cell membrane primarily contains phospholipids and proteins. It also contains enzymes associated with DNA biosynthesis, cell wall polymers, and membrane lipids. Bacterial plasma membranes usually have a higher proportion of protein than eukaryotic membranes. They usually differ from eukaryotic membranes in lacking sterols, such as cholesterol, except in Mycoplasma. The cell membrane has following functions: ■ ■ It acts as a semipermeable membrane regulating the inflow and outflow of metabolites to and from the protoplasm. It helps in electron transport and oxidative phosphorylation. ■ ■ They serve as the sites of protein synthesis; matrix ribosomes synthesize proteins destined to remain within the cell, whereas plasma membrane ribosomes make proteins for transport to the outside. They are also the sites of actions of several antibiotics, such as amino glycosides, macrolides, and tetracyclines. Mesosomes: These are vesicular convoluted or multilaminated structures formed as invagination of the plasma membrane into the cytoplasm. Mesosomes are of two types—septal and lateral. The septal mesosome attached to the bacterial DNA is believed to coordinate nuclear and cytoplasmic divisions during binary fission. The function of lateral mesosomes still remains to be known. Mesosomes are analogous to the mitochondria of eukaryotes and are the principal sites of respiratory enzymes in bacteria. Intracytoplasmic inclusion bodies: Intracytoplasmic inclusion bodies are present in the protoplasm of bacteria. Their main function is believed to be of storage. This occurs when their main constituent element is present in excess in the culture medium. Since inclusion bodies are used for storage, their quantity can vary depending on the nutritional status of the cell. They are the sources of carbon, inorganic substances, and energy. Some inclusion bodies also function to reduce osmotic pressure. They may be of two types: (i) organic inclusion bodies, which usually contain either glycogen or polyhydroxybutyrate, and (ii) inorganic inclusion bodies, which may be of polyphosphate granules or sulfur granules. Examples of intracytoplasmic inclusion bodies include metachromatic granules or volutin granules, starch inclusions, and lipid inclusions. Volutin granules, typically present in C. diphtheriae, can be demonstrated by Albert’s stain. Similarly, starch granules present in the bacteria can be demonstrated on staining with iodine. Lipid inclusion found in Mycobacteria is demonstrated by Sudan black dye. Chapter 43 ◗ Ribosomes: The cytoplasmic matrix often is packed with ribosomes. Ribosomes look like small, featureless particles at low magnification in electron micrographs. They are smaller than their eukaryotic counterpart with sedimentation of 70S, compared with 80S in eukaryotes. They consist of two subunits of 30S and 50S, giving a net 70S. Ribosomes are important because: Section III 2. Pleomorphic bacteria: Pleomorphic bacteria (e.g., Yersinia pestis) may show considerable variation in size and shape called pleomorphism. 3. Involution forms: The involution forms are those that on ageing of culture show swollen and aberrant forms, especially in high salt concentration. Bacterial cytoplasm is a colloidal suspension of a variety of organic and inorganic solutes in a viscous watery solution. The matrix is largely formed by nearly 70% water. Cytoplasm contains all the biosynthetic components required by a bacterium for growth and cell division, together with genetic material. Prokaryotic cytoplasm, unlike that of eukaryotes, lacks endoplasmic reticulum and mitochondria. It also does not show any protoplasmic streaming. Bacteria lack a true cytoskeleton. The cytoplasm consists of ribosomes, mesosomes, and intracytoplasmic inclusions bodies. Chapter 2 ■ Protoplasts: These are defective unstable forms of bacteria with an intact cytoplasmic membrane but without any cell wall. In hypertonic media, these are produced from Grampositive cells on treatment with lysozyme. Spheroplasts: These are defective forms derived from Gramnegative bacteria in the presence of EDTA (ethylenediaminetetraacetic acid). The EDTA disrupts the outer membrane allowing access of lysozyme and resulting in formation of spheroplasts. Spheroplasts are osmotically fragile and still retain outer membrane and entrapped peptidoglycan. Mycoplasma: These are naturally occurring bacteria without cell wall. They do not possess any definite shape. They are very minute in size measuring 50–300 nm in diameter. L-forms: This is named after Lister Institute, London, where the abnormal form of Streptobacillus moniliformis was first demonstrated. The L-forms do not exhibit any regular size and shape. They may be spherical or disc shaped, about 0.1– 20 ␮m in diameter. They are difficult to cultivate and usually require a medium that is solidified with agar as well as having the right osmotic strength. They are produced more readily with exposure to penicillin than with lysozyme. Some bacterial species produce L-forms spontaneously. L-forms in the host may produce chronic infections and are relatively resistant to antibiotic treatment. Cytoplasm Section I without cell walls or with deficient cell walls may be of various types, such as—protoplasts, spheroplasts, mycoplasma, and L-forms. 17 18 GENERAL MICROBIOLOGY Chapter 43 Section III Chapter 2 Section I Nucleus The bacterial nucleus is neither enclosed in a nuclear membrane nor associated with any nucleolus. It is haploid and replicates by simple fission. The nucleus of the bacteria consists of a single circle of double-stranded deoxyribonucleic acid (DNA), arranged in a supercoiled circular structure. It measures about 1000 ␮m when straightened. The chromosome is located in an irregularly shaped region called nucleoid, but often referred to as bacterial chromosome because of the analogy with the eukaryotic structure. The nucleoid is visible through the light microscope after staining with the Feulgen stain, which specifically reacts with DNA. In actively growing bacteria, the bacterial DNA can account for up to 20% of the volume of the bacterium and has projections that extend into the cytoplasmic matrix. Careful electron microscopic studies often have shown the nucleoid to be in contact with either the mesosome or the plasma membrane. Many bacteria also possess smaller circles of extrachromosomal DNA called plasmids. The plasmids are double-stranded DNA molecules, usually circular, that can exist and replicate independently. Plasmids are not required for host growth and reproduction, although they may carry genes that confer the bacterium with properties such as antibiotics resistance or the capacity to produce toxins or enzymes. Capsule and Slime Layer Many bacteria, both Gram-positive and Gram-negative, possess a gel-like layer outside the envelope when growing in their natural environments. When a gel-like layer forms a welldefined condensed layer around the bacterial envelope, it is called a capsule and is demonstrable by a light microscope. When this gel-like layer is narrower, detectable only by indirect serological methods or by electron microscope but not by light microscope, it is called a microcapsule. An amorphous viscid colloidal material secreted by some bacteria extracellularly is termed as loose or free slime or glycocalyx. ◗ Capsule The capsule is mostly made up of polysaccharides, often referred to collectively as exopolysaccharides. Exopolysaccharides are sometimes neutral homopolysaccharides (e.g., the glucans and fructans of many oral streptococci) or negatively charged (Table 2-4). TABLE 2-4 However, Bacillus anthracis has a capsule comprising of polyamino acids, such as D-glutamic acid. The D-glutamic acid is probably analogous to the negatively charged polysaccharide capsule. Key Points The capsule has various functions: ■ ■ ■ ■ ■ ■ It contributes to invasiveness of bacteria by protecting the bacteria from phagocytosis. It also prevents bacteria from generating immune response in infected hosts. It facilitates adherence of bacteria to surfaces. Streptococcus mutans, for example, owes its capacity to the glycocalyx to adhere tightly to tooth enamel to its glycocalyx. It plays a role in the formation of biofilms. The glycocalyx layer of the capsule may also play a role in resistance to desiccation. Demonstration of capsule The capsule is fully hydrated and can be demonstrated by light microscopy in either living or stained bacteria as follows: Special capsular staining methods: These include Welch method and M’Faydean capsule stain. Welch method uses copper as mordant. This involves treatment of fixed smear with hot crystal violet solution followed by rinsing with copper sulfate solution. The latter is used to remove excess stain because conventional washing with water would dissolve the capsule. The copper salt also gives color to the background, with the result that the cell and background appear dark blue and the capsule of the bacteria a much paler blue. M’Fadyean capsule stain, using polychrome methylene blue stain, is a frequently used method for demonstration of capsule of B. anthracis. Negative staining with India ink: Also known as wet India ink method. It is the simplest way to demonstrate capsule. It is carried out by mixing a suspension of bacteria with an equal volume of Indian ink on a slide, covering with a cover slip, and then examining it under microscope. The capsule appears as a clear zone around the cell. This method is useful for improving visualization of encapsulated bacteria in clinical samples, such as blood or cerebrospinal fluid. Serologic methods: Since capsules are antigenic, they can be demonstrated by serologic methods. Quellung’s reaction is Chemical composition of capsules of various bacteria Organism Polymer Chemical subunits Bacillus anthracis Polypeptide D-glutamic Enterobacter aerogenes Complex polysaccharide Glucose, fucose, glucuronic acid acid Neisseria meningitidis Homopolymers and heteropolymers Partially O-acetylated N-acetylmannosaminephosphate Streptococcus pneumoniae Complex polysaccharide (many types) Rhamnose, glucose, glucuronic acid Streptococcus pyogenes (group A) Hyaluronic acid N-acetylglucosamine, glucuronic acid Streptococcus salivarius Levan Fructose MORPHOLOGY AND PHYSIOLOGY OF BACTERIA Filament ◗ Periplasmic space Flagella ■ Monotrichous (single polar flagellum), e.g., Vibrio cholerae. Lophotrichous (multiple polar flagella), e.g., Spirilla. Peritrichous (flagella distributed over the entire cell), e.g., Salmonella Typhi, E. coli, etc. Amphitrichous (single flagellum at both the ends), e.g., Spirillum minus (Fig. 2-11). Structure: The flagella are 3–20 ␮m in length and 0.01–0.03 ␮m in diameter. The main part of the filament is made up of protein subunits called flagellin arranged in several helices around a central hollow core. The flagellum is attached to the bacterial cell Peritrichous Basal plates FIG. 2-12. FIG. 2-11. Lophotrichous Arrangement of the bacterial flagella. Structure of a bacterial flagellum. body by a complex structure consisting of a hook and a basal body. The basal body bears a set of rings, one pair in Grampositive bacteria and two pairs in Gram-negative bacteria, through which the bacteria rotates either in a clockwise or an anticlockwise direction. Above the base of filament is the hook, a short curved structure between the external filament and basal body. This part produces a propeller-like repulsion from the revolving f lagellum (Fig. 2-12). Spirochetes are motile bacteria but without any external flagella. They are motile due to the presence of an axial filament. Axial filament consists of a bundle of flagellum-like structures that lie between the cell surface and an outer sheath, and connects one end of the cell to the other. They are sometimes called the endoflagellates. Function: Flagella have the following functions: ■ ■ They are primarily responsible for motility of bacteria by chemotaxis. They may play a role in bacterial survival and pathogenesis. They are highly antigenic, they possess H antigens, and some of the immune responses to infection are directed against these proteins. The flagella of different bacteria differ antigenically. Flagellar antibodies are not protective but help in serodiagnosis. Demonstration of flagella: The flagella can be demonstrated by direct and indirect methods. The direct methods include direct demonstration of capsule by electron microscope. These also include demonstration of capsule after staining by special staining methods, such as Ryu’s method and Hugh–Leifson’s method. Since flagella are very thin structures, these staining methods are used to demonstrate flagella by increasing their thickness by mordanting with tannic acid. Indirect methods of demonstration of flagella include demonstration of motility of the bacteria by (a) dark-ground microscopy, (b) hanging drop method, or (c) observing spreading type growth on semisolid media, such as mannitol motility medium. ◗ Monotrichous Plasma membrane Pili (fimbriae) Pili or fimbriae are synonymous for most purposes. They are hair-like filaments that extend from cell surface and are found almost exclusively on Gram-negative bacteria. They are Chapter 43 Bacterial flagella are thread-like appendages intricately embedded in the cell envelope. These structures are responsible for conferring motility to the bacteria. The arrangement of flagella varies between different bacterial species. Depending on the arrangement, flagella can be of the following types: ■ Rod Section III The surface appendages of the bacteria include flagella and fimbriae or pili. ■ Peptidoglycan Chapter 2 Surface Appendages ■ Outer membrane Slime layer Slime layer (S-layer) is a structured paracrystalline protein layer shown by electron microscopy. These are generally composed of a single kind of protein molecule, sometimes with carbohydrates attached. They are resistant to proteolytic enzymes and protein-denaturing agents. The slime layer protein protects the cell from wall-degrading enzymes and bacteriophages. It plays an important role in the maintenance of cell shape, and it may be involved in cell adhesion to host epidermal surfaces. ◗ Flagellar hook Section I such a serological method for demonstration of capsule. When a suspension of bacterium is mixed with its specific anticapsular serum and methylene blue and examined under microscope, the capsule becomes very prominent and appears swollen due to increase in refractoriness. This method is useful for rapid identification of capsular serotypes of S. pneumoniae, N. meningitidis, H. influenzae, Yersinia, Bacillus, etc. 19 Chapter 2 Section I 20 composed of structural protein subunits termed pilins. Minor proteins termed adhesins are located at the tips of pili and are responsible for the attachment properties. Structure: The pili are shorter and straighter than flagella, although the basic structure is same. Like flagella, it consists of helics of protein called pilins, arranged around a hollow core but without a motor. They are 0.5 ␮m long and 10 nm thick. They are antigenic in nature. Pili hemagglutinate RBCs of guinea pigs and are specifically inhibited by mannose, on the basis of which they are classified into four types as follows: ■ ■ ■ Section III ■ Chapter 43 GENERAL MICROBIOLOGY (i) F pili: They specifically adsorb male specific RNA and DNA bacteriophages. They are encoded by sex factor F and fertility inhibition–positive resistance factors (fi ⫹ R factors). (ii) I pili: They adsorb male specific filamentous DNA phages, encoded by col factor and fi ⫺ R factor. Function: Pili play a major role in the adherence of symbiotic and pathogenic bacteria to host cells, which is a necessary step in initiation of infection. Transfer of bacterial DNA takes place through sex pili during the process of conjugation. Demonstration of pili: The pili can be detected: ■ Directly by electron microscope and By agglutination of RBCs of guinea pigs, fowl, horses, and pigs. They agglutinate human and sheep RBCs weakly. The hemagglutination can be specifically inhibited by D-mannose. Some of the Gram-positive bacteria do not possess typical pili but instead possess a fine fibrillar arrangement of proteins on their surfaces known as fibrils. These fibrils bind to the host surfaces. M-protein of S. pyogenes is an example of Grampositive bacteria possessing fibrils. Sporulation Sporulation is a primitive process of differentiation with formation of endospores, a highly resistant resting phase of some of the bacteria (e.g., spores of aerobic Bacillus spp. and anaerobic Clostridium spp.). The organism survives in spores, a dormant state, for longer period of starvation and other adverse conditions. Sporulation process begins in nutrition deprived conditions. It begins with the formation of an axial filament. The process continues with infolding of the membrane so as to produce a Septum Endospore coat Type 1: These occur in E. coli, Klebsiella, Shigella, and Salmonella. They are mannose sensitive. Type 2: These are present in Salmonella Gallinarum and Salmonella Pullorum, devoid of any hemagglutinating or adhesive properties. Type 3: These are present in some strains of Klebsiella, Serratia, etc. They agglutinate RBC only after heating and are mannose resistant. Type 4: These are mannose resistant and occur in Proteus. Sex pili: A specialized kind of pili called sex pili is responsible for the attachment of donor and recipient cells in bacterial conjugation. These pili are longer (10–20 ␮m) and vary 1–4 in number. The sex pili are of two types: ■ DNA Peptidoglycan Cytoplasmic membrane Endospore released free FIG. 2-13. Schematic diagram showing process of bacterial sporulation. double membrane structure whose facing surfaces correspond to the cell wall-synthesizing surface of the cell envelope. The growing points move progressively toward the pole of the cell so as to engulf the developing spore. Two spore membranes then engage in the active synthesis of special layers that form the cell envelope: the spore wall and the cortex, lying outside the facing membranes. In the newly isolated cytoplasm, or core, many vegetative cell enzymes are degraded and are replaced by a set of unique spore constituents. During the process of sporulation, each cell forms a single internal spore; the spore germinates to produce a single vegetative cell (Fig. 2-13). ◗ Spores Morphology: The endospores are a highly resistant resting phase of bacteria. The spore shows following structures. 1. Core: The core contains a complete nucleus (chromosome), all of the components of the protein-synthesizing apparatus, and an energy-generating system based on glycolysis. The heat resistance of spores is due in part to their dehydrated state and in part to the presence of large amounts (5–15% of the spore dry weight) of calcium dipicolinate in the core. 2. Spore wall: This is the innermost layer surrounding the inner spore membrane. It contains normal peptidoglycan and becomes the cell wall of the germinating vegetative cell. 3. Cortex: It is the thickest layer of the spore envelope containing unusual peptidoglycan. It is extremely sensitive to lysozyme, and its autolysis plays a role in spore germination. 4. Protein coat: It is composed of a keratin-like protein containing many intramolecular disulfide bonds; this layer confers relative resistance to antibacterial chemical agents due to its impermeability (Fig. 2-14). MORPHOLOGY AND PHYSIOLOGY OF BACTERIA 21 Exosporium Section I 1 Protein coat Outer membrane Core 2 Cortex Spore wall FIG. 2-14. Chapter 2 Inner membrane 4 Diagrammatic representation of bacterial spore. 8 The spores have certain uses in clinical microbiology: ■ ■ The spores of Bacillus stearothermophilus are used as indicator of proper sterilization by autoclaving. These spores are destroyed at a temperature of 121⬚C for 10–20 minutes, the time required for autoclaving. The spores of certain bacteria, such as B. anthracis are misused as agents of bioterrorism. Growth and Multiplication of Bacteria Bacterial growth can be defined as an orderly increase of all the chemical components of the cell. Cell multiplication is a consequence of growth that leads to an increase in the number of bacteria making up a population or culture. Most bacteria divide by binary fission in which the bacteria undergo cell division to produce two daughter cells identical to the parent cell. Bacterial growth can be equated to cell number: one bacterium divides into two, these two produce four, and then eight, and so on Schematic diagram showing binary fission of the bacteria. (Fig. 2-15). The growth rate of a bacterium is therefore measured by measuring the change in bacterial number per unit time. Generation Time Generation time is the time required for a bacterium to give rise to two daughter cells under optimum conditions. The generation time for most of the pathogenic bacteria, such as E. coli, is about 20 minutes. The generation time is longer (i.e., 20 hours) for M. tuberculosis and longest (i.e., 20 days) for M. leprae. A bacterium replicates and multiplies rapidly producing millions of cells within 24 hours. For example, E. coli in about 7 hours can undergo 20 generations and produce 1 million cells, in about 10 hours undergo 30 generations and produce 1 billion cells, and in 24 hours produces 1021 cells (Fig. 2-16). However, in actual practice, the multiplication of bacteria is arrested after a few cell divisions due to exhaustion of nutrients and accumulation of toxic products. Bacterial Count Microbial concentrations can be measured in terms of (i) cell concentration (the number of viable cells per unit volume of culture) or (ii) biomass concentration (dry weight of cells per unit volume of culture). The number of bacteria at a given time can be estimated by performing a total count or viable count. Total count: This denotes the total number of bacteria in the sample, irrespective of whether they are living or dead. This is done by counting the bacteria under the microscope using counting chamber or by comparing the growth with standard opacity tubes. Viable count: This usually indicates the number of living or viable bacteria. This count can be obtained by dilution or plating method. ■ In dilution method, several tubes with liquid culture media are incubated with varying dilutions of sample and the viable count is calculated from the number of tubes showing bacterial growth. This method is widely used in Chapter 43 Key Points FIG. 2-15. Section III The spores may vary among different species depending on the position, shape, and relative size of the spores. For example, spores may be central, subterminal or terminal; may be oval or spherical in shape; and may be bulging or nonbulging. Demonstration of spores: Spores are most simply observed as intracellular refractile bodies in unstained cell suspensions or as colorless areas in cells stained by conventional methods, such as Gram staining. Spores are commonly stained with malachite green or carbol fuchsin. On staining by modified ZN stain (using 0.25–0.5% sulfuric acid instead of 20% sulfuric acid), the spores appear as red acid-fast bodies. Properties of spores: Bacterial spores are resistant to ordinary boiling, disinfectants, and heating. Spores of all medically important bacteria are destroyed by autoclaving at 121⬚C for 15 minutes. The process of conversion of a spore into vegetative cell under suitable conditions is known as germination. The germination process occurs in three stages: activation, initiation, and outgrowth. GENERAL MICROBIOLOGY Section I Single bacterial cell Time (hour) Lag phase 0 Agar plate Chapter 2 Log phase Stationary phase Number of microorganism 22 4 Phase of decline Total count Viable count Time FIG. 2-17. Bacterial growth curve. 8 Chapter 43 Section III Visible bacterial colony TABLE 2-5 12 FIG. 2-16. ■ Generation time of the bacteria. microbiological testing of water for presumptive coliform count in drinking water. In the plate method, a sample is diluted and small volume of it is spread on the surface of an agar plate. The number of colonies that grow after a suitable incubation time indicates viable count of the bacteria. Bacterial Growth Curve When a broth culture is inoculated with a small bacterial inoculum, the population size of the bacteria increases showing a classical pattern. The bacterial growth curve shows the following four distinct phases (Fig. 2-17): 1. Lag phase: After a liquid culture broth is inoculated, the multiplication of bacteria does not start immediately. It takes some time to multiply. The time between inoculation and beginning of multiplication is known as lag phase. In this phase, the inoculated bacteria become acclimatized to the environment, switch on various enzymes, and adjust to the environmental temperature and atmospheric conditions. During this phase, there is an increase in size of bacteria but no appreciable increase in number of bacterial cells. The cells are active metabolically. The duration of the lag phase varies with the bacterial species, nature of culture medium, incubation temperature, etc. It may vary from 1 hour to several days. 2. Log phase: This phase is characterized by rapid exponential cell growth (i.e., 1 to 2 to 4 to 8 and so on). The bacterial Growth rate during different phases of bacterial growth curve Phase Growth rate Lag Zero Log or exponential Constant Stationary Zero Decline Negative (death) population doubles during every generation. They multiply at their maximum rate. The bacterial cells are small and uniformly stained. The microbes are sensitive to adverse conditions, such as antibiotics and other antimicrobial agents. 3. Stationary phase: After log phase, the bacterial growth almost stops completely due to lack of essential nutrients, lack of water oxygen, change in pH of the medium, etc. and accumulation of their own toxic metabolic wastes. Death rate of bacteria exceeds the rate of replication of bacteria. Endospores start forming during this stage. Bacteria become Gram variable and show irregular staining. Many bacteria start producing exotoxins. 4. Decline phase: During this phase, the bacterial population declines due to death of cells. The decline phase starts due to (a) accumulation of toxic products and autolytic enzymes and (b) exhaustion of nutrients. Involution forms are common in this stage. Growth rate during different phases of bacterial growth curve is summarized in Table 2-5. The continuous culture is a method of culture useful for industrial and research purpose. This is achieved by using a special device for replenishing nutrients and removing bacterial population continuously so that bacteria growth is not inhibited due to lack of nutrients or due to accumulation of toxic bacterial metabolites. Factors Affecting Growth of Bacteria A variety of factors affect growth of bacteria. These are discussed below: MORPHOLOGY AND PHYSIOLOGY OF BACTERIA ◗ Oxygen ■ ■ ◗ Carbon dioxide ◗ Temperature The optimum temperature for most of the pathogenic bacteria is 37⬚C. The optimal temperature, however, is variable; depending on their temperature range, growth of bacteria is grouped as follows: ■ ■ ■ ◗ Psychrophiles: These bacteria are cold loving microbes that grow within a temperature range of 0–20⬚C. Most of soil and water saprophytes belong to this group. Mesophiles: These are moderate temperature loving microbes that grow between 25⬚C and 40⬚C. Most of pathogenic bacteria belong to this group. Thermophiles: These are heat loving microbes. They can grow at a high temperature range of 55–80⬚C. B. stearothermophilus is an example. pH Most pathogenic bacteria grow between pH 7.2 and 7.6. Very few bacteria, such as lactobacilli, can grow at acidic pH below 4.0. Many food items, such as pickles and cheese, are prevented from spoilage by acids produced during fermentation. V. cholerae is an example of the bacteria that can grow at an alkaline (8.2–8.9) pH. Osmotic pressure Microbes obtain almost all their nutrients in solution from surrounding water. Hence factors such as osmotic pressure and salt concentration of the solution affect the growth of bacteria. Bacteria by virtue of mechanical strength of their cell wall are able to withstand a wide range of external osmotic variations. Organisms requiring high osmotic pressures are called osmophilic bacteria. Sudden exposure of bacteria to hypertonic solution may cause osmotic withdrawal of water, leading to osmotic shrinkage of the protoplasm ( plasmolysis). On the other hand, sudden transfer of bacteria from concentrated solution to distilled water may cause excessive imbibition of water leading to swelling and bursting of cell ( plasmoptysis). Bacterial Nutrition The minimum requirements for growth of bacteria include water, a source of carbon, a source of nitrogen, and certain inorganic salts. These are required for synthesis of proteins, enzymes, etc. For example, ■ ■ ■ Nitrogen is required for synthesis of proteins, DNA, RNA, and ATP. Sulfur is required for certain amino acids and vitamins, and phosphorous is required for nucleic acids, ATP, and phospholipids. In addition, inorganic ions, such as potassium, sodium, iron, magnesium, calcium, and chloride are required to facilitate enzymatic catalysis and to maintain chemical gradients across the cell membrane. Some bacteria grow in a variety of simple media. E. coli and other members of the family Enterobacteriaceae are examples of bacteria that can grow in a variety of simple media containing the inorganic salts and with a source of energy, the simplest being glucose. The inorganic salts in the media provide major essential elements of carbon, hydrogen, oxygen, nitrogen, phosphate, and sulfur. These chemicals are usually present in the media and are not added specifically. Some bacteria, such as H. influenzae and other related bacteria, on other hand, are very fastidious and have certain growth requirements. They require certain amino acids, vitamins, and other growth factors that are supplied by adding yeast extract and meat digests to the media. They also require addition of blood or serum for their growth. Certain lower forms of bacteria even fail to grow in cell-free culture media and require living cells for their growth. T. pallidum and M. leprae are two pathogenic bacteria that cannot grow in any artificial culture media, but can only be cultured when inoculated into living animals. Chapter 43 The organisms that require higher amounts of carbon dioxide (CO2) for their growth are called capnophilic bacteria. They grow well in the presence of 5–10% CO2 and 15% O2. In candle jar, 3% CO2 can be achieved. Examples of such bacteria include H. influenzae, Brucella abortus, etc. ◗ Section III Some fermentative organisms (e.g., Lactobacillus plantarum) are aerotolerant but do not contain the enzyme catalase or superoxide dismutase. Oxygen is not reduced, and therefore hydrogen peroxide (H2O2) and nascent oxygen (O2⫺) are not produced. Anaerobic bacteria: Obligate anaerobes are the bacteria that can grow only in the absence of oxygen (e.g., Clostridium botulinum Clostridium tetani, etc.). These bacteria lack superoxide dismutase and catalase; hence oxygen is lethal to these organisms. Light Chapter 2 ■ Obligate aerobes—which can grow only in the presence of oxygen (e.g., P. aeruginosa). Facultative aerobes—which are ordinary aerobes but can also grow without oxygen (e.g., E. coli). Most of the pathogenic bacteria are facultative aerobes. Microaerophilic bacteria—those bacteria that can grow in the presence of low oxygen and in the presence of low (4%) concentration of carbon dioxide (e.g., Campylobacter jejuni). ◗ Depending on the source of energy bacteria make use of, they may be classified as phototrophs (bacteria deriving energy from sunlight) or chemotrophs (bacteria deriving energy from chemical sources). Section I Bacteria on the basis of their oxygen requirements can be classified broadly into aerobic and anaerobic bacteria. Aerobic bacteria: They require oxygen for their growth. They may be: 23 3 Sterilization and Disinfection Introduction Microbes are ubiquitous and many microorganisms are associated with undesirable consequences, such as food spoilage and disease. Therefore, it is essential to kill a wide variety of microorganisms or inhibit their growth to minimize their destructive effects. The goal is twofold: (a) to destroy pathogens and prevent their transmission and (b) to reduce or eliminate microorganisms responsible for the contamination of water, food, and other substances. Definition of Frequently Used Terms Sterilization is defined as a process by which an article, surface, or medium is freed of all living microorganisms either in the vegetative or in the spore state. Any material that has been subjected to this process is said to be sterile. These terms should be used only in the absolute sense. An object cannot be slightly sterile or almost sterile; it is either sterile or not sterile. Although most sterilization is performed with a physical agent, such as heat, a few chemicals called sterilants can be classified as sterilizing agents because of their ability to destroy spores. A germicide, also called a microbicide, is any chemical agent that kills pathogenic microorganisms. A germicide can be used on inanimate (nonliving) materials or on living tissue, but it ordinarily cannot kill resistant microbial cells. Any physical or chemical agent that kills “germs” is said to have germicidal properties. Disinfection refers to the use of a chemical agent that destroys or removes all pathogenic organisms or organisms capable of giving rise to infection. This process destroys vegetative pathogens but not bacterial endospores. It is important to note that disinfectants are normally used only on inanimate objects because they can be toxic to human and other animal tissue, when used in higher concentrations. Disinfection processes also remove the harmful products of microorganisms (toxins) from materials. Examples of disinfection include (a) applying a solution of 5% bleach to examining table, (b) boiling food utensils used by a sick person, and (c) immersing thermometers in an isopropyl alcohol solution between use. In modern usage, sepsis is defined as the growth of microorganisms in the body or the presence of microbial toxins in blood and other tissues. The term asepsis refers to any practice that prevents the entry of infectious agents into sterile tissues and thus prevents infection. Chemical agents called antiseptics are applied directly to the exposed body surfaces (e.g., skin and mucous membranes), wounds, and surgical incisions to destroy or inhibit vegetative pathogens. Examples of antisepsis include (a) preparing the skin before surgical incisions with iodine compounds, (b) swabbing an open root canal with hydrogen peroxide, and (c) ordinary hand washing with a germicidal soap. Sanitization is any cleansing technique that mechanically removes microorganisms (along with food debris) to reduce the level of contaminants. A sanitizer is a compound (e.g., soap or detergent) that is used to perform this task. Cooking utensils, dishes, bottles, cans, and used clothing that have been washed and dried may not be completely free of microbes, but they are considered safe for normal use. Air sanitization with ultraviolet lamps reduces airborne microbes in hospital rooms, veterinary clinics, and laboratory installations. It is often necessary to reduce the numbers of microbes on the human skin through degerming procedures. This process usually involves scrubbing the skin or immersing it in chemicals, or both. It also emulsifies oils that lie on the outer cutaneous layer and mechanically removes potential pathogens from the outer layers of the skin. Examples of degerming procedures are (a) surgical hand scrub, (b) application of alcohol wipes to the skin, and (c) cleansing of a wound with germicidal soap and water. The concepts of antisepsis and degerming procedures clearly overlap, since a degerming procedure can be simultaneously treated as an antiseptic and vice versa. Sterilization Methods of sterilization can be broadly classified as: 1. Physical methods of sterilization, and 2. Chemical methods of sterilization. Physical Methods of Sterilization Physical methods of sterilization include the following: 1. 2. 3. 4. 5. ◗ Sunlight Heat Filtration Radiation Sound (sonic) waves Sunlight Direct sunlight is a natural method of sterilization of water in tanks, rivers, and lakes. Direct sunlight has an active germicidal STERILIZATION AND DISINFECTION ◗ Heat Sterilization by moist heat 1. 2. 3. 4. Sterilization at a temperature ⬍100°C Sterilization at a temperature of 100°C Sterilization at a temperature ⬎100°C Intermittent sterilization Key Points Two methods of pasteurization are followed: flash method and holder method. ■ ■ In the flash method, milk is exposed to heat at 72°C for 15–20 seconds followed by a sudden cooling to 13°C or lower. In the holder method, milk is exposed to a temperature of 63°C for 30 minutes followed by cooling to 13°C or lower, but not less than 6°C. The flash method is preferable for sterilization of milk because it is less likely to change the flavor and nutrient content, and it is more effective against certain resistant pathogens, such as Coxiella and Mycobacterium. Although pasteurization inactivates most viruses and destroys the vegetative stages of 97–99% of bacteria and Chapter 43 1. Sterilization at a temperature ⬍100°C: Pasteurization is an example of sterilisation at a temperature ⬍100°C. Pasteurization: Fresh beverages (such as milk, fruit juices, beer, and wine) are easily contaminated during collection and processing. Because microbes have potential for spoiling these foods or causing illness, heat is frequently used to reduce the microbial load and to destroy pathogens. Pasteurization is a technique in which heat is applied to liquids to kill potential agents of infection and spoilage, while at the same time retaining the liquid’s flavor and food value. This technique is named after Louis Pasteur who devised this method. This method is extensively used for sterilization of milk and other fresh beverages, such as fruit juices, beer, and wine which are easily contaminated during collection and processing. Section III Moist heat occurs in the form of hot water, boiling water, or steam (vaporized water). In practice, the temperature of moist heat usually ranges from 60 to 135°C. Adjustment of pressure in a closed container can regulate the temperature of steam. Moist heat kills microorganisms by denaturation and coagulation of proteins. Sterilization by moist heat can be classified as follows: 2. Sterilization at a temperature of 100°C: Sterilsation at a temperature of 100ºC includes (a) boiling and (b) steam sterilizer at 100°C. Boiling: Simple boiling of water for 10–30 minutes kills most of the vegetative forms of bacteria but not bacterial spores. Exposing materials to boiling water for 30 minutes kills most nonspore-forming pathogens including resistant species, such as the tubercle bacillus and staphylococci. Sterilization by boiling is facilitated by addition of 2% sodium bicarbonate to water. Since boiling only once at 100°C does not kill all spores, this method cannot be used for sterilization but only for disinfection. Hence, it is not recommended for sterilizing instruments used for surgical procedure. The greatest disadvantage of this method is that the items sterilized by boiling can be easily recontaminated when removed from water after boiling. Steam sterilizer at 100°C: Usually, Koch’s or Arnold’s steam sterilizer is used for heat-labile substances that tend to degrade at higher temperatures and pressure, such as during the process of autoclaving. These substances are exposed to steam at atmospheric pressure for 90 minutes during which most vegetative forms of the bacteria except for the thermophiles are killed by the moist heat. 3. Sterilization at a temperature > 100°C: This method is otherwise known as sterilization by steam under pressure. A temperature of 100°C is the highest that steam can reach under normal atmospheric pressure at sea level. This pressure is measured at 15 pounds per square inch ( psi), or 1 atmosphere. In order to raise the temperature of steam above this point, it must be pressurized in a closed chamber. This phenomenon is explained by the physical principle that governs the behavior of gases under pressure. When a gas is compressed, its temperature rises in direct relation to the amount of pressure. So, when the pressure is increased to 5 psi above normal atmospheric pressure, the temperature of steam rises to 109°C. When the pressure is increased to 10 psi above normal, its temperature will be 115°C and at 15 psi (a total of 2 atmospheres), it will be 121°C. It is not the pressure by itself that is killing microbes, but the increased temperature it produces. This forms the principle of sterilization by steam under pressure. Such pressure–temperature combinations can be achieved only with a special device that can subject pure steam to pressures greater than 1 atmosphere. Health and commercial industries use an autoclave for this purpose and a comparable home appliance is the pressure cooker. Chapter 3 Heat is the most dependable method of sterilization and is usually the method of choice unless contraindicated. As a rule, higher temperatures (exceeding the maximum) are microbicidal, whereas lower temperatures (below the minimum) tend to have inhibitory or microbistatic effects. Two types of physical heat are used in sterilization—moist and dry heat. fungi, it does not kill endospores or thermoduric species (mostly nonpathogenic lactobacilli, micrococci, and yeasts). Milk is not sterile after regular pasteurization. In fact, it can contain 20,000 microbes per milliliter or more, which explains why even an unopened carton of milk will eventually spoil on prolonged storage. Newer techniques have now been used to produce sterile milk that has a storage life of 3 months. In this method, milk is processed with ultrahigh temperature (UHT) of 134°C for 1–2 seconds. Section I effect due to its content of ultraviolet and heat rays. Bacteria present in natural water sources are rapidly destroyed by exposure to sunlight. 25 Chapter 43 Section III Chapter 3 Section I 26 GENERAL MICROBIOLOGY Autoclave: It is a cylindrical metal chamber with an airtight door at one end and racks to hold materials. The lid is fastened by screw clamp and rendered airtight by an asbestos washer. It has a discharge tap for air and steam at the upper side, a pressure gauge and a safety valve that can be set to blow off at any desired pressure. Heating is usually carried out by electricity. Steam circulates within the jacket and is supplied under pressure to the inner chamber where materials are loaded for sterilization (Fig. 3-1). The water in the autoclave boils when its vapor pressure equals that of surrounding atmosphere. Following the increase of pressure inside the closed vessel, the temperature at which the water boils inside the autoclave also increases. The saturated steam that has a higher penetrative power, on coming in contact with a cooler surface condenses to water and releases its latent heat to that surface. For example, nearly 1600 mL steam at 100°C and at atmospheric pressure condenses into 1 mL of water at 100°C and releases 518 calories of heat. The gross reduction in volume of steam sucks in more steam to the area and this process continues till the temperature of that surface is elevated to that of the steam. Sterilization is achieved when the steam condenses against the objects in the chamber and gradually raises their temperature. The condensed water facilitates moist conditions that ensures killing of microbes. Sterilization condition: Experience has shown that the most efficient pressure–temperature combination for achieving sterilization by autoclave is 15 psi, which yields 121°C. It is possible to use higher pressure to reach higher temperatures (for instance, increasing the pressure to 30 psi raises the temperature by 11°C), but doing so will not significantly reduce the exposure time and can harm the items being sterilized. It is important to avoid over packing or haphazardly loading the chamber, because it prevents steam from circulating freely around the contents and impedes the full contact that is necessary. The holding time varies from 10 minutes for light loads to 40 minutes for heavy or bulky ones; the average time being 20 minutes. Key Points Uses of autoclave: The autoclave has many uses, which are given below: ■ ■ ■ It is a good method to sterilize heat-resistant materials, such as glassware, cloth (surgical dressings), rubber (gloves), metallic instruments, liquids, paper, some media, and some heat-resistant plastics. It is also useful for sterilization of heat-sensitive items, such as plastic Petri plates that need to be discarded. It is useful for sterilization of materials that cannot withstand the higher temperature of the hot-air oven. However, the autoclave is ineffective for sterilizing substances that repel moisture (oils, waxes, or powders). Types and uses of various moist heat sterilization methods are summarized in Table 3-1. Sterilization controls: Various sterilization controls are used to determine the efficacy of sterilization by moist heat. These include (a) thermocouples, (b) chemical indicators, and (c) bacteriological spores as mentioned below: (a) Thermocouples are used to record temperatures directly in autoclaves by a potentiometer. (b) Brown’s tube is the most commonly used chemical indicator of moist heat sterilization in the autoclave. It contains red solution that turns green when exposed to temperature of 121°C for 15 minutes in an autoclave. (c) Bacillus stearothermophilus spores are used as the indicators of moist heat sterilization in the autoclave. This is a thermophilic bacterium with an optimum temperature of 55–60°C, and its spores require an exposure of 12 minutes at 121°C to be destroyed. The efficacy of the autoclave is carried out by placing paper strips impregnated with 106 spores in envelopes and keeping those envelopes in different parts of the load inside the autoclave. These strips after sterilization are inoculated into a suitable recovery medium and TABLE 3-1 FIG. 3-1. Autoclave. Types and uses of moist heat sterilization Method Uses Comments Water bath below 100oC Water bath at 100oC Arnold steamer: steaming at 100oC Autoclave: steam under pressure For sterilization of serum, body fluids, and vaccines For sterilization of glass, metal, and rubber items For sterilization of culture media containing sugar and gelatin For sterilization of culture media and operation theater as well as laboratory materials Only disinfection possible. Spores would be spared Some spores will still be spared at this temperature Preserves properties of media Kills all the vegetative as well as spore forms of bacteria STERILIZATION AND DISINFECTION 27 Chapter 3 FIG. 3-2. Sterilization by dry heat makes use of air with a low moisture content that has been heated by a flame or electric heating coil. In practice, the temperature of dry heat ranges from 160°C to several thousand degrees Celsius. The dry heat kills microorganisms by protein denaturation, oxidative damage, and the toxic effect of increased level of electrolytes. Dry heat is not as versatile or as widely used as moist heat, but it has several important sterilization applications. The temperature and time employed in dry heat vary according to the particular method, but in general they are greater than with moist heat. Sterilization by dry heat includes sterilization by (a) flaming, (b) incineration , and (c) hot air oven: 1. Flaming: Sterilization of inoculating loop or wire, the tip of forceps, searing spatulas, etc., is carried out by holding them in the flame of the Bunsen burner till they become red hot. Glass slides, scalpels, and mouths of culture tubes are sterilized by passing them through the Bunsen flame without allowing them to become red hot. 2. Incineration: Incineration is an excellent method for safely destroying infective materials by burning them to ashes. It has many uses: ■ ■ Incinerators are used to carry out this process and are regularly employed in hospitals and research labs to destroy hospital and laboratory wastes. The method is used for complete destruction and disposal of infectious material, such as syringes, needles, culture material, dressings, bandages, bedding, animal carcasses, and pathology samples. This method is fast and effective for most hospital wastes, but not for metals and heat-resistant glass materials. Key Points Hot-air oven is used in laboratories and clinics for heatresistant items that are not sterilized well by moist heat. They are used for sterilization of: ■ ■ ■ ■ Glasswares (syringes, Petri dishes, flasks, pipettes, test tubes, etc.). Surgical instruments (scalpels, scissors, forceps, etc.). Chemicals (liquid paraffin, sulfonamide powders, etc.); and Oils that are not penetrated well by steam used in moist heat sterilization. Thermocouples, chemical indicators, and bacteriological spores of Bacillus subtilis are used as sterilization controls to determine the efficacy of sterilization by hot-air oven. ◗ Filtration Filtration is an excellent way to reduce the microbial population in solutions of heat-labile material by use of a variety of filters. Filters are used to sterilize these heat-labile solutions. Chapter 43 3. Hot-air oven: The hot-air oven provides another means of dry heat sterilization and is the most widely used method. The hot-air oven is electrically heated and is fitted with a fan to ensure adequate and even distribution of hot air in the chamber. It is also fitted with a thermostat that ensures circulation of hot air of desired temperature in the chamber. Heated, circulated air transfers its heat to the materials inside the chamber. While sterilizing by hot-air oven, it should be ensured that the oven is not overloaded. The materials should be dry and arranged in a manner which allows free circulation of air inside the chamber. It is essential to fit the test tubes, flasks, etc., with cotton plugs and to wrap Petri dishes and pipettes in a paper. Sterilization by hot-air oven requires exposure to 160–180°C for 2 hours and 30 minutes, which ensures thorough heating of the objects and destruction of spores (Fig. 3-2). Sterilization by dry heat ■ Hot-air oven. Section III 4. Intermittent sterilization: Certain heat-labile substances (e.g., serum, sugar, egg, etc.) that cannot withstand the high temperature of the autoclave can be sterilized by a process of intermittent sterilization, known as tyndallization. Tyndallization is carried out over a period of 3 days and requires a chamber to hold the materials and a reservoir for boiling water. Items to be sterilized are kept in the chamber and are exposed to free-flowing steam at 100°C for 20 minutes, for each of the three consecutive days. On the first day, the temperature is adequate to kill all the vegetative forms of the bacteria, yeasts, and molds but not sufficient to kill spores. The surviving spores are allowed to germinate to vegetative forms on the second day and are killed on re-exposure to steam. The third day re-ensures killing of all the spores by their germination to vegetative forms. Intermittent sterilization is used most often to sterilize heat-sensitive culture media, such as those containing sera (e.g., Loeffler’s serum slope), egg (e.g., Lowenstein–Jensen’s medium), or carbohydrates (e.g., serum sugars) and some canned foods. Section I incubated at 55°C for 5 days. Spores are destroyed if the sterilizing condition of the autoclave is proper. Chapter 43 Section III Chapter 3 Section I 28 GENERAL MICROBIOLOGY Filters simply remove contaminating microorganisms from solutions rather than directly destroying them. The filters are of two types: (a) depth filters and (b) membrane filters. 1. Depth filters: Depth filters consist of fibrous or granular materials that have been bonded into a thick layer filled with twisting channels of small diameter. The solution containing microorganisms is sucked in through this layer under vacuum and microbial cells are removed by physical screening or entrapment and also by adsorption to the surface of the filter material. Depth filters are of the following types: ■ Candle filters: These are made up of (a) diatomaceous earth (e.g., Berkefeld filters) or (b) unglazed porcelain (e.g., Chamberlain filters). They are available in different grades of porosity and are used widely for purification of water for drinking and industrial uses. ■ Asbestos filters: These are made up of asbestos such as magnesium silicate. Seitz and Sterimat filters are the examples of such filters. These are disposable and single-use discs available in different grades. They have high adsorbing capacity and tend to alkalinize the filtered fluid. Their use is limited by the carcinogenic potential of asbestos. ■ Sintered glass filters: These are made up of finely powdered glass particles, which are fused together. They have low absorbing property and are available in different pore sizes. These filters, although can be cleaned easily, are brittle and expensive. 2. Membrane filters: Membrane filters are made up of (a) cellulose acetate, (b) cellulose nitrate, (c) polycarbonate, (d) polyvinylidene fluoride, or (e) other synthetic materials. These filters are now widely used and have replaced depth filters for last many years. These filters are circular porous membranes and are usually 0.1 mm thick. Although a wide variety of pore sizes (0.015–12 ␮m) are available, membranes with pores about 0.2 ␮m are used, because the pore size is smaller than the size of bacteria. These filters are used to remove most vegetative cells, but not viruses, from solutions to be filtered. In the process of filtration, the membranes are held in special holders and often preceded by depth filters made of glass fibers to remove larger particles that might clog the membrane filter. The solution is then pushed or forced through the filter with a vacuum or with pressure from a syringe, peristaltic pump, or nitrogen gas bottle, and collected in previously sterilized containers. Key Points Membrane filters remove microorganisms by screening them out in the way a sieve separates large sand particles from small ones. These filters have many uses: ■ ■ ■ They are used to sterilize pharmaceutical substances, ophthalmic solutions, liquid culture media, oils, antibiotics, and other heat-sensitive solutions. They are used to obtain bacterial free filtrates of clinical specimens for virus isolation. They are used to separate toxins and bacteriophages from bacteria. Types and uses of radiation for sterilization TABLE 3-2 Types Uses Comments Ionizing radiation administered using Cobalt-60-based instruments For sterilization of pharmaceuticals like antibiotics, hormones, sutures; and prepacked disposable items, such as syringes, infusion sets, catheters, etc. Though expensive and fraught with safety risks, it is very effective due to better penetration power Nonionizing radiation administered through UV lamps Only for disinfection of clear surfaces in OTs, laminar flow hoods, etc. Hazardous and not as effective as ionizing radiation Air also can be sterilized by filtration. Two common examples are surgical masks and cotton plugs on culture vessels that let air in but keep microorganisms out. Laminar flow biological safety cabinets are most widely used air filtration systems in hospitals and industries. In this method, air is passed through high-efficiency particulate air (HEPA) filters that remove nearly 99.97% of 0.3 ␮m particles from the filtered air. ◗ Radiations The ionizing and nonionizing radiations are the two types of radiation used for sterilization (Table 3-2). 1. Ionizing radiations: Ionizing radiation is an excellent sterilizing agent with very high penetrating power. These radiations penetrate deep into objects and destroy bacterial endospores and vegetative cells, both prokaryotic and eukaryotic. These are, however, not that effective against viruses. Ionizing radiations include (a) X-rays, (b) gamma rays, and (c) cosmic rays. Gamma radiation from a cobalt-60 source is used for sterilization of antibiotics, hormones, sutures, catheters, animal feeds, metal foils, and plastic disposables, such as syringes. This has also been used to sterilize and “pasteurize” meat and other food items. Irradiation usually kills Escherichia coli O157:H7, Staphylococcus aureus, Campylobacter jejuni, and other pathogens. Since there is no detectable increase of temperature in this method, this method is commonly referred to as “cold sterilization.” Both the Food and Drug Administration and the World Health Organization have approved food irradiation and declared it safe. Key Points Gamma radiation from a cobalt-60 source is used for sterilization of antibiotics, hormones, sutures, catheters, animal feeds, metal foils, and plastic disposables such as syringes. This has also been used to sterilize and ‘pasteurize’ meat and other food items. STERILIZATION AND DISINFECTION ■ ■ UV radiation is used primarily for disinfection of closed areas in microbiology laboratory, inoculation hoods, laminar flow, and operating theaters. It kills most vegetative bacteria but not spores, which are highly resistant to these radiations. However, it does not penetrate glass, dirt films, water, and other substances very effectively. Since UV radiations on prolonged exposure tend to burn the skin and cause damage to the eyes, UV lamps should be switched off while people are working in such areas. ◗ Key Points Sound (sonic) waves As a general rule, chilling, freezing, and desiccation are not considered as methods of disinfection or sterilization because their antimicrobial effects are erratic and uncertain, and one cannot be sure that pathogens subjected to these procedures have been killed. Disinfection Disinfection is the process of inactivating microorganisms by direct exposure to chemical or physical agents. Differences between sterilization and disinfection have been summarized in Table 3-4. ■ Chemical Methods of Sterilization Several chemical agents are used as antiseptics as well as disinfectants. All these chemical agents (e.g., alcohols, aldehydes, etc.) are described later in detail under disinfection. The effects of cold and desiccation: The main benefit of cold treatment is to slow the growth of cultures and microbes in food during processing and storage. It is essential to know that TABLE 3-3 Biological controls used for testing efficacy of sterilization techniques Technique Control organism Autoclave Geobacillus stearothermophilus Hot-air oven Bacillus subtilis Ionizing radiations Bacillus pumilus Ethylene oxide Bacillus globigii Bacillus subtilis ■ Disinfectants are products or biocides that destroy or inhibit the growth of microorganisms on inanimate objects or surfaces. Disinfectants can be sporistatic but are not necessarily sporicidal. Antiseptics are biocides or products that destroy or inhibit the growth of microorganisms in or on living tissue. Antiseptics and disinfectants are used extensively in hospitals for a variety of topical and hard surface applications. They are an essential part of infection control practices and aid in the prevention of nosocomial infections. Properties of Ideal Disinfectant An ideal disinfectant or antiseptic has the following characteristics: 1. Ideally, the disinfectant should have a wide spectrum of antimicrobial activity. It must be effective against a wide variety of infectious agents (Gram-positive and Gramnegative bacteria, acid-fast bacteria, bacterial endospores, fungi, and viruses) at high dilutions. Chapter 43 High-frequency sound (sonic) waves beyond the sensitivity of the human ear are known to disrupt cells. Sonication transmits vibrations through a water-filled chamber (sonicator) to induce pressure changes and create intense points of turbulence that can stress and burst cells in the vicinity. Sonication also forcefully dislodges foreign matter from objects. Heat generated by the sonic waves (up to 80°C) also appears to contribute to the antimicrobial action. Gram-negative rods are most sensitive to ultrasonic vibrations, while Gram-positive cocci, fungal spores, and bacterial spores are resistant to them. Ultrasonic devices are used in dental and some medical offices to clear debris and saliva from instruments before sterilization and to clean dental restorations. However, most sonic machines are not reliable for regular use in disinfection or sterilization. Biological controls used for testing the efficacy of sterilization techniques are summarized in Table 3-3. Section III Lyophilization is a process of freezing and drying. It is the most common method of preserving microorganisms and other cells in a viable state for many years. Pure cultures are frozen instantaneously and exposed to a vacuum that rapidly removes the water (it goes right from the frozen state into the vapor state). This method avoids the formation of ice crystals that would damage the cells. Although not all cells survive this process, lot many of them survive after reconstitution of lyophilized culture. Chapter 3 Infrared radiations are used for rapid and mass sterilization of disposable syringes and catheters. Ultraviolet (UV) radiation with wavelength of 240–280 nm is quite lethal and has a marked bactericidal activity. It acts by denaturation of bacterial protein and also interferes with replication of bacterial DNA. cold merely retards the activities of most microbes. Although it is true that cold temperatures kill some microbes, gradual cooling, long-term refrigeration, or deep-freezing does not adversely affect most of the microorganisms. In fact, freezing temperatures, ranging from ⫺70 to ⫺135°C, provide an environment that can preserve cultures of bacteria, viruses, and fungi for longer periods. Some psychrophiles grow very slowly even at freezing temperatures and can continue to secrete toxic products. S. aureus, Clostridium species (spore formers), Streptococcus species, and several types of yeasts, molds, and viruses are the pathogens that can survive for several months in the refrigerated food items. Section I 2. Nonionizing radiations: Nonionizing radiations include infrared and ultraviolet radiations. 29 Chapter 3 Section I 30 GENERAL MICROBIOLOGY TABLE 3-4 Definition Uses Chapter 43 Section III Examples Differences between sterilization and disinfection Sterilization Disinfection Freeing an article, surface, or medium from all living organisms including viruses, bacteria and their spores, and fungi and their spores. Process that reduces the number of contaminating microorganisms, liable to cause infection to a level which is deemed no longer harmful to health. Spores are not killed. Objects or instruments coming in direct contact with a break in skin or mucous membrane or entering a sterile body area. Objects or instruments coming in direct contact with mucous membrane but tissue is intact or via intact skin. Surgical instruments, needles, syringes, parenteral fluid, arthroscopes, media, reagents and equipments in laboratory use. Endotracheal tubes, aspirators, gastroscopes, bed pans, urinals, etc. 2. It should act in the presence of organic matter. 3. It should not be toxic to human or corrosive. In practice, this balance between effectiveness and low toxicity for animals is hard to achieve. Some chemicals are used despite their low effectiveness, because they are relatively nontoxic. 4. It should be stable upon storage and should not undergo any chemical change. 5. It should be odorless or with a pleasant odor. 6. It should be soluble in water and lipids for penetration into microorganisms. 7. It should be effective in acidic as well as in alkaline media. 8. It should have speedy action. 9. If possible, it should be relatively inexpensive. Action of Disinfectants ■ ■ ■ Types of Disinfectants Disinfectants include the following: (a) phenolic compounds, (b) halogens, (c) alcohols, (d) aldehydes, (e) gases, (f) surface active agents, (g) oxidizing agents, (h) dyes, (i) heavy metals, and (j) acids and alkalis. ◗ ■ ■ ■ Various conditions influencing the efficiency of disinfectant are as follows: ■ Temperature: Increase in temperature increases the efficiency of disinfectants. Phenol: It is effective against vegetative forms of bacteria, Mycobacterium tuberculosis, and certain fungi. It is an excellent disinfectant for feces, blood, pus, sputum, etc. It has a low degree of activity as compared to other derivatives. It is not suitable for application to skin or mucous membrane. Cresol: Cresols are more germicidal and less poisonous than phenol but corrosive to living tissues. They are used for cleaning floors (1% solution), for disinfection of surgical instruments, and for disinfection of contaminated objects. Lysol is a solution of cresols in soap. Halogenated diphenyl compounds: These compounds include hexachlorophene and chlorhexidine. They are highly effective against both Gram-positive and Gram-negative bacteria. They are used as skin antiseptics and for the cleaning of wound surfaces. Hexachlorophene has been one of the most popular antiseptics because once applied it persists on the skin and reduces growth of skin bacteria for longer periods. However, it can cause brain damage and is now used in hospital nurseries only after a staphylococcal outbreak. ◗ Factors Influencing Activity of Disinfectants Phenolic compounds In 1867, Joseph Lister employed phenolic compounds to reduce the risk of infection during operations. Phenolic compounds are the most widely used antiseptics and disinfectants in laboratories and hospitals worldwide. They are bactericidal or bacteriostatic and some are fungicidal also. They act by denaturing proteins and disrupting cell membranes. They are effective in the presence of organic material and remain active on surfaces long after application. Different phenolic compounds are as follows: Disinfectants act in many ways as discussed below. 1. They produce damage to the cell wall and alter permeability of the cell membrane, resulting in exposure, damage, or loss of the cellular contents. 2. They alter proteins and form protein salts or cause coagulation of proteins. 3. They inhibit enzyme action and inhibit nucleic acid synthesis or alter nucleic acid molecules. 4. They cause oxidation or hydrolysis. Type of microorganism: Vegetative cells are more susceptible than spores. Spores may be resistant to the action of disinfectants. Physiological state of the cell: Young and metabolically active cells are more sensitive than old dormant cells. Nongrowing cells may not be affected. Environment: The physical or chemical properties of the medium or substance influence rate as well as efficiency of disinfectants, e.g., pH of the medium and presence of extraneous materials. Halogens Halogens are fluorine, bromine, chlorine, and iodine—a group of nonmetallic elements that commonly occur in minerals, sea water, and salts. Although they can occur either in the ionic (halide) or nonionic state, most halogens exert their antimicrobial activity primarily in their nonionic state, but not in the halide state (e.g., chloride, iodide). STERILIZATION AND DISINFECTION ■ ■ Key Points ■ Sanitization and disinfection of food equipment in dairies, restaurants, and canneries; Treatment of swimming pools, spas, drinking water, and even fresh foods; Treatment of wounds; and ■ Disinfection of equipments, beddings, and instruments. ■ ■ Common household bleach is a weak solution (5%) of sodium hypochlorite that is used as an all-around disinfectant, deodorizer, and stain remover. It is frequently used as an alternative to pure chlorine in treating water supplies. However, the major limitations of chlorine compounds are that they are: (a) Ineffective if used at an alkaline pH, (b) Less effective in the presence of excess organic matter, and (c) Relatively unstable, especially if exposed to light. Iodine and its compounds: Iodine is a pungent black chemical that forms brown-colored solutions when dissolved in water or alcohol. Iodine rapidly penetrates the cells of microorganisms, where it apparently disturbs a variety of metabolic functions. It acts by interfering with the hydrogen and disulfide bonds of proteins (similar to chlorine). It kills all types of microorganisms if optimum concentrations and exposure times are used. Iodine activity, unlike chlorine, is not as adversely affected by organic matter and pH. The two primary iodine preparations are free iodine in solution and iodophors. Free iodine in solution: Aqueous iodine contains 2% free iodine in solution and 2.4% sodium iodide. It is used as a topical ◗ Alcohols Alcohols are among the most widely used disinfectants and antiseptics. They are bactericidal and fungicidal but not sporicidal. They have no action against spores and viruses. Ethyl alcohol and isopropyl alcohol are the two most popular alcohol germicides. They are effective at a concentration of 60–70% in water. They act by denaturing bacterial proteins and possibly by dissolving membrane lipids. They are used as skin antiseptics. Isopropyl alcohol is used for disinfection of clinical thermometers. A 10–15 minute soaking is sufficient to disinfect thermometers. Methyl alcohol is effective against fungal spores. ◗ Aldehydes Formaldehyde and glutaraldehyde are the two most commonly used aldehydes that are used as disinfectants. They are highly reactive molecules that combine with nucleic and alkylating molecules. They are sporicidal and can also be used as chemical sterilants. Formaldehyde: Formaldehyde is usually dissolved in water or alcohol before use. In aqueous solution, it is bactericidal, sporicidal, and also effective against viruses. Formalin solution is 40% aldehyde in aqueous solution. It is used to: ■ ■ ■ ■ ■ Preserve fresh tissue specimens, Destroy anthrax spores in hair and wool, Prepare toxoids from toxins, Sterilize bacterial vaccines, and Kill bacterial cultures and suspensions. Chapter 43 Hypochlorites are perhaps the most extensively used of all chlorine compounds. They are used for: Section III ■ Betadine, povidone, and isodine are the common iodophor compounds that contain 2–10% of available iodine. They are used to prepare skin and mucous membranes for surgery and in surgical hand scrubs. They are also used to treat burns and to disinfect equipments. A recent study has shown that betadine solution is an effective means of preventing eye infections in newborn infants, and it may replace antibiotics and silver nitrate as the method of choice. Chapter 3 antiseptic before surgery and also occasionally as a treatment for burnt and infected skin. A stronger iodine solution (5% iodine and 10% potassium iodide) is used primarily as a disinfectant for plastic items, rubber instruments, cutting blades, and thermometers. Iodine tincture is a 2% solution of iodine and sodium iodide in 70% alcohol that can be used in skin antisepsis. Because iodine can be extremely irritating to the skin and toxic when absorbed, strong aqueous solutions and tinctures (5–7%) are no longer considered safe for routine antisepsis. Iodine tablets are available for disinfecting water during emergencies or for destroying pathogens in impure water supplies. Iodophors: Iodophors are complexes of iodine and a neutral polymer, such as polyvinyl alcohol. This formulation permits the slow release of free iodine and increases its degree of penetration. These compounds have largely replaced free iodine solutions in medical antisepsis because they are less prone to staining or irritating tissues. Section I These agents are highly effective disinfectants and antiseptics, because they are microbicidal and not just microbistatic. They are also sporicidal with longer exposure. For these reasons, halogens are the active ingredients in nearly one-third of all antimicrobial chemicals currently marketed. Chlorine and iodine are the only two routinely used halogens because fluorine and bromine are dangerous to handle. Chlorine and its compounds: Chlorine has been used for disinfection and antisepsis for approximately 200 years. The major forms used in microbial control are (a) liquid and gaseous chlorine and (b) hypochlorites. In solution, these compounds combine with water and release hypochlorous acid (HOCl), which oxidizes the sulfhydryl (S–H) group on the amino acid cysteine and interferes with disulfide (S–S) bridges on numerous enzymes. The resulting denaturation of the enzymes is permanent. Gaseous and liquid chlorine are used almost exclusively for large-scale disinfection of drinking water, sewage, and wastewater from sources, such as agriculture and industry. Chlorine kills not only bacterial cells and endospores but also fungi and viruses. Treatment of water with chlorine destroys many pathogenic vegetative microorganisms without unduly affecting its taste. Chlorination at a concentration of 0.6–1.0 part of chlorine per million parts of water makes water potable and safe to use. 31 Chapter 43 Section III Chapter 3 Section I 32 GENERAL MICROBIOLOGY Glutaraldehyde: A 2% buffered solution of glutaraldehyde is an effective disinfectant. It is less irritating than formaldehyde and is used to disinfect hospital and laboratory equipments. Glutaraldehyde usually disinfects objects within time frame of 10 minutes but may require as long as 12 hours to destroy all spores. Glutaraldehyde is especially effective against tubercle bacilli, fungi, and viruses. It can be used for cleaning cystoscopes and bronchoscopes, corrugated rubber anesthetic tubes and face masks, plastic endotracheal tubes, metal instruments, and polythene tubing. ◗ 1. Cationic surface active agents: The cationic detergents are effective disinfectants. Cationic detergents like benzalkonium chloride and cetylpyridinium chloride kill most bacteria but not M. tuberculosis, endospores, or viruses. They do have the advantages of being stable and nontoxic, but they are inactivated by hard water and soap. These are often used as skin antiseptics and also as disinfectants for disinfection of food utensils and small instruments. Quaternary ammonium compounds, such as cetrimide are the most popular cationic detergents. They act by disrupting microbial membranes and possibly by denaturing proteins. 2. Anionic surface active agents: These include soaps prepared either from saturated or unsaturated fatty acids, which act better at acidic pH. The soaps prepared from saturated fatty acids are more effective against Gram-negative organisms, whereas those prepared from unsaturated fatty acids are more active against Gram-positive bacilli and Neisseria. 3. Nonionic surface active agents: These are nontoxic and some of them may even promote the growth of bacteria. 4. Amphoteric or ampholytic compounds: These are active against a wide range of Gram-positive and Gram-negative bacteria and also against a few viruses. These are known as “Tego” compounds. Gases Various gaseous agents are used for sterilization of large volume of heat-sensitive disposable items and also instruments. Ethylene oxide, formaldehyde gas, and betapropiolactone are frequently used gaseous agents. Ethylene oxide: Ethylene oxide is a colorless liquid used for gaseous sterilization. It is active against all kinds of bacteria, spores, and viruses. It kills all types of microorganisms by inhibiting proteins and nucleic acids. It is both microbicidal and sporicidal. It is a highly effective sterilizing agent because it rapidly penetrates packing materials, including plastic wraps. It is used to sterilize disposable plastic Petri dishes, sutures, syringes, heart-lung machine, respirators, and dental equipments. Ethylene oxide is highly inflammable and carcinogenic. Extensive aeration of the sterilized materials is necessary to remove residual ethylene oxide gas, which is toxic. Formaldehyde gas: The formaldehyde gas is used for (a) the fumigation of operation theaters, wards, sick rooms, and laboratories; and (b) the sterilization of instruments and heat-sensitive catheters, clothing and bedding, furniture, books, etc. The formaldehyde gas is produced by adding 150 gm of potassium permanganate to 280 mL formalin in 1000 cu ft of room volume. The room to be sterilized is completely closed and sealed at least for 48 hours after fumigation with formalin gas. Sterilization is achieved by condensation of gas on exposed surface. The gas is toxic when inhaled and is irritant to eye, hence its effect should be nullified by exposure to ammonia. It is highly inflammable and carcinogenic. Beta-propiolactone: Beta-propiolactone (BPL) is a condensation product of ketone and formaldehyde. It is active against all microorganisms and viruses. It is more efficient than formaldehyde for fumigation purpose. In the liquid form, it has been used to sterilize vaccines and sera. BPL destroys microorganisms more readily than ethylene oxide but does not penetrate materials well and may be carcinogenic. For these reasons, BPL has not been used as extensively as ethylene oxide. Recently, vapor-phase hydrogen peroxide has been used to decontaminate biological wastes. ◗ ends. Due to their amphipathic nature, detergents solubilize and are very effective cleansing agents. They are different from soaps, which are derived from fats. Surface active agents are of four types: Surface active agents Surface active agents, such as detergents are the substances that alter energy relationship at interfaces producing a reduction in surface tension. Detergents are organic molecules that serve as wetting agents and emulsifiers because they have both polar hydrophilic and nonpolar hydrophobic ◗ Oxidizing agents This group includes halogens, hydrogen peroxide, potassium permanganate, and sodium perborate. They are good disinfectants and antiseptics but are less effective in the presence of organic matter. Hydrogen peroxide, used as 3% solution, is a weak disinfectant. It is useful for cleaning of the wounds and for mouth wash or gargle. Potassium permanganate is bactericidal in nature and active against viruses also. ◗ Dyes The dyes that have been used extensively as skin and wound antiseptics include (a) acridine dyes and (b) aniline dyes. The acridine dyes include acriflavine, euflavine, proflavine, and aminacrine. They show more activity against Gram-positive bacteria than against Gram-negative organisms. They act by interfering with the synthesis of nucleic acids and proteins in bacterial cells. The yellow acridine dyes, acriflavine and proflavine, are sometimes used for antisepsis and wound treatment in medical and veterinary clinics. Aniline dyes (such as gentian violent, crystal violet, and malachite green) are also more active against Gram-positive bacteria than against Gram-negative organisms. They are also effective against various fungi, hence are incorporated into solutions and ointments to treat fungal skin infections, such as ringworm. The dyes, nevertheless, have limited applications because they stain and have a narrow spectrum of antimicrobial activity. They also have no activity against tubercle bacilli. Their actions are also inhibited by the presence of organic matter. STERILIZATION AND DISINFECTION ◗ Heavy metals ◗ Acids and alkalis Key Points ◗ Chick Martin test Testing of Disinfectants It is a modification of Rideal–Walker test, in which the disinfectant acts in the presence of organic contaminants (e.g., dried yeast, feces, etc.) to simulate the natural conditions. The efficiency of disinfectants can be determined with the help of several tests. These are: ◗ ■ ■ ■ ■ Phenol coefficient (Rideal–Walker) test Chick Martin test Capacity (Kelsey–Sykes) test In-use (Kelsey–Maurer) test TABLE 3-5 Activities of commonly used disinfectants Disinfectant Fungi Bacteria Spores Enveloped Nonenveloped viruses viruses Phenol Good Good Nil Good Variable Hypochlorite Good Good Fair Good Good Alcohols Good Good Nil Good Variable Aldehydes Good Good Good Good Good Glutaraldehyde Good Good Good Good Good Nil Good Good Iodophor Good Good Capacity (Kelsey–Sykes) test The capacity (Kelsey–Sykes) test determines the appropriate use of dilutions of the disinfectants. It measures the capacity of a disinfectant to retain its activity when repeatedly used microbiologically. The disinfectant is assessed by its ability to kill bacteria by demonstrating growth or no growth on recovery culture media but not by comparison with phenol. The test is performed under both clean and dirty conditions, hence shows the effectiveness of a disinfectant in the presence of organic material. ◗ In-use (Kelsey–Maurer) test The “in-use” (Kelsey–Maurer) test is a test that determines whether the chosen disinfectant is effective, in actual use, in hospital practice and also for the period of its use. The effectiveness of the disinfectant is determined by its ability to inactivate a known number of standard strain of a pathogenic staphylococci on a given surface within a certain given time. In-use test allows a more accurate determination of effectiveness of a disinfectant compared to phenol coefficient test. Chapter 43 The higher the phenol coefficient value, the more effective the disinfectant under these test conditions. A value greater than 1 means that the disinfectant is more effective than the phenol. The test, however, does not show the action of disinfectant in natural conditions, i.e., in the presence of organic contaminants. Section III Acids (such as sulfuric acid, nitric acid, hydrochloric acid, and benzoic acid) and alkalis (like potassium and sodium hydroxide and ammonium hydroxide) are germicidal in nature. They kill microorganisms by hydrolysis and altering the pH of the medium. They are rarely used as disinfectants. Organic acids are widely used in food preservation because they prevent spore germination and bacterial and fungal growth, and because they are generally regarded as safe to eat. Acetic acid, in the form of vinegar, is a pickling agent that inhibits bacterial growth. Propionic acid is commonly added into breads and cakes to retard molds; lactic acid is added to sauerkraut and olives to prevent growth of anaerobic bacteria, especially the clostridia; and benzoic and sorbic acids are added to beverages, syrups, and margarine to inhibit yeasts. Activities of commonly used disinfectants against various microorganisms are summarized in Table 3-5. Rideal and Walker designed the phenol coefficient test to compare the performance of a disinfectant with that of phenol for the ability to kill Salmonella typhi. Phenol coefficient is determined by dilution of the disinfectant in question which sterilizes the suspension of S. typhi in a given time divided by the dilution of phenol which sterilizes the suspension in the same time. In this test, a series of dilutions of phenol and the experimental disinfectant are inoculated with the test bacteria S. typhi and S. aureus, then placed in a 20°C or 37°C water bath. These inoculated disinfectant tubes are then subcultured on a regular fresh medium at 5 minute intervals, and the subcultures are incubated for two or more days. The highest dilutions that kill the bacteria after a 10 minute exposure, but not after 5 minutes, are used to calculate the phenol coefficient. The reciprocal of the appropriate test disinfectant dilution is divided by that for phenol to obtain the coefficient. Suppose that the phenol dilution was 1/90 and maximum effective dilution for disinfectant X tested was 1/450, then the phenol coefficient of X would be 5. Chapter 3 ◗ Phenol coefficient (Rideal–Walker) test Section I Soluble salts of mercury, silver, copper, arsenic, and other heavy metals have antibacterial activity, both bactericidal and bacteriostatic. They combine with proteins, often with their sulfhydryl groups and inactivate them. They may also precipitate cell proteins. Silver compounds are widely used as antiseptics. Silver sulfadiazine is used for burns. Silver nitrate is used as a prophylactic agent in ophthalmia neonatorum in newborn infants. Copper sulfate is an effective algicide in lakes and swimming pools. Mercuric chloride is used as disinfectant. These compounds, however, are increasingly replaced by other less toxic and more effective germicides. 33 4 Culture Media Introduction Laboratory diagnosis of an infection is usually confirmed by isolating and culturing microorganisms in artificial media. Bacteria and fungi are cultured in either liquid (broth) or on solid (agar) artificial media. Koch pioneered the use of agar as a base for culture media. He developed the pour plate method and was the first to use solid culture media for culture of bacteria. At first, Koch cultured bacteria on the sterile surfaces of cut, boiled potatoes. This was unsatisfactory, because bacteria would not always grow well on potatoes. He then tried to solidify regular liquid media by adding gelatin. Separate bacterial colonies developed after the surface had been streaked with a bacterial sample. The sample could also be mixed with liquefied gelatin medium. When the gelatin medium hardened, individual bacteria produced separate colonies. Despite its advantages, gelatin was not an ideal solidifying agent because it was digested by many bacteria and melted when the temperature rose above 28°C. A better alternative was provided by Fannie Eilshemius Hesse, the wife of Walther Hesse, one of Koch’s assistants. She suggested the use of agar as a solidifying agent—she had been using it successfully to make jellies for sometime. Agar was not attacked by most bacteria and did not melt until it reaches a temperature of 100°C. One of Koch’s assistants, Richard Petri, developed the Petri dish (plate), a container for solid culture media. Ingredients of Culture Media The basic constituents of culture media include the following: Agar Agar or Agar Agar is the main component that is used universally for preparation of solid media. It is obtained from a variety of sea weeds and after necessary processing is usually available as powder or as long shreds. Agar is chiefly composed of: ■ ■ ■ A long-chain polysaccharide, consisting of D-galactopyranose units; A variety of inorganic salts, minute quantities of protein-like materials, traces of long-chain fatty acids; and Minerals, such as calcium and magnesium. Agar is usually used in a concentration of 2–3%. Agar is hydrolyzed at high temperatures and at high acid or alkaline pH. Key Points Agar has a unique property of melting at 98°C and solidifying at 42°C. The jellifying property of agar varies depending on the type of agar used (e.g., New Zealand agar is more jellifying than Japanese agar). Agar does not have any nutritive value and is also not affected by growth of bacteria. Peptone Peptone is another important ingredient of culture media. It is a complex mixture of partially digested proteins. It is obtained by digestion of lean meat or other protein materials (such as heart muscle, casein, fibrin, or soya flour) with proteolytic enzymes (such as pepsin, trypsin, or papain). Key Points ■ ■ ■ Peptone is an important source of nutrition for bacteria to grow. It contains peptones, proteoses, amino acids, inorganic salts (phosphates, potassium, and magnesium), and certain accessory factors, such as nicotinic acid and riboflavin. Neopeptone and proteose peptone are special types of peptone with high nutritive value. Other Ingredients Other common ingredients of media include water, sodium chloride and other electrolytes, meat extract, yeast extract, malt extract, blood, and serum. Meat extract is available commercially as Lab-Lemco and contains inorganic salts, carbohydrates, certain growth factors, and protein degradation products. Blood or serum is usually used for enriching culture of bacteria. Usually, 5–10% defibrinated sheep or human blood is used. Types of Culture Media Culture media can be classified in several ways: Liquid Media, Semisolid Media, and Solid Media ◗ Liquid media Liquid media provide greater sensitivity for the isolation of small numbers of microorganisms. Examples of liquid media CULTURE MEDIA ■ ■ ◗ Semisolid media Addition of reduced concentration of agar (0.2–0.4%) makes the medium semisolid, which facilitates spread of the bacteria in the medium. Simple, Complex, Defined, and Special Media Simple, complex, defined, and special media are different types of media used for culture of the bacteria. Chapter 4 ■ Identification of mixed cultures growing in liquid media requires subculture onto solid media so that isolated colonies can be processed separately for identification. Growth in liquid media also cannot ordinarily be quantitated. Bacteria grown in liquid cultures often form colloidal suspensions. ◗ Section I include nutrient broth, sugar media, and enrichment media. Composition and uses of some common liquid media are summarized in Table 4-1. Liquid media have the following disadvantages: 35 Solid media Nutrient agar showing colonies of Staphylococcus spp. Composition and uses of some common liquid media Medium Composition Uses Peptone water Peptone, sodium chloride, water Routine culture, base for sugar fermentation test, indole test TABLE 4-2 Composition and uses of some common solid media Medium Composition Uses Nutrient agar Nutrient broth, agar 2% Routine culture Culture of GramPeptone, lactose, sodium taurocholate, agar, neutral negative bacteria, such as Escherichia coli red Nutrient broth Peptone water, meat extract Routine culture MacConkey medium Glucose broth Nutrient broth, glucose Blood culture, culture of fastidious organisms, such as streptococci Blood agar Nutrient agar, 5% sheep or human blood Routine culture, culture of fastidious organisms, such as Streptococcus spp. Chocolate agar Heated blood agar Culture of Haemophilus influenzae and Neisseria Deoxycholate citrate agar Nutrient agar, sodium deoxycholate, sodium citrate, lactose, neutral red, etc. Culture of Shigella spp. and Salmonella spp. Thiosulfate citrate bile salt sucrose agar Thiosulfate, citrate, Culture of Vibrio bile salt, sucrose, cholerae bromothymol blue, thymol blue Brain heart infusion broth Sodium citrate, sodium chloride, Whole blood, bone marrow, body fluid sodium phosphate, dextrose culture peptone, brain and heart infusion broth (ox), sodium polyanethol sulfonate (SPS) Alkaline peptone water Peptone water (pH 8.6) Enrichment medium for Vibrio Selenite-F broth Peptone water, sodium selenite Enrichment medium for feces for Salmonella and Shigella Tetrathionate broth Culture of feces for Nutrient broth, sodium thiosulfate, calcium carbonate, Salmonella iodine solution Loeffler’s serum Nutrient broth, glucose, slope horse serum Robertson’s cooked meat (RCM) broth Nutrient broth, predigested cooked meat of ox heart Lowenstein– Jensen medium Anaerobic bacterial culture Coagulated hen’s egg, mineral salt solution, asparagine, malachite green Culture of Corynebacterium diphtheriae Culture of Mycobacterium tuberculosis Chapter 43 TABLE 4-1 FIG. 4-1. Section III Agar is used as a solidifying agent in most culture media. By varying the concentration of agar, it is possible to make the medium solid or semisolid. Solid media, though somewhat less sensitive than liquid media, provide isolated colonies that can be quantified and identified. Some genera and species can be recognized on the basis of their colony morphologies. Nutrient agar (Fig. 4-1) prepared by adding 2% agar to nutrient broth is the simplest and most common medium used routinely in diagnostic laboratories. Other examples of solid media include blood agar, chocolate agar, MacConkey agar, etc. Composition and uses of some common solid media are summarized in Table 4-2. 36 Section III Chapter 4 Section I ◗ GENERAL MICROBIOLOGY Simple media The simple or basal media include nutrient broth and peptone water, which form the basis of other media. ■ ■ ◗ Nutrient broth is an example of a simple liquid medium that consists of peptone, meat extract, sodium chloride, and water. Addition of 0.5% glucose to nutrient broth makes it glucose broth. Nutrient agar is an example of a simple solid medium. The medium is used routinely for isolation of many bacteria from clinical specimens. ■ ■ ■ Key Points Enrichment media are useful for isolation of wanted bacteria from stool and other specimens containing more than one species of bacteria. Complex media Most of the media other than basal media are usually known as complex media [e.g., chocolate agar, MacConkey agar, Robertson’s cooked meat (RCM) medium, Lowenstein–Jensen (LJ) medium, etc.]. Complex media have some complex ingredients, which consist of a mixture of many chemicals in unknown proportions. This is an undefined medium, because the amino acid source contains a variety of compounds with the exact composition unknown. The complex media contain: ■ Enrichment media: Enrichment media are liquid media that stimulate the growth of certain bacteria or suppress the growth of others for isolation of desired pathogenic bacteria. Commensal bacteria, such as Escherichia coli present in feces, tend to overgrow pathogenic ones in stool specimen. In such situations, enrichment media (such as selenite-F broth or tetrathionate broth) are used for the isolation of Salmonella Typhi and Shigella spp. from feces. Water, A carbon source such as glucose for bacterial growth, Various salts needed for bacterial growth, and A source of amino acids and nitrogen (e.g., beef and yeast extract). Selective media: These are solid media that contain substances that inhibit the growth of all but a few bacteria but at the same time facilitate isolation of certain bacteria. Some examples of selective media include: ■ ■ ■ ■ ■ Chapter 43 ■ ◗ Defined media A defined medium, also known as synthetic medium, contains known quantities of all ingredients. All the chemicals used are known, and it does not contain any animal, yeast, or plant tissue. These media consist of: ■ ■ Trace elements and vitamins; A defined carbon source and nitrogen source required by certain microbes. Glucose or glycerol is often used as carbon sources and ammonium salts or nitrates as inorganic nitrogen sources. ■ Some selective media used in routine microbiology laboratories are summarized in Table 4-3. Differential or indicator media: Differential or indicator media distinguish one microorganism from another growing on the same media by their growth characteristics. Key Points Dubos’ medium with Tween 80 is an example of this medium. ◗ Differential or indicator media depend on the biochemical properties of a microorganism growing in the presence of specific nutrients or indicators, such as neutral red, phenol red, eosin, or methylene blue. The medium changes color when a bacterium grows in them. For example, S. typhi grows as black colonies on Wilson and Blair medium containing sulfite. Lactose fermenting bacteria, such as E. coli produce pink colonies (Color Photo 2), whereas nonlactose fermenting bacteria, such as Salmonella spp. form pale or colorless colonies on MacConkey agar. Fermentation of lactose in the medium makes it acidic and leads to the formation of pink colonies in the presence of neutral red. Special media These include (a) enriched media, (b) enrichment media, (c) selective media, (d) indicator or differential media, (e) transport media, and ( f ) sugar media. Enriched media: The enriched media are invariably solid media that facilitate growth of certain fastidious bacteria. These media are prepared by adding substances like blood, serum, and egg to the basal media in order to meet the nutritional requirements of more exacting and more fastidious bacteria. Blood agar (Color Photo 1), chocolate agar, Loeffler’s serum slope (LSS), and LJ medium are some examples of enriched media. Blood agar is an enriched medium in which nutritionally rich whole blood supplements constitute the basic nutrients. Chocolate agar is enriched with heat-treated blood (80°C), which turns brown and gives the medium the color for which it is named. Thiosulfate citrate bile salt sucrose agar (TCBS) (Color Photo 35) selective for the isolation of Vibrio cholerae. Deoxycholate citrate agar (DCA) selective for enteric bacilli, such as Salmonella spp. and Shigella spp. LJ medium selective for Mycobacterium tuberculosis. Hektoen enteric (HE) agar selective for Gram-negative bacteria. Mannitol salt agar (MSA) selective for Gram-positive bacteria. Xylose lysine desoxycholate (XLD) agar selective for Gram-negative bacteria. Buffered charcoal yeast extract agar selective for certain Gram-negative bacteria, such as Legionella pneumophila. Examples of differential media include: ■ ■ Eosin methylene blue (EMB), differential for lactose and sucrose fermentation; MacConkey, differential for lactose fermentation; CULTURE MEDIA Medium Some selective media used in routine microbiology laboratories Colony characteristics Small pink colonies of Staphylococcus epidermidis Organisms inhibited Streptococcus spp. Gray colonies of Neisseria meningitidis and Neisseria gonorrhoeae MacConkey agar medium Lactose fermenters: red colonies, e.g., Escherichia coli Lactose nonfermenters: colorless colonies of Salmonella spp. Gram-positive cocci Thiosulfate citrate bile salt sucrose agar Sucrose fermenter: yellow colonies of Vibrio cholerae Sucrose nonfermenters: green colonies of Vibrio parahaemolyticus Enteric bacilli Charcoal yeast extract Cut glass colonies of Legionella spp. Lowenstein– Jensen medium Rough, tough, and buff colonies of Mycobacterium tuberculosis Sabouraud’s dextrose agar Most fungi Gram-positive cocci It consists of 1% sugar in peptone The indicator used in sugar media is Andrade’s indicator that consists of 0.005% acid fuchsin in 1 N NaOH. The production of acid after fermentation of sugar is indicated by the change of color of the medium to pink due to the presence of indicator. Durham’s tube is kept inverted inside the sugar tube to demonstrate the production of gas. Production of gas is indicated by the demonstration of gas bubble in Durham’s tube. Nowadays, dehydrated media are available for wide use in diagnostic laboratories because of simplicity of procedure and being less cumbersome to prepare. These dehydrated media are prepared by simply reconstituting in distilled water and sterilizing it before use. Chapter 43 Thayer– Martin medium Transport media: Transport media are used to maintain the viability of certain delicate organisms in clinical specimens during their transport to the laboratory. They typically contain only buffers and salt. They lack carbon, nitrogen, and organic growth factors, hence do not facilitate microbial multiplication. Examples of transport media are Stuart’s transport medium for Neisseria gonorrhoeae. Sugar media: Sugar media, basically contains 1% “sugar”, which in microbiology denotes any fermentable substance, such as glucose, sucrose, lactose, and mannitol that is routinely used for fermentation tests. The sugar media shows the following characteristics: Section III Big yellow colonies of Staphylococcus aureus ■ ■ ■ Gram-positive cocci Smooth and pigmented colonies of atypical Mycobacterium spp. Cocci Most bacteria Mannitol salt agar (MSA), differential for mannitol fermentation; and X-gal plates, differential for lac operon mutants for detection of recombinant strains of bacteria for study in molecular biology. Chapter 4 Mannitol salt agar ■ Section I TABLE 4-3 37 ■ 5 Culture Methods Introduction Culture methods are very crucial in a microbiology laboratory. Various culture methods are carried out to: 1. Isolate bacteria in pure culture and identify the same by performing various tests. 2. Demonstrate biochemical, antigenic, and other phenotypic and genomic properties of the isolated colonies. 3. Demonstrate susceptibility of the isolated bacteria to antibiotics, bacteriophages, bacteriocins, etc. 4. Prepare antigens for various uses. 5. Maintain stock culture. Methods of Culture Various methods are used for culturing of bacteria. These include (a) streak culture, (b) lawn culture, (c) pour-plate culture, (d ) stroke culture, (e) stab culture, and ( f ) liquid culture. Streak Culture Streak culture is the most useful method for obtaining discrete colonies of the bacteria. It is carried out by streaking on the surface of a solid media plate using a platinum or nichrome loop of 2–4 mm diameter. In this method, a loopful of the inoculum is placed near the peripheral area of the plate. The inoculum is then spread with the loop to about one-fourth of the plate with close parallel strokes. From the primary inoculum, it is spread thinly over the plate by streaking with the loop in parallel lines. The loop is flamed and cooled in between the streaks to obtain isolated colonies. The inoculated culture plate is incubated at 37°C overnight for demonstration of colonies. Confluent growth occurs at the primary inoculum, but becomes progressively thinner, and well-separated colonies are demonstrated on the final streaks of the inoculum (Fig. 5-1). Single isolated colonies obtained by this method are very useful to study various properties of bacteria. Streak culture is the most useful method for obtaining discrete colonies of the bacteria. Lawn Culture The lawn culture provides a uniform layer of bacterial growth on a solid medium. It is carried out by flooding the surface of the solid media plate with a liquid culture or suspension of bacteria, pipetting off the excess inoculum, and finally incubating the plate overnight at 37°C. Alternatively, the culture plate may be inoculated by a sterile swab soaked in liquid bacterial culture or suspension and incubating overnight for demonstration of the bacterial colonies. Key Points Lawn culture method is useful: ■ ■ ■ To carry out antibiotic sensitivity testing by disc diffusion method; To carry out bacteriophage typing; and To produce a large amount of bacterial growth required for preparation of bacterial antigens and vaccines. Pour-Plate Culture The pour-plate culture is used to determine approximate number of viable organisms in liquids, such as water or urine. It is used to quantitate bacteria in urine cultures and also to estimate the viable bacterial count in a suspension. This method is carried out in tubes, each containing 15 mL of molten agar. The molten agar in tubes is left to cool in a water bath at 45°C. The inoculum to be tested is diluted in serial dilution. Then 1 mL each of diluted inoculum is added to each tube of molten agar and mixed well. The contents of tubes are poured into sterile Petri dishes and allowed to set. After overnight incubation of these Petri dishes at 37°C, colonies are found to be distributed throughout the depth of the medium, which can be counted using a colony counter. Stroke Culture Stroke culture provides a pure growth of bacteria for carrying out slide agglutination and other diagnostic tests. It is carried out in tubes usually containing nutrient agar slopes. Stab Culture Stab culture is prepared by stabbing the medium in tubes with a long, straight wire and incubating at 37°C. Key Points Stab culture is frequently used for: ■ ■ ■ Maintaining stock cultures. Demonstration of oxygen requirement of bacteria. Demonstration of gelatin liquefaction of bacteria. CULTURE METHODS The inoculum is smeared thoroughly to form a well Loop is resterilized and parallel lines are drawn from the well Section I Loop is resterilized and parallel lines are drawn from primary strokes Loop is resterilized and parallel lines are drawn from secondary strokes Chapter 5 Loop is resterilized and zigzag lines are drawn from tertiary strokes Appearance of isolated colonies towards the tapering end Diagrammatic representation showing streak culture. Liquid Culture Key Points Liquid culture is specifically used: ■ ■ ■ For blood culture and for sterility tests, where the concentration of bacteria is expected to be small; For culture of specimens containing antibiotics and other antibacterial substances, as these are rendered ineffective by dilutions in the medium; and When large yields of bacteria are required. A major disadvantage of liquid culture is that it does not provide pure culture of the bacteria and also the bacterial growth does not exhibit special characteristic appearances. Anaerobic Culture Obligate anaerobes are bacteria that can live only in the absence of oxygen. These anaerobes are killed when exposed to the atmosphere for as briefly as 10 minutes. Some anaerobes are tolerant to small amounts of oxygen. Facultative anaerobes are those anaerobes that grow with or without oxygen. Anaerobic bacterial culture is a method used to grow anaerobes from a clinical specimen. Culture and identification of anaerobes is essential for initiating appropriate treatment. Specimen Collection Specimens frequently used for anaerobic culture include: ■ Blood, bile, bone marrow, cerebrospinal fluid, direct lung aspirate, and tissue biopsy from a normally sterile site; ■ Fluid aspirated from a normally sterile site, such as a joint; ■ Pus specimens from dental abscess, burn wound, abdominal or pelvic abscess; and ■ Specimens from knife, gunshot, or surgical wounds. Collection of a contamination-free specimen and protecting it from oxygen exposure during collection form the mainstay of anaerobic culture. The specimens need to be obtained from an appropriate site without contaminating the sample with bacteria from the adjacent skin, mucous membrane, or tissue. Abscesses or fluids are usually collected by using a sterile syringe and is then tightly capped to prevent entry of air. Tissue samples are placed into a degassed bag and sealed, or into a gassed out screw top vial that may contain oxygen-free prereduced culture medium and tightly capped. The specimens need to be plated as rapidly as possible onto culture media for isolation of bacteria. Key Points ■ Swabs are always avoided when collecting specimens for anaerobic culture because cotton fibers may be detrimental to anaerobes. ■ Coughed throat discharge (sputum), rectal swab, nasal or throat swab, urethral swab, and voided urine are some of the specimens that are not suitable for processing anaerobic cultures. Chapter 43 Liquid culture is prepared in a liquid media enclosed in tubes, flasks, or bottles. The medium is inoculated by touching with a charged loop or by adding the inoculum with pipettes or syringes and incubating at 37°C, followed by subculture on to solid media for final identification. The failure to do so may have serious consequences, such as amputation, organ failure, sepsis, meningitis, and even death. Section III FIG. 5-1. 39 40 GENERAL MICROBIOLOGY Chapter 5 Section I Culture Media The commonly used media for anaerobic culture include Robertson cooked meat broth, thioglycollate broth, Willis and Hobbs’ media, and neomycin blood agar. Robertson cooked meat (RCM) broth is the most widely used medium in an anaerobic culture. It consists of nutrient broth and pieces of fat-free minced cooked meat of ox heart with a layer of sterile liquid paraffin over it. Unsaturated fatty acids and even glutathione and cysteine present in the meat extract utilize oxygen for autooxidation. The medium before inoculation is usually boiled at 80°C in a water bath to make the medium free of oxygen. The media after inoculation and incubation allows the growth of even strict anaerobes and also indicates their saccharolytic or proteolytic activities as meat is turned red or black, respectively. Chapter 43 Section III Methods of Anaerobic Culture Anaerobic cultures are carried out in an environment that is free of oxygen, followed by incubation at 95°F (35°C) for at least 48 hours before the plates are examined for growth. The cultures of anaerobic bacteria are carried out as follows: 1. McIntosh–Fildes anaerobic jar: It is the most widely used and dependable method of anaerobiosis (Color Photo 3). It consists of a glass or metal jar with a metal lid that can be clamped air tight with the help of a screw. The lid has one inlet tube and another outlet tube. The outlet tube is connected to a vacuum pump by which the air is evacuated out of the jar. The inlet tube is connected to a source of hydrogen supply. The lid has two electric terminals also that can be connected to an electric supply. The underside of the lid contains a catalyst (e.g., alumina pellets coated with palladium) that catalyzes the combination of hydrogen with residual oxygen present in the air. This method ensures complete anaerobiosis. The culture media are inoculated with the specimens suspected to contain anaerobic bacteria. The inoculated media are then kept inside the jar, and the lid is closed air tight. The anaerobiosis in the jar is carried out by first evacuating the air from the jar through outlet tube with the help of a vacuum pump. The outlet tube is closed, then the sealed jar containing the culture plates is replaced with hydrogen gas passed through inlet tube till reduced atmospheric pressure is brought to normal atmospheric pressure, which is monitored on the vacuum gauge as zero. The electrical terminals are then switched on to heat the catalyst that catalyzes combination of hydrogen with residual oxygen and ensures complete anaerobiosis in the jar. Reduced methylene blue is used as the indicator of anaerobiosis in the jar. If anaerobiosis is complete, it remains colorless; if anaerobiosis is not complete, it turns blue on exposure to oxygen. Gas pack system is a simple and effective method of production of hydrogen gas for anaerobiosis. It does not require the cumbersome method of evacuation and filing up of gases after evacuation. Carbon dioxide, which is also generated, is required for growth by some anaerobes. Water activates the gas pack system, resulting in the production of hydrogen and carbon dioxide. Hydrogen combines with oxygen in the air in the presence of catalyst and maintains anaerobiosis. In this method, the inoculated plates are kept along with the gas pack envelope with water added in the air tight jar. Key Points It is the method of choice for preparing anaerobic jars. It is commercially available as a disposable envelope containing chemicals that produce hydrogen and carbon dioxide on addition of water. 2. Anaerobic glove box: The anaerobic glove box is another innovation developed for isolating anaerobic bacteria. It is essentially a large clear-vinyl chamber with attached gloves, containing a mixture of 80% nitrogen, 10% hydrogen, and 10% carbon dioxide. A lock at one end of the chamber is fitted with two hatches, one leading to outside and the other to the inside of the chamber. Specimens are placed in the lock, the outside hatch is closed, and the air in the lock is evacuated and replaced with the gas mixture. The inside hatch is then opened to introduce the specimen into the chamber. 3. Anoxomat: This is a fully automated system that evacuates a portion of the jar contents and refills the jar with an anaerobic gas mixture. During this procedure, the oxygen concentration in the air is rarefied. For anaerobic atmosphere, this procedure is repeated three times, after which the oxygen concentration is rarefied to 0.16%. A small catalyst removes this very small percentage of oxygen content. Anoxomat is capable of producing microaerophilic conditions also. The method is being increasingly used for processing clinical specimens for isolation of anaerobic bacteria. Laboratory Identification of Bacteria and Taxonomy Introduction It is not always possible to identify microorganisms by microscopic methods alone, due to similarity of bacteria in their morphology and staining characters. Hence, further study of microorganisms is essential for their identification. For this purpose, organisms have been grown artificially in the laboratory. Identification of Bacteria The complete identification of bacteria involves the following steps: (a) morphology of bacterial colony on solid medium, (b) growth in liquid medium, (c) biochemical reactions, (d ) antigenic structures, (e) animal pathogenicity, ( f ) antibiotic sensitivity, ( g ) typing of bacterial strains, (h) rapid identification methods, and (i ) molecular methods. Morphology of Bacterial Colony on Solid Medium Morphology of the bacterial colony on solid medium depends on a number of factors, such as nature of culture medium, temperature and time of incubation, age of culture, and number of subcultures done. The characteristics noted are shape, size, surface, edge, elevation, opacity, color, and hemolysis of the colonies, as follows: ■ ■ ■ ■ ■ ■ The colonies may be a few millimeters in size: pinhead size (Staphylococcus aureus) or pinpoint (streptococci). The shape may be circular or irregular, and surface of the colonies may be smooth, rough, or granular. The colonies on the medium may be flat, raised, low convex, convex, or umbonate. The edge may be entire, lobate, crenated, undulate, or ciliate. The colonies may be transparent, translucent, or opaque. Certain bacterial colonies are associated with production of pigments and hemolysis around them. 6 at the bottom of the liquid medium, whereas most of the Gram-negative bacteria produce uniform turbidity. Pseudomonas spp. and other aerobic bacteria tend to produce surface pellicles in liquid media. The pigment production of some bacteria, such as Pseudomonas aeruginosa can be appreciated in liquid media. Smears prepared on glass slides from bacterial colonies grown on either solid or liquid media are examined for bacteria by appropriate staining. Gram staining is most widely used to differentiate between Gram-positive and Gram-negative bacteria. Ziehl–Neelsen stain differentiates acid-fast bacilli (e.g., Mycobacterium tuberculosis, Mycobacterium leprae, etc.) from nonacid-fast bacilli (Fig. 6-1). Albert’s stain is used to demonstrate metachromatic granules in Corynebacterium diphtheriae. Biochemical Reactions Biochemical reactions are very important in the identification of bacterial isolates and in the identification of different bacterial species. These tests depend on the presence of certain enzymes, such as catalase, oxidase, urease, gelatinase, etc., produced by the bacteria. Commonly used biochemical tests are described below: ◗ Catalase test Catalase test is used to detect the presence of enzyme catalase in a bacterium. The enzyme catalase catalyzes the breakdown of hydrogen peroxide with the release of free oxygen. It is found in most aerobic and facultative anaerobic bacteria. The main exception is Streptococcus spp. It is not found in anaerobes. Growth in Liquid Medium Nutrient broth and peptone water are frequently used as liquid media for growth of bacteria. The characteristics of bacterial growth in liquid media provide some clue in presumptive identification. For example, streptococci produce granular deposits FIG. 6-1. Ziehl-Neelsen staining to differentiate acid-fast bacilli from nonacid-fast bacilli. Chapter 43 Section III Chapter 6 Section I 42 GENERAL MICROBIOLOGY Red blood cells contain catalase and their presence, therefore, gives a false positive result. Catalase test is primarily used to differentiate Staphylococcus and Streptococcus. In this test, a small amount of culture to be tested is picked up from a nutrient agar plate with a sterile platinum loop or glass rod and this is inserted into hydrogen peroxide solution (3%) held on a slide or in a tube. Immediate production of air bubbles in the solution denotes a positive test and no bubbles indicate a negative test. Positive ◗ This test determines the presence of enzyme oxidase in many bacteria. The enzyme oxidase catalyzes the oxidation of reduced cytochrome by molecular oxygen. Kovac’s oxidase reagent that contains tetramethyl-p-phenylenediamine dihydrochloride is the main reagent used in the oxidase test. The dye serves as an alternate substrate for the cytochrome oxidase reaction. In the reduced state, the reagent is colorless but when oxidized, it becomes purple. Oxidase test can be performed by several methods that include: FIG. 6-2. The dry filter paper method is performed by impregnating strips of filter paper with 1% Kovac’s oxidase reagent. The paper is smeared with the bacterial colonies to be tested by a glass rod. In a positive test, the smeared area on the filter paper turns deep purple within 10 seconds. No color change indicates negative test (Color Photo 4). Key Points Oxidase-positive bacteria include Neisseria spp., Vibrio spp., Aeromonas spp., Plesiomonas spp., Pseudomonas spp., and Moraxella spp. Oxidase-negative bacteria include all members of the family Enterobacteriaceae. Indole test Indole test is used to detect the ability of bacteria to decompose amino acid tryptophan to indole, which accumulates in the medium. Tryptophan or peptone broth is the medium used for indole test (Color Photo 5). The test is performed by inoculating the medium with bacteria, incubating at 37°C for 24–48 hours. Then, 5 drops of Kovac’s reagent containning amyl or isoamyl alcohol, p-dimethyl amino benzaldehyde, and concentrated hydrochloric acid are added to the inoculated medium. Positive test is indicated by formation of a red ring at the surface of the medium. No color change indicates a negative test (Fig. 6-2). Precautions in interpretation of indole test are as follows: (a) cultures to be tested for indole production need to be incubated aerobically and (b) the optimum pH for tryptophanase activity is alkaline (pH 7.4–7.8), hence a decrease in pH results in decreased indole production and gives a possible false negative reaction. Indole test. Key Points (a) Dry filter paper method, (b) Wet filter paper method, and (c) Plate method. ◗ Negative Oxidase test Indole-positive bacteria are Escherichia coli, Vibrio cholerae, Proteus vulgaris, and Edwardsiella spp. Indole-negative bacteria are Salmonella typhi, Klebsiella spp., and P. mirabilis. ◗ Carbohydrate fermentation test Carbohydrate fermentation test is done to determine the ability of a bacterium to ferment a specific carbohydrate incorporated in a basal medium, producing acid or acid with visible gas. The carbohydrates include glucose, lactose, sucrose, maltose, etc. The sugar medium contains 1% sugar. Andrade’s indicator is a solution of acid fuchsin to which sodium hydroxide is added. It is a pH indicator that is added to the basal medium, which indicates the formation of organic acids. The test is performed by inoculating the sugar media with bacteria, incubating at 37°C for 18–24 hours. The change of the color to pinkish red (acidic) is considered as a positive test result, whereas yellow to colorless (alkaline) sugar indicates negative test result. Production of gas is indicated by appearance of gas bubbles in Durham’s tube. Key Points All members of the family Enterobacteriaceae ferment glucose, whereas glucose and mannitol are fermented by Salmonella spp., and glucose and lactose are fermented by E. coli and Klebsiella spp. ◗ Oxidation–fermentation test Oxidation–fermentation test (OF test) determines the oxidative or fermentative metabolism of a carbohydrate by a bacterium. The OF test is used to determine whether a bacterium has the enzymes necessary for the aerobic breakdown of glucose LABORATORY IDENTIFICATION OF BACTERIA AND TAXONOMY ◗ Kligler’s iron agar/triple sugar iron agar test Urease test is useful for screening the production of urease by Proteus spp., Morganella spp., and some Providencia spp. Proteus spp. is rapidly urease positive—usually within 6 hours. Another example of urease producing organism is Helicobacter Pylori, the production of which is essential for its survival in the acidic pH of the stomach. ◗ Citrate test Citrate test is used to demonstrate the ability of an organism to utilize citrate as the sole source of carbon for metabolism. Koser’s citrate medium (a liquid medium) and Simmon's citrate medium (a solid medium) are used for the test. In this test, either of the medium is inoculated with the bacteria and then incubated at 37°C overnight. Growth on the Simmon's medium is accompanied by a rise in pH to change the medium from its initial green color to deep blue. Hence, growth with blue color on the slant indicates positive test and no growth without any color change indicates negative test. The medium needs to be lightly inoculated (from plate cultures, not from a broth) to avoid a carryover of nutrients, which may lead to a false positive result. In Koser’s liquid medium, turbidity indicates positive test and absence of turbidity indicates negative test. ◗ Phenylalanine deaminase test Phenylalanine deaminase test indicates the ability of an organism to deaminate phenylalanine to phenylpyruvic acid (PPA), which reacts with ferric salts to give green color. This test is also called PPA test. The test is performed by inoculating the medium containing phenylalanine by the bacteria. After overnight incubation at 37°C, a few drops of 10% ferric chloride solution are added to the inoculated medium. If PPA is produced, the medium becomes green in color due to combination of ferric chloride with PPA, which indicates a positive test. Absence of any color change indicates a negative test. PPA-positive bacteria are Proteus spp., Providencia spp., and Morganella spp. ◗ Nitrate reduction test Nitrate reduction test is used to determine the presence of enzyme nitrate reductase in the bacteria. The enzyme reduces nitrate to nitrites or free nitrogen gas. The test is carried out by inoculating the broth containing 1% potassium nitrate (KNO3) and incubating at 37°C up to 5 days. Then 1–2 drops of a reagent that consists of a mixture of 1 mL of naphthylamine Chapter 43 H2S production and carbohydrate fermentation patterns are generally characteristic for specific bacterial groups, especially the Enterobacteriaceae. Key Points Section III 1. Fermentation of glucose only (alkali/acid or K/A reaction; K denotes alkaline reaction and A denotes acidic reaction): After 18–24 hours, the low glucose concentration is completely used up, and the organism starts to utilize the peptones present in the medium, resulting in an alkaline pH in slant (red). In the butt, a yellow color exists due to anaerobic degradation of glucose. 2. Fermentation of lactose and glucose (acid/acid or A/A reaction): Lactose is present in 10 times the amount of glucose. In 18–24 hours, the lactose is not depleted and therefore acidic conditions exist in the butt and slant. 3. Neither lactose nor glucose fermented (alkali/alkali or K/K reaction): Many Gram-negative, nonenteric bacilli are unable to ferment glucose or lactose. A reaction of K/K is a result of aerobic catabolism of peptone by the organism. Urease test Chapter 6 Kligler’s iron agar (KIA) and triple sugar iron agar (TSI) tests are used to determine the ability of an organism to attack a specific carbohydrate incorporated into a basal growth medium, with or without the production of gas, along with production of hydrogen sulfide. KIA medium contains two carbohydrates: lactose and glucose in a ratio of 10:1. TSI contains a third carbohydrate, i.e., sucrose, in addition to lactose and glucose. The test is performed by inoculating KIA or TSI with an inoculating needle by stabbing the butt and streaking the slant and then incubating at 37°C for 18–24 hours. During incubation, the bacterium first utilizes glucose and then lactose and sucrose. After 18–24 hours, the glucose concentration is depleted in the slant and in the butt. The bacteria start oxidative degradation of the peptone present in the slant, resulting in the production of alkaline by-products, thereby changing the indicator to a red color. Anaerobic fermentation of glucose in the butt produces a large volume of acid, which neutralizes the alkalinity caused by peptone degradation; hence the butt continues to remain yellow. Yellow color (acidic), therefore, indicates fermentation of carbohydrates and red color (alkaline) indicates no fermentation. Presence of bubble in the butt indicates production of gas during fermentation of carbohydrates. Certain bacteria produce H2S, which is detected as black precipitate that blackens the slant and butt of the medium. The medium is turned black due to combination of H2S with ferric ions, from ferric salts present in the medium, to form ferrous sulfide as black precipitates. Three basic fermentation patterns are observed on KIA medium after incubation at 37°C for 24 hours: ◗ Urease test is used to determine the ability of an organism to split urea to ammonia by the enzyme urease. Production of ammonia makes the medium alkaline; thus the indicator phenol red changes to red or pink in color. The test is performed in Christensen’s urease medium. The medium is inoculated with the bacterial colony and incubated at 37°C. Urease-positive bacteria produce a pink color. Section I (i.e., oxidation) and/or for the fermentation of glucose. The test differentiates Enterobacteriaceae (fermenters) from the members of the family Pseudomonadaceae (the nonfermenters). 43 GENERAL MICROBIOLOGY and 1 mL of sulfanilic acid is added. Red color developing within a few minutes signifies positive reaction, while absence of color indicates negative reaction. Key Points Nitrate reduction test is used mainly for the identification of members of the family Enterobacteriaceae, which are usually positive for nitrate reduction test. ◗ Methyl red test Methyl red (MR) test detects the ability of an organism to produce and maintain stable acid end products during the fermentation of glucose, thereby maintaining a sustained pH below 4.5. The test is performed by inoculating a glucose phosphate broth and incubating it at 37°C for 2–5 days. Five drops of 0.04% of MR solution are then added to the inoculated medium, mixed, and the result is read immediately. Development of red color denotes a positive test, and yellow color indicates negative test. E. coli, Yersinia spp., and Listeria monocytogenes are the examples of MR-test-positive bacteria. It should be noted that if the MR test is performed too early, the results may produce a false positive reaction. This is because MR-negative organisms may not have adequate time to completely metabolize the initial acid products that have been produced during the fermentation of glucose. Key Points Chapter 43 Section III Chapter 6 Section I 44 Methyl red test is commonly used to differentiate E. coli (MR positive) from Enterobacter (MR negative) and Yersinia spp. (MR positive) from other Gram-negative, non-enteric bacteria (MR negative). ◗ Voges–Proskauer test Voges–Proskauer (VP) test detects the production of acetyl methyl carbinol (acetoin), a natural product formed from pyruvic acid in the course of glucose fermentation. The acetoin, in the presence of alkali and atmospheric oxygen, is oxidized to diacetyl that reacts with alpha-naphthol to produce red color. This test is performed by inoculating the glucose phosphate broth with the organism and incubating at 37°C for 48 hours. Then approximately 3 drops of alpha-naphthol is added followed by addition of 1 drop of 40% potassium hydroxide. The reagents are mixed well and are allowed to stand for 30 minutes. In positive test, pink color appears in 2–5 minutes, deepening to magenta in half an hour. The solution remains colorless for 30 minutes in negative test. Staphylococcus spp., V. cholerae biotype eltor, Klebsiella spp., and Enterobacter spp. are the common examples of VP-test-positive bacteria. Key Points Indole, MR, VP, and citrate test commonly referred to as “IMViC tests” are the most frequently used tests in the identification of enteric, Gram-negative bacteria. ◗ Hydrogen sulfide production These tests are carried out to demonstrate H2S produced by certain bacteria. Production of H2S is demonstrated by inoculating the bacteria in the media containing lead acetate, ferric ammonium citrate, or ferrous acetate and incubating overnight at 37°C. H2S-producing bacteria by their enzymatic actions produce H2S from sulfur-containing amino acids in the medium. Production of sulfur produces black color in the media, visible to the naked eye. Filter paper strip is another method of demonstration of production of H2S. In this method, filter paper strip impregnated with lead acetate is kept between the cotton plug and the medium in the tube. Production of H2S is indicated by blackening of the paper. P. mirabilis, P. vulgaris, and Salmonella spp. are some examples of the H2S-producing bacteria. Antigenic Structures Agglutination of biochemically confirmed bacteria with specific antisera facilitates further identification of the isolated bacteria. Agglutination test is used in the identification of presumptive isolates of pathogens (e.g., Salmonella) from clinical samples. It is also used for the grouping of betahemolytic streptococci. Animal Pathogenicity Some pathogenic bacteria and their bacterial metabolites produce characteristic lesions in laboratory animals. The most commonly employed animals are rats, guinea pigs, mice, and rabbits. These animals, depending on the organisms to be tested, may be inoculated by subcutaneous, intramuscular, intravenous, intraperitoneal, or intracerebral routes. The identification of the bacteria is made depending on the postmortem findings and cultural properties of the bacteria. For example, guinea pigs are commonly used for performing animal pathogenicity of C. diphtheriae, Clostridium perfringens, and M. tuberculosis. Antibiotic Sensitivity Determination of antibiotic sensitivity of an isolate from a patient is essential for the choice of drug therapy. In some cases, sensitivity of an organism to a particular agent helps in the identification, e.g., Streptococcus pyogenes is sensitive to bacitracin and Streptococcus pneumoniae to optochin. The sensitivity to the antibiotic can be determined by disc diffusion test, serial dilution tests, and E-test, as described in detail in Chapter 9. Typing of Bacterial Strains The ability to discriminate between similar strains of bacteria may be important in tracing sources or modes of spread of infection in a community. Typing methods are widely used for epidemiological studies. These include (a) phenotypic techniques and (b) genotypic techniques. LABORATORY IDENTIFICATION OF BACTERIA AND TAXONOMY ◗ Phenotypic techniques ■ ■ ■ ◗ Biotyping: It relies on a set of biochemical reactions to distinguish different strains within a given species. Antimicrobial susceptibility testing is an example of this type. Serotyping: Different strains of organisms of the same species can be differentiated based on the difference in the expression of antigenic determinants on the cell surface. Bacteriocin typing: This is used in case of bacterial species for which a number of lytic bacteriophages have been identified. Phage typing: This has been the mainstay of strain discrimination and is widely used in epidemiological studies. Bacterial classification may be defined as the arrangement of organisms into taxonomic groups (taxa) on the basis of their phenotypic (observable) and genotypic (genetic) similarities and differences. It allows proper and systematic grouping of microorganisms. Organisms are classified into three main kingdoms: Animals, Plants, and Protista. The Protista contains unicellular microorganisms including eukaryotes and prokaryotes. Although no universally accepted bacterial classification system is available, three main approaches are usually followed. These include (a) phylogenetic, (b) Adansonian, and (c) genetic classifications, which are discussed below: Genotypic techniques ◗ Genotypic techniques depend on differences related to the genome of bacteria. Genotypic techniques employed to differentiate strains of bacteria include plasmid profile analysis and restriction endonuclease analysis of chromosomal DNA. The phylogenetic classification is a type of hierarchical classification that represents a branching tree-like arrangement, one characteristic being employed for divisions at each branch or level. It is called phylogenetic classification, because it denotes an evolutionary arrangement of species. This classification groups together the types that are related on evolutionary basis where several groups are used, such as Divisions, Classes, Orders, Families, Tribes, Genera, and Species. Some characters of special importance, such as Gram staining properties, lactose fermentation, spore formation, etc., are used to differentiate major groups, whereas less important properties, such as nutritional requirements for growth of bacteria, production of certain enzymes by bacteria, etc., are employed to distinguish minor groups, such as the genera and species. As per the classification, the full taxonomical position of a bacterium (say, E. coli) can be described as follows: Division: Class: Order: Family: Tribe: Genus: Species: Molecular Methods Molecular methods, such as nucleic acid probes, polymerase chain reaction (PCR), and other amplification procedures are also used increasingly nowadays for identification of microorganisms. Genetic probes are based on the detection of unique nucleotide sequences with the DNA or RNA of a microorganism. Hybridization of the sequence with a complementary sequence of DNA or RNA follows cleavage of the double-stranded DNA of the microorganism in the specimen. PCR has major applications in the detection of infections due to microorganisms that are difficult to culture (e.g., the human immunodeficiency virus) or that have not as yet been successfully cultured (e.g., the Whipple’s disease bacillus). Bacterial Taxonomy Bacterial taxonomy comprises: (a) bacterial classification of organism and (b) nomenclature or naming of the microbial isolates. Protophyta Schizomycetes Eubacteriales Enterobacteriaceae Escherichiae Escherichia coli Bergey’s Manual of Systematic Bacteriology is an authoritative published compilation that describes a phylogenetic classification of bacteria. The manual is a useful compilation of names and descriptions of bacteria and is the most standard reference book accepted worldwide. The book is extremely useful for identification of newly isolated bacterial types. A minimum number of important characters, such as morphology of the bacteria, staining properties, cultural characteristics, biochemical reactions, antigenic structure, and guanine to cytosine ratio of DNA, etc., are used for identification and classification of bacteria. ◗ Adansonian classification The Adansonian classification makes no phylogenetic assumption, but considers all the characteristics expressed at the time of Chapter 43 Automated methods are now available, which take only hours for characterization of isolates. These include detection of specific enzymes, toxins, antigens, or metabolic end products. Obligate anaerobes can be identified by gas liquid chromatography of short-chain fatty acids produced by them. Latex particle agglutination, coagglutination, direct fluorescent antibody test, and dot enzyme-linked immunosorbent assay (ELISA) are the most frequently used techniques in the clinical laboratory for rapid detection of microbial antigens directly in clinical specimens. Antibody to a specific antigen is bound to latex particles or to a heat-killed and treated protein A-rich strain of S. aureus to produce agglutination. Phylogenetic classification Section III Rapid Identification Methods Chapter 6 ■ Bacterial Classification Section I Phenotyping techniques depend on various observable properties of bacteria, which are discussed as follows: 45 Chapter 6 Section I 46 the study. Hence it is called a phonetic system. The Adansonian classification was first proposed by Michael Adanson in the eighteenth century. It avoids the use of weighted characteristics. This classification gives equal weight to all measurable features and groups of bacteria on the basis of similarities of several characteristics. Recently, availability of computer facilities has expanded the scope of phonetic classification by permitting comparison of very large number of properties of several organisms at the same time. The computer analysis of large number of characteristics of a bacterium facilitates the identification of several broad subgroups of bacterial strains that are further subdivided into species. This type of classification, based on the properties of large number of properties, is known as numerical taxonomy. Section III ◗ Chapter 43 GENERAL MICROBIOLOGY Genetic classification The genetic or molecular classification is based on homology of the DNA base sequences of the microorganisms. DNA relatedness of the microorganisms is tested first by extracting DNA from the organism to be studied, and then studying the nucleotide sequence of DNA by DNA hybridization or recombination methods. The degree of hybridization can be assessed by many methods, such as by using labeled DNA preparations. The study of messenger RNA (mRNA) also provides useful information on genetic relatedness among bacteria. The analysis of ribosomal RNA (rRNA) has proved to be of immense value. Study of the nucleotide sequence of 16S ribosomal RNA from different biologic sources has shown evolutionary relationships among widely divergent organisms and has contributed to the understanding of new groups of bacteria, such as the archaebacteria. Genetic classification is now increasingly used for study of viruses. ◗ Intraspecies classification Intraspecies classification makes an attempt to subclassify species of a bacteria based on biochemical properties (biotypes), antigenic properties (serotypes), susceptibility to bacteriophage (phage types), and production of bacteriocins (colicin types). Recently, molecular methods have increasingly been used for intraspecies classification of microorganisms, especially viruses. Key Points Molecular methods of intraspecies classification are broadly of two types—phenotypic and genotypic. Phenotypic methods are based primarily on the study of expressed characteristics by microorganisms and are carried out by performing electrophoretic typing of bacterial proteins and immunoblottings. Genotypic methods include direct analysis of genes and chromosomal and extrachromosomal DNA. These genotypic methods include plasmid profile analysis, restriction endonuclease analysis of chromosomal DNA with Southern blotting, PCR, and nucleotide sequence analysis. Nomenclature of Microorganisms Nomenclature refers to the naming of microorganisms. The nomenclature of microorganisms is governed by the International Committee on Systematic Bacteriology and published as Approved List of Bacterial Names in the International Journal of Systematic Bacteriology. This confers and maintains uniformity for use of names of microorganisms accepted internationally. Similarly, the nomenclature and classification of viruses are governed by the International Committee on Taxonomy of Viruses. Two kinds of names are usually given to bacteria—common name and scientific name: (a) The common or casual name for a microorganism varies from country to country and is usually known in the local language. For example tubercle bacillus, typhoid bacillus, gonococcus are common names for communication at the local level. (b) The scientific name is the international name that is accepted throughout the world. By accepted taxonomic conventions, the order names end in ales (e.g., the order Eubacteriales), family names end in aceae (e.g., the family Enterobacteriaceae), and the tribe names end in eae (e.g., the tribe Proteae). The order, family, and tribe names begin with capital letters. The genus name also begins with capital letter, but species name (e.g., coli) begins with running letter and not capital letter. Both the genus (e.g., Escherichia) and species names are either italicized or underlined when written in the text. The scientific name of the bacterium when written for the first time, is written in full (e.g., Escherichia coli), but later mentioned in an abbreviated form (e.g., Escherichia coli). When bacteria are referred to as a group, their names are neither capitalized nor italicized or underlined (e.g., streptococci). 7 Bacterial Genetics Introduction Genetics is the study of heredity and variation to understand the cause of resemblance and differences between parents and their progeny. The term genetics was coined by William Bateson, a British biologist, in 1906. The unit of heredity is the gene, a segment of deoxyribonucleic acid (DNA) that carries in its nucleotide sequence information for a specific biochemical or physiologic property. All hereditary properties are encoded in DNA. Hence, the chromosomal DNA plays an important role in the maintenance of character from generation to generation. Genes carry the information to code for all the necessary components and the actions of life. The genes at each cell division are replicated and a copy is transmitted to each daughter cell. Although heritability and variations in bacteria have been observed from the early days of bacteriology, it was not known then that bacteria too obey the laws of genetics. It was not until the 1950s that DNA was recognized as the building material of genes. Bacteria unlike eukaryotic cells (such as human cells) are haploid (1n), which means they have a single copy of each gene. In contrast, eukaryotic cells are diploid (2n); in other words, they have a pair of each chromosome and therefore two copies of each gene. The genotype of an organism is the specific set of genes it possesses. Chromosomal Substances Structure of DNA The DNA is the key basic component of gene, which carries the genetic information that is transcribed onto ribonucleic acid and then translated as the particular polypeptide. The basic structure of DNA molecule was first described by Watson and Crick for which they were honored with the Nobel Prize in Medicine. The DNA molecule is composed of two strands of complementary nucleotides wound together in the form of a double helix. The double helix has a diameter of 2 nm. Each full turn of the double helix contains 10 nucleotide pairs and is 3.4 nm in length. ◗ DNA strand Each DNA strand has a backbone of deoxyribose (sugar) and phosphate group residues arranged alternately. It has a sugar– phosphate backbone substituted with purine and pyrimidine bases. It contains four nitrogenous bases, two purines (adenine and guanine), and two pyrimidines (thymine and cytosine). The two complementary strands are held together by hydrogen bonds between the nitrogenous bases on the opposite strands. The hydrogen bonding follows a specific binding manner in which hydrogen bonds are formed only between guanine and cytosine and between adenine and thymine. Guanine and cytosine form a complementary base pair and adenine and thymine form another base pair. A molecule of DNA therefore contains as many units of adenine as thymine and of guanine as cytosine. For example, when the arrangement of bases along one strand is AGCTAG, the arrangement on the other strand will be TCGATC. The ratio of adenine and thymine to guanine and cytosine is constant for each species, but varies widely from one bacterial species to another. During replication, the DNA molecule replicates, first by unwinding at one end to form a fork and then by separation of strands at the other end. Each strand then acts as a template for the synthesis of a complementary strand with which it then forms a double helix. ◗ Gene It is a segment of DNA that carries codons specifying for a particular polypeptide. A DNA molecule consists of a large number of genes, each of which contains hundreds of thousands of nucleotides. The DNA of a bacterial chromosome is usually arranged in a circular form and when straightened, it measures around 1000 μ. The length of DNA is usually expressed as kilobases (1 kbp = 1000 base pairs, or bp). Bacterial DNA measures usually 4000 kbp and the human genome about 3 million kbp. Key Points The DNA of Escherichia coli is most extensively studied. Typically, it consists of a single circular DNA molecule consisting of approximately 5 ⫻ 106 bp. It has a molecular weight of 2 ⫻ 109. This amount of genetic information in the bacteria can code for about 2000 proteins with an average molecular weight of 50,000 kDa. Structure of RNA Basically, the structure of RNA is similar to that of DNA except for two major differences: (a) In DNA, the sugar is D-2-deoxyribose; in RNA, the sugar is D-ribose. GENERAL MICROBIOLOGY (b) The RNA contains the nitrogenous base uracil instead of thymine that is present in DNA. On the basis of structure and function, the RNA can be differentiated into three types: (a) Messenger RNA (mRNA), (b) Ribosomal RNA (rRNA), and (c) Transfer RNA (tRNA). The RNA molecules range in size from the small tRNAs (which contain fewer than 100 bases) to mRNAs (which may carry genetic messages extending to several thousand bases). Bacterial ribosomes contain three kinds of rRNA with respective sizes of 120, 1540, and 2900 bases and a number of proteins. Corresponding rRNA molecules in eukaryotic ribosomes are somewhat larger. A few RNA molecules have been shown to function as enzymes (ribozymes). For example, the 23S RNA in the 50S ribosomal subunit catalyzes the formation of the peptide bond during protein synthesis. Some small RNA molecules (sRNA) function as regulators either (a) by binding near the 5' end of an mRNA, preventing ribosomes from translating that message, or (b) by base pairing directly with a strand of DNA near the promoter, preventing transcription. Key Points The most common function of RNA is communication of DNA gene sequences in the form of mRNA to ribosomes. For the synthesis of mRNA, DNA acts as a template; hence adenine, guanine, cytosine, and uracil in mRNA become complementary to the thymine, cytosine, guanine, and adenine, respectively, in DNA. The ribosomes, which contain rRNA and proteins, translate this message into the primary structure of proteins via aminoacyl-tRNAs. Chapter 43 Section III Chapter 7 Section I 48 Mutations Mutation is a random, undirected, and heritable variation seen in DNA of the cell. This is caused by a change in base sequence of DNA due to addition, deletion, or substitution of one or more bases in the nucleotide sequence of DNA. It can involve any of the genes present in the bacterial chromosome. Mutation results in insertion of a different amino acid into a protein, resulting in the appearance of an altered phenotype. Types of Mutations Mutations are a natural event occurring in dividing cells. The frequency of mutations ranges from 10−2 to 10−10 per bacterium per division. These occur spontaneously or are enhanced by different mutagens. Mutations are of three types: (a) base substitution, (b) frame-shift mutation, and (c) mutations due to transposons or insertion sequences. ◗ Mutation due to base substitution This type of mutation occurs when one base in the nucleotide sequence is inserted in place of another. This occurs during replication of DNA either due to an error in the function of DNA polymerase or due to a mutagen that alters the hydrogen bonding of the base being used as a template in such a manner that the wrong base is inserted. The base substitution mutation may be of two types: missense mutation and nonsense mutation. A. Missense mutation: It is one in which the base substitution results in a codon that specifies a different amino acid to be inserted. B. Nonsense mutation: It is another type of mutation in which the base substitution produces a terminal codon that stops synthesis of protein prematurely. Entire protein function is destroyed during the process of nonsense mutation. ◗ Frame-shift mutation It is the second type of mutation. This occurs when one or more base pairs are added or deleted in the DNA. This, therefore, leads to shifting of the reading frame of the ribosome that results in incorporation of the wrong amino acids downstream from the mutation. Result of the frame-shift mutation ends in production of an inactive protein. ◗ Mutation due to transposons or insertion sequence This is the third type of mutation that occurs when transposons or insertion sequences are integrated into the DNA. These newly inserted pieces of DNA cause profound changes in the gene into which they are inserted and also causes changes in the adjacent genes. Causative Agents of Mutation Mutation can be caused by (a) viruses, (b) radiation, or (c) chemicals. ◗ Viruses Bacterial viruses (mutator bacteriophage) are an example of viruses that cause a high frequency of mutation by inserting their DNA into the bacterial chromosome. Mutations can occur in various genes as viral DNA can insert bacterial chromosome at many different sites. The mutations caused by these viruses may be either frame-shift mutations or deletions. ◗ Radiations X-rays and ultraviolet light are the examples of radiation that can cause mutation in chromosomal DNA. ■ ■ X-rays: X-rays damage DNA in many ways. They cause damage by producing free radicals that can attack the bases or alter them in the strand, thereby changing their hydrogen bonding. They also damage DNA by breaking the covalent bonds that hold the ribose phosphate together. Ultraviolet light: Ultraviolet radiation causes damage in DNA by cross-linking of the adjacent pyrimidine bases to form dimers. For example, the cross-linking of adjacent thymine to form thymine dimers results in the inability of DNA to replicate properly. BACTERIAL GENETICS ◗ Chemicals ■ ■ Plasmids Plasmids are extrachromosomal DNA substances. They are replicons that are maintained as discrete, extrachromosomal genetic elements in bacteria. They are usually much smaller than the bacterial chromosome, varying from less than 5 to more than several 100 kbp. However, plasmids as large, as 2 million base pairs can occur in some bacteria. Plasmids are circular and doublestranded DNA molecules that encode traits that are not essential for bacterial viability. They are capable of replicating independently of the bacterial chromosomes. The plasmids can also be present as integrated with bacterial chromosomes, and plasmids integrated with host chromosome are known as episomes. Plasmids are present in both Gram-positive and Gram-negative bacteria. ◗ Types of plasmids Conditional Lethal Mutation Lethal mutations occur when some mutations involve vital functions, resulting in production of nonviable mutants. On the other hand, a conditional lethal mutation is a form of lethal mutation, in which mutation is expressed only under certain conditions, resulting in production of viable mutants. This is of medical importance, because it is made use for preparation of vaccine strains. Temperature-sensitive strains are the most common example of conditional lethal mutations. The temperature-sensitive organisms have the unique property of replicating at a low permissive temperature, such as 32⬚C but cannot grow at a higher restrictive temperature, such as 37⬚C. This is due to a mutation that causes changes in an amino acid in an essential protein, allowing these organisms to function at 32⬚C but not at 37⬚C. Temperaturesensitive influenza virus strain used in experimental vaccine is an example of conditional lethal mutations. This influenza vaccine contains a virus that can grow at 32⬚C and infect nose and can replicate and induce immunity. But the virus cannot grow at 37⬚C, hence cannot infect the lungs and does not cause pneumonia. 1. Transmissible plasmids: They can be transferred from cell to cell by a process of genetic transfer known as conjugation. They are large (mol. wt. 40–100 million) plasmids. They contain more than a dozen genes responsible for synthesis of the sex pilus and for the synthesis of enzymes required for their transfer. Usually, one to three copies of the plasmid are present in a cell. 2. Nontransmissible plasmids: These cannot be transferred from cell to cell, because they do not contain the transfer genes. They are small (mol. wt. 3–20 million), usually nonconjugative, and have high copy numbers (typically 10–60 per chromosome). They depend on their bacterial host to provide some functions required for replication and are distributed randomly between daughter cells at division. Nature of factors Depending on the nature of factors, plasmids are of the following types: (a) the F factor, (b) the R factor, and (c) the Col factor. 1. The F factor: The F plasmid, also called F factor, is a transfer factor that contains the genetic information, essential for controlling mating process of the bacteria during conjugation. The F plasmid of Escherichia coli is the prototype for fertility plasmids in Gram-negative bacteria. Strains of E. coli with an extrachromosomal F plasmid are called F⫹ and function as donors, whereas strains that lack the F plasmid are F− and behave as recipients. The conjugative functions of the F plasmid are determined by a cluster of at least 25 transfer (tra) genes. These genes determine (a) expression Chapter 43 Mutations in the bacteria cause a lot of changes in their various properties. Mutation alter drug susceptibility, antigenic structure, and virulence of mutant bacteria. It also alter susceptibility of bacteria to bacteriophages, alter their colony morphology and pigment productions, and affect their ability to produce capsule or flagella. Development of drug resistance due to mutations in bacteria is a major health concern. Plasmids, depending on transmissibility are of two types: (a) transmissible plasmids and (b) nontransmissible plasmids. Section III Plasmids depending on their transmissibility and nature of the factor can be of the following types: Transmissibility of plasmids Effects of Mutations Chapter 7 ■ Benzpyrene: This is commonly present in tobacco smoke that binds to existing DNA bases and causes frame-shift mutations. The benzpyrene, which is a carcinogen as well as a mutagen, intercalates between the adjacent bases, thereby distorting and offsetting the nucleotide sequence in the DNA. Nitrous acid and alkylating agents: They act by altering the existing base in the DNA. This results in formation of a hydrogen bond with a wrong base. For example, adenine does not form bond with thymine but makes wrong pair with cytosine. Base analogs: Base analogs, such as 5-bromouracil, have less hydrogen bonding capacity than thymine, so they bind to guanine with better frequency. This results in a mutation due to a transition from AT base pair to a GC base pair. Iododeoxyuridine, an antiviral drug, also acts as a base-pair analog. Extrachromosomal DNA Substances Section I Various chemicals, such as nitrous acid, alkylating agents, benzpyrene, and base analogs, such as 5-bromouracil cause mutation in several different ways: 49 GENERAL MICROBIOLOGY of pili, (b) synthesis and transfer of DNA during mating, (c) interference with the ability of F⫹ bacteria to serve as recipients, and (d) other functions. The F plasmid in E. coli can occur as an extrachromosomal genetic element or be integrated into the bacterial chromosome. Both the F plasmid and the bacterial chromosome are circular DNA molecules. Hence, reciprocal recombination between them produces a larger DNA circle consisting of F-plasmid DNA inserted linearly into the chromosome. 2. The R factor: Resistance factors, also called R factors, are extrachromosomal plasmids. They are circular with double-stranded DNA. R factors occur in two sizes: large plasmids (mol. wt. 60 million) and small plasmids (mol. wt. 10 million). The large plasmids are conjugative “R” factors, which contain extra DNA to code for the conjugation process. The small plasmids contain only the “r” genes and are not conjugative. R factor consists of two components: the resistance transfer factor (RTF) and resistant determinant (r). The RTF is responsible for conjugational transfer, while each r determinant carries resistance for one of the several antibiotics. Key Points Functions of R factor: ■ Chapter 43 Section III Chapter 7 Section I 50 ■ ■ The R factors are responsible for spread of multiple-drug resistance among bacteria. They carry the genes for a variety of enzymes that can destroy antibiotics and modify membrane transport system. The R factor may carry one antibiotic resistance gene or may carry two or more of these genes. The R factor carrying more than two genes has many clinical implications: ● First and foremost, a bacterium carrying such genes will show resistance to more than one type of antibiotics, such as penicillins and aminoglycosides. ● The second importance is that the use of an antibiotic that selects an organism for a bacterium resistant to one antibiotic (such as penicillin) will select for a bacterium resistant to other antibiotics (such as tetracyclines, aminoglycosides, chloramphenicol, erythromycin, etc.). They may also carry genes for resistance to metal ions. For example, the genes code for an enzymes that reduce mercuric ions to elementary mercury, thereby making the bacteria resistant to action of mercuric ions. They also carry resistance to certain bacteriophages by coding for the enzymes, e.g., restriction endonucleases that degrade the DNA of the infecting bacteriophages. 3. Colicinogenic (Col) factor: Col factor is a plasmid that resembles the F factor in promoting conjugation, leading to self-transfer and also at times transfer of segments of chromosomes. ■ ■ The Col factor encodes for production of colicins, which are antibiotics-like substances that are specifically and selectively lethal to other enteric bacteria. They also encode for production of diphthericin and pyocyanin produced by Corynebacterium diphtheriae and Pseudomonas pyocyanea, respectively, which are substances similar to colicins. Box 7-1 Plasmid-determined properties Plasmids carry genes for the following: 1. Resistance to antibiotics that is mediated by a variety of enzymes. 2. Resistance to ultraviolet light that is mediated by DNA repair enzymes. 3. Resistance to heavy metals such as mercury and silver that is mediated by enzyme reductase. 4. Pili that mediate the adherence of bacteria to epithelial cells. Examples include K88, K99 found in uropathogenic E. coli. 5. Exotoxins including many enterotoxins. Representative toxins encoded by plasmids include heat-labile and heat-stable enterotoxins of E. coli, exfoliative toxin of Staphylococcus aureus, hemolysins of Clostridium perfringens, and tetanus toxin of Clostridium tetani. 6. Bacteriocins produced by certain Gram-negative bacteria are lethal for other bacteria. Examples include pyocin produced by P. pyocyanea and diphthericin produced by C. diphtheriae. 7. A variety of enzymes such as urease of Helicobacter pylori and proteases of Pseudomonas spp. have degradative properties. The degradative enzymes produced by Pseudomonas spp. are capable of cleaning of oil spills and toxic chemical wastes in the environment. ◗ Functions of plasmids Many plasmids control medically important properties of pathogenic bacteria. These include (a) resistance to one or several antibiotics, (b) production of toxins, and (c) synthesis of cell surface structures required for adherence or colonization. Plasmiddetermined properties are summarized in Box 7-1. Some plasmids are cryptic and have no recognizable effects on the bacterial cells that harbor them. Comparing plasmid profiles is a useful method for assessing possible relatedness of individual clinical isolates of a particular bacterial species for epidemiological studies. Transfer of DNA Within Bacterial Cells Transfer of DNA within the bacterial cells can occur by (a) transposons (b) integrative conjugating elements, and (c) programed rearrangement. Transposons Transposons are a type of mobile DNA of 2000–20,000 bp. They can transfer DNA from one site of the bacterial chromosome to another site or to a plasmid. The idea of transposons or jumping genes was first given by Barbara McClintock, a geneticist working in the field of maize genetics. The mode of genetic transfer by transposon is called transposition. The transposition differs from recombination in that a segment of DNA can be transferred from one to another molecule that has no genetic homology with either the transposable element or the donor DNA. Transposons do not occur independently but have the characteristic of jumping from one part of a chromosome to another or to a plasmid. They can also jump from one plasmid to another or back to the chromosome, hence, are called as jumping genes. Transposition in prokaryotes usually involves two steps—selfreplication and recombination. They jump from one part to BACTERIAL GENETICS ■ ■ ■ Integrative conjugative elements (ICEs) are ways of horizontal gene transfer and self-transmissible mobile genetic elements. The elements exchange by conjugation, but need to integrate into chromosome to propagate. They cannot replicate autonomously. ICEs integrate into and replicate along with the host cell chromosome, whereas plasmids exist as extra-chromosomal (usually circular) autonomously replicating DNA molecules. Key Points When purified DNA is injected into the nucleus of a bacterial cell, the process is called as transfection. Transfection is frequently used in genetic engineering studies. Programed Rearrangements The transfer of DNA within bacteria can also occur by programed rearrangement. In this programed rearrangement, there is a movement of a gene from a silent site where the gene is not expressed to an active site where transcription and translation occur. Many silent genes are present in the DNA that encode variants of the antigens. Presentation of the new gene into the active site occurs in a sequential and repeated manner, which then manifests in antigenic variations in the bacteria and parasites. This mechanism is responsible for antigenic variations seen in Neisseria gonorrhoeae, Borrelia recurrentis, and Trypanosoma brucei. Transfer of DNA Between Bacterial Cells The genetic information can be transferred from one bacterium to another. There are three general methods for genetic exchange in bacteria: (a) transformation, (b) transduction, and (c) conjugation. Transduction The transfer of a portion of DNA from one bacterium to another mediated by a bacteriophage is known as transduction. During replication of virus within the cell, a piece of bacterial DNA is incorporated into the bacteriophage and is carried into the recipient bacterium at the time of infection. The phage DNA within the recipient bacterial cell integrates into the cell DNA during a process called lysogenic conversion. The process of lysogenic conversion confers a new property to the bacterial cell; for example, by lysogenic conversion nonpathogenic bacteria can become pathogenic. Bacteriophages encode diphtheria toxin, botulinum toxin, cholera toxin, and erythrogenic toxin and can be transferred from one bacterium to another by transduction (Fig. 7-2). Transduction is of two types: (a) generalized transduction and (b) specialized transduction. Chapter 43 Integrative Conjugative Elements Section III Plasmid can contain several transposons carrying drugresistance genes. The transposons code for drug-resistance enzymes, toxins, or a variety of metabolic enzymes. These transposons can either cause mutations in the gene into which they insert or alter the expression of nearby genes. Insertion sequences: The simplest form of transposons is the insertion sequences. These are a type of transposons that have a few bases varying from 800 to 1500 bp. They are found as multiple copies at the end of larger transposon units. These cause mutation by moving from one side to another in DNA sequence and are believed to control various cellular responses. Transformation is a process of the transfer of DNA itself from one bacterium to another. This may occur either in nature or in a laboratory. In nature, DNA is released from a bacterium by lysis, which may be taken up by recipient bacterium that must be competent. This natural process of transfer of genetic material appears to play no role in disease. In laboratory conditions, DNA may be extracted from one type of bacterium and introduced into genetically different bacteria. The cell walls of bacteria in vitro are made more permeable for DNA uptake by using substances, such as calcium chloride. Griffith (1922) in his classical experiment on mice demonstrated that neither of the mice died when injected separately with a live, noncapsulated Pneumococcus (nonvirulent) and heat-killed, capsulated Pneumococcus (nonvirulent), but the mice died when they were injected with a mixture of both these strains. From the dead mice, he could isolate live, capsulated pneumococci, which were virulent. He demonstrated that some factor in heat-killed, capsulated pneumococci had transferred the material for capsule synthesis in the noncapsulated strains of the bacteria, making them virulent (Fig. 7-1). McLeod and McCarthy in 1944 demonstrated that DNA extracted from encapsulated, smooth pneumococci could transform nonencapsulated, rough pneumococci into capsulated, smooth organisms. They demonstrated the transforming principle of DNA. The experimental use of transformation was the first experiment to reveal important information about DNA and was the first example of genetic exchange in bacteria. Another bacterium where transformation is observed is Haemophilus influenzae. Chapter 7 ■ The first domain is a short DNA sequence of inserted repeats that are present at the end. This domain mediates the integration of the transposons into the recipient DNA. The second domain is the gene for the enzyme transposases. These enzymes mediate the excision and integration process. The third domain is the gene for the repressor, which regulates the synthesis of both transposase and the gene products of the fourth domain. The fourth domain is the gene for the expression of enzymes. Transformation Section I another by synthesizing a copy of their DNA and inserting the copy at another site in the bacterial chromosome or the plasmid. Transposons, unlike plasmids, are not self-replicating and depend on chromosomal or plasmid DNA for replication. Transposons do not require homology with the recipient site for its transfer. Transposons typically consist of four domains: 51 GENERAL MICROBIOLOGY Chapter 43 Section III Chapter 7 Section I 52 1 2 3 Living encapsulated bacteria injected into mouse Mouse died Colonies of encapsulated bacteria were isolated from dead mouse 1 2 3 Living nonencapsulated bacteria injected into mouse Mouse healthy Few colonies of nonencapsulated bacteria were isolated from mouse 1 2 3 Heat-killed encapsulated bacteria injected into mouse Mouse healthy No colonies were isolated from mouse 1 2 3 Mouse died Colonies of encapsulated bacteria were isolated from dead mouse Living nonencapsulated bacteria and heat-killed encapsulated bacteria injected into mouse FIG. 7-1. ◗ A schematic diagram showing transmission of DNA by transformation. Generalized transduction This occurs when a small fraction of the phage virions produced during lytic cycle are aberrant and contain a random fragment of the bacterial genome instead of phage DNA. Each individual transducing phage carries a different set of closely linked genes, representing a small segment of the bacterial genome. Transduction mediated by populations of such phages is called generalized transduction. Each part of the bacterial genome has approximately the same probability of being transferred from donor to recipient bacteria. Generalized transduction involves any segment of the donor DNA at random. This occurs because the cell DNA is fragmented after such infection and pieces of same DNA, the same size as viral DNA, are incorporated into the bacterial DNA. This occurs at a frequency of about 1 in every 1000 viruses. Generalized transduction may be complete or abortive: ■ Complete transduction is characterized by production of stable recombinants that inherit donor genes and retain the ability to express them. ■ Abortive transduction refers to the transient expression of one or more donor genes without formation of recombinant progeny. The donor DNA fragment does not replicate in abortive transduction, and only one bacterium contains the donor DNA fragment among the progeny of the original transductant. The donor gene products become progressively diluted in all other progeny after each generation of bacterial growth until the donor phenotype can no longer be expressed. On selective medium, abortive transductants produce minute colonies that can be distinguished easily from colonies of stable transductants. The frequency of abortive transduction is typically one to two times more than the frequency of generalized transduction. This indicates that most cells infected by generalized transducing phages do not produce recombinant progeny. ◗ Specialized transduction Specialized transduction results from lysogenization of the recipient bacterium by the specialized transducing phage and expression of the donor genes. Specialized transducing phages BACTERIAL GENETICS 1 Infection Gene A positive Bacterial cell Integration 2 3 Lysis 4 5 Infection Transduction: Gene A from transducing phage is inserted into gene A negative bacterium by recombination, the resulting bacteria are gene A positive Induction Phage genome Phage genome with gene A FIG. 7-2. A schematic diagram showing transmission of genetic material by bacteriophage-mediated transduction. Sex pilus F+ Conjugation process is initiated F+ F+ 5⬘ 3⬘ Key Points Transduction is not only confined to transfer of chromosomal DNA but also to plasmids and episomes. Transformation involving transfer of plasmids from one bacterium to another by transduction is responsible for penicillin resistance in staphylococci. Transduction appears to be the most widespread mechanism of gene transfer among bacteria. This method also provides an excellent tool for the genetic mapping of the bacteria. It can occur in many bacteria for which bacteriophages are known. It may occasionally occur in eukaryotic cells. Conjugation Conjugation is a process of transfer of DNA from the donor bacterium to the recipient bacterium during the mating of two F− F plasmid Separation of cell after the F plasmid transfer Initiation of rolling circle replication by binding protein at 5⬘ end 3⬘ 5⬘ Transfer of single stranded DNA and synthesis of complimentary strand FIG. 7-3. A schematic diagram showing transmission of genetic material by conjugation. Chapter 43 are formed only when lysogenic donor bacteria enter the lytic cycle and release phage progeny. The specialized transducing phages are rare recombinants that lack part of the normal phage genome. They contain part of the bacterial chromosome present adjacent to the site of prophage attachment. Many specialized transducing phages are defective. They cannot complete the lytic cycle of phage growth in infected cells unless helper phages are present to provide missing phage functions. Specialized transduction differs from generalized transduction in many ways. The former is mediated only by specific temperate phages and only a few specific donor genes can be transferred to recipient bacteria. Section III Gene A negative Chapter 7 Lysogen Gene A positive bacterial cells. In conjugation, direct contact between the donor and recipient bacteria leads to formation of a cytoplasmic bridge between them and transfer of part or all of the donor genome to the recipient (Fig. 7-3). Conjugation takes place between two closely related species and occurs mostly in Gramnegative bacteria. Conjugation also occurs in Gram-positive bacteria. Donor ability of bacteria is determined by specific conjugative plasmids called fertility (F⫹) plasmids or sex plasmids. The F plasmid controls the mating process of bacteria. Pilus is the most important protein that forms the sex pilus or conjugation tube. The sex pilus produces a bridge between conjugating cells in Gram-negative bacteria. Mating occurs between the donor male bacterium carrying the F factor (F⫹) and the recipient female bacterium that does not contain F factor (F⫺). It begins when the pilus of F⫹ bacterium attaches to a receptor on the surface of a female (F⫺) bacterium. The cells are then brought into direct contact by the link in the pilus. This is followed by an enzymatic cleavage of the F factor DNA in which one strand of bacterial DNA is transferred into the recipient cell through the conjugation bridge. The synthesis of the complementary strand to form a double-stranded F-factor plasmid in both the donor and recipient cells completes the process of conjugation. The recipient cell becomes F⫹ male that is capable of transmitting the plasmid to other F⫺ cells. High-frequency recombination (Hfr): Long length of DNA can be transferred by process of conjugation. Hfr strain is a type of F+ cells that have an F plasmid integrated into the bacterial DNA. Hence they acquire the capability of transferring the Section I Transducing bacteriophage 53 Chapter 43 Section III Chapter 7 Section I 54 GENERAL MICROBIOLOGY TABLE 7-1 Methods of transfer Comparison of transformation, transduction, and conjugation Mechanism Transformation Recipient cell uptake of free DNA released into the environment Nature of DNA transferred Any gene Transduction Transfer of DNA from one bacterium to another by bacteriophage Any gene in generalized transduction; only selected genes in specialized transduction Conjugation Transfer of DNA from one living bacterium to another through the sex pilus Chromosomal or plasmid DNA chromosome to another cell. A whole chromosome can be transferred if it is integrated with F plasmid. In this process, the single strand of DNA that enters the recipient F⫺ cell contains a part of the F factor at one end, followed by the bacterial chromosome, and then by the remainder of the F factor. The bacterial genes adjacent to the leading piece of F factor are the most frequently transferred. The newly acquired DNA recombines with the recipient DNA and becomes an integral component of genetic material. The complete transfer of the bacterial DNA is usually completed in approximately 100 minutes. In matings between F+ and F− bacteria, only the F plasmid is transferred with high efficiency to recipients. Chromosomal genes are transferred with very low efficiency, which is mediated by the spontaneous Hfr mutants in F⫹ populations. In matings between Hfr and F⫺ strains, the segment of the F plasmid containing the tra region is transferred last, after the entire bacterial chromosome has been transferred. Most recombinants produced after matings between Hfr and F⫺ cells fail to inherit the entire set of F-plasmid genes and are phenotypically F⫺. In matings between F⫹ and F⫺ strains, the F plasmid spreads rapidly throughout the bacterial population and most recombinants are F⫹. Conjugation also occurs in Gram-positive bacteria. Grampositive donor bacteria produce adhesions that cause them to aggregate with recipient cells, but sex pili are not involved. In some Streptococcus spp., recipient bacteria produce extracellular sex pheromones that facilitate conjugation. Table 7-1 shows a comparison of transformation, transduction, and conjugation. Key Points Transfer of plasmids during conjugation is responsible for the spread of multiple drug resistance among bacteria. The plasmid responsible for drug resistance consists of two components, namely, RTF and a resistance determinant (r) for each of the several drugs. Transferable drug resistance occurs widely among pathogenic as well as commensal bacteria of humans and animals. Plasmid is also responsible for production of colicins, the antibiotic-like substances lethal to other Gram-negative bacteria. The plasmid that encodes for production of colicins is known as col factor and is also transferred by conjugation. Recombination After the DNA is transferred from one donor bacterium to the recipient through transformation, transduction, or conjugation, it combines with the chromosome of the bacterium by a process called recombination. Recombination is of two types: homologous and nonhomologous. Homologous recombination takes place between two pieces of DNA showing extensive homologous regions. This results in pairing up and exchange of pieces by the processes of breakage and reunion. The nonhomologous recombination takes place between two pieces of DNA showing little or no homology. Genetic Engineering and Molecular Methods Introduction The deliberate modification of an organism’s genetic information by directly changing its nucleic acid genome is called genetic engineering and is achieved by a group of methods known as recombinant DNA technology. DNA: An Amazing Molecule The structure of deoxyribonucleic acid (DNA) provides a complex code that encodes for synthesis of proteins. The DNA as a molecule exhibits many intriguing features. One useful property of DNA is that it readily anneals, meaning that it changes its binding properties in response to heating and cooling. Exposure to temperatures just below boiling (90–95°C) causes DNA to become temporarily denatured. When heat breaks open the hydrogen bonds that keep the double helix together, it separates longitudinally into two strands. Each strand displays its nucleotide code so that the DNA in this form can be subjected to tests or replicated. When heating is followed by gradual cooling, two single DNA strands rejoin (renature) by hydrogen bonds at complementary sites. Annealing is a necessary feature of the polymerase chain reaction (PCR) and nucleic acid probes described later. Genetic Engineering Genetic engineering is the application of science to social needs. In recent years, engineering based on bacterial genetics has transformed biology. Specified DNA fragments can be isolated and amplified, and their genes can be expressed at high levels. The nucleotide specificity, required for cleavage by restriction enzymes, allows fragments containing genes or parts of genes to be covalently bound to plasmids (vectors) that can then be inserted into bacterial hosts. Bacterial colonies or clones carrying specified genes are identified by hybridization of DNA or RNA with chemical or radiochemical probes. Alternatively, protein products encoded by the genes are recognized either by enzyme activity or by immunologic techniques. Thus, genetic engineering techniques are used to isolate virtually any gene with a biochemically recognizable property. 8 Preparation of DNA Fragments with Restriction Enzymes The genetic diversity of bacteria is reflected in their remarkable range of restriction enzymes, which possess remarkable selectivity that allows them to recognize specific regions of DNA for cleavage. DNA sequences recognized by restriction enzymes are predominantly palindromes (inverted sequence repetitions). GAATTC is a typical sequence palindrome, recognized by the frequently used restriction enzyme EcoRI. The inverted repetition, inherent in the complementarity of the G–C and A–T base pairs, results in the 5⬘ sequence TTC being reflected as AAG in the 3⬘ strand. Most restriction enzymes recognize 4, 6, or 8 base sequences; however, other restriction enzymes recognize 10, 11, 12, or 15 base sequences. Restriction enzymes that recognize 8 bases produce fragments with a typical size of 64,000 bp and are useful for analysis of large genetic regions. Restriction enzymes that recognize more than 10 bases are useful for construction of a physical map and for molecular typing by pulse-field gel electrophoresis. Physical Separation of Differently Sized DNA Fragments Much of the simplicity underlying genetic engineering techniques lies in the fact that gel electrophoresis permits DNA fragments to be separated on the basis of size. The smaller the fragment, the more rapid the migration. Overall rate of migration and optimal range of size for separation are determined by the chemical nature of the gel and by the degree of its crosslinking. Highly cross-linked gels optimize the separation of small DNA fragments. The dye ethidium bromide forms a brightly fluorescent color as it binds to DNA, and so small amounts of separated DNA fragments can be photographed on gels. Specific DNA fragments can be recognized by probes containing complementary sequences. Pulsed-field gel electrophoresis allows the separation of DNA fragments containing up to 100,000 bp (100 kilobase pairs, or kbp). Characterization of such large fragments has allowed construction of a physical map for the chromosomes from several bacterial species. Enzymes for Dicing, Splicing, and Reversing Nucleic Acids Restriction endonucleases: The polynucleotide strands of DNA can also be clipped crosswise at selected positions by Chapter 43 Section III Chapter 8 Section I 56 GENERAL MICROBIOLOGY means of enzymes called restriction endonucleases. These enzymes recognize foreign DNA and are capable of digesting or hydrolyzing DNA bonds. Presence of enzyme in the bacterial cell protects bacteria against the incompatible DNA of bacteriophages or plasmids. In a laboratory, restriction endonuclease enzymes can be used to cleave DNA at desired sites and are a must for the techniques of recombinant DNA technology. So far, hundreds of restriction endonucleases have been discovered in bacteria. Each type has a known sequence of 4–10 bp as its target, so sites of cutting can be finely controlled. Endonucleases are named by combining the first letter of the bacterial genus, the first two letters of the species, and the endonuclease number. For example, EcoRI is the first endonuclease found in Escherichia coli and HindIII is the third endonuclease discovered in Haemophilus influenzae typed. Restriction fragment length polymorphisms: The pieces of DNA produced by restriction endonucleases are termed restriction fragments. Because genomes of members of the same species can vary in the cutting pattern by specific endonucleases, it is possible to detect genetic differences by restriction fragment length polymorphisms (RFLPs). Hundreds of cleavage sites that produce RFLPs are distributed throughout genomes. Because RFLPs serve as a type of genetic marker, they can help locate specific sites along a DNA strand. The RFLPs are thus useful in preparation of gene maps and DNA profiles, and also in analysis of genetic relationships. Ligase: It is an enzyme necessary to seal the sticky ends together by rejoining the phosphate–sugar bonds cut by endonucleases. Its main application is in final splicing of genes into plasmids and chromosomes. Reverse transcriptase: It is an enzyme, best known for its role in the replication of the AIDS virus and other retroviruses. This enzyme is used by geneticists as a valuable tool for converting RNA into DNA. Complementary DNA: The copies called complementary DNA, or cDNA, can be made from messenger, transfer, ribosomal, and other forms of RNA. The technique provides a valuable means of synthesizing eukaryotic genes from mRNA transcripts. The advantage is that the synthesized gene will be free of the intervening sequences (introns) that can complicate the management of eukaryotic genes in genetic engineering. Complementary DNA can also be used to analyze the nucleotide sequence of RNAs, such as those found in ribosomes and transfer RNAs. kb. Humans have approximately 3.5 billion base pairs (Bbp) arrayed along 46 chromosomes. Oligonucleotides are very short pieces of DNA or RNA. They vary in length from 2 to 200 bp, although the most common ones are about 20–30 bp. They can be isolated from cells or prepared tailor-made by a DNA synthesizer that limits the length to about 200 nucleotides. Hybridization probes are used routinely in the cloning of DNA. The amino acid sequence of a protein is used to deduce the DNA sequence from which a probe may be constructed and employed to detect a bacterial colony containing the cloned gene. cDNA, encoded by mRNA, is used to detect the gene that encodes the mRNA. ◗ Types of hybridization Hybridization is of the following types: ■ ■ ■ Northern blot: Hybridization of DNA to RNA is known as Northern blot, which provides quantitative information about RNA synthesis. Southern blot: Hybridization of DNA to DNA is known as Southern blot. This method is useful to detect specific DNA sequences in restriction fragments separated on gels. These blots can be used to detect overlapping restriction fragments. Western blot: It is a technique used for detection of genes, in which antibodies are used to detect cloned genes by binding to their protein products. Cloning of these fragments makes it possible to isolate flanking regions of DNA by a technique known as chromosomal walking. ◗ DNA sequencing DNA sequencing shows gene structure that helps research workers to find out the structure of gene products. Key Points DNA sequencing has many applications as follows: ■ ■ ■ ■ Methods Used to Size, Synthesize, and Sequence DNA The relative sizes of nucleic acids are usually known by the number of base pairs or nucleotides they contain. For example, the palindromic sequences recognized by endonucleases are usually 4–10 bp in length. An average gene in E. coli is approximately 1300 bp, or 1.3 kilobases (kb), and its entire genome is approximately 4.7 million base pairs (Mb). The Epstein–Barr virus, cause of infectious mononucleosis, has a gene of 172 Information obtained by DNA sequencing makes it possible to understand or alter the function of genes. DNA sequence analysis demonstrates regulatory regions that control gene expression and genetic “hot spots” particularly susceptible to mutation. Comparison of DNA sequences shows evolutionary relationships that provide a framework for definite classification of microorganisms including viruses. Comparison of DNA sequences facilitates identification of conserved regions, which are useful for development of specific hybridization probes to detect microorganisms including viruses in clinical samples. Maxam–Gilbert technique and Sanger (dideoxy termination) method are two methods used routinely for DNA sequence determination. Maxam–Gilbert technique depends on the relative chemical liability of different nucleotide bonds, whereas the Sanger method interrupts elongation of DNA sequences by incorporating dideoxynucleotides into the sequences. GENETIC ENGINEERING AND MOLECULAR METHODS Key Points Applications of DNA Probes DNA probes are nonamplified methods; they only detect DNA in specimens but without any amplification of the same (Fig. 8-1). DNA probes have many applications as follows: Nucleic Acid Probes ■ Polymerase Chain Reaction In 1983, Kary Mullis developed a new technique that made it possible to synthesize large quantities of a DNA fragment without cloning it. This technique is called polymerase chain Stool 1 Stool 2 Stool 3 Stool 4 DNA extracted from stool specimen was blotted on nitrocellulose paper Shigella fragment DNA cloned in E. coli DNA is denatured to single strand of DNA Cloned DNA fragment marked with fluorescent dye and separated into single strands, forming DNA probes Fluorescent probe DNA probes are added to the DNA stool specimens No Shigella DNA present in stool specimens FIG. 8-1. A schematic diagram showing the principle of DNA probe. DNA probes hybridize with Shigella DNA from stool samples—fluorescence indicate presence of Shigella in stool specimens Chapter 43 Shigella colonies on growth medium ■ Section III Nucleic acid probes are segments of DNA and RNA labeled with radioisotopes or enzymes that can hybridize to complementary nucleic acids with high degree of specificity. Hybridization probes have practical value, because they can detect specific nucleotide sequences in unknown samples. The probes carry reporter molecules, such as radioactive labels, which are isotopes that emit radiation, or luminescent labels, which give off visible light. Reactions can be revealed by placing photographic film in contact with the test reaction. Fluorescent probes contain dyes that can be visualized with ultraviolet light. Probes can be used in a broad range of analytic procedures. Two different nucleic acids can hybridize by uniting at their complementary sites. All different combinations are possible: single-stranded DNA can unite with other single-stranded DNA or RNA, and RNA can hybridize with other RNA. This property forms the basis of specially formulated oligonucleotide tracers called gene probes. Chapter 8 A number of DNA probes have been developed for identification of culture isolates and for various uses in clinical microbiology by (a) direct detection of microbes in clinical specimens and (b) by identification of organisms after isolation of culture (Box 8-1). Some regions of human DNA exhibit substantial variability in the distribution of restriction sites. This variability is termed restriction fragment length polymorphism. Oligonucleotide probes that hybridize with RFLP DNA fragments can be used to trace DNA from a small sample to its human donor. Thus, the technique is valuable to forensic science. Applications of RFLP to medicine include identification of genetic regions that are closely linked to human genes with dysfunctions coupled to genetic disease. This information will be a valuable aid in genetic counseling. ■ Section I Shotgunning: The study of biology has been revolutionized by the development of technology that allows sequencing and analysis of entire genomes ranging from viruses to unicellular prokaryotic and eukaryotic microorganisms to humans. This has been facilitated by use of the procedure known as shotgunning. In this procedure, the DNA is broken into random smaller fragments to create a random fragment library. 57 58 Box 8-1 Section I Chapter 8 Section III Chapter 43 GENERAL MICROBIOLOGY Applications of DNA probes 1. Direct detection of microbes in clinical specimens (e.g., Mycobacterium tuberculosis, Streptococcus pyogenes, Candida albicans). 2. Diagnosis of uncultivable bacteria (e.g., chlamydia, rickettsia, etc.). 3. Identification of culture isolates. 4. Strain identification for epidemiological typing. 5. Identification of virulence factors of microbial agents. 6. Identification of toxins (e.g., diphtheria toxin). 5⬘ 3⬘ 3⬘ 5⬘ Denaturation 94°C 5⬘ 3⬘ Separation of DNA strands 5⬘ Annealing of primers reaction (PCR) and has great practical importance and impact on biotechnology. By this technique, large quantities of a particular DNA sequence can be prepared. In PCR, oligonucleotide sequences identical to those flanking the targeted sequence are first synthesized. These synthetic oligonucleotides are usually about 20 nucleotides long and serve as primers for DNA synthesis. Pieces ranging in size from less than 100 bp to several 1000 bp in length can be amplified, and only 10–100 pmol primer is required. The concentration of target DNA can be as low as 10–15 ␮L. The reaction mix for PCR contains (a) the target DNA (b) a very large excess of the desired primers, (c) a thermostable DNA polymerase, and (d ) four deoxyribonucleoside triphosphates. Only DNA polymerases that are able to function at the high temperatures can be employed in the PCR technique. Taq polymerase from the thermophilic bacterium Thermus aquaticus and the Vent polymerase from Thermococcus litoralis are the two popular enzymes used in the PCR. The PCR cycle takes place in three steps as follows (Fig. 8-2): ■ ■ ■ Step 1: The target DNA containing the sequence to be amplified is heat denatured to separate its complementary strands. Normally the target DNA is between 100 and 5000 bp in length. Step 2: The temperature is lowered so that the primers can anneal to the DNA on both sides of the target sequence. Because the primers are present in excess, the targeted DNA strands normally anneal to the primers rather than to each other. Step 3: DNA polymerase extends the primers and synthesizes copies of the target DNA sequence using the deoxyribonucleoside triphosphates. At the end of one cycle, the targeted sequences on both strands are copied. When the three-step cycle is repeated, the four strands from the first cycle are copied to produce eight fragments. The third cycle yields 16 products. Theoretically, 20 cycles will produce about one million copies of the target DNA sequence, and 30 cycles yield around one billion copies. The PCR technique has now been automated and is carried out through a specially designed machine called thermocycler. Currently, a thermocycler or PCR machine can carry out 25 cycles and amplify DNA 105 times in as little as 57 minutes. During a typical cycle, the DNA is denatured at 94°C for 15 seconds; then the primers are annealed and extended (steps 2 and 3) at 68°C for 60 seconds. PCR technology is improving continually and undergoing many changes as follows: 1. Nowadays, RNA can be efficiently used in PCR procedures. The Tth DNA polymerase, a recombinant Thermus Target DNA sequence 40–60°C 3⬘ 5⬘ Primer 1 3⬘ 5⬘ A TCGCA T TAGCGT A 3⬘ DNA synthesis (elongation) Binding of primers 1 and 2 C GTCAGG GCAGTCC 3⬘ 5⬘ Primer 2 5⬘ Taq polymerase 72°C dATP, dCTP, dGTP, TTP Target DNA molecules are doubled Approximately 30 repetitive cycles DNA products (amplicons) up to 109 fold amplification FIG. 8-2. A schematic diagram showing the principle of polymerase chain reaction. thermophilus DNA polymerase, will transcribe RNA to DNA and then amplify the DNA. Cellular RNAs and RNA viruses may be studied even when the RNA is present in very small amounts (as few as 100 copies can be transcribed and amplified). 2. Also, PCR can quantitate DNA products without the use of isotopes. This allows one to find the initial amount of target DNA in less than an hour using automated equipment. Quantitative PCR is quite valuable in virology and gene expression studies. 3. As mentioned earlier, the target DNA to be amplified is normally less than about 5000 bp in length. A long PCR technique has been developed that will amplify sequences up to 42 kilo bases long. It depends on the use of errorcorrecting polymerases because Taq polymerase is error-prone. 4. Multiplex PCR is another modification of PCR in which two or more target sequences can be demonstrated simultaneously in a single specimen at the same time. This method uses two or more primer sets designed for amplification of different targets. Multiplex is now increasingly evaluated for simultaneous demonstration of two or more pathogens in a clinical specimen. GENETIC ENGINEERING AND MOLECULAR METHODS ■ ■ Applications of PCR The PCR and other molecular techniques have already proven exceptionally valuable in many areas of molecular biology, medicine, and biotechnology. These methods are useful to: ■ ■ ■ ■ ■ Recombinant DNA Technology In recombinant DNA technology, first, the DNA responsible for a particular phenotype is identified and isolated. Once purified, the gene or genes are fused with other pieces of DNA to form recombinant DNA molecules. These are propagated (gene cloning) by insertion into an organism that need not even be in the same kingdom as the original gene donor. Cloning Vectors and Hosts A good recombinant vector has two indispensable qualities: it must be capable of carrying a significant piece of the donor DNA and it must be readily accepted by the cloning host. ◗ Cloning vectors Cloned vectors include: (a) plasmids, (b) bacteriophages, and (c) hybrid vectors. Cloning hosts E. coli is the traditional cloning host that is still used in the majority of experiments. This is because this bacterium was the original recombinant host and the protocols using it are well established, relatively easy, and reliable. Hundreds of specialized cloning vectors have been developed for it. The main disadvantage with E. coli is its lack of versatility in correctly expressing eukaryotic genes. The yeast Saccharomyces cerevisiae is another alternative host used for certain industrial processes and research. This host being eukaryotic already possesses mechanisms for processing and modifying eukaryotic gene products. Certain techniques may also employ different bacteria (Bacillus subtilis), animal cell culture, and even live animals and plants to serve as cloning hosts. Biological Products of Recombinant DNA Technology Recombinant DNA technology is used by pharmaceutical companies to manufacture medicines that cannot be manufactured by any other means. Diseases, such as diabetes and dwarfism, caused by lack of an essential hormone are now being treated by replacing the genes of missing hormone. Porcine and bovine insulin were once the only forms available to treat diabetes, even though such animal products used to cause allergic reactions in certain sensitive individuals. In contrast, dwarfism that cannot be treated with animal growth hormones was treated only with human growth hormone (HGH) obtained from the pituitaries of cadavers. At one time, not enough HGH was available to treat thousands of children in need. However, now the scenario is changed by advent of recombinant HGH. Recombinant technology has changed the outcome of these and many other conditions by enabling large-scale manufacture of lifesaving hormones and enzymes of human origin. Chapter 43 ■ ◗ Section III ■ Amplify very small quantities of a specific DNA and provide sufficient material for accurately sequencing the fragment or cloning it by standard techniques. Detect previously unrecognized or uncultivable microorganisms. Detect the genes responsible for drug resistance. They supplement conventional antimicrobial susceptibility testing for the detection of methicillin resistance in staphylococci, rifampicin resistance in M. tuberculosis, etc. Predict and monitor response of individuals chronically infected with hepatitis B virus (HBV), hepatitis C virus (HCV), or HIV to antiviral therapy. Provide useful information that may predict progression of the disease. Determination of HIV-1 viral load as a predictor of progression to AIDS is an example of such use. Diagnose AIDS, Lyme disease, chlamydia, tuberculosis, hepatitis, the human papilloma virus, and other infectious agents. Detect genetic diseases, such as sickle cell anemia, phenylketonuria, and muscular dystrophy. Plasmids are excellent vectors because they are small, well characterized, easy to manipulate, and they can be transferred into appropriate host cells through transformation. E. coli plasmid carries genetic markers for resistance to antibiotics, although it is restricted by the relatively small amount of foreign DNA it can accept. Bacteriophages are also good vectors because they have natural ability to inject DNA into bacterial hosts through transduction. The Charon2 phage is a modified phage vector that lacks a large part of its genome; hence it can carry a fairly large segment of foreign DNA. Hybrid vectors have been developed by splicing two different vectors together. A cosmid is an example of a hybrid vector that combines a plasmid and a phage and is capable of carrying relatively large genomic sequences. A hybrid E. coli–yeast vector can be inserted in both bacterial and yeast cloning hosts. Chapter 8 Key Points ■ Section I 5. Real-time PCR is the most recent development. It is so named, because the PCR amplicons can be detected in real time. In fact, “real time” refers to the detection of amplicons after each PCR cycle. Several commercial instruments are available that combine PCR amplification of target DNA with detection of amplicons in the same closed vessel. Probe detection formats involve detecting fluorophores. Results are semiquantitative and can be obtained in considerably less time than it takes to perform a conventional PCR assay. 59 Chapter 8 Section I 60 GENERAL MICROBIOLOGY Genetically Modified Organisms The process of artificially introducing foreign genes into organisms is termed transfection, and the recombinant organisms produced in this way are called transgenic or genetically modified organisms. Foreign genes have been inserted into a variety of microbes, plants, and animals through recombinant DNA techniques developed especially for them. Transgenic “designer” organisms are available for a variety of biotechnological applications. Because they are unique life forms that would never have otherwise occurred, they can be patented. Chapter 43 Section III Gene Therapy Gene therapy is a technique for replacing a faulty gene with a normal one in individuals with fatal or extremely debilitating genetic diseases. The inherent benefit of this therapy is to permanently cure the physiological dysfunction by repairing the genetic defect. There are two strategies for this therapy: the ex vivo therapy and the in vivo therapy. In ex vivo therapy, the normal gene is cloned in vectors, such as retroviruses (e.g., mouse leukemia virus) or adenoviruses that are infectious but relatively harmless. Tissues removed from the patient are incubated with these genetically modified viruses to transfect them with the normal gene. The transfected cells are then reintroduced into patient’s body by transfusion. In contrast, the in vivo type of therapy does not have the intermediate step of incubating excised patient tissue. Instead, the naked DNA or a virus vector is directly introduced into the patient’s tissues. The first gene therapy experiment in humans was started in 1990 by researchers at the National Institutes of Health, USA. The subject was a 4-year-old girl suffering from a severe immunodeficiency disease caused by the lack of enzyme adenosine deaminase (ADA). She was transfused with her own blood cells that had been engineered to contain a functional ADA gene. Later, other children were given the same type of therapy. So far, the children have shown remarkable improvement and continue to be healthy, but the treatment is not permanent and must be repeated. Proper scientific controlled trials are required before induction of it as a routine clinical practice. Antimicrobial Agents: Therapy and Resistance Introduction Any chemical used in treatment, relief, or prophylaxis of disease is defined as a chemotherapeutic drug or agent. When chemotherapeutic drugs are given as a means to control infection, the practice is termed antimicrobial chemotherapy. Antimicrobial drugs (also termed anti-infective drugs) are a special class of compounds capable even in high dilutions of destroying or inhibiting microorganisms. The origin of modern antimicrobial drugs is varied. The antibiotics are substances produced by the natural metabolic processes of some microorganisms that can inhibit or destroy other microorganisms. Synthetic antimicrobial drugs are derived in laboratory from dyes or other organic compounds through chemical reactions. Although division into these two categories has been traditional, they tend to overlap, because most antibiotics, termed semisynthetic antibiotics, are now chemically altered in laboratory. The current trend is to use the term antimicrobic for all antimicrobial drugs, regardless of origin Antimicrobial drugs vary in their spectrum of activities. They may be broad-spectrum or narrow-spectrum antibiotics. ■ ■ Broad-spectrum or extended-spectrum antibiotics are active against a wider range of different microbes. For example, tetracyclines are active against a variety of Gram-positive and Gramnegative bacteria, rickettsiae, mycoplasmas, and even protozoa. Narrow-spectrum antibiotics are effective against one or very few microbes. For example, vancomycin is active against certain Gram-positive bacteria (such as staphylococci and enterococci) or griseofulvin, which is used only against fungal skin infections. Antibiotics act against bacteria by the following mechanisms: 1. 2. 3. 4. Penicillins, cephalosporins, and vancomycin are the antibiotics that act against bacteria by interfering with their cell wall synthesis. Penicillins and cephalosporins are called -lactam antibiotics because they possess an intact -lactam ring essential for antimicrobial activity. ◗ ■ Penicillins The penicillins are called -lactam antibiotics because they have a common chemical nucleus (6-aminopenicillanic acid) that contains a -lactam ring. They are bactericidal antibiotics that act by inhibiting bacterial cell wall synthesis. They act primarily against Gram-positive organisms. Other penicillins, such as ampicillin, amoxicillin, carbenicillin, ticarcillin, piperacillin, etc., act against both Gram-positive and Gram-negative organisms. Penicillins kill bacteria by the following mechanisms: ■ ■ Antimicrobial drugs may be bactericidal or bacteriostatic. A bactericidal drug kills bacteria, whereas a bacteriostatic drug inhibits the growth of bacteria, but does not kill them. Bactericidal drugs are very much useful in (a) life-threatening situations, (b) endocarditis, (c) patients with low polymorphonuclear count (below 500/ L), and (d) conditions in which bacteriostatic drugs do not cause a cure. The bacteriostatic drugs depend on the host defense mechanisms, such as phagocytes to kill the bacteria. Hence, these drugs are not used when the patient has too few neutrophils. Inhibition of cell wall synthesis Inhibition of protein synthesis Inhibition of nucleic acid synthesis Alteration of cell membrane function Inhibition of Cell Wall Synthesis Mechanisms of Action of Antimicrobial Drugs ■ 9 ◗ Penicillins kill bacteria during their growing stage and are more active against replicating bacteria during the log phase than the lag phase of the bacterial growth curve. The intact peptidoglycan in the cell wall of the bacteria has chains of N-acetyl muramic acid (NAM) and N-acetyl glucosamine (NAG) glycans cross-linked by peptide bridges. Penicillins and cephalosporins act by inhibiting penicillinbinding proteins (PBPs), also known as transpeptidases, that link the cross-bridges between NAMs, thereby, greatly weakening the cell wall meshwork. Streptococcus pneumoniae is an example of bacteria that show resistance against penicillins due to mutations that occur in the genes encoding PBPs. Cephalosporins Cephalosporins like penicillins are -lactam antibiotics. However, they differ from penicillins in having 7-amino-cephalosporanic acid instead of 6-aminopenicillanic acid in their structure. They exhibit bactericidal activity similar to that of penicillin. They are effective against a wide variety of bacterial pathogens. GENERAL MICROBIOLOGY TABLE 9-1 Second generation Third generation Fourth generation Drugs Cephalexin, cephalothin, cephradine, cephaloridine Cefoxitin, cefaclor, cefamandole, cefuroxime, cefprozil, cefmetazole Cefoperazone, cefotaxime, ceftazidime, ceftizoxime, ceftriaxone, cefixime Cefpirome, cefepime Antibacterial activity Gram-positive bacteria: Staphylococcus aureus, Streptococcus spp. except enterococci Gram-negative bacteria: Escherichia coli, Klebsiella, Haemophilus influenzae, Proteus mirabilis First-generation spectrum  Enterobacter spp., Serratia spp., Proteus vulgaris, Citrobacter spp.  Gram-negative anaerobes Second-generation spectrum  Pseudomonas aeruginosa, Neisseria gonorrhoeae including -lactamase–producing strains Third-generation spectrum  extended Gram-negative coverage including Citrobacter spp. and Enterobacter spp. resistant to third-generation cephalosporins ■ Section III ■ Chapter 43 List of cephalosporins used in clinical practice First generation Chapter 9 Section I 62 First-generation cephalosporins are active mainly against Gram-positive cocci. Second-, third-, and fourth-generation cephalosporins are active primarily against Gram-negative bacilli. The important cephalosporins are listed in Table 9-1. Carbapenems (such as imipenem) and monobactams (such as aztreonam) are other examples of -lactam antibiotics, but these are structurally different from penicillins and cephalosporins. ◗ Vancomycin Vancomycin is a glycopeptide, but its mode of action is very similar to that of -lactam antibiotics, such as penicillins and cephalosporins. It kills bacteria by inhibiting their cell wall synthesis. Vancomycin is a bactericidal antibiotic and is used: ■ ■ ■ ◗ Most widely against Clostridium spp. and Staphylococcus spp. infections; Orally for treatment of antibiotics-associated colitis; and Also for treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. Teicoplanin Teicoplanin is a glycopeptide antibiotic extracted from Actinoplanes teichomyceticus, with a similar spectrum of activity to vancomycin. Its mechanism of action is to inhibit bacterial cell wall synthesis. Teicoplanin is used in: ■ ■ Prophylaxis and treatment of serious infections caused by Gram-positive bacteria, including MRSA and Enterococcus faecalis. Treatment of pseudomembranous colitis and Clostridium difficile-associated diarrhea, with comparable efficacy with vancomycin. Its strength is considered to be due to the length of the hydrocarbon chain. Inhibition of Protein Synthesis Bacteria have 30S and 50S ribosomal units, whereas mammalian cells have 80S ribosomes. The subunits of each type of ribosome, their chemical composition, and their functional specificities are sufficiently different, which explains why these antimicrobial drugs can inhibit protein synthesis in bacterial ribosomes without having a major effect on mammalian ribosomes. Aminoglycosides and tetracyclines act at the level of 30S ribosomal subunits, whereas erythromycins, chloramphenicol, and clindamycins act at the level of 50S ribosomal subunits (Fig. 9-1). ◗ Aminoglycosides Aminoglycosides are a family of drugs that include streptomycin, gentamicin, tobramycin, amikacin, and neomycin, which are increasingly used in different clinical conditions. They kill bacteria by inhibiting protein synthesis. They do so by binding to the 30S subunit ribosome, which blocks the initiation complex, leading to no formation of peptide bonds or polysomes. They act mostly against Gram-negative bacteria. ◗ Tetracyclines Tetracyclines are a family of drugs that include dimethylchlorotetracyclines, doxycycline, and rolitetracycline. They are bacteriostatic drugs. They act by inhibiting protein synthesis of the bacteria by blocking the binding of aminoacyl t-RNA to the 30S ribosomal subunits. Therefore, they prevent introduction of new amino acids to the nascent peptide chain. The action is usually inhibitory and reversible upon withdrawal of the drug. They are variably effective in infections caused by both cocci and bacilli. They are also highly effective against rickettsial and chlamydial infections. Doxycycline, dimethylchlorotetracycline, and rolitetracycline are the examples of semisynthetic tetracyclines. ◗ Macrolides Macrolides (such as erythromycin, azithromycin, clarithromycin, and roxithromycin) have a macrolide ring to which sugars are attached. They are bacteriostatic antibiotics. They act by inhibiting protein synthesis of the bacteria by blocking the release of the t-RNA after it has transferred its amino acids to the growing polypeptide. Erythromycin is effective against Grampositive cocci including MRSA, Neisseria spp., and Haemophilus influenzae. ANTIMICROBIAL AGENTS: THERAPY AND RESISTANCE 30 Ribosome 50 mRNA Metabolites RNA synthesis Rifampin Rifabutin 50 Protein synthesis (50S ribosome) Chloramphenicol Macrolides Streptogramins Schematic diagram showing various mechanisms of actions of antibiotics. Chloramphenicol Clindamycin Trimethoprim (3,4,5-trimethoxybenzylpyrimidine) is a bacteriostatic drug active against both Gram-positive and Gramnegative organisms. The compound inhibits the enzyme dihydrofolic acid reductase 50,000 times more efficiently in bacteria than in mammalian cells. This enzyme reduces dihydrofolic to tetrahydrofolic acid, leading to decreased synthesis of purines and ultimately of DNA. Sulfonamides and trimethoprim inhibit the synthesis of tetrahydrofolic acid, the main donor of the methyl groups that are essential to synthesize adenine, guanine, and cytosine. The mode of action of clindamycin is similar to that of erythromycin. It inhibits bacterial protein synthesis by blocking the release of t-RNA after it has transferred its amino acids to the growing polypeptide. Key Points ■ ■ Inhibition of Nucleic Acid Synthesis Sulfonamides, trimethoprim, quinolones, and rifampin are examples of drugs that act by inhibition of nucleic acid synthesis. ◗ Sulfonamides and trimethoprim, each can be used alone to inhibit bacterial growth. If used together, they produce sequential blocking, resulting in a marked enhancement (synergism) of activity. Such mixtures of sulfonamide (five parts) plus trimethoprim (one part) have been used in the treatment of pneumocystis pneumonia, malaria, shigella enteritis, systemic salmonella infections, urinary tract infections, and many others. Sulfonamides and trimethoprim Sulfonamides and trimethoprim inhibit nucleic acid synthesis by inhibiting nucleotide synthesis. Sulfonamides are structural analogs of para-aminobenzoic acid (PABA). Due to structural similarity to PABA, sulfonamide competes with the latter during bacterial metabolism. Sulfonamides enter into the reaction in place of PABA and compete for the active center of the enzyme. As a result, nonfunctional analogs of folic acid are formed, preventing further growth of the bacterial cell. The inhibitory action of sulfonamides on bacterial growth can be counteracted by an excess of PABA in the environment (competitive inhibition). Many other bacteria, however, synthesize folic acid, as mentioned above, and consequently are susceptible to action by sulfonamides. Sulfonamides are bacteriostatic drugs effective against a variety of Gram-negative and Gram-positive bacteria. ◗ Quinolones Quinolones are synthetic analogs of nalidixic acid. They are a family of drugs that include ciprofloxacin, ofloxacin, and levofloxacin. They are bactericidal and act by inhibiting bacterial DNA synthesis by blocking DNA gyrase. DNA gyrase is the enzyme that unwinds DNA strands, so they can be replicated. Quinolones are effective against both Gram-positive and Gram-negative organisms. ◗ Rifampin Rifampin inhibits bacterial growth by binding strongly to the DNAdependent RNA polymerase of bacteria. Thus, it inhibits bacterial RNA synthesis. The mechanism of rifampin action on viruses is different. It blocks a late stage in the assembly of poxviruses. Chapter 43 Chloramphenicol is mainly a bacteriostatic agent, and growth of bacteria resumes when the drug is withdrawn. It acts by binding to the 50S subunit of the ribosome and blocking peptidyl transferase, the enzyme that delivers the amino acid to the growing polypeptide, resulting in the inhibition of bacterial protein synthesis. Chloramphenicol is used less frequently nowadays. ◗ Antimetabolites Sulfonamides Dapsone Trimethoprim Para-aminosalicylic acid Section III ◗ 50 50 50 Protein synthesis (30S ribosome) Aminoglycosides Tetracyclines Oxazolidinone FIG. 9-1. 30 30 30 30 Chapter 9 Cell wall synthesis Beta-lactams Vancomycin Isoniazid Ethambutol Cycloserine Ethionamide Bacitracin Polymyxin DNA replication Quinolones Metronidazole Section I DNA 63 64 GENERAL MICROBIOLOGY Chapter 9 Section I Alteration of Cell Membrane Function The cytoplasm of all living cells is surrounded by the cytoplasmic membrane, which serves as a selective permeability barrier. The cytoplasmic membrane carries out active transport functions, and thus controls the internal composition of the cell. If the functional integrity of the cytoplasmic membrane is disrupted, macromolecules and ions escape from the cell, and cell damage or death ensues. The cytoplasmic membrane of bacteria and fungi has a structure different from that of animal cells and can be more readily disrupted by certain agents. Consequently, selective chemotherapy is possible. Antifungal drugs act by altering the cell membrane function of the fungi. They show selective toxicity because cell membrane of the fungi contains ergosterol, while human cell membrane has cholesterol. Bacteria with the exception of Mycoplasma do not have sterols in their cell membranes, hence are resistant to action of these drugs. Chapter 43 Section III Key Points ■ ■ Amphotericin B and azoles are the frequently used antifungal drugs. Amphotericin B acts against fungi by disrupting the cell membrane by binding at the site of ergosterol in the membrane. Azoles (such as ketoconazole, fluconazole, itraconazole, and clotrimazole) inhibit synthesis of ergosterol, hence are toxic to fungi. Polymyxin B is an antibiotic primarily used for resistant Gram-negative infections. It is derived from the bacterium Bacillus polymyxa. Polymyxin B is a mixture of two closely related compounds—polymyxin B1 and polymyxin B2. It has a bactericidal action against almost all Gram-negative bacilli except the Proteus group. Polymyxins bind to the cell membrane and alter its structure, making it more permeable. The resulting water uptake leads to cell death. They are cationic, basic proteins that act like detergents (surfactants). Side-effects include neurotoxicity and acute renal tubular necrosis. It is commonly used in the topical first-aid preparation. Cellular target sites of antimicrobial drugs are listed in Table 9-2. TABLE 9-2 Cellular target sites of antibiotics Drug Target site Mechanism of action -lactams Cell wall Bactericidal, interfere with crosslinking of cell wall peptidoglycan molecules Erythromycin, fusidic acid, tetracycline Ribosomes Bacteriostatic or bacteriocidal, interfere with translocation and attachment of t-RNA, thus inhibiting protein synthesis Polyenes Cytoplasmic membrane Bacteriostatic, disrupt yeast cell membrane Metronidazole, idoxuridine, acyclovir Nucleic acid replication Bactericidal, interfere with DNA replication Resistance to Antimicrobial Drugs Bacterial resistance to drugs is a condition in which the bacteria that were earlier susceptible to antibiotics develop resistance against the same antibiotics and are not susceptible to the action of these antibiotics. Antibiotic resistance among bacteria is a major concern in the treatment of a patient. Emergence of antibiotic resistance to the old as well as new antibiotics by bacteria is posing a major challenge in the treatment of infections caused by bacteria. Antibiotic resistance is seen more commonly in hospitalacquired infections than in community-acquired infections. The antibiotic-resistant bacteria are more commonly seen in hospital environment due to widespread use of antibiotics in hospitals that select for these bacteria. These hospital strains of bacteria are characterized by developing resistance to multiple antibiotics at the same time. Common examples of such strains of bacteria showing drug resistance include hospital strains of S. aureus and Gram-negative enteric bacteria, such as Escherichia coli and Pseudomonas aeruginosa. Resistance to multiple antibiotics is mediated by plasmid-carrying several genes that encode enzymes responsible for the resistance. Mechanisms of Antibiotic Resistance There are many different mechanisms by which microorganisms might exhibit resistance to drugs (Fig. 9-2). These are (a) production of enzymes, (b) production of altered enzymes, (c) synthesis of modified targets, (d) alteration of permeability of cell wall, (e) alteration of metabolic pathways, and (f) efflux pump as follows: ◗ Production of enzymes Bacteria produce enzymes that inactivate antibiotics. For example, penicillin-resistant staphylococci produce an enzyme -lactamase that destroys the penicillins and cephalosporins by splitting the -lactam ring of the drug. Gram-negative bacteria resistant to aminoglycosides, mediated by a plasmid, produce adenylating, phosphorylating, or acetylating enzymes that destroy the drug. ◗ Production of altered enzymes Certain microorganisms develop an altered enzyme that can still perform its metabolic function, but is much less affected by the drug. For example, in trimethoprim-resistant bacteria, the dihydrofolic acid reductase is inhibited far less efficiently than in trimethoprim-susceptible bacteria. ◗ Synthesis of modified targets Certain bacteria produce modified targets against which the antibiotic has no effect. For example, a methylated 23S ribosomal RNA can result in resistance to erythromycin, and a mutant protein in the 50S ribosomal subunits can result in resistance to streptomycin. Penicillin resistance in S. pneumoniae and enterococci is caused by the loss or alteration of PBPs. ANTIMICROBIAL AGENTS: THERAPY AND RESISTANCE Antibiotic sensitive cells Section I Antibiotic target 65 Altered target Antibiotic Inactivated antibiotic Antibiotic resistant cells Chapter 9 Decreased permeability FIG. 9-2. ◗ Alteration of target Schematic diagram showing the mechanisms of drug resistance in antibiotics. Alteration of permeability of cell wall Nongenetic Basis of Resistance Nongenetic basis of resistance plays a less important role in the development of drug resistance: Alteration of metabolic pathways Bacteria may develop resistance by altering metabolic pathway that bypasses the reaction inhibited by the drug. For example, certain sulfonamide-resistant bacteria do not require extracellular PABA but, like mammalian cells, can utilize preformed folic acid. ◗ Efflux pumps Efflux pumps have been found to be responsible for conferring resistance to many groups of antibiotics including aminoglycosides, quinolones, etc. The major family of bacterial efflux pumps include ABC (ATP-binding cassette) multidrug efflux pump, multidrug resistance and toxic compound extrusion (MATE) efflux pumps, major facilitator superfamily efflux (MFSE) pumps, etc. Genetic Basis of Drug Resistance The genetic basis of drug resistance, mediated by genetic change in bacteria, is most important in the development of drug resistance in bacteria. This is of three types as follows: (a) chromosome-mediated resistance, (b) plasmid-mediated resistance, and (c) transposons-mediated resistance. ◗ Basis of Resistance Resistance by bacteria against antibiotic may be classified as: 1. Nongenetic basis 2. Genetic basis Chromosome-mediated resistance Chromosome-mediated resistance occurs as a result of spontaneous mutation. This is caused by mutation in the gene that codes for either the target of drug or the transport system in the membrane of the cell wall, which controls the entry of drugs into cells. The frequency of chromosomal mutation is much less than the plasmid-mediated resistance. It varies between 107 and 109. Chapter 43 1. Certain bacteria under ordinary circumstances are usually killed by penicillins. But these bacteria, if lose their cell wall and become protoplast, become nonsusceptible to the action of cell wall–acting drug such as penicillins. 2. In certain conditions, such as in the abscess cavity, bacteria can be walled off, which prevents drugs to penetrate effectively into bacteria. Surgical drainage of pus, however, makes these bacteria again susceptible to the action of antibiotics. 3. Presence of foreign bodies (such as surgical implants and catheters) and penetration injury caused by splinters and sharpeners make successful antibiotic treatment more difficult. 4. Nonreplicating bacteria in their resting stage are less sensitive to the action of cell wall inhibitors such as penicillin and cephalosporins. This is particularly true for certain bacteria such as Mycobacterium tuberculosis that remains in resting stage in tissues for many years, during which it is insensitive to drugs. However, when these bacteria begin to multiply, they become susceptible to antibiotics. Section III Some bacteria develop resistance to antibiotic by changing their permeability to the drug in such a way that an effective intracellular concentration of the antibiotic is not achieved inside the bacterial cell. For example, P. aeruginosa develops resistance against tetracyclines by changing its porins that can reduce the amount of tetracycline entering the bacteria, thereby developing resistance to the antibiotics. Resistance to polymyxins is also associated with a change in permeability to the drugs. Streptococci have a natural permeability barrier to aminoglycosides. This can be partly overcome by the simultaneous presence of a cell wall–active drug, e.g., penicillin. ◗ Inactivation of antibiotic 66 GENERAL MICROBIOLOGY Section I Key Points Mutational resistance in M. tuberculosis is of much importance in the treatment of tuberculosis: ■ Chapter 9 ■ Section III ◗ Chapter 43 TABLE 9-4 Treatment of tuberculosis with two or more antitubercular drugs is carried out to prevent emergence of multidrug resistance in the tuberculosis. This is based on the principle that it is most unlikely that the bacterium, M. tuberculosis, becomes concurrently resistant to all the antibiotics given simultaneously. There is less likely resistant mutation because on treatment with antitubercular drugs, a mutant of M. tuberculosis resistant to one drug, if appears, will be destroyed by the other drug. Plasmid-mediated resistance Plasmid-mediated drug resistance in bacteria occurs by transfer of plasmid and genetic materials. It is mediated by resistance plasmid, otherwise known as R factor. R factors: These are circular, double-stranded DNA molecules that carry the genes responsible for resistance against variety of antibiotics. These factors may carry one or even two or more resistant genes. The genes encode for a variety of enzymes that destroy the antibiotics by degrading antibiotics or modify membrane transport system. For example, the genes code for enzymes like -lactamases that destroy -lactam ring (which is responsible for the antibactericidal action of -lactam antibiotics, such as penicillins and cephalosporins). Table 9-3 lists different mechanisms of plasmid-mediated resistance in bacteria. Key Points Plasmid-mediated resistance plays a very important role in antibiotics usage in clinical practice. This is because: ■ ■ ■ ◗ A high rate of transfer of plasmids from one bacterium to another bacterium takes place by conjugation, Plasmids mediate resistance to multiple antibiotics, and Plasmid-mediated resistance occurs mostly in Gramnegative bacteria. Transposons-mediated drug resistance Drug resistance is also mediated by transposons that often carry the drug resistance genes. Transposons are small pieces of DNA TABLE 9-3 Plasmid-mediated antibiotic resistance Differences between mutational and transferable drug resistance Mutational drug resistance Transferable drug resistance Chromosome mediated Plasmid mediated Resistance to one drug Resistance to multiple drugs Resistance nontransferable Resistance transferable Virulence of organism lowered Virulence of organism not lowered Low-degree resistance High-degree resistance Due to decreased permeability, development of alternate metabolic pathway or inactivation of drug Due to production of many degrading enzymes that move from one site of the bacterial chromosome to another and from bacterial chromosome to plasmid DNA. Many R factors carry one or more transposons. Differences between chromosomal and transferable drug resistance is listed in Table 9-4. Specific Mechanisms of Resistance ◗ Penicillins and cephalosporins Resistance to penicillin is mainly mediated by three mechanisms: (a) production of penicillin-destroying enzymes, (b) mutation genes coding for PBP, and (c) reduced permeability to drug. 1. Production of penicillin-destroying enzymes (␤-lactamases): Resistance to penicillins may be determined by the organism’s production of penicillin-destroying enzymes (-lactamases). -lactamases, such as penicillinases and cephalosporinases, open the -lactam ring of penicillins and cephalosporins and abolish their antimicrobial activity. -lactamases have been described for many species of Gram-positive and Gram-negative bacteria. Some -lactamases are plasmid-mediated (e.g., penicillinase of S. aureus), while others are chromosomally mediated (e.g., many species of Gram-negative bacteria such as Enterobacter spp., Citrobacter spp., Pseudomonas spp., etc). There is one group of -lactamases that is occasionally found in certain species of Gram-negative bacilli, usually Klebsiella pneumoniae and E. coli. These enzymes are termed extended-spectrum ␤-lactamases because they confer upon bacteria an additional ability to hydrolyze the -lactam rings of cefotaxime, ceftazidime, or aztreonam. 2. Mutation in genes coding for PBP: This form of resistance occurs due to the absence of some penicillin receptors (PBP) and occurs as a result of chromosomal mutation. This mechanism is responsible for both low-level and high-level resistance seen in S. pneumoniae to penicillin G and in S. aureus to nafcillin. 3. Reduced permeability to drug: Low-level resistance of Neisseria gonorrhoeae to penicillin is caused by poor permeability of the drug. However, high-level resistance is mediated by a plasmid coding for penicillinase. Cephalosporins are resistant to -lactamases in varying degrees. Antibiotic Mechanism of resistance ␤-lactams -lactamases break down the -lactam ring to an inactive form Aminoglycosides Aminoglycosides modifying enzymes: acetyltransferases, phosphotransferases, and nucleotidyltransferases Erythromycin and clindamycin Induced enzymatic activity due to methylating ribosomal RNA Chloramphenicol Acetylation of the antibiotic to an inactive form ◗ Tetracycline Alteration of cell membrane, decreases permeability to the antibiotic Resistance to vancomycin is mediated by change in D-ALA-DALA part of peptide in the peptidoglycan to D-ALA-D-lactate. Vancomycin ANTIMICROBIAL AGENTS: THERAPY AND RESISTANCE 1. Plasmid-dependent resistance to aminoglycosides enzymes is the most important mechanism. It depends on the production of plasmid-mediated phosphorylating, adenylating, and acetylating enzymes that destroy the drugs. 2. Chromosomal resistance of microbes to aminoglycosides is the second mechanism. Chromosomal mutation in genes results in the lack of a specific protein receptor on the 30S subunit of the ribosome, essential for binding of drug. 3. A “permeability defect,” is the third mechanism of resistance. This leads to an outer membrane change that reduces active transport of the aminoglycoside into the cell so that the drug cannot reach the ribosome. Often this is plasmid-mediated. ◗ Tetracyclines ◗ Macrolides Resistance to macrolides, such as erythromycin, is caused by a plasmid-encoded enzyme that methylates the 23S ribosomal RNA, thereby blocking binding of the drug. ◗ Sulfonamides and Trimethoprim Resistance to sulfonamide is caused by plasmid-mediated transport system that actively exports the drug out of bacteria. It is also caused by chromosomal mutation in the gene that codes for the target enzyme dihydrofolate synthetase, resulting in reduced binding affinity of the drug. Resistance to trimethoprim is caused by a chromosomal mutation in the gene coding for dihydrofolate reductase, the enzyme that reduces dihydrofolate to tetrahydrofolate. ◗ Quinolones Resistance to quinolone occurs mainly due to chromosomal mutations that modify the bacterial DNA gyrase. Resistance is also caused by changes in the outer membrane proteins of the bacteria, which results in reduced uptake of drug into bacteria. ◗ Metronidazole Metronidazole is a bactericidal drug that acts by inhibiting DNA synthesis. It is effective against anaerobes and protozoa. Chapter 43 Resistance to tetracyclines occurs by three mechanisms: (a) efflux, (b) ribosomal protection, and (c) chemical modification. The first two are the most important. Efflux pumps, located in the bacterial cell cytoplasmic membrane, are responsible for pumping the drug out of the cell. Tet gene products are responsible for protecting the ribosome, possibly through mechanisms that induce conformational changes. These conformational changes either prevent binding of the tetracyclines or cause their dissociation from the ribosome. This is often plasmid controlled. Section III Vancomycin-resistant Staphylococcus aureus (VRSA): In 1997, the first clinical isolate of S. aureus with diminished susceptibility to vancomycin (strain Mu50) was described in Japan. This strain displayed a vancomycin MIC of 8 g/mL, which is in the range of intermediate susceptibility (4–8 g/mL) as per the current Clinical and Laboratory Standards Institute (CLSI) breakpoints, and thus was referred to as vancomycin-intermediate S. aureus (VISA) or glycopeptide-intermediate S. aureus (GISA). This initial report was followed by others from various countries. Precursors of these VISA isolates are the heteroresistant-VISA (hVISA or hGISA) with subpopulations of cells that are able to grow at a vancomycin concentration of 8 g/mL. As of late 2008, nine clinical isolates of vancomycin-resistant S. aureus (VRSA) harboring the enterococcal vanA gene have been described in the United States, with MICs ranging from 32 g/mL to 1024 g/mL. Aminoglycosides Chapter 9 Key Points ◗ Resistance to aminoglycosides is mediated by three important mechanisms as follows: Section I This results in the inability of vancomycin to bind to the bacteria. Vancomycin resistance in Enterococcus spp. is been increasingly documented in different clinical conditions. Vancomycin-resistant enterococci (VRE): VRE were first detected in Europe (United Kingdom and France) in 1986 and soon after, a Van B Enterococcus faecalis clinical isolate was reported in the United States. These have now been reported from Australia, Belgium, Canada, Denmark, Germany, Italy, Malaysia, Netherland, Spain and Sweden. But the incidence of human VRE infections in European countries is relatively low (1–3%) compared with the high and rising rate in the US. Seven types of glycopeptide resistance have been described among enterococci (VanA, VanB, VanC, VanD, VanE, VanG, and VanL), which are named based on their specific ligase genes (e.g.,vanA, vanB, etc.). Related gene clusters have been found in non-pathogenic organisms. The common endpoint of these phenotypes is the formation of a peptidoglycan precursor with decreased affinity for glycopeptides, resulting in decreased inhibition of peptidoglycan synthesis. Peptidoglycan precursors ending in the depsipeptide d-alanyl-d-lactate are produced in VanA, VanB, and VanD strains, whereas VanC, VanE, and VanL (recently described in an E. faecalis strain) isolates produce precursors terminating in d-alanyl-d-serine, instead of the normally occurring d-alanyl-d-alanine. The vanA gene cluster was originally detected in the Tn1546 transposon, and this or related genetic elements are usually carried by plamids and occasionally by host chromosome; this plamid-borne transposon also has been found in clinical isolates of S. aureus (vancomycin-resistant S. aureus strains). Glycopeptide resistance in enterococci is classified as either intrinsic (as a species characteristic) or acquired. The former is a characteristic of the motile species Enterococcus gallinarum and Enterococcus casseliflavus/flavescens, members of which all carry the naturally occurring vanC-1 and vanC-2/vanC-3 genes, respectively. These enterococci show variable MICs of vancomycin, with many falling in the susceptible range, and clinical failures have been reported following vancomycin use. In general, the isolation of these species does not require strict infection control isolation procedures, unless they are highly resistant, suggesting the added presence of potentially transferable vanA or vanB genes. 67 68 Chapter 9 Section I ◗ ◗ Rifampin Rifampin resistance results from a change in RNA polymerase due to a chromosomal mutation that occurs with high frequency. ◗ Antitubercular drugs Antitubercular drugs include isoniazid, ethambutol, rifampicin, pyrazinamide, and streptomycin. ■ Section III Chloramphenicol Microorganisms resistant to chloramphenicol produce the enzyme chloramphenicol acetyltransferase that destroys drug activity. Production of this enzyme is usually mediated by a plasmid. ■ Chapter 43 GENERAL MICROBIOLOGY ■ ■ Isoniazid is a bacteriostatic agent. It penetrates well into tissue, fluid, and also acts on intracellular organisms. Resistance to isoniazid is mainly due to loss of enzyme catalase that activates isoniazid to active metabolites that inhibit synthesis of mycolic acid. Ethambutol acts by interfering RNA metabolism. The bacteria develop resistance due to mutation in the gene coding for arabinosyl transferase, which synthesizes arabinogalactan in the mycobacterial cell wall. Rifampin inhibits RNA synthesis. The bacteria develop resistance due to mutation in the gene coding for DNAdependent RNA polymerases. Pyrazinamide resistance is due to mutation of gene coding for bacterial amidase, which converts it to its active form, pyrazinoic acid. Combination therapy in tuberculosis is, therefore, essential to prevent emergence of drug resistance. ◗ Carbapenems Production of carbapenemases including NDM metallobetalactamase: New Delhi metallo-beta-lactamase-1 (NDM-1) is an enzyme that makes bacteria resistant to a broad range of beta-lactam antibiotics. These include the antibiotics of carbapenem family, which are a mainstay for the treatment of antibiotic-resistant bacterial infections. The gene for NDM-1 is one member of a large gene family that encodes beta-lactamase enzymes called carbapenemases. Bacteria that produce carbapenemases are often referred to in the news media as “superbugs” because infections caused by them are difficult to treat. Such bacteria are usually susceptible only to polymyxins and tigecycline. NDM-1 was first detected in a K. pneumoniae isolate from a Swedish patient of Indian origin in 2008. It was later detected in bacteria in India, Pakistan, the United Kingdom, the United States, Canada, Japan, and Brazil. The most common bacteria that make this enzyme are Gram-negative such as E. coli and K. pneumoniae, but the gene for NDM-1 can spread from one strain of bacteria to another by horizontal gene transfer. The NDM-1 enzyme was named after New Delhi, the capital city of India, as it was first described in December 2009 in a Swedish national who fell ill with an antibiotic-resistant bacterial infection that he acquired in India. The infection was unsuccessfully treated in a New Delhi hospital, and, after the patient’s repatriation to Sweden, a carbapenem-resistant K. pneumoniae strain bearing the novel gene was identified. It was concluded that the new resistance mechanism “clearly arose in India, but there are few data arising from India to suggest how widespread it is.” Its exact geographical origin, however, has not been conclusively verified. In March 2010, a study in a hospital in Mumbai found that most carbapenem-resistant bacteria isolated from patients carried the blaNDM-1 gene. In May 2010, a case of infection with E. coli expressing NDM-1 was reported in Coventry in the United Kingdom. The patient was a man of Indian origin who had visited India 18 months previously, where he had undergone dialysis. In initial assays, the bacterium was fully resistant to all antibiotics tested, while later tests found that it was susceptible to tigecycline and colistin. It is believed that international travel and patients’ use of multiple countries’ healthcare systems could lead to the “rapid spread of NDM-1 with potentially serious consequences”. Antibiotic Sensitivity Testing Antibiotic sensitivity testing is carried out to determine the appropriate antibiotic agent to be used for a particular bacterial strain isolated from clinical specimens. Antibiotic sensitivity testing can be carried out by two broad methods, as follows: 1. Disc diffusion tests 2. Dilution tests Disc Diffusion Tests Disc diffusion tests are the most commonly used methods in a laboratory to determine susceptibility of bacteria isolates to antibiotics. In this method, as the name suggests, discs impregnated with known concentrations of antibiotics are placed on agar plate that has been inoculated with a culture of the bacterium to be tested. The plate is incubated at 37°C for 18–24 hours. After diffusion, the concentration of antibiotic usually remains higher near the site of antibiotic disc, but decreases with distance. Susceptibility to the particular antibiotic is determined by measuring the zone of inhibition of bacterial growth around the disc (Fig. 9-3). ◗ Selection of media The medium that supports both test and control strains is selected for carrying out antibiotic susceptibility testing of the bacteria. For example, Mueller–Hinton agar is used for testing Gram-negative bacilli and Staphylococcus spp., blood agar for Streptococcus spp. and Enterococcus spp. species, chocolate agar for Haemophilus influenzae, and Wellcotest medium for sulfonamides and cotrimoxazole. The medium is prepared by pouring onto the flat horizontal surface of Petri dishes of 100 mm to a depth of 4 mm. The pH of the medium is maintained at 7.2–7.4. More alkaline pH increases the activity of tetracyclines, novobiocin, and fusidic acid, whereas an acidic pH reduced the activity of aminoglycosides and macrolides, such as erythromycin. The plates after preparation may be stored at 4°C for up to 1 week. ANTIMICROBIAL AGENTS: THERAPY AND RESISTANCE 69 Section I Different antibiotic disc Organism to be cultured Chapter 9 Incubate for 18–24 hours at 37°C (antibiotic will diffuse in agar radially) Sterile nutrient agar No zone of inhibition of growth (organism is resistant to antimicrobial agent) Large zone of inhibition of growth (organism is sensitive to antimicrobial agent) FIG. 9-3. ◗ Schematic diagram showing the performance of antibiotics sensitivity testing by disc diffusion method. Preparation of the inoculum Antibiotic discs Only the clinically relevant antibiotics are tested in antibiotic susceptibility tests. Antibiotic discs (6-mm filter paper discs) can be prepared from pure antimicrobial agents in laboratories or can be obtained commercially. The discs are applied with sterile forceps, a sharp needle, or a dispenser onto the surface of the medium, streaked with test strains, and the reading is reported after incubating the plate for 18–24 hours at 37°C aerobically. ◗ Types of disc diffusion tests Disc diffusion tests are of the following types: 1. Kirby–Bauer disc diffusion method 2. Stokes disc diffusion method 3. Primary disc diffusion test Kirby–Bauer disc diffusion method: Kirby–Bauer disc diffusion method is the most common method used routinely for determination of antibiotic sensitivity of bacteria isolated from clinical specimens. In this method, both the test strains and the control strains are tested in separate plates. The test is performed by inoculating the test organism in a suitable broth solution, followed by incubation at 37°C for 2–4 hours. Then 0.1 mL of the broth is inoculated on the surface Antibiotic disc A Cf Ak G Zone of inhibition Ca Cl FIG. 9-4. Schematic diagram showing Kirby-Bauer disc diffusion method of antibiotic sensitivity. Chapter 43 ◗ Section III For testing antibiotic sensitivity, the bacteria are first isolated in pure culture on a solid medium. At least three to four morphologically similar colonies of the bacteria to be tested are touched and inoculated into appropriate broth and incubated at 37°C for 4–6 hours. The density of bacterial suspension in the broth is adjusted to 1.5  108 cfu/mL by comparing its turbidity with that of 0.5 McFarland opacity standard tube. The broth is inoculated on the medium by streaking with sterile swabs. A sterile cotton swab is dipped into the broth and excess broth is removed by rotation of the swab against the sides of the tube above the fluid level. of the agar medium by streaking with a sterile swab. In this method, either nutrient agar or Mueller–Hinton agar in Petri dishes is used. The inoculated medium is incubated overnight at 37°C. The susceptibility of drug is determined from the zones of inhibition of bacterial growth surrounding the antibiotic discs. The diameters of the zone of inhibition are calculated with vernier calipers or a thin transparent millimeter scale to the nearest millimeter. The point of abrupt diminution of the zone is considered as the zone edge. A maximum of six antibiotic discs are tested in a Petri dish of 85 mm size (Fig. 9-4). Interpretation of the zone size is done as per the interpretation chart. Depending on the zone size, bacteria can be considered sensitive, intermediate or resistant to antibiotics. Stokes method: This is a disc diffusion method, which makes use of inbuilt controls against many variables. In this method, the Petri dish containing the Mueller–Hinton agar is divided horizontally into three parts. The test strain is inoculated in the central area and the control strains on the upper and lower third of the plate. In modified Stokes method, control strain is inoculated in the central part but test strains are inoculated on the upper and lower third of the plate. The plates are incubated at 37°C and observed for zones of bacterial inhibition around the discs (Fig. 9-5). A maximum of six antibiotic discs can be applied on a 100 mm Petri dish. 70 GENERAL MICROBIOLOGY Section I Control strain Zone of inhibition Test strain FIG. 9-5. Cl G A Cl Chapter 9 Test strain Section III Chapter 43 ■ ■ Control strain Control strain Test strain Stokes disc diffusion method Modified Stokes disc diffusion method Schematic diagram showing Stokes method of antibiotic sensitivity. Sensitive (S): The zone of test bacterium is equal to or more than that of control strain. The differences between the zone sizes of control and test strains should not be more than 3 mm if the zone size of the test bacterium is smaller than that of control. Intermediate (I): The zone size of the test bacterium should be at least 2 mm, and the differences between the zone of test and control strain should be at least 3 mm. Resistant (R): The zone size of the test bacterium is 2 mm or less. edge of the zone and without any gradual fading away of the growth of the bacteria toward the disc. In such condition, the zones of inhibition should not be considered, and it should be reported resistant irrespective of the zone size. 4. Trimethoprim and sulfamethoxazole should be tested separately to know whether the bacterium is sensitive to both or only to one of these. These should never be tested in combination, the way these two drugs are used in combination in clinical practice. Key Points Primary disc diffusion test Primary disc diffusion test is a method carried out directly on clinical specimens unlike Kirby–Bauer or Stokes diffusion method that are performed on pure cultures of bacterial isolates from clinical specimens. In this method, the clinical specimen (e.g., urine) is inoculated uniformly on the surfaces of the agar to which antibiotic discs are applied directly. The plate is incubated overnight at 37°C for demonstration of zones of inhibition. This method is useful to know the antibiotic sensitivity results urgently, but results of this primary disc diffusion test should always be confirmed by testing the isolates subsequently by Kirby–Bauer or Stokes diffusion method. Interpretation of disc diffusion tests: Results of disc diffusion tests, such as Kirby–Bauer and Stokes method, are interpreted as follows: ■ ■ ■ G Cf Cf Reporting of the result is carried out by comparing the zones of inhibition of test and control bacteria. The zone sizes are measured from the edge of the disc to edge of the zone. It is interpreted as follows: ■ A Sensitive (S): Infection treatable by the normal dosage of the antibiotic. Intermediate (I): Infection may respond to higher dosage. Resistant (R): Unlikely to respond to usual dosage of the antibiotics. However, for certain bacteria and antibiotic discs, the following may be kept in mind while interpreting results of disc diffusion tests: 1. Proteus mirabilis, Proteus vulgaris, and other bacteria producing swarming produce a thin film on agar surface often extending into the zones of inhibition. In such situations, the zones of swarming should be ignored and the outer clear margin should be measured to determine the zones of inhibition. 2. Many strains of MRSA grow very slowly in the presence of methicillin. They produce growth within the zone of inhibition on incubation for more than 48 hours. This problem can be overcome by incubating the bacteria at 30°C or by using 5% salt agar and incubating at 37°C. 3. Penicillinase producing strains of Staphylococcus often fail to secrete enough enzymes to neutralize penicillin close to the antibiotic disc. In such situation, it may show a zone of inhibition, but with the presence of large colonies at the Dilution Tests Dilution tests are performed to determine the minimum inhibitory concentration (MIC) of an antimicrobial agent. MIC is defined as the lowest concentration of an antimicrobial agent that inhibits the growth of organisms. Estimation of the MIC is useful to: ■ ■ Regulate the therapeutic dose of the antibiotic accurately in the treatment of many life-threatening situations, such as bacterial endocarditis. Test antimicrobial sensitivity patterns of slow-growing bacteria, such as M. tuberculosis. Following methods are carried out to determine the MIC: 1. Broth dilution method 2. Agar dilution method 3. Epsilometer test (E-test) ANTIMICROBIAL AGENTS: THERAPY AND RESISTANCE ◗ Broth dilution method Agar dilution method is useful: ■ ■ To test organisms from serious infections like bacterial endocarditis or To verify equivocal results of disc diffusion test. ◗ Epsilometer test (E-test). Epsilometer test (E test) Epsilometer test (E test), based on the principle of disc diffusion, is an automated system for measuring MIC of a bacterial isolate. In this method, an absorbent plastic strip with a continuous gradient of antibiotic is immobilized on one side. MIC interpretative scale corresponding to 15 twofold MIC dilutions is used on the other side. The strip is placed on the agar plate inoculated with the test organism with the MIC scale facing toward the opening side of the plate. An elliptical zone of growth inhibition is seen around the strip after incubation at 37°C overnight. The MIC is read from the scale at the intersection of the zone with the strip. The end point is always read at complete inhibition of all growth including hazes and isolated colonies. E test is a very useful test for easy interpretation of the MIC of an antibiotic (Fig. 9-6). Antibacterial Assays in Body Fluids Mueller–Hinton agar is used in this method. Serial dilution of the antibiotic are made in agar and poured onto Petri dishes. Dilutions are made in distilled water and added to the agar that has been melted and cooled to not more than 60°C. One control plate is inoculated without antibiotics. Organism to be tested is inoculated and incubated overnight at 37°C. Plates are examined for presence or absence of growth of the bacteria. The concentration at which bacterial growth is completely inhibited is considered as the MIC of the antibiotic. The organisms are reported sensitive, intermediate, or resistant by comparing the test MIC values with that given in CLSI guidelines. The main advantage of the method is that a number of organisms can be tested simultaneously on each plate containing an antibiotic solution. Antibacterial assays are carried out to demonstrate the toxic and therapeutic level of the antibiotics in blood and other body fluids. In this method, the MIC of the antibiotic for the test organism is done first by standard broth dilution method. The test with patient’s serum is done concurrently. Serial dilutions of the patients’ serum in nutrient broth are carried out and to each a standard drop of culture is added and incubated for 18 hours. The highest dilution of serum which has inhibited growth of the assay organism is noted. This is equivalent to the MIC of the antibiotic in question. Multiplication of the MIC by the serum dilution gives the concentration of the antibiotic which was present in the patient’s undiluted serum. Chapter 43 Key Points FIG. 9-6. Section III Agar dilution method is a quantitative method for determining the MIC of antimicrobial agent against the test organism. Chapter 9 Agar dilution method Section I The broth dilution method is a quantitative method for determining the MIC of an antimicrobial agent that inhibits the growth of organisms in vitro. In this method, the antimicrobial agent is serially diluted in Mueller–Hinton broth by doubling dilution in tubes and then a standard suspension of the broth culture of test organism is added to each of the antibiotic dilutions and control tube. This is mixed gently and incubated at 37°C for 16–18 hours. An organism of known susceptibility is included as a control. The MIC is recorded by noting the lowest concentration of the drug at which there is no visible growth as demonstrated by the lack of turbidity in the tube. The main advantage of this method is that this is a simple procedure for testing a small number of isolates. The added advantage is that using the same tube, the minimum bactericidal concentration (MBC) of the bacteria can be determined. The MBC is determined by subculturing from each tube, showing no growth on a nutrient agar without any antibiotics. Subcultures are made from each tube showing no growth into the nutrient agar plates without any antibiotics. The plates are examined for growth, if any, after incubation overnight at 37°C. The tube containing the lowest concentration of the drug that fails to show any growth on subculture plate is considered as the MBC of the antibiotic for that strain. Broth dilution may be of two types—macrodilution and microdilution. Broth microdilution is done using microtiter plates and is considered the “gold standard.” ◗ 71 10 Microbial Pathogenesis Introduction Host–parasite relationship is determined by the interaction between host factors and the infecting microorganisms. Outcome of any microbial infection depends on the interaction between the host and the parasite. The relationship of existence between the host and parasite may be (a) symbiosis, (b) commensalism, or (c) disease process: ■ ■ ■ Symbiosis describes a situation where both the microorganisms and host species live together with mutual benefit. There is an element of symbiosis in the relationship between the human host and the gut flora; humans provide the bacteria with a warm, moist environment for their survival and gut flora provides a natural barrier against many invading pathogens. Commensalism is an association in which only the microorganism derives benefit, without causing any injury to the host. Most human microbes are commensals. They are present as bacterial flora of the skin and mucous membranes, including the upper respiratory tract, the lower gastrointestinal tract, and the vagina. Disease is caused by certain microorganisms known as pathogens. Microorganisms vary in their ability to cause disease in humans. Types of Microorganisms Microorganisms may be of the following types: 1. Saprophytes: These are free-living microorganisms that live on dead or decaying organic matter. They are usually present in soil and water. They are generally unable to invade the living body. 2. Parasites: These are microorganisms that live on a living host and derive nutrition from the host without any benefit to the host and causes harm to the infected host. 3. Commensals: These are microorganisms that live on a living host without causing any injury to the host. Most human microbes are commensals. 4. Pathogens: A microorganism capable of causing disease, especially if it causes disease in immunocompetent people, is called as a pathogen. These pathogens, however, represent a very small proportion of the microbial species. 5. Opportunistic pathogens: A microbe that is capable of causing disease only in immunocompromised people is known as opportunistic pathogen. These organisms can cause disease only if one or more of the usual defense mechanisms of humans are reduced or altered by accident, by intent (e.g., surgery), or by an underlying metabolic disorder or an infectious disease (e.g., AIDS). There are two major differences between primary pathogens and opportunistic ones: 1. The first and foremost being that the primary pathogens regularly cause overt disease, whereas opportunistic ones take the opportunity offered by reduced host defenses to cause disease. 2. Long-term survival of a microbe is another difference. Long-term survival in a primary pathogen is absolutely dependent on its ability to replicate and to be transmitted in a particular host. However, this is not necessarily the case for a number of the opportunistic pathogens that infect humans. Infection This is a process when an organism enters the body, increases in number, and causes damage to the host. All infections do not invariably result in the disease. The term infection has more than one meaning: (a) the presence of microbes in the body and (b) the symptoms of the disease. The presence of microbes in the body does not always result in symptoms of the disease. Bacteria cause symptoms of disease by two main mechanisms: (a) production of toxins, both endotoxin and exotoxin and (b) production of inflammation. The words “virulence” and “virulent” are derived from the Latin word virulentus, meaning “full of poison.” The term virulentus is derived from the Latin words virus (poison) and lentus (fullness), and, in turn, the term virus may be related to the Sanskrit word visham, meaning “poison.” Virulence is a measure of a microbe’s ability to cause disease. It is a quantitative measure of pathogenicity and is measured by the number of organisms required to cause disease. It means that a highly virulent microbe requires fewer organisms to cause disease than a less virulent one; hence it is directly dependent on the infectious dose of the organism. The 50% lethal dose (LD50) is the number of organisms required to kill half of the hosts, whereas 50% infectious dose (ID50) is the number of microbes needed to cause infection in half of the hosts. The infectious dose of an organism required to cause disease varies among the pathogenic bacteria. MICROBIAL PATHOGENESIS ■ Important bacterial surface virulence Virulence factors Bacteria ■ Capsule Polysaccharide capsule Pili protein Escherichia coli Protein A Staphylococcus aureus M protein Streptococcus pyogenes V and W proteins Yersinia pestis ■ ■ Endemic: The infection that occurs at a persistent, usually low level in a certain geographical area is called endemic. Epidemic: The infection that occurs at a much higher rate than usual is known as epidemic. Pandemic: Infection that spreads rapidly over large areas of the world is known as a pandemic. Types of Infections Infections may be of the following types: ■ ■ ■ ■ ■ Primary infection: This condition denotes an initial infection with an organism in a host. Reinfection: This condition denotes subsequent infection with the same organism in the same host. Secondary infection: This condition denotes an infection with a new organism in a host whose body resistance is already lowered by a pre-existing infectious disease. Cross-infection: This condition denotes an infection with a new organism from another host or another external source in a patient who is already suffering from a disease. Nosocomial infection: Cross-infections acquired in hospitals are called hospital-acquired, hospital-associated, or nosocomial infections. Infectious diseases are complex. The outcome of infection depends on a variety of factors of the microbe and host as follows: 1. The ability of the organism to break host barriers and to evade destruction by innate local and tissue host defenses. 2. The ability of the organism to replicate, to spread, to establish infection, and to cause disease. 3. The ability of the organism to transmit to a new susceptible host. 4. The innate and adaptive immunologic ability of the host to control and eliminate the invading microorganism. The infection process involves the following stages: (a) transmission of infection, (b) entry of the organisms and evasion of the local defenses, (c) adherence to cell surfaces, (d) growth and multiplication of the bacteria at the site of adherence, (e) manifestations of disease, and ( f ) termination of disease. Transmission of Infection There are three important components that play an important role in successful transmission of microbial diseases. These are (a) reservoir, (b) mode of transmission, and (c) susceptible host. ◗ Reservoir Reservoirs of microbial infections are human, animal, plant, soil, or inanimate matter in which organisms usually live, multiply, and cause the infections with or without overt clinical manifestations. Humans are usually the common reservoirs of many of the microbial infections. Animals are reservoirs of zoonotic infections, such as plague (e.g., rats), rabies (e.g., dogs), cysticercosis (e.g., pigs), etc. Sources of infections: The sources of infections may be endogenous and exogenous: ■ Endogenous sources: The source of infection is the normal bacterial flora present in the human body. These bacteria are usually nonpathogenic but in certain situations become pathogenic and cause infections at different sites in the same host. For example, Escherichia coli present as normal flora of the intestine may cause urinary tract infection in the same host. Similarly, viridans streptococci present as a part of the normal flora of the mouth may cause infective endocarditis. Chapter 43 ■ Stages of Pathogenesis of Infections Section III For example, the infectious dose of Shigella to cause dysentery is less than 100 organisms, whereas that of Salmonella to cause diarrhea is more than 100,000 organisms. The virulence of a microbe is determined by virulence factors, such as capsules, exotoxins, or endotoxins (Table 10-1). Pathogenicity is the capacity of a pathogen species to cause disease, while virulence is used to describe the sum of disease causing properties of a population (strain) within the species. Pathogens can be distinguished from their avirulent counterparts by the presence of specific genes or gene clusters in the genome known as pathogenicity islands. The diseases that can be spread from one person to another are called communicable diseases. Most microbial infections are communicable diseases. Three epidemiological terms are often used to describe infection: endemic, epidemic, and pandemic: ■ Chapter 10 Polypeptide capsule Streptococcus pneumoniae Klebsiella pneumoniae Haemophilus influenzae Salmonella Typhi Neisseria meningitidis Bacillus anthracis Iatrogenic infection: This condition denotes a physicianinduced infection as a result of therapy with drugs or investigation procedures. Subclinical infection: Inapparent clinical infections are called subclinical infections. Latent infections: This denotes a condition in which some organisms may remain in a latent or hidden stage in host and subsequently they multiply to produce clinical disease when host resistance is lowered. Section I TABLE 10-1 73 74 Chapter 10 Section I ■ ■ ■ Section III Exogenous sources: The source of infection is from outside the host’s body. Most of the microbial infections are exogenous in nature. The exogenous sources include the following: 1. Humans: Humans are the most common sources of infections caused by the microorganisms. They may be either patients or carriers. The patient suffering from an active infection is an important source of infection to others. A carrier is a person who harbors pathogenic microorganisms without showing any signs and symptoms of disease. Carriers are also important sources of infections. A carrier may be (a) healthy carrier, (b) convalescent carrier, (c) temporary carrier, and (d) chronic carrier. ■ Chapter 43 GENERAL MICROBIOLOGY ■ Healthy carrier is the host who harbors the microorganism without ever suffering from the disease caused by that microorganism. Convalescent carrier is the host who continues to harbor the microorganism even after recovering from the clinical disease caused by the same pathogen. Temporary carrier is the host who harbors the microorganism up to 6 months after recovering from the disease caused by the same pathogen. Chronic carrier is the host who harbors the microorganism for many years after recovering from the clinical disease caused by the same pathogen. TABLE 10-2 2. Animals: Animals are also important sources of infection for humans. The symptomatic as well as asymptomatic animals can transmit infections to humans. Asymptomatic animals act as a reservoir of human infections. These are called as reservoir hosts. Infections transmitted from animals to humans are called zoonotic infections. The examples of zoonotic infections include bacterial (e.g., plague, anthrax, bovine tuberculosis, etc.), viral (e.g., rabies, Japanese encephalitis, etc.), fungal (e.g., dermatophytic infections), and parasitic (e.g., toxoplasmosis, cysticercosis, hydatid disease, etc.). 3. Insects: Insects, such as mosquitoes, ticks, mites, flies, fleas, and lice may transmit a wide variety of microorganisms to the humans (Table 10-2). The diseases transmitted by the insects are collectively referred to as arthropod-borne diseases and the insects transmitting these pathogens are called vectors. Insect vectors may transmit the infection in two ways: mechanical transmission and biological transmission. ■ ■ Mechanical vectors: Insects (e.g., domestic flies) carry enteric bacteria (Salmonella typhi, Shigella spp., etc.) mechanically on their legs, wings, and surface of the body and transfer them to food. Biological vectors: These are the vectors in which the microorganisms multiply or undergo a part of their life Microbial diseases transmitted by insects Organism Disease Reservoir Insect Yersinia pestis Plague Rodents, especially rats Rat fleas Francisella tularensis Tularemia Rabbits and other rodents Ticks Borrelia recurrentis Relapsing fever Humans Lice Borrelia burgdorferi Lyme disease Rodents and deer Ixodes ticks Bacteria Rickettsia rickettsii Rocky mountain spotted fever Dogs, rodents, and ticks Ticks Rickettsia prowazekii Epidemic typhus Humans Lice Ehrlichia chaffeensis Ehrlichiosis Deer and rodents Ticks Leishmania spp. Leishmaniasis Humans, dogs, and wild canine Sandfly (Phlebotomus) Trypanosoma spp. Trypanosomiasis Humans, armadillos, antelope, and cattle Reduviid bug, tsetse fly Plasmodium spp. Malaria Humans Anopheles mosquitoes Wuchereria bancrofti and Brugia malayi Filaria Humans Mosquitoes (Culex, Aedes, Mansonia spp.) Japanese encephalitis virus Japanese encephalitis Birds Culex tritaeniorhynchus mosquitoes West Nile virus West Nile fever Birds Culex mosquitoes Parasites Viruses Chikungunya virus Chikungunya Humans and monkeys Aedes aegypti mosquitoes Dengue virus Dengue Humans Aedes aegypti mosquitoes Kyasanur forest disease virus Kyasanur forest disease Forest birds and small mammals Tick Yellow fever virus Yellow fever Monkeys and humans Aedes aegypti mosquitoes MICROBIAL PATHOGENESIS ◗ Modes of transmission Modes of transmission Different modes of transmission Disease Causative agents Direct contact Gonorrhea Neisseria gonorrhoeae ◗ Indirect contact Dysentery Shigella dysenteriae Blood-borne Syphilis Treponema pallidum Transplacental Congenital syphilis Treponema pallidum The infective agent enters the body by four main routes: (a) genital tract, (b) respiratory tract, (c) gastrointestinal tract, and (d) skin. The pathogens can be transmitted either as vertical or horizontal transmission. Vertical transmission: Certain bacteria (Treponema pallidum), viruses (rubella and cytomegalovirus), and parasites (Toxoplasma gondii) can be transferred from mother to fetus by a process called vertical transmission (Table 10-4). The organisms can be transmitted vertically by three ways: Human to human Nonhuman to human (animal origin) Direct contact Cat-scratch disease Bartonella henselae Lyme disease Borrelia burgdorferi Hemolytic uremic syndrome Enterohemorrhagic Escherichia coli (EHEC) Handling of fomites Skin infection Staphylococcus aureus Soil borne Tetanus Clostridium tetani Water borne Legionnaire’s disease Legionella pneumophila Through animal excreta Susceptible host (a) Across the placenta, (b) Within birth canal during birth, and (c) Through breast milk. Chapter 43 TABLE 10-3 Section III Microbial pathogens causing various infectious diseases are transmitted from one host to another by many ways: (a) contact, (b) inoculation, (c) ingestion, (d) inhalation, and (e) vectors (Table 10-3). 1. Contact: Transmission of microorganisms from person to person occurs by direct or indirect contact. Transmission by direct contact occurs through the acts of touching, kissing, sex, etc. Hence, this mode of transmission is also known as personto-person transmission. The diseases transmitted by direct contact include common cold, staphylococcal infections, and sexually transmitted infections (e.g., gonorrhea, syphilis, and AIDS, etc.). The term contagious disease was used earlier for the disease acquired by direct contact. Microorganisms can also be transmitted by indirect contact through inanimate objects, such as clothings, handkerchief, toys, etc., called fomites. The fomites, contaminated by microbial pathogens, act as a vehicle for their transmission. Influenza, tuberculosis, and certain superficial fungal infections are examples of diseases transmitted by fomites. Chapter 10 Insects, besides acting as vectors, also act as reservoir hosts (e.g., ticks in relapsing fever). 4. Food: Food items contaminated with pathogens also act as source of infection and cause diarrhea, dysentery, food poisoning, and gastroenteritis. 5. Water: Water contaminated with microorganisms also acts as a source of infection and transmits water-borne diseases, such as leptospirosis, cholera, dysentery, hepatitis A infection, etc. 2. Inoculation: Infections can be transmitted by inoculation of microorganisms directly into tissues of the host. For example, tetanus is transmitted by direct inoculation of Clostridium tetani spores present in soil to the injured tissues in the host. These spores then germinate to vegetative forms of bacteria and migrate along the neural tissues to cause tetanus. Similarly, rabid dogs through their act of biting inoculate rabies virus directly to host tissue and cause rabies in humans. Iatrogenic infection occurs following the use of unsterile syringes and equipment in a hospital. Hepatitis B and C and HIV infections are the examples of iatrogenic infections caused by use of contaminated syringes and that of contaminated blood and blood products. 3. Ingestion: Ingestion of water and food contaminated with microorganisms can transmit a wide variety of microbial infections. For example, food poisoning caused by Bacillus cereus is transmitted by rice contaminated with bacterial spores that survive boiling. If the rice is cooled and reheated, the spores of the bacteria may germinate to the bacteria that produce a heat-stable toxin that induces vomiting. Cholera, typhoid, food poisoning, hepatitis A, poliomyelitis, and many parasitic infections are the other examples of diseases transmitted by ingestion of contaminated food and water. 4. Inhalation: Infections are transmitted by inhalation of droplet nuclei that are discharged into the air by coughing, sneezing, or talking. Respiratory pathogens are shed into the environment by patients in secretions from the nose or throat during coughing, sneezing, or talking. Small droplets (less than 0.1 mm in diameter) become airborne as minute particles or droplet nuclei (1–10 ␮m in diameter), while large droplets fall down to the ground. Infections are transmitted by inhalation of these droplet nuclei containing respiratory pathogen, which remain suspended in air for a long period of time. Measles, influenza, whooping cough, tuberculosis, aspergillosis, etc. are few examples of infectious diseases acquired by inhalation. 5. Vectors: Mosquitoes, flies, fleas, ticks, mite, and lice are the vectors that transmit many diseases as mentioned earlier. Section I cycle before being transmitted to humans. Rat flea and female Anopheles mosquitoes are the examples of biological vectors that transmit plague and malaria, respectively, to humans by biting. 75 GENERAL MICROBIOLOGY Modes of transmission (e.g., Polio, influenza, typhoid) Horizontal transmission Vertical transmission of microbial agents TABLE 10-4 Organism Disease Bacteria Staphylococcus aureus Skin or oral infection Viruses Human T-cell leukemia virus Asymptomatic Cytomegalovirus Asymptomatic Air, water, food, contact, vectors, etc. Breast milk Chapter 10 Section I 76 Asymptomatic infection Human immunodeficiency virus (HIV) Sperm, ovum, placenta, milk, contact First generation During passage through birth canal Bacteria Chapter 43 Section III Viruses Group B streptococcus Neonatal sepsis and meningitis Neisseria gonorrhoeae Conjunctivitis Chlamydia trachomatis Pneumonia or conjunctivitis HIV Asymptomatic infection Hepatitis B virus Hepatitis B Herpes simplex virus-2 Skin or CNS infection; sepsis Listeria monocytogenes Neonatal sepsis and meningitis Treponema pallidum Congenital syphilis Cytomegalovirus Congenital abnormalities Parvovirus B19 Hydrops fetalis Toxoplasma gondii Congenital toxoplasmosis Transplacental Bacteria Viruses Parasite Table 10-4 summarizes a list of diseases transmitted vertically. Horizontal transmission: Unlike vertical transmission, horizontal transmission occurs from person to person and is not from mother to offspring (Fig. 10-1). Entry of Organisms and Evasion of Local Defenses Skin, mucus, ciliated epithelium, and secretions containing antibacterial substances (e.g., lysozyme) are the natural barriers of the human and animal hosts that prevent microbial entry. However, these barriers are sometimes broken (e.g., a break in the skin, an ulcer in the intestine, or a tumor, etc.), thereby allowing the entry of microbes into the host (Table 10-5). On entry, the microbes spread through blood circulation to other sites in the body (Fig. 10-2). Skin: The stratified squamous epithelium of the skin with its superficial cornified anucleate layers is a simple and efficient mechanical barrier to prevent microbial invasion. Organisms gain access to the underlying tissues only by breaks or by way of hair follicles, sebaceous glands, and sweat glands that traverse the stratified layers. (e.g., Hepatitis B virus, rubella virus, leukemia virus, Vertical transmission commensal bacteria, reactivating persistent infection) Second generation FIG. 10-1. Schematic diagram showing horizontal and vertical transmission of infections. The surface of the skin continuously desquamates and thereby tends to shed contaminating organisms. The skin also inhibits the growth of most extraneous microorganisms due to its low moisture, low pH, and the presence of substances with an antibacterial activity. Mucus: Viscous mucus secreted by goblet cells protects the epithelium lining the respiratory and gastrointestinal tracts and urogenital system. Microorganisms become trapped in the mucus layer and may be swept away before they reach the epithelial cell surface. Secretory IgA, secreted into the mucus, and other secreted antimicrobials (such as lysozyme and lactoferrin) facilitate this cleansing process. Ciliated epithelial cells: These cells constantly move the mucus away from the epithelial surfaces. For example, mucus in the respiratory tract—particles larger than 5 ␮m are washed and trapped in the mucus. Similarly, the multilayered transitional epithelium of the urinary tract uses the flushing effect of urine, and its relatively low pH acts as an additional defense mechanism to limit microbial entry and growth. Secretions: The high level of hydrochloric acid and gastric enzymes in the normal stomach kills many ingested bacteria. Others are susceptible to pancreatic digestive enzymes or to the detergent effect of bile salts. Adherence to Cell Surfaces Adherence of bacteria to body surface is the most important event in the pathogenesis of disease. Once bacteria enter the body of the host, they must adhere to the cells of a tissue surface. If they do not adhere, they will be swept away by mucus and other fluids that bathe the tissue surface. MICROBIAL PATHOGENESIS Bacteria Virus Fungi Skin and mucus membrane Clostridium tetani, Leptospira Hepatitis B virus (HBV), human immunodeficiency virus (HIV) Dermatophytes Respiratory tract Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Mycobacterium tuberculosis Rhinovirus, RSV (respiratory syncytial virus), Epstein–Barr virus, influenza virus Cryptococcus neoformans, Histoplasma capsulatum, Pneumocystis jirovecii Gastrointestinal tract Shigella spp., Salmonella spp., Vibrio spp. Hepatitis A or E virus, poliovirus Candida albicans Genital tract Neisseria gonorrhoeae, Treponema pallidum HIV, human papilloma virus Candida albicans Portal of entry* FIG. 10-2. process. Portal of exit Capsule Cell wall component Enzymes Siderophores Antigenic variation Cytoskeleton ■ Direct damage Toxins Exotoxins Endotoxin Lysogenic conversion ■ Generally the same as the portals of entry for a given microbe Schematic diagram showing various stages of infection Most pathogenic microorganisms have more than a single mechanism of host cell attachment. Adherence is important not only during the initial encounter between the pathogen and its host but also throughout the infection cycle. Adherence requires participation of two factors: bacterial adhesins and a receptor on the host cell. ◗ Key Points Bacterial adhesins Bacterial adherence to the cell surface is mediated by specialized molecules. The various molecules that mediate adherence to the cell surface are called adhesins. These adhesins allow the bacteria to adhere to the surface of human cell, thereby promoting their ability to cause disease. Microorganisms that lack this mechanism are nonpathogenic. Bacterial adhesins can be divided into two major groups: pili (fimbriae) and nonpilus adhesins (afimbrial adhesins). Most E. coli strains that cause pyogenic nephritis produce an adhesin protein known as P-pili (pyelonephritis-associated pili) encoded by pap genes. Many of these adhesin proteins are present at the tip of the pili that bind specific receptors on the surface of the urinary bladder. The binding prevents the bacteria from being washed away from the urinary bladder by the flushing action of the urine. Similarly, the pili of Neisseria gonorrhoeae mediate the attachment of the organism by binding to oligosaccharide receptors on epithelial cells of the urethra. The gonococci use pili as primary adhesins and opacity-associated proteins (Opa) as secondary adhesins to host cells. Certain Opa proteins mediate adherence to polymorphonuclear cells. Some gonococci survive after phagocytosis by these cells. In uterine (fallopian) tube organ cultures, the gonococci adhere to the microvilli of nonciliated cells and appear to induce engulfment by these cells. The gonococci multiply intracellularly and migrate to the subepithelial space by an unknown mechanism. The pili of the Gram-negative bacteria have been classified into five different types and are extremely important in the pathogenesis of infections caused by them. Further antigenic variation in the actual structural pilin protein can be an important source of antigenic diversity for the pathogen. ■ Nonpilus adhesins: These include glycocalyx and other adhesins present on the bacterial surfaces. Glycocalyx is a polysaccharide “slime layer” secreted by some strains of bacteria that mediates strong adherence to certain structures, such as catheters, prosthetic implants, and heart valves. For example, the glycocalyx of Staphylococcus epidermidis and that of certain viridans streptococci allows the bacteria to adhere strongly to the endothelium of the heart valve. The matrix formed by these adhesins forming proteins is called a biofilm. The biofilms are important in pathogenesis because they protect the bacteria by host defense and antibiotics. Chapter 43 Damage to host cells/ cytopathic effects Pili: These are the main mechanisms by which bacteria adhere to human cells. They are the fibers that extend from the bacterial surfaces and mediate attachment of bacteria to specific receptor on the host cells. The pili of many Gram-negative bacteria bind directly to sugar residues that are part of glycolipids or glycoproteins on the host cells. They also act as a protein scaffold to which another more specific adhesive protein is affixed. Section III Penetration or evasion of host defenses Mucus membranes Respiratory tract Gastrointestinal tract Genitourinary tract Conjunctiva Skin Parenteral route Chapter 10 Portal of entry Section I Routes of entry of microbial pathogens TABLE 10-5 • Also affected by number of invading microbes • Adherence 77 Chapter 10 Section I 78 ■ ■ Chapter 43 Section III ◗ GENERAL MICROBIOLOGY The biofilms facilitate colonization of bacteria, especially of surgical appliances, such as artificial valve or indwelling catheters. Streptococcus pyogenes makes use of nonpilus adhesins (such as lipoteichoic acid, protein F, and M protein) to bind to epithelial cells. The lipoteichoic acid and protein F cause adherence of the streptococci to buccal epithelial cells. M protein acts as an antiphagocytic molecule. Recently, it has been shown that certain strains of E. coli and Shigella spp. have surface proteins called curli, which help in the binding of bacteria to the host endothelium as well as to extracellular proteins. Receptor on the host cell Certain receptors are present on the host cells to which pathogens adhere and initiate infections. For example, many adhesion proteins are present at the tip of the pili of E. coli. These bind specific receptors on the surface of the urinary bladder to initiate urinary tract infections. Similarly, the gonococci adhere to the microvilli of nonciliated cells and start disease process. Growth and Multiplication of Bacteria at the Site of Adherence Bacteria cause diseases by three main mechanisms: (a) invasion of tissues followed by inflammation, (b) toxin production, and (c) immunopathogenesis. Table 10-6 summarizes a list of bacteria with their virulence factors. ◗ role for the host cells. For many disease-causing bacteria, invasion of the host’s epithelium is central to the infectious process. Some bacteria (e.g., Salmonella spp.) invade tissues through the intracellular junctions in the cytoplasm. Some bacteria (e.g., Shigella spp.) multiply within host cells, whereas other bacteria do not. Shigella spp. initiate infection process by adhering to host cells in the small intestine. There are multiple proteins, including the invasion plasmid antigens (IpA-D), that contribute to the process. Once inside the cells, the shigellae either are lysed or escape from the phagocytic vesicle, where they multiply in the cytoplasm. Other bacteria (e.g., Yersinia species, N. gonorrhoeae, Chlamydia trachomatis) invade specific types of the host’s epithelial cells and may subsequently enter the tissue. Once inside the host cell, the bacteria may remain enclosed in a vacuole composed of the host cell membrane, or the vacuole membrane may dissolved and bacteria may disperse within the cell and from one cell to another. Invasion of tissues followed by inflammation is enhanced by many factors, which include: (a) enzymes, (b) antiphagocytic factors, (c) biofilms, (d) inflammation, and (e) intracellular survival. 1. Enzymes: Invasion of bacteria is enhanced by many enzymes. Many species of bacteria produce enzymes that are not intrinsically toxic but do play important roles in the infectious process. Some of these enzymes are discussed below: ■ Invasion of tissues followed by inflammation Invasiveness refers to the ability of an organism to invade the host cells after establishing infection. “Invasion” is the term commonly used to describe the entry of bacteria into host cells, implying an active role for the organisms and a passive TABLE 10-6 Bacterial virulence factors Organism Virulence factors Staphylococcus aureus Coagulase, protein A Streptococcus pyogenes M protein Streptococcus pneumoniae Capsular polysaccharide Enterococcus faecalis Cytolysin, biofilm formation Neisseria gonorrhoeae Pili, opacity-associated proteins (Opa), IgA proteases Neisseria meningitidis Capsular polysaccharide Bacillus anthracis Capsule, edema factor, lethal factor, protective antigen Listeria monocytogenes Internalin Escherichia coli Heat-labile and heat-stable enterotoxins, pili Haemophilus influenzae Capsular polysaccharide Vibrio cholerae Cholera toxin Mycobacterium tuberculosis Mycolic acid cell wall ■ ■ Hyaluronidases and collagenase: Hyaluronidases and collagenase are the enzymes that hydrolyze hyaluronic acid and degrade collagen, respectively; thereby allowing the bacteria to spread through subcutaneous tissues. Hyaluronidases are produced by many bacteria (e.g., staphylococci, streptococci, and anaerobes) and aid in their spread through tissues. For example, hyaluronidase produced by S. pyogenes degrades hyaluronic acid in the subcutaneous tissue, thereby facilitating the organism to spread rapidly. Clostridium perfringens produces the proteolytic enzyme collagenase, which degrades collagen (the major protein of fibrous connective tissue), and promotes the spread of infection in tissue. Coagulase: Staphylococcus aureus produces the enzyme coagulase, which in association with blood factors coagulates the plasma. Coagulase contributes to the formation of fibrin walls around staphylococcal lesions, which protects bacteria from phagocytosis by walling off the infected area. The enzyme also causes deposition of fibrin on the surfaces of individual staphylococci, which may help protect them from phagocytosis or from destruction within phagocytic cells. Streptokinase ( fibrinolysin): Many hemolytic streptococci produce enzyme streptokinase, which activates a proteolytic enzyme of plasma. This enzyme is then able to dissolve coagulated plasma and thereby possibly aids in the rapid spread of streptococci through tissues. Streptokinase has been used in the treatment of acute myocardial infarction to dissolve fibrin clots. MICROBIAL PATHOGENESIS FIG. 10-3. Schematic diagram showing inhibition of phagocytosis by the capsule of Streptococcus pneumoniae. Examples Bacteria Mycobacterium spp. Listeria monocytogenes Brucella spp. Legionella pneumophila Francisella spp. Yersinia pestis Salmonella Typhi Shigella dysenteriae Rickettsia Chlamydia Viruses All viruses Parasites Leishmania spp. Trypanosoma cruzi Plasmodium spp. Babesia spp. Toxoplasma gondii Cryptosporidium parvum Microsporidium spp. Fungus Histoplasma capsulatum Biofilms are important in human infections that are persistent and difficult to treat. A few such infections include: (a) S. epidermidis and S. aureus infections of central venous catheters; (b) Eye infections that occur with contact lenses and intraocular lenses; (c) Infections in dental plaque; and (d) Pseudomonas aeruginosa airway infections in cystic fibrosis patients. Key Points Biofilm confers an inherent resistance to antimicrobial agents, whether these antimicrobial agents are antibiotics, disinfectants, or germicides. The mechanisms of resistance are: ■ ■ ■ Delayed penetration of antimicrobial agent through the biofilm matrix; Altered growth rate of biofilm organisms; and Other physiological changes due to biofilm mode of growth. 4. Inflammation: Inflammation is an important host defense induced by the presence of bacteria in the body. It is of two types: pyogenic and granulomatous. Pyogenic inflammation is the host defense seen primarily against pyogenic or pusproducing bacteria, such as S. pyogenes. It typically consists of neutrophils and the production of specific antibodies and elevated level of complement. Granulomatous inflammation is the host defense seen primarily against intracellular granuloma-producing bacteria, such as Mycobacterium tuberculosis, Mycobacterium leprae, etc. The response consists of production of macrophages and CD4+ T cells. Chapter 43 Phagocyte Organism Section III Streptococcus pneumoniae Intracellular pathogens Chapter 10 Inhibition of phagocytosis by capsule TABLE 10-7 Section I IgA1 proteases: Certain pathogenic bacteria produce enzymes IgA1 proteases that split IgA1 at specific proline–threonine or proline–serine bonds in the hinge region and inactivate its antibody activity. IgA1 protease is an important virulence factor of the pathogens, such as N. gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae. Production of IgA1 protease allows the pathogens to inactivate the primary antibody found on mucosal surfaces and thereby facilitates the attachment of these bacteria to the mucous membrane. 2. Antiphagocytic factors: Many bacterial pathogens are rapidly killed once they are ingested by polymorphonuclear cells or macrophages. Some pathogens evade phagocytosis or leukocyte microbicidal mechanisms by several antiphagocytic factors; the most important being (a) capsule, (b) cell wall proteins, (c) cytotoxins, and (d) surface antigens. ■ Capsule: The capsule surrounding bacteria, such as S. pneumoniae (Fig. 10-3) and N. meningitidis, is the most important antiphagocytic factor. It retards the phagocytosis of bacteria by preventing the phagocytes from adhering to the bacteria. ■ Cell wall proteins: The cell wall proteins, such as the protein A and protein M, of S. aureus and S. pyogenes especially are antiphagocytic. For example, protein A of S. aureus binds to IgG and prevents the activation of complement. M protein of S. pyogenes is antiphagocytic. ■ Cytotoxins: Certain bacteria produce cytotoxins that interfere with chemotaxis or killing of phagocytes. For example, S. aureus produces hemolysins and leukocidins that lyse and damage RBCs and WBCs. ■ Surface antigens: Surface antigens of bacteria, such as Vi antigen of S. typhi and K antigen of E. coli make the bacteria resistant to phagocytosis and lytic activity of complement. A list of intracellular pathogens is given in Table 10-7. 3. Biofilms: The biofilm is an aggregate of interactive bacteria attached to a solid surface or to each other and encased in an exopolysaccharide matrix. Biofilms consist of single cells and microcolonies of bacteria, all found together in a highly hydrated, predominantly anionic exopolymer matrix. This is distinct from planktonic or free-living bacterial growth, in which interactions of the microorganisms do not occur. Biofilms form a slimy coat on solid surfaces and occur throughout the nature. A single species of bacteria may be involved, or more than one species may coaggregate to form a biofilm. Fungi, including yeasts, are occasionally involved. ■ 79 80 GENERAL MICROBIOLOGY Section I 5. Intracellular survival: A few mechanisms that are suggested for intracellular survival of bacteria include (a) inhibition of phagolysosome fusion, (b) resistance to action of lysosomal enzymes, and (c) adaptation to cytoplasmic replication as follows: Chapter 10 ■ ■ Chapter 43 Section III ■ ◗ ■ Bacteria (such as Chlamydia, M. tuberculosis) that interfere with the formation of phagolysosomes in a phagocyte can survive intracellularly and evade host defense process. These bacteria live within cells and are protected from attack by macrophages and neutrophils. The bacteria that do not interfere with the formation of phagolysosomes are otherwise killed inside the phagocytes. Presence of capsular polysaccharide in Mycobacterium lepraemurium and mycoside in M. tuberculosis makes these bacteria resistant to action of lysosomal enzymes. Certain bacteria, such as rickettsiae, escape from the phagosome into the cytoplasm of the host cell before the fusion of phagosome with lysosome takes place and hence continue to remain intracellular. Toxin production Toxins produced by bacteria are generally classified into two groups: exotoxins and endotoxins. 1. Exotoxins: Exotoxins are heat-labile proteins that are produced by several Gram-positive and Gram-negative bacteria. These are bacterial products, which are secreted into tissues and directly harm tissues or trigger destructive biological activities (Fig. 10-4). The genes coding for these proteins are frequently encoded on plasmid or on bacteriophage DNA. Some important toxins encoded by plasmids are tetanus toxin of C. tetani and heat-labile and heat-stable toxins of enterotoxigenic E. coli. Toxins encoded by bacteriophage DNA are cholera toxins, diphtheria toxins, and botulinum toxin. Key Points Exotoxins show the following features: ■ ■ ■ ■ Exotoxins are good antigens; they induce the synthesis of protective antibody called antitoxins. Some of these antitoxins are useful in the treatment of botulism, tetanus, and other diseases. Exotoxins treated with formaldehyde or acid or heat can be converted into toxoid. The toxoids lack toxicity but retain antigenicity. Hence, these are used in protective vaccines. Exotoxins are some of the most toxic substances known. They are highly potent even in minute amounts. Botulinum toxin is the most potent one, and it has been estimated that 3 kg of botulinum toxin can kill all persons in the world. Similarly, the fatal dose of tetanus toxin for a human is estimated to be less than 1 ␮g. Many toxins have a dimeric A–B subunit structure. Diphtheria toxin, tetanus toxin, cholera toxins, and the enterotoxin of E. coli are some of the examples of exotoxins that have an A–B subunit structure. A is the active subunit that possesses the toxic activity, and B is the binding subunit that is responsible for the binding of exotoxin to specific receptors of the membrane of human cell (Fig. 10-5). These toxins are very specific in their mechanism of action and act at specific sites of a tissue. The biochemical targets of A–B toxin include ribosomes, transport mechanisms, and intracellular signaling (cyclic adenosine monophosphate, CAMP; G protein production); all these cause diarrhea, loss of neuronal functions, or even death. The exotoxins have specific pharmacological activities and do not produce fever, unlike endotoxins. Cell wall Exotoxin Endotoxin FIG. 10-4. Schematic diagram showing release of exotoxin and endotoxin. Superantigens: Superantigens are special group of toxins. These molecules activate T-cell nonspecifically by binding simultaneously to a T-cell receptor and major histocompatibility complex class II (MHC II) molecules on another cell, without requiring antigen. Nonspecific activation of T cells results in a life-threatening autoimmune-like response by producing a large amount of interleukins, such as IL-1 and IL-2. Furthermore, stimulation of T cells by superantigen can also lead to the death of activated T cell, resulting in loss of specific T-cell clones and that of their immune response. Staphylococcal enterotoxin (toxic shock syndrome toxin) of S. aureus and erythrogenic toxin of type A or C of S. pyogenes are examples of superantigens. 2. Endotoxins: The term endotoxin was coined in 1893 by Pfeiffer to distinguish the class of toxic substances released after lysis of bacteria from the toxic substances (exotoxins) secreted by bacteria. Key Points Endotoxins show the following properties: ■ ■ ■ ■ ■ ■ They are produced by Gram-negative bacteria, but not by Gram-positive bacteria. They are lipopolysaccharide (LPS) components of the outer membrane of Gram-negative bacteria. These form an integral part of the cell wall unlike exotoxins, which are actively released from the cells. The genes that encode the enzymes that produce the LPS are present on the bacterial chromosome, but not on plasmids or bacteriophage DNA, which usually encodes the exotoxins. They are heat stable, and they are released from the bacterial cell surface by disintegration of the cell wall. They are weakly antigenic and do not induce, or poorly induce, protective antibodies. Hence, their action is not neutralized by the protective antibodies. They cannot be toxoided. MICROBIAL PATHOGENESIS Nucleus Exotoxin coding mRNA Exotoxin Altered exotoxin 2 Exotoxin is released from bacteria Exotoxin enters cell by binding to host cell receptor through binding component (B) Mechanisms of endotoxin-mediated toxicity Fever Interleukin-1 Inflammation Activation of alternative pathway of complement (C3a, C5a) Disseminated intravascular coagulation (DIC) Activation of Hageman factor Shock (hypotension) Bradykinin, nitric oxide Leukopenia, thrombocytopenia, decreased peripheral circulation, and perfusion to organs Secondary to DIC Biological activity of endotoxin: Gram-negative bacteria produce endotoxin during infection. The toxicity of endotoxin is low in comparison with exotoxins. All endotoxins usually produce the same generalized effect of fever and shock. The lipid A protein of LPS is responsible for endotoxin activities (Table 10-8). The endotoxin binds to specific receptors, such as CD14 and TLR4, present on macrophages, B cells, and other cells. Endotoxin exerts profound biological effects on the host and may be lethal. Biological activities of the endotoxins include the following: ■ Mitogenic effects on B lymphocytes that increase resistance to viral and bacterial infections. Production of gamma interferon by T lymphocytes, which may enhance the antiviral state, promote the rejection of tumor cells, and activates the macrophages and natural killer cells. Activation of the complement cascade with the formation of C3a and C5a. Production and release of acute-phase cytokines, such as IL-1, TNF-␣ (tumor necrosis factor-alpha), IL-6, and prostaglandins. Endotoxic shock: Endotoxins at low concentration induce a protective response, such as fever, vasodilation, and activation of immunity and inflammatory response. However, endotoxins at very high concentration, as seen in blood of patients with Gram-negative bacterial sepsis, cause a syndrome of endotoxic shock. Endotoxic shock is characterized by fever, leukopenia, thrombocytopenia, sudden fall of blood pressure, circulatory collapse, and sudden death. This is because high concentration of endotoxin can activate the alternative pathway of complement and cause vasodilatation and capillary leakage, resulting in high fever, hypertension, and shock. It also causes activation of blood coagulation pathway, leading to disseminated intravascular coagulation. Endotoxins are not destroyed by autoclaving; hence infusion of sterile solution containing endotoxins can cause serious illness. Detection of endotoxins in medical solutions: Endotoxins are omnipresent in the environment. Solutions for human use (such as intravenous fluids) are prepared under carefully controlled conditions to ensure sterility and to remove endotoxin. Representative samples of every manufacturing batch are checked for endotoxins by one of two procedures: (a) limulus lysate test or (b) rabbit pyrogenicity test. ■ ■ Limulus lysate test: The test depends on the ability of endotoxin to induce gelation of lysates of amoebocyte cells from the horseshoe crab Limulus polyphemus. Test kits are commercially available. It is simple, fast, and sensitive to detect endotoxin at a level of 1 ng/mL. Rabbit pyrogenicity test: The test depends on the exquisite sensitivity of rabbits to the pyrogenic effects of endotoxin. In this test, a sample of the solution to be tested is injected intravenously into the ear veins of adult rabbits, while the rectal temperature of the animal is monitored. Careful monitoring of the temperature responses provides a sensitive and reliable indicator of the presence of endotoxin in the solution. Table 10-9 summarizes differences between exotoxins and endotoxins. Chapter 43 Mechanism ■ Active component (A) of exotoxin alters cell function by inhibiting protein synthesis Section III Clinical features ■ 3 Chapter 10 1 Schematic diagram showing mode of action of exotoxin. TABLE 10-8 ■ Section I FIG. 10-5. DNA 81 Chapter 43 Section III Chapter 10 Section I 82 GENERAL MICROBIOLOGY TABLE 10-9 Differences between endotoxins and exotoxins Feature Endotoxin Exotoxin Nature Lipopolysaccharide Protein (polypeptide) Source Gram-negative bacterial cell wall Gram-positive bacteria and some Gram-negative bacteria Location of genes Chromosome Plasmid or bacteriophage Nature of secretion Not secreted by the bacterial cell Actively secreted by the bacteria Release of toxin Cell lysis Filtration of bacterial cultures Heat stability Highly stable (withstand even 100°C for an hour) Heat-labile, destroyed mostly at 60°C Mode of action Mediated by interleukins (IL-1) and tumor necrosis factor Mostly enzyme-like action Effect Nonspecific (fever, shock, etc.) Specific pharmacological effect Tissue affinity No Specific affinity for certain tissues Diseases Gram-negative bacterial sepsis, meningococcemia Botulism, diphtheria, and staphylococcal toxic shock syndrome Fatal dose Only large doses are fatal Small doses (even a few micrograms) are fatal Antigenicity Poorly antigenic Highly antigenic Neutralization by antibodies Ineffective Neutralized by specific antibodies Vaccine No effective vaccine Specific toxoids are available ◗ Immunopathogenesis In certain diseases, the symptoms are caused not by the organism itself, but due to immune response to the presence of organisms. For example, immune complexes deposited in the glomerulus of the kidney cause poststreptococcal glomerulonephritis. Antibodies that are produced against the M proteins of S. pyogenes cross-react with joint, heart, and brain tissues producing disease manifestations of rheumatic fever. Similarly, the host immune response is an important cause of disease symptoms in patients suffering from syphilis caused by T. pallidum, Lyme disease caused by Borrelia, and other diseases. Manifestations of Disease Diseases caused by various pathogenic as well as opportunistic pathogens cause a variety of clinical manifestations in infected human hosts. Many of these manifestations are caused either due to the inflammation or toxin production by the bacteria. Termination of Disease Termination of an infectious disease may occur by resolution or continuation of the disease. This depends on a complex interaction of host immunity with pathogens and host response to treatment with specific antimicrobial agents. Stages of an Infectious Disease There are four discrete stages of an infectious disease, which are as follows: 1. Incubation period: This denotes the time interval between the entry of infective agent and the onset of clinical manifestations of disease. During this period, the infective agent after reaching the selective tissue undergoes multiplication. 2. Prodrome period: It is the time during which only nonspecific symptoms of disease occur. 3. Specific illness period: The time during which the characteristic features of disease occur. 4. Recovery period: The time during which symptoms resolve and health is restored. Not all the cases that recover become free of organism. Some become chronic carriers and act as sources of infection for others. Though some cases may not develop infection, they can act as a link in the transmission of infection. This is called subclinical infection. “This page intentionally left blank" “This page intentionally left blank" 43 11 Mycobacterium Leprae Immunity Introduction The term immunity is derived from the Latin word “immunis” (exempt), which was originally referred to the protection from legal prosecution offered to the Roman senators during their tenures in office. This term was adopted subsequently to designate the naturally acquired protection against diseases, such as measles or smallpox. It indicated that an individual can develop lifelong resistance to a certain disease after having contracted it only once. The cells and molecules responsible for immunity constitute the immune system, and their collective and coordinated response to foreign substances is called the immune response. The concept of immunity has existed since the ancient times. An example is the Chinese custom of making children resistant to smallpox by making them inhale powders made from the skin lesions of patients recovering from the disease. The first European mention of immunity is recorded by Thucydides in Athens during the fifth century BC. In describing plague in Athens, he wrote in 430 BC that only those who had recovered from plague could nurse the sick, because they would not contract the disease a second time. Once the concept of existence of immunity was established, it was not long before manipulation of immunity under controlled conditions followed. First, it was Edward Jenner who in a successful experiment injected the material from a cowpox pustule into the arm of an 8-year-old boy and demonstrated the lack of development of disease after subsequent exposure to smallpox. This was based on his observations that milkmaids who had suffered from cowpox never contracted the more serious smallpox. Jenner’s technique of inoculating with cowpox to protect against smallpox spread quickly throughout Europe. However, for many reasons, including lack of knowledge of obvious disease targets and their causes, it was after nearly hundred years that this technique was applied to prevent smallpox. The experimental work of Emil von Behring and Shibasaburo Kitasato in 1890 gave the first insights into the mechanism of immunity, earning von Behring the Nobel Prize in Medicine in 1901. Von Behring and Kitasato demonstrated that serum (the liquid, noncellular component of coagulated blood) from animals previously immunized to diphtheria could transfer the immune state to unimmunized animals. Since then, immunology as a field of study has come a long way. It has been and remains one of the hottest fields of research as shown by the statistic that about 17 Nobel Prizes have been awarded to scientists involved in immunological research. Types of Immunity The main function of the immune system is to prevent or limit infections by pathogenic microorganisms, such as bacteria, viruses, parasites, and fungi. The recognition of microorganisms and foreign substances is the first event in immune responses of a host. The body’s defense mechanisms can be divided into: (a) innate (natural) immunity and (b) acquired (adaptive) immunity. Innate Immunity Innate immunity is the resistance that an individual possesses by birth. Innate immunity may be classified as (a) individual immunity, (b) racial immunity, and (c) species immunity. Individual immunity: Individual immunity denotes resistance to infection, which varies within different individuals in the same race and species and is genetically determined. For example, if one homozygous twin develops tuberculosis, there is a very high possibility that the other twin will also develop tuberculosis. But in heterozygous twins, there is a very low possibility of the other twin suffering from tuberculosis. Racial immunity: Racial immunity denotes a difference in susceptibility or resistance to infection among different races within a same species. For example, races with sickle cell anemia prevalent in Mediterranean coast are immune to infection caused by malaria parasite Plasmodium falciparum. This is due to a genetic abnormality of erythrocytes, resulting in sickleshaped erythrocytes that prevent parasitization by P. falciparum. Similarly, individuals with a hereditary deficiency of glucose6-phosphatase dehydrogenase are also less susceptible to infection by P. falciparum. Species immunity: Species immunity denotes a total or relative resistance to a pathogen shown by all members of a particular species. For example, chickens are resistant to Bacillus anthracis, rats are resistant to Corynebacterium diphtheriae, whereas humans are susceptible to these bacteria. The exact reason for such type of immunity is not known. 86 IMMUNOLOGY Key Points Innate immunity shows the following features: ■ ■ Section II ■ ■ Chapter 11 ◗ It is due to the genetic and constitutional makeup of an individual. Prior contact with microorganisms or their products is not essential. It acts as the first line of defense of the host immune system. The mechanisms involved in innate immunity are present in place even before exposure to the foreign agent. They are not specific to any infectious agent and do not seem to improve response on repeated exposures. Phagocytic cells (e.g., macrophages and neutrophils), barriers (e.g., skin and mucous membrane), and a variety of antimicrobial compounds synthesized by the host, all play important roles in innate immunity. Factors influencing innate immunity The factors that may influence innate immunity of the host include age and nutritional status of the host. Age: Extremes of age make an individual highly susceptible to various infections. This is explained in part by the immature immune system in very young children and waning immunity in older individuals. The fetus-in-utero is usually protected from maternal infections by the placental barrier. However, human immunodeficiency virus (HIV), rubella virus, cytomegalovirus, and Toxoplasma gondii cross the placental barrier and cause congenital infections. Very old people are susceptible to suffer more than young people from a disease (e.g., pneumonia) and have high mortality. Measles, mumps, poliomyelitis, and chicken pox are few examples of the diseases that cause more severe clinical illness in adults than in young children. This may be due to more active immune response in an adult causing greater tissue damage. Nutritional status: Nutritional status of the host plays an important role in innate immunity. Both humoral and cellmediated immunities are lowered in malnutrition. Examples are: ■ ■ Neutrophil activity is reduced, interferon response is decreased, and C3 and factor B of the complement are decreased in protein–calorie malnutrition. Deficiency of vitamin A, vitamin C, and folic acid makes an individual highly susceptible to infection by many microbial pathogens. Hormonal levels: Individuals with certain hormonal disorders become increasingly susceptible to infection. For example, individuals suffering from diabetes mellitus, hypothyroidism, and adrenal dysfunction are increasingly susceptible to staphylococcal infection, streptococcal infection, candidiasis, aspergillosis, zygomycosis and many other microbial infections. Similarly, pregnant women are more susceptible to many infections due to higher level of steroid during pregnancy. ◗ Mechanisms of innate immunity Innate immunity of the host performs two most important functions: it kills invading microbes and it activates acquired (adaptive) immune processes. Innate immunity unlike adaptive immunity, however, does not have any memory and does not improve after re-exposure to the same microorganism. The innate immunity is primarily dependent on four types of defensive barriers: (a) anatomic barriers, (b) physiologic barriers, (c) phagocytosis, and (d) inflammatory responses. Anatomic barriers: Anatomic barriers include skin and mucous membrane. They are the most important components of innate immunity. They act as mechanical barriers and prevent entry of microorganisms into the body. The intact skin prevents entry of microorganisms. For example, breaks in the skin due to scratches, wounds, or abrasion cause infection. Bites of insects harboring pathogenic organisms (e.g., mosquitoes, mites, ticks, fleas, and sandflies), introduce the pathogens into the body and transmit the infection. Skin secretes sebum, which prevents growth of many microorganisms. The sebum consists of lactic acid and fatty acids that maintain the pH of skin between 3 and 5, and this pH inhibits the growth of most microorganisms. Mucous membranes form a large part of outer covering of gastrointestinal, respiratory, genitourinary, and many other tracts of human host. A number of nonspecific defense mechanisms act to prevent entry of microorganisms through mucous membrane. ■ ■ ■ ■ Saliva, tears, and mucous secretions tend to wash away potential invading microorganisms, thereby preventing their attachment to the initial site of infections. These secretions also contain antibacterial or antiviral substances that kill these pathogens. Mucus is a viscous fluid secreted by the epithelial cells of mucous membranes that entraps invading microorganisms. In lower respiratory tract, mucous membrane is covered by cilia, the hair-like protrusions of the epithelial cell membranes. The synchronous movement of cilia propels mucusentrapped microorganisms from these tracts. In addition, nonpathogenic organisms tend to colonize the epithelial cells of mucosal surfaces. These normal flora generally compete with pathogens for attachment sites on the epithelial cell surface and for necessary nutrients. Physiologic barriers: The physiologic barriers that contribute to innate immunity include the following: ■ ■ ■ Gastric acidity is an innate physiologic barrier to infection because very few ingested microorganisms can survive the low pH of stomach contents. Lysozyme, interferon, and complement are some of the soluble mediators of innate immunity. Lysozyme has antibacterial effect due to its action on the bacterial cell wall. Interferons are secreted by cells in response to products of viral infected cells. These substances have a general antiviral effect by preventing the synthesis of viral structural proteins. Complement is a group of serum-soluble substances that when activated damage the cell membrane. There are certain types of molecules that are unique to microbes and are never found in multicellular organisms. The ability of the host to immediately recognize and combat invaders displaying such molecules is a strong feature of innate immunity. IMMUNITY ■ Mediators of inflammatory reactions: Histamine, kinins, acutephase proteins, and defensin are the important mediators of inflammatory reactions. ■ ■ Histamine: It is a chemical substance produced by a variety of cells in response to tissue injury. It is one of the principal mediators of the inflammatory response. It binds to receptors on nearby capillaries and venules, causing vasodilatation and increased permeability. Kinins: These are other important mediators of inflammatory response. They are normally present in blood plasma in an inactive form. Tissue injury activates these small peptides, which then cause vasodilatation and increased TABLE 11-1 Feature Adaptive (Acquired) Immunity Adaptive immunity is also called acquired immunity, since the potency of immune response is acquired by experience only. Differences between innate and acquired immunity are summarized in Table 11-1. Differences between innate and acquired immunity Innate immunity Acquired immunity Definition The resistance to infection that an individual possesses by virtue of genetic and constitutional makeup The resistance that an individual acquires during life Types Nonspecific and specific Active and passive Time taken to develop Hours Days Specificity For structures shared by groups of related microbes For antigens of microbes and for nonmicrobial antigens Memory None; repeated exposure brings response like primary response Yes; secondary response much faster than primary response Physical and chemical barriers Skin, mucosal epithelia, and antimicrobial chemicals Lymphocytes in epithelia and antibodies secreted at epithelial surfaces Blood and tissue antimicrobial substances Complement; leukins from leukocytes, plakins from platelets, lactic acid found in muscle tissue, lactoperoxidase in milk, and interferons (antiviral) Antibodies Cells Phagocytes (macrophages and neutrophils) and natural killer cells Lymphocytes Components Chapter 11 Inflammatory responses: Tissue damage caused by a wound or by an invading pathogenic microorganism induces a complex sequence of events, collectively known as the inflammatory responses. The end result of inflammation may be the activation of a specific immune response to the invasion or clearance of the invader by components of the innate immune system. The four cardinal features of inflammatory responses are rubor (redness), calor (rise in temperature), dolor (pain), and tumor (swelling). ■ permeability of capillaries. Bradykinin also stimulates pain receptors in the skin. This effect probably serves a protective role because pain normally causes an individual to protect the injured area. Acute-phase proteins: These include C-reactive proteins and mannose-binding proteins that form part of the innate immunity. These proteins are produced at an increased concentration in plasma during acute-phase reaction, as a nonspecific response to microorganisms and other forms of tissue injury. They are synthesized in the liver in response to cytokines called proinflammatory cytokines, namely, interleukin-1 (IL-1), interleukin-6 (IL6), and tissue necrosis factor (TNF). They are called proinflammatory cytokines because they enhance the inflammatory responses. Defensins: They are another important component of the innate immunity. They are cationic peptides that produce pores in membrane of the bacteria and thereby kill them. These are present mainly in the lower respiratory tract and gastrointestinal tract. The respiratory tract contains ␤-defensins, whereas the gastrointestinal tract contains ␣-defensins. The ␣-defensins also exhibit antiviral activity. They bind to the CXCR4 receptors and block entry of HIV virus into the cell. How these defensins differentiate microbes from some cells is not known. Section II Phagocytosis: Phagocytosis is another important defense mechanism of the innate immunity. Phagocytosis is a process of ingestion of extracellular particulate material by certain specialized cells, such as blood monocytes, neutrophils, and tissue macrophages. It is a type of endocytosis in which invading microorganisms present in the environment are ingested by the phagocytic cells. In this process, plasma membrane of the cell expands around the particulate material, which may include whole pathogenic microorganisms to form large vesicles called phagosomes. 87 88 IMMUNOLOGY Key Points Adaptive immunity shows the following features: ■ ■ ■ Chapter 11 Section II ■ ■ ◗ It is the resistance acquired by an individual during life. It occurs after exposure to an agent and is mediated by antibodies as well as T lymphocytes (helper T cells and cytotoxic T cells). It has immunologic memory and a remarkable capability of discriminating between self and nonself antigens. Once an antigen has been recognized by the cells of acquired immune system, the response to it is specific and can be repeated. In most cases, the acquired immune response improves with repeated exposure. The immune response to the second challenge occurs more quickly than the first, is stronger, and is often more effective in neutralizing and clearing the pathogen. Types of acquired immunity Acquired immunity against a microbe may be induced by the host’s response to the microbe or by transfer of antibodies or lymphocytes specific for the microbes. It is of two types: active immunity and passive immunity. Active immunity The immunity induced by exposure to a foreign antigen is called active immunity. Active immunity is the resistance developed by an individual after contact with foreign antigens, e.g., microorganisms. This contact may be in the form of: ■ ■ ■ clinical or subclinical infection, immunization with live or killed infectious agents or their antigens, or exposure to microbial products, such as toxins and toxoids. In all these circumstances, the immune system of the host is stimulated to elicit an immune response consisting of antibodies and activated helper T (TH) cells and cytotoxic T lymphocytes/cells (CTLs). Active immunity develops after a latent period, during which immunity of the host is geared up to act against the microorganism. Hence it is slow in onset, especially during this primary response. However, once the active immunity develops, it is long-lasting and this is the major advantage of the active immunity. The active immunity is of two types: natural active immunity and artificial active immunity. ■ ■ Natural active immunity: It is acquired by natural clinical or subclinical infections. Such natural immunity is longlasting. For example, individuals suffering from smallpox become immune to second attack of the disease. Artificial active immunity: It is induced in individuals by vaccines. There is a wide range of vaccines available against many microbial pathogens. These may be live vaccines, killed vaccines, or vaccines containing bacterial products (Table 11-2). Mediators of active immunity: Active immunity is mediated by humoral immunity and cell-mediated immunity. These two TABLE 11-2 Differences between cell-mediated and humoral immunity Cell-mediated immunity Humoral immunity Immune response mediated by cells Immune response mediated by antibodies Protects against fungi, viruses, and facultative intracellular bacterial pathogens Protects against extracellular bacterial pathogens and viruses infecting respiratory or intestinal tract; and prevents recurrence of viral infections Mediates delayed (type IV) hypersensitivity Mediates immediate (types I, II, and III) hypersensitivity Only T-cell-dependent antigens lead to cellmediated immunity B cells directly bind soluble antigens resulting in production of antibodies Both CD4⫹ and CD8⫹ T cells are involved Only TH cells are involved Provides immunological surveillance and immunity against cancer No major role in immunological surveillance Participates in rejection of homografts and graftversus-host reaction May be involved in early graft rejection due to preformed antibodies types of immunities are mediated by different components of the immune system and function in different ways to kill different types of pathogens. ■ ■ Humoral immunity: It is mediated by molecules in the blood and mucosal secretions called antibodies. The antibodies are secreted by a subset of lymphocytes known as B cells. The antibodies recognize microbial antigens, combine specifically with the antigens, neutralize the infectivity of microbes, and target microbes for elimination by various effector mechanisms. Humoral immunity is the principal defense mechanism against extracellular microbes. Cell-mediated immunity: It is mediated by both activated TH cells and CTLs. Cytokines secreted by TH cells activate various phagocytic cells, enabling them to phagocytose and kill microorganisms. This type of cell-mediated immune response is especially important against a host of bacterial and protozoal pathogens. CTLs play an important role in killing virus-infected cells and tumor cells. They act by killing altered self-cells. Differences between humoral and cell-mediated immunities are summarized in Table 11-2. Antigen recognition: Antigens, which are generally very large and complex, are not recognized in their entirety by lymphocytes. Instead, both B and T lymphocytes recognize discrete sites on the antigens called antigenic determinants, or epitopes. Epitopes are the immunologically active regions on a complex antigen, the regions that actually bind to B-cell or T-cell receptors. IMMUNITY When immunity is conferred by transfer of serum or lymphocytes from a specifically immunized individual, it is known as passive immunity. This is a useful method for conferring resistance rapidly, i.e., without waiting for the development of an active immune response. Passive immunity may be natural or artificial. Natural passive immunity: It is observed when IgG is passed from mother to fetus during pregnancy. This forms the basis of prevention of neonatal tetanus in neonates by active immunization of pregnant mothers. It is achieved by administering tetanus toxoid to pregnant mothers during the last trimester of pregnancy. This induces production of high level of antibodies in mother against tetanus toxin, which are subsequently transmitted from mother to fetus through placenta. The antibodies subsequently protect neonates after birth against the risk of tetanus. Natural passive immunity is also observed by passage of IgA from mother to newborn during breast feeding. Artificial passive immunity: It is induced in an individual by administration of preformed antibodies, generally in the form of antiserum, raised against an infecting agent. Administration of these antisera makes large amounts of antibodies available in the recipient host to neutralize the action of toxins. The preformed antibodies against rabies and hepatitis A and B viruses, etc. given during incubation period prevent replication of virus, and hence alter the course of infection. Immediate availability of large amount of antibodies is the main advantage of passive immunity. However, short lifespan of these antibodies and the possibility of hypersensitivity reaction, if antibodies prepared in other animal species are given to individuals who are hypersensitive to these animal globulins (e.g., serum sickness), are the two noted disadvantages of passive immunity. Differences between passive and active immunity Passive immunity Active immunity No active host participation; received passively Produced actively by host’s immune system Antibodies transferred directly Antibodies induced by infection or by immunogens Passive immunity is due to readymade antibodies Active immunity often involves both the cell-mediated and humoral immunity Types: Natural—transfer of maternal antibodies through placenta; Artificial—injection of immunoglobulins Types: Natural—clinical or inapparent infection; Artificial— induced by vaccines Immediate immunity; no lag period Immunity effective only after lag period Transient; less effective Durable; effective protection No immunological memory Immunological memory present Subsequent dose less effective due to immune elimination Booster effect on subsequent dose No negative phase Negative phase may occur Applicable even in immunodeficient Not applicable in immunodeficient Differences between active and passive immunity are summarized in Table 11-3. Combined passive–active immunity is carried out by giving both preformed antibodies (antiserum) and a vaccine to provide immediate protection and long-term protection, respectively, against a disease. This approach is followed for prevention of certain infectious conditions, namely, tetanus, rabies, and hepatitis B. Local Immunity The immunity at a particular site, generally at the site of invasion and multiplication of a pathogen, is referred to as local immunity. Local immunity is conferred by secretory IgA antibodies in various body secretions. These antibodies are produced locally by plasma cells present on mucosal surfaces or in secretory glands. Natural infection or attenuated live viral vaccines given orally or intranasally induces local immunity at gut mucosa and nasal mucosa, respectively. Herd Immunity Herd immunity refers to an overall level of immunity in a community. Eradication of an infectious disease depends on the development of a high level of herd immunity against the pathogen. Epidemic of a disease is likely to occur when herd immunity against that disease is very low indicating the presence of a larger number of susceptible people in the community. Chapter 11 Passive immunity TABLE 11-3 Section II B cells and T cells differ in their mechanisms of antigen recognition. While B cells recognize the antigen by interacting with the epitope on their own, T cells recognize the antigen only when the epitope is “presented” by one of the specialized antigen-presenting cells. Once the antigen has been recognized, these cells then go on to diversify by several intricate mechanisms. This diversification helps in conferring the specificity, one of the cardinal characteristics of the immune system. Major histocompatibility complex (MHC): It is a large genetic complex with multiple loci. The MHC loci encode two major classes of membrane-bound glycoproteins: class I and class II MHC molecules. Class II molecules present antigens to the TH cells, while class I molecules do the same for CTLs. In order for a foreign protein antigen to be recognized by a T cell, it must be degraded into small antigenic peptides that form complexes with class I or class II MHC molecules. This conversion of proteins into MHC-associated peptide fragments is called antigen processing and presentation. 89 12 Antigen Introduction Molecules that can be recognized by the immunoglobulin receptor of B cells or by the T-cell receptor when complexed with major histocompatibility complex (MHC) are called antigens. The word antigen is a shortened form of the words “antibody generator.” Antigens are substances that react with antibodies, while immunogens are molecules that induce an immune response. In most cases, antigens are immunogens, and the terms are used interchangeably. The antigens that are not immunogenic but can take part in immune reactions are termed as haptens. The term immunogenicity means the ability of an antigen to elicit an immune reaction in the form of a B-cell or T-cell response, whereas the term antigenicity means just the ability to combine specifically with the products of the above responses. All molecules that are immunogenic are antigenic too, but all antigenic molecules cannot be considered immunogenic. Thus, haptens can be said to lack immunogenicity. Foreignness To be immunogenic, a molecule must be recognized as nonself, i.e., foreign. The molecule is considered self or nonself by the immune system depending on whether or not the molecule was exposed to the immune system during fetal development. Foreignness implies ability of the host to tolerate selfantigens. Tolerance to self-antigens develops by contact with them in the initial phases of the development of immune system, particularly during the development of lymphocytes. In general, the more distantly related two species are, the greater the immunogenicity of a molecule from one species will be when exposed to the other. For example, the bovine serum albumin is more immunogenic in a chicken than in a goat. A graft from an unrelated human will be rejected within about 2 weeks unless immunosuppressive drugs are used, but a graft from a chimpanzee will be rejected within hours even if drugs are used. In contrast, a kidney graft from an identical twin will be accepted readily. Chemical-Structural Complexity Determinants of Antigenicity A number of factors have been identified that make a substance immunogenic. Some of the important determinants of antigenicity include: 1. 2. 3. 4. 5. Molecular size Foreignness Chemical-structural complexity Stability Other factors Molecular Size In general, protein molecules with large molecular weight are highly antigenic. Substances with molecular weights of about 100,000 Da and more are highly immunogenic, while substances with molecular weights of less than 5000 Da are generally not immunogenic. This property has been exploited in experimental studies by using high molecular weight proteins like bovine gamma globulin (MW 150,000 Da) to induce an immune reaction. Substances with low molecular weight may be made antigenic by adsorbing these on carrier particles, such as bentonite, kaolin, and other inert particles. Proteins are the most potent immunogens followed by polysaccharides. Nucleic acids and lipids are not efficient in eliciting a good immune reaction, although they may act as haptens. Structural complexity of a protein contributes to its immunogenicity. Chains of single amino acids or single sugars are poorly immunogenic, but if different amino acids or sugars are combined in the same molecule, the immunogenicity is greatly enhanced. In cell-mediated immunity, the response of T cells to the peptide component of the proteins depends on how the peptide is recognized and presented by the MHC cells. Therefore, the structure of protein plays an important role in its immunogenicity, especially in inducing cellular immunity. The lipid-specific antibodies are not easily produced; hence, they do not play a major role in immunity. However, these antibodies have a role in the measurement of certain lipid-based molecules and drugs. These antibodies are produced first by treating lipids with haptens and then conjugating with suitable carrier molecules, such as the proteins (e.g., hemocyanin or bovine serum albumin). Stability Highly stable and nondegradable substances (e.g., some plastics, metals, or chains of D-amino acids) are not immunogenic. ANTIGEN Other Factors ◗ Adjuvants Adjuvants are the substances that when mixed with an antigen and injected with it boost the immunogenicity of the antigen. Adjuvants increase both the strength and the duration of immune response. Adjuvants boost immunogenicity of antigens in several ways: ■ Adjuvants like aluminum potassium sulfate (alum) and Freund’s water-in-oil adjuvant prolong the persistence of antigen by forming a depot at the injection site. Alum Antigenic Specificity Antigenic specificity of the antigen depends on antigenic determinants or epitopes. Epitopes An epitope is defined as the immunologically active region of an immunogen that binds to antigen-specific membrane receptors on lymphocytes or secreted antibodies. The interaction between cells of the immune system and antigens takes place at many levels and the complexity of any antigen is mirrored by its epitope. There are two types of epitopes: B-cell epitopes and T-cell epitopes. ◗ B-cell epitopes B-cell epitopes are antigenic determinants recognized by B cells. B-cell epitope can combine with its receptor only if the antigen molecule is in its native state. The complementary surfaces of the antibody and the antigen molecules appear to be relatively flat. Smaller molecules often fit nicely within a particular depression or groove in the antigen-binding site of the antibody molecule. The B-cell epitope is about six or seven sugar residues or amino acids long. B-cell epitopes tend to be hydrophilic and are often located at bends in the protein structure. They are also often found in regions of proteins, which have a higher mobility; this may make it possible for an epitope to shift just a bit to fit into an almost-right site. ◗ T-cell epitopes T cells recognize amino acids in proteins but do not recognize polysaccharide or nucleic acid antigens. This is the reason why polysaccharides are considered as T-independent antigens and proteins as T-dependent antigens. The primary sequence of amino acids in proteins determines the antigenic determinants recognized by T cells. Free peptides are not recognized by T cells, while the complex of MHC molecules and peptide are recognized by T cells. Thus for a T-cell response, it should Chapter 12 Dosage and route of the antigen The dose of antigen and the route by which it comes into contact with the immune system also influence immunogenicity of the antigen. Very low doses of antigen do not stimulate immune response, either because too few lymphocytes are contacted or because a nonresponsive state is elicited. Conversely, an extremely high dose also fails to elicit tolerance. Repeated administration of antigens (booster doses) may be required to enhance immune response of the host to certain antigens. This is particularly important in case of vaccines where a prerequisite immune level needs to be attained. Hence the booster doses of vaccines, such as DPT (Diphtheria, Pertussis, Tetanus), DT (Diphtheria, Tetanus), etc., are given to ensure good protective levels of antibodies. Generally, antigens are administered by the parenteral route to produce good level of antibodies. The antigens can be given by (a) intravenous, (b) subcutaneous, (c) intradermal, (d) intramuscular, (e) intraperitoneal, and (f) mucosal routes. Usually, the subcutaneous route of administration proves to be better than intravenous routes at eliciting an immune response. ◗ ■ Biological system Biological system also plays an important role in determining the immunological efficiency of an antigen. Some substances are immunogenic in one individual but not in others (i.e., responders and nonresponders). This is due to the fact that individuals may lack or have altered genes that code for the receptors for antigen on B cells and T cells, or they may not have the appropriate genes needed for the APC to present antigen to the helper T (TH) cells. ◗ ■ precipitates the antigen and releases it a little at a time. The water-in-oil emulsion forms small droplets with the antigen and also releases these slowly over time. Freund’s complete adjuvant contains, in addition to the emulsifying factors, heat-killed mycobacteria. The bacterial components activate macrophages and increase both the production of IL-1 and the level of B7 membrane molecules, which enhances the immune response. The increased expression of class II MHC increases the ability of APC to present antigen to TH cells. B7 molecules on the APC bind to CD28, a cell-surface protein on TH cells, triggering costimulation, an enhancement of the T-cell immune response. Some adjuvants, like synthetic polyribonucleotides and bacterial lipopolysaccharides, stimulate nonspecific lymphocyte proliferation and bring about their action. Section II This is because internalization, processing, and presentation by antigen-presenting cells (APCs) are always essential to mount an immune response. Therefore, very stable substances (such as silicon) have been successful as nonimmunogenic materials for reconstructive surgeries, such as breast implants. On the other hand, if a substance is very unstable, it may break up before an APC can be internalized, and hence become immunogenic. In addition, large, insoluble complexes are more immunogenic than smaller, soluble ones. This is because macrophages find it easier to phagocytose, degrade, and present the insoluble complexes than the soluble complexes. 91 IMMUNOLOGY recognize both the antigenic determinant and also the MHC, and therefore it is said to be MHC restricted. In general, T-cell epitopes or antigenic determinants are small and are only 8–15 amino acids long. The antigenic determinants are limited to those parts of the antigen that can bind to MHC molecules. Since the MHC molecules are subjected to genetic variability, there can be difference among individuals in their T-cell response to the same stimulus. Each MHC molecule can bind several, but not all, peptides. Therefore, for a peptide to be immunogenic in a particular individual, that individual must have MHC molecules that can bind to it. Key Points Processing of an antigen by APCs is absolutely essential for a T cell to recognize it. Two different types of processing can prepare a protein antigen for antigen presentation. These include: Chapter 12 Section II 92 ■ ■ Externally derived antigens’ processing: In this process, phagocytosed bacteria are killed and lysed by phagocytic cells, such as macrophages. Pieces of the bacteria are then processed and presented in the context of class II MHC molecules. Endogenously derived antigens’ processing: In this process, virus proteins synthesized in a cell are processed and then presented in the context of class I MHC molecules. ■ Providing valuable evidence in paternity disputes, the results of which are supplemented by m ore recent DNA fingerprinting tests. Histocompatibility Antigens Histocompatibility antigens are the cellular determinants specific for each individual of a species. These antigens are associated with the plasma membrane of tissue cells. Human leukocyte antigen (HLA) is the major histocompatibility antigen that determines the homograft rejection. Therefore, HLA typing is absolutely essential before carrying out transplantation of tissue or organ from one individual to another. Autospecificity Self-antigens are generally nonantigenic. Sequestrated antigens (such as eye lens protein and sperm) are, however, exceptions, because these are not recognized as selfantigens. This is because corneal tissue and sperm are never encountered by the immune system during the development of tolerance to self-antigens. Therefore, these tissues become immunogenic if accidentally or experimentally released into the blood or tissues. Species Specificity Tissues of all individuals in a species possess certain speciesspecific antigens. However, some degree of cross-reaction occurs between antigens from related species. The species specificity shows phylogenetic relationship. The phylogenetic relationship is useful in: ■ ■ Tracing evolutionary relationship between species. The species identification from blood and seminal stains in forensic medicine. Isospecificity Organ Specificity Antigens characteristic of an organ or tissues are called organ-specific antigens. These antigens found in the brain, kidney, and lens tissues, even of different animal species, share the same antigen specificity. Organ-specific antigens, such as brain-specific antigens, shared by human and sheep brain are one such example. The antirabies vaccines prepared from sheep brain, when given, may induce immune response in some humans, causing damage to neural tissues of the recipient. This may result in neuroparalytic complications in some individuals. Isospecificity is determined by the presence of isoantigens or histocompatibility antigens. Isoantigens Isoantigens are antigens found in some, but not all, members of a species. A species may be grouped depending on the presence of different isoantigens in its members. These are genetically determined. Human erythrocyte antigens, based on which individuals are classified into different blood groups, are the best examples of isoantigens in humans. The blood groups are of primary importance in: ■ ■ Transfusion of blood and blood products, Isoimmunization during pregnancy, and Heterophile Specificity Heterophile specificity is determined by the presence of heterophile antigens. The same or closely related antigens, sometimes present in tissues of different biological species, classes, or kingdoms are known as heterophile antigens. Antibodies against the heterophile antigens produced by one of the species cross-react with the antigens of other species. This property is exploited for diagnosis of many infectious diseases. Weil–Felix reaction, Paul-Bunnell test, and cold agglutination tests are the examples of serological tests that use such heterophile antigens. ANTIGEN 93 Key Points ■ ■ Haptens Hapten molecule FIG. 12-1. Carrier molecule Complete antigen Hapten–carrier conjugate. epitopes on the carrier protein, and (c) new epitopes formed by combined parts of both the hapten and carrier. In fact, the hapten–carrier molecule is bound to surface immunoglobulins on B cells via the hapten epitopes. The hapten–carrier molecule is then taken in, processed, and pieces of the carrier are presented by these B cells and TH cells. In the body, the formation of hapten–carrier conjugates is the basis for development of allergic responses to drugs, such as penicillin. Superantigens Superantigens are a class of molecules that can interact with APCs and T lymphocytes in a nonspecific way. The superantigens act differently by interacting with MHC class II molecules of the APC and the Vb domain of the T-lymphocyte receptor. This interaction results in the activation of a larger number of T cells (10%) than conventional antigens (1%), leading to massive cytokine expression and immunomodulation. Examples of superantigens are staphylococcal enterotoxins, toxic shock syndrome toxin, exfoliative toxins, and also some viral proteins. Chapter 12 Haptens are small organic molecules that are antigenic but not immunogenic. They are not immunogenic because they cannot activate helper T cells. Failure of hapten to activate helper T cells is due to their inability to bind to MHC proteins; they cannot bind because they are not proteins and only proteins can be presented by MHC proteins. Moreover, haptens are univalent hence cannot activate B cells by themselves. The haptens, however, can activate B cells when covalently bound to a “carrier” protein. When bound with a carrier molecule, they form an immunogenic hapten–carrier conjugate (Fig. 12-1). In this process, the haptens combine with an IgM receptor on the B cells, and the hapten–carrier protein complex is internalized. A peptide of the carrier protein is presented in association with class II MHC protein to the helper T cells. The activated helper T cells then produce interleukins, which stimulate the B cells to produce antibodies to hapten. Animals immunized with such a conjugate produce antibodies specific for (a) the hapten determinant, (b) unaltered + Section II ■ Weil–Felix reaction is a test used for diagnosis of rickettsial infections, in which the strains of Proteus species (such as OX 19, OX 2, and OX K) are used to detect heterophile antibodies produced against rickettsial pathogens. Paul–Bunnell test is used for diagnosis of infectious mononucleosis caused by Epstein–Barr virus infection by demonstration of heterophile antibodies that agglutinate sheep erythrocytes. Cold agglutinin test is performed for diagnosis of primary atypical pneumonia caused by Mycoplasma pneumoniae by demonstration of heterophilic antibodies. 13 Antibodies Introduction Antibodies are globulin proteins (immunoglobulins) that are synthesized in serum and tissue fluids, which react specifically with the antigen that stimulated their production. Three types of globulins are present in the blood: alpha, beta, and gamma. The antibodies are the gamma globulins. Antibodies are one of the major plasma proteins, and against infection often referred to as “first line of defense”. The most important function of antibodies is to confer protection against microbial pathogens. Antibodies confer protection in the following ways: 1. They prevent attachment of microbes to mucosal surfaces of the host. 2. They reduce virulence of microbes by neutralizing toxins and viruses. 3. They facilitate phagocytosis by opsonization of microbes. 4. They activate complement, leading to complement-mediated activities against microbes. Von Behring and Kitasato performed the first experiments that proved the physical existence of antibodies in 1890. They demonstrated that serum obtained from rabbits immunized with tetanus or diphtheria toxins could prevent disease in mice infected with such pathogens. The unknown substance that was present in serum and that provided protection on transfer was named “antitoxin” by Tizzoni and Cattani in 1891. Subsequently, experimental works by Paul Ehrlich and Jules Bordet demonstrated that a protective response could be generated even against whole cells (erythrocytes). The more inclusive term antibody subsequently replaced the term antitoxin. Tiselius and Kabat accomplished the first successful attempt to identify antibody molecules in 1939. They demonstrated that hyperimmunization increased the concentration of -globulins in serum and that this fraction contained antibody activity. Because -globulins are large-molecular-weight proteins, it was suggested that further characterization of antibodies requires breaking them into smaller and easily handled fragments. Porter in 1959, succeeded in digesting rabbit immunoglobulin G (IgG) with the proteolytic enzyme papain. These produced two distinct fragments: a monovalent fragment with antigenbinding activity, termed Fab (fragment antigen binding) and a second fragment that retained the antibody’s effector functions and crystallized readily into a lattice, termed Fc (fragment crystallizable). Edelman and Poulik using a similar method splitted myeloma globulins into two distinct components, which subsequently were termed heavy (H) and light (L) chains. The World Health Organization (WHO) in 1964 coined the term “immunoglobulin (Ig)” for the term antibody. The immunoglobulin includes not only antibody globulins but also the cryoglobulins, macroglobulins, and abnormal myeloma proteins. Thus, all antibodies are immunoglobulins but not all immunoglobulins may be antibodies. Immunoglobulins There are five classes of immunoglobulins: (i) immunoglobulin G (IgG), (ii) immunoglobulin M (IgM), (iii) immunoglobulin A (IgA), (iv) immunoglobulin E (IgE), and (v) immunoglobulin D (IgD). Myeloma proteins were first used for the amino acid sequencing of immunoglobulins. These proteins were also the first immunoglobulins that were subjected to crystallographic studies. They provided the first glimpses of the domain structure of the prototypic immunoglobulin. Structure of Immunoglobulins Immunoglobulins show the following properties: ■ ■ ◗ They are glycoproteins. They are a complex structure of four polypeptide chains: two identical heavy (typically 55 kDa each) chains and two identical light chains (25 kDa each). This gives immunoglobulin an overall ‘Y’ or ‘T’ shape, which is the most widely recognized feature of immunoglobulin structure. ● The terms “heavy” and “light” refer to the molecular weights of the chains. The heavy chains have a molecular weight of 50,000–70,000 Da, while light chains have a molecular weight of 25,000 Da. The heavy chains are longer, and light chains are shorter (Fig. 13-1). Heavy chains An immunoglobulin molecule has two heavy chains. Each heavy chain is made up of 420–440 amino acids. The two heavy chains are held together by one to five disulfide (S—S) bonds. Each heavy chain is bound to a light chain by a disulfide bond and by noncovalent bonds, such as salt linkages, hydrogen bonds, and hydrophobic bonds to form a heterodimer (H–L). Similar noncovalent interactions and disulfide bridges link the two identical heavy and light (H–L) chains to each other to form the basic four-chain (H–L)2 antibody structure. ANTIBODIES Heavy chain NH s NH H 3+ L V H1 CH 3 COO− L s C CHO COO− Schematic diagram of monomer of the immunoglobulin. Classes of immunoglobulins and their heavy chains and subclasses Class Heavy chain Subclasses IgG Gamma 1, 2, 3, 4 IgM Mu None IgA Alpha 1, 2 IgE Epsilon None IgD Delta None The heavy chains of a given antibody molecule determine the class of that antibody. For example, IgM contains mu (), IgG contains gamma (), IgA contains alpha (), IgD contains delta (), and IgE contains epsilon () heavy chains (Table 13-1). These heavy chains are structurally and antigenically distinct for each class of immunoglobulin. They differ in their size, carbohydrate content, and as antigens. ◗ Light chains An immunoglobulin molecule has two light chains. Each light chain is made up of 220–240 amino acids. Light chain is attached to the heavy chain by a disulfide bond. The light chains are structurally and chemically similar in all classes of immunoglobulins. They are of two types: kappa () and lambda (). These two types differ in their amino acids present in constant regions. Each immunoglobulin has either two  or two  chains but never both. The  and  chains are present in human serum in a ratio of 2:1. ◗ Variable and constant regions Each polypeptide chain of an immunoglobulin molecule contains an amino terminal part and a carboxy terminal part. The amino terminal part is called the variable region (V region) and the carboxy terminal part is called the constant region (C region). Both heavy and light chains contain variable and constant regions. These regions are composed of three-dimensional folded structures with repeating segments, which are called domains. Each heavy chain consists of one variable (VH) and Treatment of Immunoglobulins with Proteolytic Enzymes The immunoglobulin molecule can be broken into a number of “sections” or “fragments” by action of proteolytic enzymes. The proteolytic enzyme papain cleaves just above the interchain disulfide bonds linking the heavy chains, whereas the enzyme pepsin cleaves just below these bonds, thereby generating different digestion products. For example, peptide bonds in the “hinge” region are broken on treatment of antibody molecule with papain, resulting in production of two identical Fab fragments and one Fc fragment. The Fab fragments produced during cleavage monovalently bind to the antigen. Treatment with pepsin cleaves immunoglobulin but at a different site, producing an Fc fragment and two Fab fragments, F (ab)2, which upon exposure to reducing conditions are separated into Fab monomeric units. Immunoglobulin Antigen Determinants There are three major types of immunoglobulin antigen determinants: isotypes, allotypes, and idiotypes. Chapter 13 TABLE 13-1 three constant (CH) domains. IgG and IgA have three CH domains (CH1, CH2, and CH3), whereas IgM and IgE have four domains (CH1, CH2, CH3, and CH4). Each light chain consists of one variable (VL) and one constant domain (CL). Variable region: The amino-terminal half of the light or heavy chain, consisting of 100–110 amino acids, is known as variable or V regions (VL in light chains and VH in heavy chains). V region is different for each class of immunoglobulin. The variable regions of both light and heavy chains consist of three highly variable regions known as hypervariable regions. The antigen combining sites Fab of the antibody molecule that consists of only 5–10 amino acids each are present in the hypervariable region of both the light and heavy chains. These antigen-binding sites are responsible for specific binding of antibodies with antigens. The high specificity of antibodies is primarily due to the presence of these hypervariable regions. Constant region: The carboxyl-terminal half of the molecule is called the constant (C) region. It consists of two basic amino acid sequences. The Fc fragment, found to crystallize under low ionic conditions, is present in the constant region of heavy chain. The constant region of the heavy chain has many biological functions. It is responsible for activation of the complement, binding to cell surface receptors, placental transfer, and many other biological activities. The constant region of the light chain has no biological function. A single antibody molecule has two identical heavy chains and two identical light chains, H2L2, or a multiple (H2L2)n of this basic four-chain structure. Subisotypes exist for  and  chains, and this leads to the existence of subclasses of the respective immunoglobulins. Section II CH2 CH 2 s s s s s s s s s C ss CH4 CL s μ,γ, α,δ, or ε s s 1 CH s κ or λ s s s s s s s s s VL s ss CHO FIG. 13-1. 3+ V s s VH NH + 3 + 3 s NH Light chain 95 96 ◗ IMMUNOLOGY Isotypes Chapter 13 Section II The isotype of an immunoglobulin refers to the particular constant region of the light- or heavy-chain of the immunoglobulin. Immunoglobulins are classified on the basis of various heavy chain isotypes. Heavy chains are distinguished by the presence of heavy chain markers, such as , , , , and  in the immunoglobulins IgM, IgG, IgA, IgD, and IgE, respectively. The light chains are also distinguished by isotype markers, such as  and . Isotypes are present in all members of a species. ◗ Allotypes The allotype refers to allelic differences in both the variable and constant regions of immunoglobulin. The allotype markers are present on the constant regions of light and heavy chains. They are Am on  heavy chains, Gm on  heavy chains, and Km on  light chains. Allotype markers are absent on , , and  heavy chains and on  light chains. More than 25 Gm types, 3 Km allotypes, and 2 Am on IgA have been described. Allotypes are present in some but not all members of a species and are inherited in a simple Mendelian fashion. ◗ Idiotypes The idiotype refers to a specificity that is associated with the variable region. Idiotype markers are found on the hypervariable region of the immunoglobulin. Idiotypes are specific for each antibody molecule. Anti-idiotypic antibodies produced against Fab fragments prevent antigen–antibody interaction. Biosynthesis of Immunoglobulins B lymphocytes and plasma cells take part in the synthesis of immunoglobulins. Resting B cells synthesize only small amounts of immunoglobulins that mainly get incorporated into cell membranes. Plasma cells, the most differentiated B cells, are specialized to produce and secrete large amounts of immunoglobulins. The synthetic capacity of the plasma cells is reflected by the abundant cytoplasm, which is extremely rich in endoplasmic reticulum. Normally, heavy and light chains are synthesized in separate polyribosomes of the plasma cell. The amounts of heavy and light chains synthesized on the polyribosomes are usually balanced and so both types of chains are combined to produce complete Ig molecules, without excess of any given chain. The assembly of a complete Ig molecule is carried out either by associating one heavy and one light chain to form an H–L hemimolecule, and then joining two H–L hemi-molecules to form a single complete molecule (H2L2), or by forming H2 and L2 dimers that later associate to form the complete molecule. While free light chains can be effectively secreted from plasma cells, free heavy chains are generally not secreted. The heavy chains are synthesized and transported to the endoplasmic reticulum, where they are glycosylated, but secretion requires combination with light chains to form a complete immunoglobulin molecule. If light chains are not synthesized or heavy chains are synthesized in excess, the free heavy chains combine through their CH1 domain with a heavy-chainbinding protein, which is believed to be responsible for their intracytoplasmic retention. Both IgM and IgA are the polymeric antibodies, which have one additional polypeptide chain, the J chain. The J chain is synthesized by all plasma cells, including those that produce IgG. However, it is only incorporated to polymeric forms of IgM and IgA. It is believed that the J chain has some role in initiating polymerization. IgM proteins are assembled in two steps. First, the monomeric units are assembled. Then, five monomers and one J chain combine via covalent bonds to produce a pentameric molecule. Metabolism of Immunoglobulins Half-life (T 1/2) of immunoglobulin is one of the most commonly used parameters to assess the catabolic rate of immunoglobulins. The half-life corresponds to the time elapsed for a reduction to half of a circulating immunoglobulin concentration after equilibrium has been reached. This is usually determined by injecting an immunoglobulin labeled with a radioisotope (131I). The IgG is the immunoglobulin class with the longest halflife (average of 21 days), with the exception of IgG3. The IgG3 has a considerably shorter half-life (average of 7 days) that is nearer to that of IgA (5–6 days) and IgM (5 days). The synthesis rate of IgA1 (24 mg/kg/day) is not very different from that of IgG1 (25 mg/kg/day), but the serum concentration of IgA1 is about one-third of the IgG1 concentration. This is explained by a fractional turnover rate three-times greater for IgA1 (24%/day). The highest fractional turnover rate and shorter half-life are those of IgE (74%/day and 2.4 days, respectively). The lowest synthesis rate is that of IgE (0.002 mg/kg/day, compared to 20–60 mg/kg/day for IgG). Immunoglobulin Classes The structure and biological functions of five classes of immunoglobulins (IgG, IgM, IgA, IgE, and IgD) are described below: ◗ Immunoglobulin G IgG is a 7S immunoglobulin with a molecular weight of 150,000 Da. It has a half-life of 23 days—longest among all the immunoglobulins. Other properties of the IgG are given in Table 13-2. IgG is the most abundant class of immunoglobulins in the serum, comprising about 80% of the total serum immunoglobulin. There are four IgG subclasses IgG1, IgG2, IgG3, and IgG4—so numbered according to their decreasing concentrations in serum. Though the differences between these subclasses are minute, their functions vary as follows: 1. IgG1, IgG3, and IgG4 are special because these are the only immunoglobulins with the ability to cross the placental barrier. They play an important role in protecting the developing fetus against infections. 2. IgG3, IgG1, and IgG2, in order of their efficiency, are effective in the activation of the complement. ANTIBODIES TABLE 13-2 97 Comparison of various properties of immunoglobulins Characteristics IgG IgA IgM IgD IgE Structure Monomer Dimer Pentamer Monomer Monomer Percentage of total serum 80% 10–13% 5–8% 0.2% 0.002% Location Blood, lymph, and intestine Blood, lymph, and B cell surface Secretions B cell surface, blood, and lymph Bound to mast and basophil cell Sedimentation coefficient 7 7 19 7 8 160 900 180 190 8 12 13 12 Serum concentration (mg/mL) 12 2 1.2 0.03 0.00004 Half-life (days) 23 6–8 5 2–8 1–5 Heavy chain 1, 2, 3, 4 1, 2  Light chain  or   or   or   or   or  Complement binding Classical pathway Alternate pathway Classical pathway None None Heterologous None None None Homologous  Placental transport Present in milk Seromucous secretion Heat stability (56oC) Binding to tissue 3. IgG1 and IgG3 bind with high affinity to Fc receptors on phagocytic cells and thus mediate opsonization. IgG4 has an intermediate affinity for Fc receptors and IgG2 has an extremely low affinity. Key Points IgG shows the following biological activities: ■ Two  chains, along with two  or  light chains, joined together by disulfide bonds, comprise an IgG molecule as follows: ■ ■ ■ ■ ■ The  chain is a 51-kDa, 450-amino acid residue heavy polypeptide chain. It consists of one variable VH domain and a constant (C) region with three domains designated CH1, CH2, and CH3. The hinge region is situated between CH1 and CH2. Proteolytic enzymes, such as papain and pepsin, cleave an IgG molecule in the hinge region to produce Fab and F (ab´) 2 and Fc fragments. There are four subclasses of IgG in humans with four corresponding  chain isotypes designated -1, -2, -3, and -4. IgG1, IgG2, IgG3, and IgG4 show differences in their hinge regions and differ in the number and position of disulfide bonds that link two  chains in each IgG molecule. There is only a 5% difference in amino acid sequence among human  chain isotypes, exclusive of the hinge region. Cysteine residues, which make it possible for interheavy () chain disulfide bonds to form are found in the hinge area. IgG1 and IgG4 have two interheavy chain disulfide bonds, IgG2 has 4, and IgG3 has 11. The IgG, is distributed equally in the intra- and extravascular compartments. ■ ■ ■ ◗ In response to infection, IgG antibodies appear late after appearance of IgM antibodies, but persists for a longer period. It confers protection against the microorganisms that are present in the blood and tissues. It is distributed equally in the intra- and extravascular compartments. It is the only immunoglobulin that crosses the placenta; hence, it confers natural passive immunity to the newborns. It takes part in precipitation, complement fixation, and neutralization of toxins and viruses. It binds to microorganisms and facilitates the process of phagocytosis of microorganisms. Immunoglobulin M IgM constitutes about 5–8% of total serum immunoglobulins. It is distributed mainly intravascularly. It is a heavy molecule (19S) with a molecular weight varying from 900,000 to 1,000,000 Da (millionaire molecule). It has a half-life of 5 days (Table 13-2). IgM is basically a pentamer, composed of five immunoglobulin subunits (monomeric subunits, IgMs) and one molecule of J chain. Each monomeric IgM is composed of two light chains ( or  light chains) and two heavy chains (). The heavy chains are larger than those of IgG by about 20,000 Da, corresponding to an extra domain on the constant region Chapter 13 150 3 Section II Molecular weight (kDa) Carbohydrate (%) 98 IMMUNOLOGY Key Points IgM IgM is pentameric. It is an effective first line defense against foreign bodies. IgM is produced in the primary immune response IgM shows the following biological activities: ■ ■ Chapter 13 Section II IgG IgG is monomeric. It is an effective defense against extravascular compartments from foreign bodies and their components IgD IgD is monomeric. It influences lymphocyte functions IgE IgE is monomeric. It gives protection against intestinal parasites and is responsible for many of symptoms of allergy IgA ■ ■ ■ IgA is dimeric. It gives protection to mucosal surfaces Pentameric IgM, because of its high valency, is more efficient than other isotypes in binding antigens with many repeating epitopes, such as viral particles and red blood cells. It is more efficient than IgG in activating complement. Complement activation requires two Fc regions in close proximity, and the pentameric structure of a single molecule of IgM fulfills this requirement. IgM is the first immunoglobulin produced in a primary response to an antigen. The immunoglobulin confers protection against invasion of blood by microbial pathogens. Deficiency of IgM antibodies is associated with septicemia. IgM antibodies are short lived and disappear early as compared to IgG. The presence of IgM antibody in serum, therefore, indicates recent infection. It is also the first immunoglobulin to be synthesized by a neonate in about 20 weeks of age. IgM is not transported across the placenta; hence, the presence of IgM in the fetus or newborn indicates intrauterine infection. The detection of IgM antibodies in serum, therefore, is useful for the diagnosis of congenital infections, such as syphilis, rubella, toxoplasmosis, etc. FIG. 13-2. Schematic diagram of immunoglobulins and their functions. ◗ (CH4). Two subclasses of IgM (IgM1 and IgM2) are described, which differ in their  chains. IgM1 consists of 1 and IgM2 consists of 2 chains (Fig. 13-2). The immunoglobulin  chain is a 72 kDa, 570-amino acid heavy polypeptide chain comprising one variable region, designated VH, and a four-domain constant region, designated CH1, CH2, CH3, and CH4. The  chain does not have a hinge region. A “tail piece” is located at the carboxy terminal end of the chain. It comprises 18-amino acid residues. A cysteine residue at the penultimate position of a carboxy terminal region of the  chain forms a disulfide bond that joins to the J chain. There are five N-linked oligosaccharides in the  chain of humans. Monomeric IgM, with a molecular weight of 180,000 Da, is expressed as membrane-bound antibody on B cells. As mentioned earlier, the J chain found in the IgM molecule was believed to play a major role in the secretion of its polymerized form. Being present on the membrane of B cells, IgM acts as the antigen-binding molecule in the antigen–antibody complex. Because of its pentameric structure with 10 antigenbinding sites, serum IgM has a higher valency than the other isotypes. An IgM molecule can bind 10 small hapten molecules; however, because of steric hindrance, only five or fewer molecules of larger antigens can be bound simultaneously. Treatment of serum with 2-mercaptoethanol destroys IgM without affecting IgG antibodies. This forms the basis for differential estimation of IgM and IgG antibodies in serum pretreated with 2-mercaptoethanol. Immunoglobulin A IgA is the second major serum immunoglobulin, comprising nearly 10–15% of serum immunoglobulin. It has a half-life of 6–8 days (Table 13-2). IgA consist of  heavy chain that confers class specificity on IgA molecules. The  chain is a 58-kDa, 470-amino acid residue heavy polypeptide chain. The chain is divisible into three constant domains, designated CH1, CH2, and CH3, and one variable domain, designated VH. Hinge region is situated between CH1 and CH2 domains. An additional segment of 18-amino acid residues at the penultimate position of the chain contains a cysteine residue where the J chain can be attached through a disulfide bond. IgA occurs in two forms: serum IgA and secretory IgA. Serum IgA: It is present in the serum and is a monomeric 7S molecule with a molecular weight of 60,000 Da. It has a halflife of 6–8 days. It has two subclasses, IgA1 and IgA2, which are two -chain isotypes -1 and -2, respectively. The -2 chain has two allotypes, A2m (1) and A2m (2), and does not have disulfide bonds linking heavy to light chains. Differences in the two  chains are found in two CH1 and five CH3 positions. Thus, there are three varieties of -heavy chains in humans. Secretory IgA: It is a dimer or tetramer and consists of a J-chain polypeptide and a polypeptide chain called secretory component, or SC, or secretory piece (Fig. 13-3). The SC is a polypeptide with a molecular weight of 70,000 Da and is produced by epithelial cells of mucous membranes. It consists of five immunoglobulin-like domains that bind to the Fc region domains of the IgA dimer. This interaction is stabilized by a disulfide bond between the fifth domain of the SC and one ANTIBODIES Secretory component Heavy chain 99 Variable region Light chain Light chain Heavy chain Disulfide bond Disulfide bonds J chain Constant regions Schematic diagram of immunoglobulin A (IgA). Key Points FIG. 13-4. an IgE molecule. The e chain is a 72-kDa, 550-amino acid residue polypeptide chain. It consists of one variable region, designated VH, and a four-domain constant region, designated CH1, CH2, CH3, and CH4. This heavy chain does not possess a hinge region. In humans, the  heavy chain has 428 amino acid residues in the constant region (Fig. 13-4). IgE does not cross the placenta or fix the complement. Secretory IgA shows the following biological activities: ■ ■ ■ ■ Key Points It protects the mucous membranes against microbial pathogens. It serves an important effector function at mucous membrane surfaces, which are the main entry sites for most pathogenic organisms. Because it is polymeric, secretory IgA can cross-link large antigens with multiple epitopes. Binding of secretory IgA to bacterial and viral surface antigens prevents attachment of the pathogens to the mucosal cells, thus inhibiting viral infection and bacterial colonization. Complexes of secretory IgA and antigen are easily entrapped in mucus and then eliminated by the ciliated epithelial cells of the respiratory tract or by peristalsis of the gut. Breast milk contains secretory IgA and many other molecules that protect the newborns against infection during the first month of life. Because the immune system of infants is not fully functional, breast-feeding plays an important role in maintaining the health of newborns. Secretory IgA has shown to provide an important line of defense against bacteria (such as Salmonella spp., Vibrio cholerae, and Neisseria gonorrhoeae) and viruses (such as polio, influenza, and reovirus). IgE shows the following biological activities: ■ ■ ■ ◗ ◗ Immunoglobulin E IgE constitutes less than 1% of the total immunoglobulin pool. It is present in serum in a very low concentration (0.3 g/mL). It is mostly found extravascularly in lining of the respiratory and intestinal tracts. IgE is an 8S molecule with a molecular weight of 190,000 Da and half-life of 2–3 days. Unlike other immunoglobulins that are heat stable, IgE is a heat-labile protein—easily inactivated at 56°C in 1 hour (Table 13-2). Two e heavy polypeptide chains, along with two  or two  light chains, fastened together by disulfide bonds, comprise Schematic diagram of immunoglobulin E (IgE). IgE is also known as reaginic antibody that mediates the type I immediate hypersensitivity (atopy) reactions. IgE is responsible for the symptoms of hay fever, asthma, and anaphylactic shock. IgE binds to Fc receptors on the membranes of blood basophils and tissue mast cells. Cross-linkage of receptor bound IgE molecules by antigen (allergen) induces basophils and mast cells to translocate their granules to the plasma membrane and release their contents to the extracellular environment—a process known as degranulation. As a result, varieties of pharmacologically active mediators are released and give rise to allergic manifestations. Localized mast-cell degranulation induced by IgE may also release mediators that facilitate a buildup of various cells necessary for antiparasitic defense. Immunoglobulin D IgD comprises less than 1% of serum immunoglobulins. It is a 7S monomer with a molecular weight of 180,000 Da. The halflife of IgD is only 2–3 days (Table 13-2). IgD has the basic fourchain monomeric structure with two  heavy chains (molecular weight 63,000 Da each) and either two  or two  light chains (molecular weight 22,000 Da each) (Table 13-2). Immunoglobulin  chain is a 64-kDa, 500-amino acid residue heavy polypeptide chain consisting of one variable region, designated as VH, and a three-domain constant region, designated as CH1, CH2, and CH3. There is also a 58-residue amino acid residue hinge region in human  chains. Two exons encode the hinge region. IgD is very susceptible to the action of proteolytic enzymes at its hinge region. Two separate exons Chapter 13 of the chains of the dimeric IgA. IgA-secreting plasma cells are concentrated along mucous membrane surfaces. The daily production of secretory IgA is greater than that of any other immunoglobulin. Secretory IgA is the major immunoglobulin present in external secretions, such as breast milk, saliva, tears, and mucus of the bronchial, genitourinary, and digestive tracts. IgA activates the complement not by classical pathway but by alternative pathway. Section II FIG. 13-3. 100 IMMUNOLOGY TABLE 13-3 Role of immunoglobulins in human defense IgG IgM IgA IgD IgE Enhances phagocytosis Especially effective against microorganisms and agglutinating antigens Localized protection on mucosal surfaces Serum function not known Allergic reaction Neutralizes toxins and viruses First antibody produced in response to initial infection Present on B cells; and function in initiation of immune response Possibly lysis of parasitic worms Chapter 13 Section II Protects fetus and newborn encode the membrane component of  chain. A distinct exon encodes the carboxy terminal portion of the human  chain that is secreted. The human  chain contains three N-linked oligosaccharides. Table 13-3 summarizes roles of various immunoglobulins in human defense. Key Points IgD is present on the surface of B lymphocytes and both IgD and IgM serve as recognition receptors for antigens. The role of IgD in immunity continues to remain elusive. Abnormal Immunoglobulins Abnormal immunoglobulins are other structurally similar proteins that are found in serum in certain pathological conditions, such as multiple myeloma, heavy chain disease, and cryoglobulinemia and sometimes in healthy individuals also. Multiple myeloma: Bence-Jones (BJ) proteins were the earliest abnormal proteins described in 1847 that were found in patients with multiple myeloma. These proteins are the light chains of immunoglobulins, hence occur as either  or  forms. In a patient, it may occur as either  or  but never in both the forms. BJ proteins have a peculiar property of coagulating at 60°C and redissolving again at a higher temperature of 80°C. In multiple myeloma, plasma cells synthesizing IgG, IgA, IgD, or IgE are affected. Myeloma involving IgM-producing plasma cells is known as Waldenström’s macroglobulinemia. This condition is characterized by excessive production of the respective myeloma proteins (M proteins) and that of their light chains (BJ proteins). The study of myeloma proteins led to a great advancement in our understanding of immunoglobulin function. These “single” or “monoclonal” antibodies obtained from the sera of patients with multiple myeloma were used in many of the serologic and biochemical studies of the 1950s and 1960s. They remained the major source of homogeneous immunoglobulins until the development of the hybridoma in 1974. The serologists injected them into animals and produced antisera that were used to study some of the basic properties of antibodies. For example, the immune sera were absorbed with other myeloma proteins and were used to identify isotypic, allotypic, and idiotypic specificities. Heavy chain disease: Heavy chain disease is a different disorder, which is a lymphoid neoplasia, characterized by an excess production of heavy chains of the immunoglobulins. Cryoglobulinemia: Cryoglobulinemia is a condition characterized by presence of cryoglobulins in blood. The condition may not be always associated with disease but is often found in patients with macroglobulinemia, systemic lupus erythematosus, and myelomas. Most cryoglobulins consist of either IgG or IgM or their mixed precipitates. In cryoglobulinemia, serum from patient precipitates on cooling and redissolves on warming. 14 Antigen–Antibody Reactions Introduction The interactions between antigens and antibodies are known as antigen–antibody reactions. The reactions are highly specific, and an antigen reacts only with antibodies produced by itself or with closely related antigens. Since these reactions are essentially specific, they have been used in many diagnostic tests for the detection of either the antigen or the antibody in vitro. The antigen and antibody reactions also form the basis of immunity against microbial diseases in vivo. In the host, it may cause tissue injury in hypersensitivity reactions and in autoimmune diseases. General Features of Antigen–Antibody Reactions Antigen and antibody bind through noncovalent bonds in a manner similar to that in which proteins bind to their cellular receptors, or enzymes bind to their substrates. But antigen– antibody reactions differ from the latter as there is no irreversible chemical alteration in either of the participants, i.e., antigen or the antibody. The antigen and antibody binding is reversible and can be prevented or dissociated by high ionic strength or extreme pH. Following are some of the general features of these interactions: Physicochemical Properties Electrostatic bonds, hydrogen bonding, van der Waals bonds, and hydrophobic interactions are the intermolecular forces involved in antigen–antibody reactions. All these types of intermolecular forces depend on the close proximity of the antigen and antibody molecules. For that reason, the “good fit” between an antigenic determinant and an antibody-combining site determines the stability of the antigen–antibody reaction. Multiple bonding between the antigen and the antibody ensures that the antigen will be bound tightly to the antibodies. Affinity Affinity denotes the intensity of attraction between antigen and antibody. ■ Low-affinity antibodies bind antigen weakly and tend to dissociate readily, whereas high-affinity antibodies bind antigen more tightly and remain bound longer. ■ High-affinity binding is believed to result from a very close fit between the antigen-binding sites and the corresponding antigenic determinants that facilitates development of strong noncovalent interactions between antigen and antibody. Avidity Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies. Avidity is a better indicator of the strength of interactions in real biological systems than affinity. Therefore, the avidity of an antigen–antibody reaction is dependent on the valencies of both antigens and antibodies and is greater than the sum total of individual affinities. Specificity The term specificity refers to the ability of an individual antibodycombining site to react with only one antigenic determinant or the ability of a population of antibody molecules to react with only one antigen. Antigen–antibody reactions usually show a high degree of specificity. Key Points Antibodies can specifically recognize differences in: ■ ■ ■ primary structure of an antigen, isomeric forms of an antigen, and secondary and tertiary structure of an antigen. Despite this, cross-reactions between antigens and antibodies, however, do occur and are sometimes responsible for causing diseases in hosts and for causing false results in diagnostic tests. Cross-Reactivity Although antigen–antibody reactions are highly specific, in some cases antibody elicited by one antigen can cross-react with an unrelated antigen. Such cross-reactivity occurs if two different antigens share an identical or very similar epitope. In the latter case, the antibody’s affinity for the cross-reacting epitope is usually less than that for the original epitope. Antisera containing polyclonal antibodies can often be found to crossreact with immunogens partially related to those used for immunization, due to the existence of common epitopes or of epitopes with similar configurations. 102 IMMUNOLOGY Stages of Antigen–Antibody Reactions The antigen–antibody reaction occurs in two stages: primary and secondary. Types of Antigen–Antibody Reactions Chapter 14 Section II Primary Stage Primary stage is the initial interaction between antigen and antibody. It is rapid and reversible, but without any visible effects. The ionic bonds, hydrogen bonds, van der Waals forces, and hydrophobic interactions are the weaker intermolecular forces that bind antigen and antibodies together in this primary stage. Covalent binding, which is a stronger intermolecular force between antigen and antibody, however, does not occur in this stage. Secondary Stage Secondary stage is an irreversible interaction between antigen and antibody, with visible effects, such as agglutination, precipitation, neutralization, complement fixation, and immobilization of motile organisms. The binding between antigen and antibody during this stage occurs by covalent binding. A single antibody is capable of causing different types of antigen–antibody reactions, and a single antigen is capable of inducing production of different classes of immunoglobulins, which differ in their biological properties. The results of agglutination, precipitation, neutralization, and other tests are usually expressed as a titer. Titer is defined as the highest dilution of serum that gives a positive reaction in TABLE 14-1 test. Higher titer means greater level of antibodies in serum. For example, a serum with a titer of 1/128 contains more antibodies than a serum with a titer of 1/8. Serological tests are widely used for detection of either serum antibodies or antigens for diagnosis of a wide variety of infectious diseases (Table 14-1). These serological tests are also used for diagnosis of autoimmune diseases and in typing of blood and tissues before transplantation. The following are the examples of antigen–antibody reactions: (a) precipitation, (b) agglutination, (c) complement-dependent serological tests, (d) neutralization test, (e) opsonization, (f ) immunofluorescence, (g) enzyme immunoassay, (h) radioimmunoassay, (i) western blotting, ( j) chemiluminescence assay, and (k) immunoelectronmicroscopic tests. Precipitation Precipitation test shows the following features: ■ ■ ■ It is a type of antigen–antibody reaction, in which the antigen occurs in a soluble form. It is a test in which antibody interacts with the soluble antigen in the presence of electrolyte at a specified pH and temperature to produce a precipitate. A lattice is formed between the antigens and antibodies; in certain cases, it is visible as an insoluble precipitate. Antibodies that aggregate soluble antigens are called precipitins. Commonly used tests in clinical microbiology Test Uses Flocculation test Detection of reaginic antibodies in syphilis by VDRL test Radial immunodiffusion Detection of fungal antigen and antibodies Counter-current immunoelectrophoresis Detection of both antigen and antibodies in bacterial, viral, fungal, and parasitic diseases Slide agglutination test Identification of bacterial isolates, such as Salmonella, Shigella, Vibrio, etc. Tube agglutination test Detection of antibodies in bacterial infections, e.g., Widal test for enteric fever Latex agglutination test Quantitation and detection of antigen and antibodies Hemagglutination test Detection of both antigens and antibodies in viral and parasitic infections Coagglutination test Detection of microbial antigens Complement fixation test Quantitation and detection of antibodies Direct immunofluorescence test Detection and localization of antigen in a cell or tissue Indirect immunofluorescence test Detection of specific antibodies in the serum Sandwich ELISA Detection of antigens and antibodies Indirect ELISA Quantitation and detection of antibodies Radioimmunoassay Quantitation of hormones, drugs, etc. Western blot Detection of antigen-specific antibody 103 ANTIGEN–ANTIBODY REACTIONS ■ ■ ■ ■ Prozone excess antibody no aggregation Dilution Prozone phenomenon Antibody Antibody Precipitate formed (optimal antigen and antibody concentration) Post zone excess antigen no aggreagation Dilution Diluted antibody Antigen Highly diluted antibody Antigen Antigen FIG. 14-2. Marrack’s lattice hypothesis. Key Points In the prozone phenomenon, there is too much antibody for efficient lattice formation. This is because antigen combines with only few antibodies and no cross-linkage is formed. In postzone phenomenon, small aggregates are surrounded by excess antigen and again no lattice network is formed. Thus, for precipitation reactions to be detectable, they must be run in the zone of equivalence (Fig. 14-2). The prozone and postzone phenomena are taken into consideration in the interpretation of serological tests, because false negative reactions can occur in either of these conditions. A false negative reaction suspected to be due to prozone phenomenon can be rectified by diluting out the antibody and performing the test. In the postzone phenomenon, excess antigen may obscure the presence of small amount of antibodies. Typically, such a test is repeated with an additional patient specimen taken about a week later. This would give time for the further production of antibodies. If the test is negative on this occasion, it is unlikely that the patient has that particular antibody. ◗ Types of precipitation reactions Precipitation reactions can be broadly of three types: 1. Precipitation in solution 2. Precipitation in agar 3. Precipitation in agar with an electric field Zone of antibody excess FIG. 14-1. Zone of equivalence Prozone phenomenon. Zone of antigen excess Precipitation in solution Ring test and flocculation test are examples of precipitation in solution. Chapter 14 Antigen and antibody reaction occurs optimally only when the proportion of the antigen and antibody in the reaction mixture is equivalent (zone of equivalence) (Fig. 14-1). On either side of the equivalence zone, precipitation is actually prevented because of an excess of either antigen or antibody. The zone of antibody excess is known as the prozone phenomenon and the zone of antigen excess is known as postzone phenomenon. Marrack in 1934 proposed the lattice hypothesis to explain the prozone phenomenon. Marrack’s hypothesis is based on the assumptions that each antibody molecule must have at least two binding sites, and antigen must be multivalent. In the zone of equivalence where optimum precipitation occurs, the number of multivalent sites of antigen and antibody are approximately equal. In this zone, precipitation occurs as a result of random, reversible reactions whereby each antibody binds to more than one antigen and vice versa, forming a stable network or lattice. As they combine, it results in a multimolecular lattice that increases in size until it precipitates out of solution. Antigen Equivalence zone proper ratio aggregation Section II ◗ When instead of sedimenting, the precipitate remains suspended as floccules, the reaction is known as flocculation. Formation of an antigen–antibody lattice depends on the valency of both the antibody and antigen. The antibody must be bivalent; a precipitate will not form with monovalent Fab fragments. The antigen must be either bivalent or polyvalent; that is, it must have at least two copies of the same epitope, or have different epitopes that react with different antibodies present in polyclonal antisera. 104 ■ Chapter 14 Section II ■ IMMUNOLOGY Ring test: In this test, antigen solution is layered over antiserum in a test tube. Precipitation between antigen and antibodies in antiserum solution is marked by the appearance of a ring of precipitation at the junction of two liquid layers. C-reactive protein (CRP) and streptococcal grouping by the Lancefield methods are the examples of the ring test. Flocculation test: Flocculation test may be performed in a slide or tube. VDRL test for detection of reaginic antibodies in syphilis is an example of a slide flocculation test. In this test, a drop of VDRL antigen suspension is added to a drop of patients’ serum on a cavity slide, and the result is recorded after shaking the slide on a VDRL shaker. In a positive test, the floccules appear, which can be demonstrated well under a microscope. Kahn test for syphilis is an example of tube flocculation test. The tube flocculation test for standardization of toxins and toxoids is another example. FIG. 14-3. the surface of gel. The antigen is then applied to a well cut into the gel. When antibody already present in the gel reacts with the antigen, which diffuses out of the well, a ring of precipitation is formed around the wells. The diameter of the ring is directly proportional to th=e concentration of antigen. The greater the amount of antigen in the well, the farther the ring will be from the well (Fig. 14-3, Color Photo 6). Precipitation in agar The precipitation test in agar gel is termed as immunodiffusion test. In this test, reactants are added to the gel and antigen– antibody combination occurs by means of diffusion. The rate of diffusion is affected by the size of the particles, temperature, gel viscosity, amount of hydration, and interactions between the matrix and reactants. An agar concentration of 0.3–1.5% allows for diffusion of most reactants. Agarose is often preferred to agar because agar has a strong negative charge, while agarose has almost none, so that interactions between the gel and reactants are minimized. Key Points Radial immunodiffusion has been used for the quantitative estimation of antibodies and antigens in the serum. It is used to measure: ■ ■ ■ Key Points Immunodiffusion reactions have the following advantages: ■ ■ In this test, the line of precipitation is visible as a band, which can also be stained for preservation. The test can be used to detect identity, cross-reaction, and nonidentity between different antigens in a reacting mixture. ■ Single diffusion in one dimension: Single diffusion in one dimension, as the name suggests, is the single diffusion of antigen in agar in one dimension. It is otherwise called Oudin procedure because this technique was pioneered by Oudin who for the first time used gels for precipitation reactions. In this method, antibody is incorporated into agar gel in a test tube and the antigen solution is poured over it. During the course of time, the antigen diffuses downward toward the antibody in agar gel and a line of precipitation is formed. The number of precipitate bands shows the number of different antigens present in the antigen solution. Single diffusion in two dimensions: Single diffusion in two dimensions is also called radial immunodiffusion. In this method, antiserum solution containing antibody is incorporated in agar gel on a slide or Petri dish. The wells are cut on IgG, IgM, IgA, and complement components in the serum, antibodies to influenza virus in sera, and serum transferrin and ␣-fetoprotein. However, the test has recently been replaced by more sensitive and automated methods, such as nephelometry and enzyme-linked immunosorbent assays (ELISAs). ■ Types of immunodiffusion reactions: Immunodiffusion reactions are classified based on the (a) number of reactants diffusing and (b) direction of diffusion, as follows: ■ Radial immunodiffusion. ■ Double diffusion in one dimension: This method is also called Oakley–Fulthrope procedure. In this method, the antibody is incorporated in agar gel in a test tube, above which a layer of plain agar is placed. The antigen is then layered on top of this plain agar. During the course of time, the antigen and antibody move toward each other through the intervening layer of plain agar. In this zone of plain agar, both antigen and antibody react with each other to form a band of precipitation at their optimum concentration. Double diffusion in two dimensions: This method is also called the Ouchterlony procedure. In this method, both the antigen and antibody diffuse independently through agar gel in two dimensions, horizontally and vertically. The test is performed by cutting wells in the agar gel poured on a glass slide or in a Petri dish. The antiserum consisting of antibodies is placed in the central well, and different antigens are added to the wells surrounding the center well. After an incubation period of 12–48 hours in a moist chamber, the lines of precipitins are formed at the sites of combination of antigens and antibodies (Color Photo 7). Three types of reactions can be demonstrated as follows (Fig. 14.4): 105 ANTIGEN–ANTIBODY REACTIONS Identical Antigen Antibody FIG. 14-4. a Nonidentical a αa a b Partially identical a αab ab Electrophoretic current +ve −ve αab Ag Ab Ouchterlony procedure. FIG. 14-6. Counter-current immunoelectrophoresis. Antibody in agarose gel Precipitation band +ve Chapter 14 Key Points −ve Double diffusion in two dimension has been used for: ■ ■ ■ demonstration of antibodies in serodiagnosis of small pox, identification of fungal antigens, and detection of antibodies to extractable nuclear antigens. Elek’s precipitation test in gel is a special test used for demonstration of toxigenicity of Corynebacterium diphtheriae. Precipitation in agar with an electric field Immunoelectrophoresis: Immunoelectrophoresis is a process of combination of immunodiffusion and electrophoresis. It is a method in which different antigens in serum are separated according to their charge under an electric field. In this method, a drop of antigen is placed into a well in agar on a glass slide. An electric current is then passed through the agar. During electrophoresis, antigens move in the electric field according to their charge and size. Following electrophoresis, a trough is cut into the agar and is filled with the antibody and diffusion is allowed to occur. As the antigen and antibody diffuse toward each other, they form a series of lines of precipitation (Fig. 14-5). The main advantage of immunoelectrophoresis is that a number of antigens can be identified in serum. The method is used to detect normal as well as abnormal proteins, such as myeloma proteins in human serum. − FIG. 14-5. + Immunoelectrophoresis. Section II 1. Line of precipitation at their junction forming an arc represents serologic identity or the presence of a common epitope in antigens. 2. A pattern of crossed lines demonstrates two separate reactions and indicates that the compared antigens are unrelated and share no common epitopes. 3. Fusion of two lines with a spur indicates cross-reaction or partial identity. In this last case, the two antigens share a common epitope, but some antibody molecules are not captured by the antigen and traverse through the initial precipitin line to combine with additional epitopes found in the more complex antigen. Antigen wells FIG. 14-7. Increasing antigen concentration Rocket electrophoresis. Counter-current immunoelectrophoresis: Counter-current immunoelectrophoresis depends on movement of antigen towards the anode and of antibody towards the cathode through the agar under electric field. The test is performed on a glass slide with agarose in which a pair of wells is punched out. One well is filled with antigen and the other with antibody. Electric current is then passed through the gel. The migration of antigen and antibody is greatly facilitated under electric field, and the line of precipitation is made visible in 30–60 minutes (Fig. 14-6). Key Points The counter-current immunoelectrophoresis has many uses: ■ ■ It is a rapid and a highly specific method for detection of both antigen and antibodies in the serum, cerebrospinal fluid, and other body fluids in diagnosis of many infectious diseases including bacterial, viral, fungal, and parasitic. It is commonly used for Hepatitis B surface antigen (HBsAg), ␣-fetoprotein, hydatid and amoebic antigens in the serum, and cryptococcal antigen in the CSF. Rocket electrophoresis: This technique is an adaptation of radial immunodiffusion developed by Laurell. It is called so due to the appearance of the precipitin bands in the shape of cone-like structures (rocket appearance) at the end of the reaction (Fig. 14-7, Color Photo 8). In this method, antibody is incorporated in the gel and antigen is placed in IMMUNOLOGY Chapter 14 Section II 106 FIG. 14-8. Photograph of rocket electrophoresis. phenomenon of light scattering by precipitates in a solution. Turbidimetry is a measurement of turbidity or cloudiness of precipitate in a solution. In this method, a detection device is placed in direct line with the incident light that collects the light after it has passed through the solution. It thus measures the reduction in the intensity of light due to reflection, absorption, or scatter. Scattering of light occurs in proportion to the size, shape, and concentration of precipitates present in solution. Nephelometry is an improvement on this technique in that it measures the light that is scattered at a particular angle from the incident beam as it passes through a suspension containing the antigen–antibody precipitate. The amount of light scattered is an index of the concentration of the solution. Beginning with a constant amount of antibody, an increasing amount of antigen would result in an increase in antigen–antibody complexes. Thus the relationship between antigen concentrations, as indicated by the antigen–antibody complex formation, and light scattering approaches linearity. By using a computer, the exact values of the antigen or antibody in the serum can be estimated through this system. To improve the sensitivity of this system, laser beams have been used as the source of incident light. wells cut in the gel. Electric current is then passed through the gel, which facilitates the migration of antigen into the agar. This results in formation of a precipitin line that is conical in shape, resembling a rocket. The height of the rocket, measured from the well to the apex, is directly in proportion to the amount of antigen in the sample (Fig. 14-8). Rocket electrophoresis is used mainly for quantitative estimation of antigen in the serum. Two-dimensional immunoelectrophoresis: Two-dimensional immunoelectrophoresis is a variant of rocket electrophoresis. It is a double diffusion technique used for qualitative as well as quantitative analysis of sera for a wide range of antigens. This test is a two-stage procedure. In the first stage, antigens in solution are separated by electrophoresis. In the second stage, electrophoresis is carried out again, but perpendicular to that of first stage to obtain rocket-like precipitation. In this test, first, a small trough is cut in agar gel on a glass plate and is filled with the antigen solution. Electric current is then passed through the gel, and the antigens migrate into the gel at a rate proportional to their net electric charge. In the second stage, after electrophoresis, the gel piece containing the separated antigens is placed on a second glass plate and the agar containing antibody is poured around the gel piece. A second electric potential is applied at right angles to the first direction of migration. The preseparated antigens then migrate into the gel containing antibodies at a rate proportional to their net charge and precipitate with antibodies in the gel, forming precipitates. This method is both qualitative, in that it identifies different antigens that are present in the serum solution, and quantitative, in that it detects the amount of different antigens present in the solution. Turbidimetry and nephelometry: Turbidimetry and nephelometry are the two methods used to detect and quantitate precipitation reactions in serum and are based on the Key Points Nephelometry is now becoming the method of choice for use in various laboratories for the measurement of plasma proteins including IgG, IgM, and IgA, complement components, RA (rheumatoid arthritis) factor, ASLO (anti-streptolysin O), etc. Agglutination Agglutination is an antigen–antibody reaction in which a particulate antigen combines with its antibody in the presence of electrolytes at a specified temperature and pH resulting in formation of visible clumping of particles. Agglutination occurs optimally when antigens and antibodies react in equivalent proportions. ■ ■ Agglutination reactions are mostly similar to precipitation reactions in their fundamentals and share similar features. This reaction is analogous to the precipitation reaction in that antibodies act as a bridge to form a lattice network of antibodies and the cells that carry the antigen on their surface. Because cells are so much larger than a soluble antigen, the result is more visible when the cells aggregate into clumps. Agglutination differs from precipitation reaction in that since the former reaction takes place at the surface of the particle involved, the antigen must be exposed and be able to bind with the antibody to produce visible clumps. In agglutination reactions, serial dilutions of the antibody solution are made and a constant amount of particulate antigen is added to serially diluted antibody solutions. After several hours of incubation at 37°C, clumping is recorded by visual inspection. The titer of the antiserum is recorded ANTIGEN–ANTIBODY REACTIONS ◗ Types of agglutination reactions Direct agglutination ■ ■ Heterophile agglutination test: This test depends on demonstration of heterophilic antibodies in serum present in certain bacterial infections: Key Points ■ Direct agglutination reactions can broadly be of the following types: (a) slide agglutination, (b) tube agglutination, (c) heterophile agglutination, and (d) antiglobulin (Coombs’) test. Slide agglutination test: It is a basic type of agglutination reaction that is performed on a slide. Identification of bacterial types represents a classic example of a direct slide agglutination that is still used today. In this test, a suspension of bacteria is prepared and is added to a drop of standardized antiserum. A positive reaction is indicated by clumping of bacteria and clearing of the background solution. Clumping occurs instantly or within seconds in a positive test. A control consisting of antigen suspension in saline without adding antiserum is included on the same slide. It is used to validate the results and also to detect possible false positives due to autoagglutination of the antigen. Key Points Slide agglutination is used: ■ ■ As a routine procedure to identify bacterial strains, such as Salmonella, Shigella, Vibrio, etc., isolated from clinical specimens. For blood grouping and cross-matching. Tube agglutination test: Tube agglutination test, as the name suggests, is performed in glass tubes. Typically, in these tests, patient’s serum is diluted in a series of tubes and bacterial antigens specific for the suspected disease are added to it. Antigen and antibody reactions are demonstrated by demonstration of visible clumps of agglutination. It is a standard method used for quantitative estimation of antibodies in the serum. Tube agglutination tests are routinely used for demonstration of antibodies in the serum for serodiagnosis of enteric fever and brucellosis, as follows: Widal test is used to diagnose enteric fever and uses different Salmonella antigens (TO, TH, AH, and BH) to detect the presence of antibodies to Salmonella typhi, S. paratyphi A, and S. paratyphi B in patient’s serum. The standard agglutination test is a commonly used test for serodiagnosis of brucellosis. The tube agglutination test for brucellosis, however, is complicated by the prozone phenomenon. This is due to high concentration of brucella antibodies in patient’s serum, resulting in false negative reactions. This problem is obviated by use of several dilutions of serum to prevent false positive reactions. The presence of blocking or incomplete antibodies in the serum is another problem. This is avoided by using antiglobulin (Coombs’ test) to detect these antibodies. ■ ■ Weil–Felix test is an example of heterophile agglutination reaction for serodiagnosis of rickettsial infections. In this test, the cross-reacting antibodies produced against rickettsial pathogen are detected by using cross-reacting related antigens (e.g., Proteus strains OXK, OX19, and OX2). Although the antibodies are produced against rickettsial organisms, they cross-react with antigens of Proteus strains OXK, OX19, and OX2. Paul–Bunnell test is another heterophile agglutination test, which is used to detect antibodies in infectious mononucleosis by using sheep erythrocytes as antigens. Streptococcus MG agglutination test is a similar test used for detection of antibodies to Mycoplasma pneumoniae causing primary atypical pneumonia. Antiglobulin (Coombs’) test: Coombs’ test was devised originally by Coombs’, Mourant, and Race for detection of incomplete anti-Rh antibodies that do not agglutinate Rh⫹ erythrocytes in saline. When serum containing incomplete anti-Rh antibodies is mixed with Rh⫹ erythrocytes in saline, incomplete antibody antiglobulin coats the surface of erythrocytes but does not cause any agglutination. When such erythrocytes are treated with antiglobulin or Coombs’ serum + Antibody bound patient’s RBCs FIG. 14-9. Antihuman immunoglobulin (Coombs reagent) Principle of the direct Coombs’ test. Agglutination of RBCs Chapter 14 Agglutination reactions where the antigens are found naturally on a particle are known as direct agglutination. This is different from passive agglutination, which employs particles that are coated with antigens not normally found on their surfaces. Key Points Section II as the reciprocal of the highest dilution that causes clumping. Since the cells have many antigenic determinants on their surface, the phenomenon of antibody excess is rarely encountered. Occasionally, antibodies are formed that react with the antigenic determinants of a cell but does not cause any agglutination. They inhibit the agglutination by the complete antibodies added subsequently. Such antibodies are called blocking antibodies. Anti-Rh antibodies and anti-brucella antibodies are few examples of such blocking antibodies. Agglutination reactions have a wide variety of applications in the detection of both antigens and antibodies in serum and other body fluids. They are very sensitive and the result of the test can be read visually with ease. 107 108 IMMUNOLOGY + Chapter 14 Section II Antibodies in patient’s serum Target RBCs Agglutination of RBCs FIG. 14-10. Antibody bound RBCs Antihuman immunoglobulin (Coombs reagent) Principle of the indirect Coombs’ test. (rabbit antiserum against human ␥ globulin), then the cells are agglutinated. Coombs’ test is of two types: (a) direct Coombs’ test and (b) indirect Coombs’ test. ■ ■ Direct Coombs’ test: In this test, the sensitization of red blood cells (RBCs) with incomplete antibodies takes place in vivo. The cell-bound antibodies can be detected by this test in which antiserum against human immunoglobulin is used to agglutinate patient’s red cells (Fig. 14-9). Indirect Coombs’ test: In this test, the sensitization of RBCs with incomplete antibodies takes place in vitro. In this test, the patient’s serum is mixed with normal red cells and antiserum to human immunoglobulin is added. Agglutination occurs if antibodies are present in the patient’s serum (Fig. 14-10). Coombs’ tests are used for detection of (a) anti-Rh antibodies and (b) incomplete antibodies in brucellosis and other diseases. Passive agglutination Passive agglutination employs carrier particles that are coated with soluble antigens. This is usually done to convert precipitation reactions into agglutination reactions, since the latter are easier to perform and interpret and are more sensitive than precipitation reactions for detection of antibodies. When the antibody instead of antigens is adsorbed on the carrier particle for detection of antigens, it is called reverse passive agglutination. Until the 1970s, erythrocytes were the major carrier particles used for coating of antigens. Recently, however, a variety of other particles including polystyrene latex, bentonite, and charcoal are used for this purpose. Particle size vary from 7 ␮ for RBCs to 0.05 ␮ for very fine latex particles. The use of synthetic beads or particles provides the advantage of consistency, uniformity, and stability. Reactions are also easy to read visually. Passive agglutination reaction, depending on the carrier particles used, can be of the following types: (i) latex agglutination test, (ii) hemagglutination test, and (iii) coagglutination test. Latex agglutination test: It is a test that employs latex particles as carrier of antigen or antibodies. In 1955, Singer and Plotz accidentally found that IgG was naturally adsorbed to the surface of polystyrene latex particles. Latex particles are inexpensive, relatively stable and are not subject to cross-reactivity with other antibodies. These particles can be coated with antibodies to detect antigen in the serum and other body fluids. Use of monoclonal antibodies has reduced the cross-reactions resulting in reduction of false positive reactions. Additionally, the large particle size of the latex facilitates better visualization of antigen–antibody reactions by the naked eye observation. The tests are usually performed on cardboard cards or glass slides and positive reactions are graded on a scale of 1⫹ to 4⫹. Key Points The latex agglutination tests have following uses: ■ ■ ■ The tests are used for rapid identification of antigens of group B Streptococcus, Staphylococcus aureus, Neisseria meningitidis, Cryptococcus neoformans, etc. The tests have also been found to be useful for detection of soluble microbial antigens in urine, spinal fluid, and serum for diagnosis of a variety of infectious diseases. These tests are being used to detect RA factor, ASLO, CRP, etc., in serum specimens. Hemagglutination test: RBCs are used as carrier particles in hemagglutination tests. RBCs of sheep, human, chick, etc. are commonly used in the test. When RBCs are coated with antigen to detect antibodies in the serum, the test is called indirect hemagglutination (IHA) test. The IHA is a most commonly used test for serodiagnosis of many parasitic diseases including amoebiasis, hydatid disease, and toxoplasmosis. When antibodies are attached to the RBCs to detect microbial antigen, it is known as reverse passive hemagglutination (RPHA). The RPHA has been used extensively in the past to detect viral antigens, such as in HBsAg in the serum for diagnosis of hepatitis B infection. The test has also been used for detection of antigens in many other viral and parasitic infections. Viral hemagglutination: Many viruses including influenza, mumps, and measles have the ability to agglutinate RBCs without antigen–antibody reactions. This process is called viral hemagglutination. This hemagglutination can be inhibited by antibody specifically directed against the virus, and this phenomenon is called hemagglutination inhibition. This forms the basis of the viral hemagglutination inhibition test, which is used to detect antibodies in patient’s sera that neutralize the agglutinating viruses. To perform this test, patient’s serum is first incubated with a viral preparation. Then RBCs that the virus is known to agglutinate are added to the mixture. If antibody is present, this will combine with viral particles and prevent ANTIGEN–ANTIBODY REACTIONS + Antigen Antibody-coated particles (S. aureus) + 109 + Complement Antigen Complement Serum containing antibody against antigen Serum without antibody against antigen Complement fixation reaction No complement fixation reaction Antigen Antibody to sheep RBC Precipitate visible Sheep RBC + No hemolysis (complement used up in antigen– antibody reaction) Positive test Principle of the coagglutination. Antibody to sheep RBC + Hemolysis (unused complement available for hemolysis) Negative test agglutination, and a lack of or reduction in agglutination indicates presence of antibody in patient’s serum. FIG. 14-12. Coagglutination test: Coagglutination is a type of agglutination reaction in which Cowan I strain of S. aureus is used as carrier particle to coat antibodies. Cowan I strain of S. aureus contains protein A, an anti-antibody, that combines with the Fc portion of immunoglobulin, IgG, leaving the Fab region free to react with the antigen present in the specimens (Fig. 14-11). In a positive test, protein A bearing S. aureus coated with antibodies will be agglutinated if mixed with specific antigen. The advantage of the test is that these particles show greater stability than latex particles and are more refractory to changes in ionic strength. promoting phagocytosis, and in immune adherence. The complement-dependent serological tests may be of the following types: Key Points Coagglutination test has been used for: ■ ■ ■ Detection of cryptococcal antigen in the CSF for diagnosis of cryptococcal meningitis; Detection of amoebic and hydatid antigens in the serum for diagnosis of amoebiasis and cystic echinococcosis, respectively; and Grouping of streptococci and mycobacteria and for typing of Neisseria gonorrhoeae. Complement-Dependent Serological Tests The complement system is a group of serum proteins that is present in normal serum. The system consists of 20 or more serum proteins that interact with one another and with cell membrane. It is a biochemical cascade that helps to clear pathogens from the body. It aids the antibodies in lysing bacteria, 1. 2. 3. 4. ◗ Complement fixation test. Complement fixation test Immune adherence test Immobilization test Cytolytic or cytocidal reactions Complement fixation test The principle of the complement fixation test is that when antigen and antibodies of the IgM or the IgG classes are mixed, complement is “fixed” to the antigen–antibody complex. If this occurs on the surface of RBCs, the complement cascade will be activated and hemolysis will occur. The complement fixation test consists of two antigen–antibody complement systems: (a) an indicator system and (b) a test system. Indicator system: It consists of RBCs that have been preincubated with a specific anti-RBC antibody, in concentrations that do not cause agglutination, and no hemolysis of RBCs occurs in the absence of complement. Such RBCs are designated as “sensitized” red cells. Test system: In the test system, patient’s serum is first heated to 56°C to inactivate the native complement. Then the inactivated serum is adsorbed with washed sheep RBC to eliminate broadly cross-reactive anti-RBC antibodies (also known as Forssman-type antibodies), which could interfere with the assay. The serum is then mixed with purified antigen and with a dilution of fresh guinea pig serum, used as source of Chapter 14 FIG. 14-11. Section II Sheep RBC IMMUNOLOGY complement. The mixture is incubated for 30 minutes at 37°C to allow antibody in the patient’s serum to form complexes with the antigen and to fix complement (Fig. 14-12). In complement fixation test, “sensitized” red cells are then added to the mixture. If the red cells are lysed, it indicates that there were no antibodies specific to the antigen in the serum of the patient. The complement therefore was not consumed in the test system and was available to be used by the anti-RBC antibodies, resulting in hemolysis. This reaction is considered negative. The test is considered positive if the red cells are not lysed. Nonlysis of the cells indicates that patient’s serum had antibodies specific to the antigen, which have “fixed” complement. Hence, no complement was available to be activated by the indicator system. ◗ Immobilization test Immobilization test is a complement-dependent test in which certain live bacteria, such as T. pallidum, are immobilized when mixed with patient’s serum in the presence of complement. This forms the basis of T. pallidum immobilization test. A positive test shows serum to contain treponemal antibodies. ◗ Cytolytic or cytocidal reactions When a live bacterium, such as V. cholerae, is mixed with its specific antibody in the presence of complement, the bacterium is killed and lysed. This forms the basis of test used to measure anti-cholera antibodies in the serum. Neutralization Tests Key Points Chapter 14 Section II 110 The complement fixation reactions were used earlier for diagnosis of many infections, such as: ■ ■ Wassermann test for syphilis and Tests for demonstration of antibodies to M. pneumoniae, Bordetella pertussis, many different viruses, and to fungi (such as Cryptococcus spp., Histoplasma, and Coccidioides immitis). Since this test is technically very cumbersome, and often difficult, it is no longer used now-a-days. Indirect complement fixation test: Indirect complement fixation test is carried out to test the sera that cannot fix guinea pig complement. These include avian sera (e.g., parrot, duck) and mammalian sera (e.g., cat, horse). The test is carried out in duplicate and after the first test, the standard antiserum known to fix the complement is added to one set. Hemolysis indicates a positive test. In a positive test, if the serum contains antibody, the antigen would have been used up in the first test, standard antiserum added subsequently would fail to fix the complement, therefore causing hemolysis. Conglutinating complement adsorption test: It is an alternative method for systems that do not fix guinea pig complement. Sheep erythrocytes sensitized with bovine serum are used as the indicator system. The bovine serum contains conglutinin, a ␤ globulin that acts as antibody to the complement. Therefore, conglutinin causes agglutination of sensitized sheep erythrocytes if these are combined with complement, which is known as conglutination. If the horse complement had been used up by the antigen–antibody reaction in the first step, the agglutination of the sensitized cells does not occur. ◗ Immune adherence test Immune adherence test is a test in which certain pathogens (e.g., Vibrio cholerae, Treponema pallidum, etc.) react with specific antibodies in the presence of complement and adhere to erythrocytes or platelets. The adherence of cells to bacteria is known as immune adherence, which facilitates phagocytosis of the bacteria. Neutralization is an antigen–antibody reaction in which the biological effects of viruses and toxins are neutralized by homologous antibodies known as neutralizing antibodies. These tests are broadly of two types: (a) virus neutralization tests and (b) toxin neutralization tests. ◗ Virus neutralization tests Neutralization of viruses by their specific antibodies are called virus neutralization tests. Inoculation of viruses in cell cultures, eggs, and animals results in the replication and growth of viruses. When virus-specific neutralizing antibodies are injected into these systems, replication and growth of viruses is inhibited. This forms the basis of virus neutralization test. Viral hemagglutination inhibition test is an example of virus neutralization test frequently used in the diagnosis of viral infections, such as influenza, mumps, and measles. If patient’s serum contains antibodies against certain viruses that have the property of agglutinating the red blood cells, these antibodies react with the viruses and inhibit the agglutination of the red blood cells. ◗ Toxin neutralization tests Toxin neutralization tests are based on the principle that biological action of toxin is neutralized on reacting with specific neutralizing antibodies called antitoxins. Examples of neutralization tests include: ■ ■ In vivo—(a) Schick test to demonstrate immunity against diphtheria and (b) Clostridium welchii toxin neutralization test in guinea pig or mice. In vitro—(a) antistreptolysin O test and (b) Nagler reaction used for rapid detection of C. welchii. Opsonization Opsonization is a process by which a particulate antigen becomes more susceptible to phagocytosis when it combines with opsonin. The opsonin is a heat-labile substance present in fresh normal sera. Unlike opsonin, bacteriotropin is heat-stable substance present in the serum but with similar activities. ANTIGEN–ANTIBODY REACTIONS The term “opsonic index” is defined as the ratio of the phagocytic activity of patient’s blood for a particular bacterium to the phagocytic activity of blood from a normal individual. It is used to study the progress of resistance during the course of disease. It is measured by incubating fresh citrated blood with the suspension of bacteria at 37°C for 15 minutes and estimating the average number of phagocytic bacteria from the stained blood films. microscope (Fig. 14-13). Direct immunofluorescence test is widely used for detection of bacteria, parasites, viruses, fungi, or other antigens in CSF, blood, stool, urine, tissues, and other specimens. Few examples include: Key Points ■ ◗ Direct immunofluorescence test Direct immunofluorescence test is used to detect unknown antigen in a cell or tissue by employing a known labeled antibody that interacts directly with unknown antigen. If antigen is present, it reacts with labeled antibody and the antibodycoated antigen is observed under UV light of the fluorescence Antibodies Fluorochrome Antibodies combined with fluorochrome Bacterial cell Antigen on cell surface The need for preparation of separate labeled antibody for each pathogen is the major disadvantage of the direct immunofluorescence test. ◗ Indirect immunofluorescence test The indirect immunofluorescence test is used for detection of specific antibodies in the serum and other body fluids for serodiagnosis of many infectious diseases. Indirect immunofluorescence is a two-stage process. In the first stage, a known antigen is fixed on a slide. Then the patient’s serum to be tested is applied to the slide, followed by careful washing. If the patient’s serum contains antibody against the antigen, it will combine with antigen on the slide. In the second stage, the combination of antibody with antigen can be detected by addition of a fluorescent dye-labeled antibody to human IgG, which is examined by a fluorescence microscope. The first step in the indirect immunofluorescence test is the incubation of a fixed antigen (e.g., in a cell or tissue) with unlabeled antibody, which becomes associated with the antigen. Next, after careful washing, a fluorescent antibody (e.g., fluorescent labeled anti-IgG) is added to the smear. This second antibody will become associated to the first, and the antigen–antibody complex can be visualized on the fluorescence microscope. The indirect method has the advantage of using a single labeled antiglobulin (antibody to IgG) as a “universal reagent” to detect many different specific antigen–antibody reactions. The test is often more sensitive than the direct immunofluorescence test. Key Points Indirect immunofluorescence test is used widely to: Bacterial cell with bound antibodies combined with fluorochrome ■ ■ ■ ■ FIG. 14-13. Direct fluorescent antibody test. Detect specific antibodies for serodiagnosis of syphilis, leptospirosis, amoebiasis, toxoplasmosis, and many other infectious diseases; Identify the class of a given antibody by using fluorescent antibodies specific for different immunoglobulin isotypes; Identify and enumerate lymphocyte subpopulations by employing monoclonal antibodies and cytofluorographs; and Detect autoantibodies, such as antinuclear antibodies in autoimmune diseases. Chapter 14 1. Direct immunofluorescence test 2. Indirect immunofluorescence test ■ Direct immunofluorescence test for antemortem diagnosis of rabies: The test is used for detection of rabies virus antigen in the skin smear collected from the nape of the neck in humans and in the saliva of dogs. Also used for detection of N. gonorrhoeae, C. diphtheriae, T. pallidum, etc. directly in appropriate clinical specimens. Section II Immunofluorescence The property of certain dyes absorbing light rays at one particular wavelength (ultraviolet light) and emitting them at a different wavelength (visible light) is known as fluorescence. Fluorescent dyes, such as fluorescein isothiocyanate and lissamine rhodamine, can be tagged with antibody molecules. They emit blue-green and orange-red fluorescence, respectively under ultraviolet (UV) rays in the fluorescence microscope. This forms the basis of the immunological test. Immunofluorescence tests have wide applications in research and diagnostics. These tests are broadly of two types: 111 112 IMMUNOLOGY The major limitation of immunofluorescence is that the technique requires (a) expensive fluorescence microscope and reagents, (b) trained personnel, and (c) have a factor of subjectivity that may result in erroneous results. Chapter 14 Section II Enzyme Immunoassays Enzyme immunoassays (EIAs) can be used for detection of either antigens or antibodies in serum and other body fluids of the patient. In EIA techniques, antigen or antibody labeled with enzymes are used. Alkaline phosphatase, horseradish peroxidase, and galactosidase are the enzymes used in the EIA tests. The commonly used EIAs are enzyme-linked immunosorbent assays (ELISAs). The ELISA technique was first conceptualized and developed by Peter Perlmann and Eva Engvall at Stockholm University, Sweden. These assays involve the use of an immunosorbent specific to either the antigen or antibody. Following the antigen–antibody reaction, chromogenic substrate specific to the enzyme (o-phenyldiamine dihydrochloride for peroxidase, p-nitrophenyl phosphate for alkaline phosphatase, etc.) is added. The reaction is detected by reading the optical density. Usually, a standard curve based on known concentrations of antigen or antibody is prepared from which the unknown quantities are calculated. There are different types of ELISAs available for the detection and quantitation of either the antigen or antibodies in serum and other body fluids. These include: (a) indirect ELISA, (b) sandwich ELISA, (c) competitive ELISA, and (d) ELISPOT assay. ◗ Indirect ELISA The indirect ELISA is used for the quantitative estimation of antibodies in the serum and other body fluids. In this method, specimens are added to microtiter plate wells coated with antigen to which specific antibodies are to be detected. After a period of incubation, the wells are washed. If antibody was present in the sample, antigen–antibody complex would have been formed and will not get washed away. On the other hand, if the specific antibody was not present in the specimen, there would not be any complex formation. Next, an anti-isotype antibody conjugated with an enzyme is added and incubated. After another washing step, a substrate for the enzyme is added. If there was complex formation in the initial step, the secondary anti-isotype antibody would have bound to the primary antibody, and there would be a chromogenic reaction between the enzyme and substrate. By measuring the optical density values of the wells, after a stop solution has been added to arrest the chromogenic reaction, one can determine the amount of antigen–antibody complex formed in the first step (Fig. 14-14). Key Points The test is extensively used for determination of serum antibodies for diagnosis of human immunodeficiency virus (HIV) infection, Japanese encephalitis, dengue, and many other viral infections. ◗ Sandwich ELISA The sandwich ELISA is used for the detection of antigen. In this test, the known antibody is coated and immobilized onto the wells of microtiter plates. The test sample containing the suspected antigen is added to the wells and is allowed to react with the antibodies in the wells. After the step of washing the well, a second enzyme-conjugated antibody specific for a different epitope of the antigen is added and allowed to incubate. After removing any free secondary antibody by rewashing, the specific substrate is added, and the ensuing chromogenic reaction is measured. The chromogenic reaction is then compared with a standard curve to determine the exact amount of the antigen present in the test sample. In a positive test, an enzyme acts on the substrate to produce a color, and its intensity can be measured by spectrophotometer or ELISA reader. The change of color can also be observed by the naked eye (Fig. 14-15). Antigen Antibody Enzyme conjugated antibody Enzyme substrate Product Antigen is adsorbed to well surface FIG. 14-14. Indirect ELISA test. Excess unbound antibody is washed off during washing Clinical specimen is added: antibody binds to antigen Excess unbound enzyme conjugated antibody is washed off during washing Enzyme conjugated anti-immunoglobulin antibody binds to antibody Enzyme substrate ( ) is added and substrate is converted to product ( ) that causes a visible color ANTIGEN–ANTIBODY REACTIONS 113 Antigen Antibody Enzyme conjugated antibody Enzyme substrate Product Enzyme substrate ( ) is added and substrate is converted to product ( ) that causes a visible color Sandwich ELISA test. Key Points The sandwich ELISA is used to detect rotavirus and enterotoxin of Escherichia coli in feces. ◗ Enzyme conjugated antibody specific for the test antigen binds to antigen forming sandwich ◗ ELISPOT Assay ELISPOT assay is a modification of ELISA. It allows the quantitative determination of number of cells in a population that are producing antibodies specific for a given antigen or an antigen for which one has a specific antibody. These tests have found application widely in the measurement of cytokines. Competitive ELISA Competitive ELISA is another technique used for the estimation of antibodies present in a specimen, such as serum. Principle of the test is that two specific antibodies, one conjugated with enzyme and the other present in test serum (if serum is positive for antibodies), are used. Competition occurs between the two antibodies for the same antigen. Appearance of color indicates a negative test (absence of antibodies), while the absence of color indicates a positive test (presence of antibodies). In this test, the microtiter wells are coated with HIV antigen. The sera to be tested are added to these wells and incubated at 37°C and then washed. If antibodies are present in the test serum, antigen–antibody reaction occurs. The antigen– antibody reaction is detected by adding enzyme-labeled-specific HIV antibodies. In a positive test, no antigen is left for these antibodies to act. Hence, the antibodies remain free and are washed away during the process of washing. When substrate is added, no enzyme is available to act on it. Therefore, positive result indicates no color reaction. In a negative test, in which no antibodies are present in the serum, antigen in the coated wells is available to combine with enzyme-conjugated antibodies and the enzyme acts on the substrate to produce color. Key Points Competitive ELISA is the most commonly used test for detection of HIV antibodies in serum in patients with HIV. Radioimmunoassay When radioisotopes instead of enzymes are used as labels to be conjugated with antigens or antibodies, the technique of detection of the antigen–antibody complex is called as radioimmunoassay (RIA). RIA was first described in 1960 for measurement of endogenous plasma insulin by Solomon Berson and Rosalyn Yalow of the Veterans Administration Hospital in New York. Yalow was awarded the 1977 Nobel Prize for Medicine for the development of the RIA for peptide hormones, but because of his untimely death in 1972, Berson could not share the award. The classical RIA methods are based on the principle of competitive binding. In this method, unlabeled antigen competes with radiolabeled antigen for binding to antibody with the appropriate specificity. Thus, when mixtures of radiolabeled and unlabeled antigen are incubated with the corresponding antibody, the amount of free (not bound to antibody) radiolabeled antigen is directly proportional to the quantity of unlabeled antigen in the mixture. In the test, mixtures of known variable amounts of cold antigen and fixed amounts of labeled antigen and mixtures of samples with unknown concentrations of antigen with identical amounts of labeled antigen are prepared in the first step. Identical amounts of antibody are added to the mixtures. Antigen–antibody complexes are precipitated either by crosslinking with a second antibody or by means of the addition of reagents that promote the precipitation of antigen–antibody complexes. Counting radioactivity in the precipitates allows Chapter 14 FIG. 14-15. Clinical specimen is added: antigen binds to antibody Excess unbound enzyme conjugated antibody is washed off during washing Section II Antibody is adsorbed to well surface Excess unbound antigen is washed off during washing 114 IMMUNOLOGY the determination of the amount of radiolabeled antigen precipitated with the antibody. A standard curve is constructed by plotting the percentage of antibody-bound radiolabeled antigen against known concentrations of a standardized unlabeled antigen, and the concentrations of antigen in patient samples are extrapolated from that curve. The extremely high sensitivity of RIA is its major advantage: Chapter 14 Section II Key Points Uses of RIA: ■ ■ The test can be used to determine very small quantities (e.g., nanogram) of antigens and antibodies in the serum. The test is used for quantitation of hormones, drugs, HBsAg, and other viral antigens. The main drawbacks of the RIA include: (a) the cost of equipment and reagents, (b) short shelf-life of radiolabeled compounds, and (c) the problems associated with the disposal of radioactive waste. Western Blotting Western blotting is called so because the procedure is similar to Southern blotting, which was developed by Edwin Southern for the detection of DNA. While Southern blotting is done to detect DNA, Western blotting is done for the detection of proteins. Western blotting is usually done on a tissue homogenate or extract. It uses SDS-PAGE (sodium dodecyl sulphatepolyacrylamide gel electrophoresis), a type of gel electrophoresis to first separate various proteins in a mixture on the basis of their shape and size. The protein bands thus obtained are transferred onto a nitrocellulose or nylon membrane where they are “probed” with antibodies specific to the protein to be detected. The antigen–antibody complexes that form on the band containing the protein recognized by the antibody can be visualized in a variety of ways. If the protein of interest was bound by a radioactive antibody, its position on the blot can be determined by exposing the membrane to a sheet of X-ray film, a procedure called autoradiography. However, the most generally used detection procedures employ enzyme-linked antibodies against the protein. After binding of the enzyme–antibody conjugate, addition of a chromogenic substrate that produces a highly colored and insoluble product causes the appearance of a colored band at the site of the target antigen. The site of the protein of interest can be determined with much higher sensitivity if a chemiluminescent compound along with suitable enhancing agents is used to produce light at the antigen site (Fig. 14-16). Key Points Western blot technique has many uses as follows: Proteins are separated through SDS-PAGE ■ Proteins separated on gel were blotted onto nitrocellulose paper Nitrocellulose paper ■ Incubation with patient’s antisera (first antibody) Incubation with enzyme conjugated antihuman serum (second antibody) Antibodies attach to specific antigen bands on nitrocellulose paper Enzyme conversion of substrate identifies the antigen Bands can be visually interpreted FIG. 14-16. Western blot test. ■ ■ It is used for identification of a specific protein in a complex mixture of proteins. In this method, known antigens of well-defined molecular weight are separated by SDS-PAGE and blotted onto nitrocellulose. The separated bands of known antigens are then probed with the sample suspected of containing antibody specifi c for one or more of these antigens. Reaction of an antibody with a band is detected by using either radiolabeled or enzyme-linked secondary antibody that is specific for the species of the antibodies in the test sample. It is also used for estimation of the size of the protein as well as the amount of protein present in the mixture. The Western blot test is most widely used as a confirmatory test for diagnosis of HIV, where this procedure is used to determine whether the patient has antibodies that react with one or more viral proteins or not. The Western blotting is also used for demonstration of specific antibodies in the serum for diagnosis of neurocysticercosis and tubercular meningitis. Chemiluminescence Assay The chemiluminescence assay uses chemiluminescent compounds that emit energy in the form of light during the antigen–antibody reactions. The emitted lights are measured and the concentration of the analyze is calculated. The assay is a fully automated method, which is used commonly for drug sensitivity testing of Mycobacterium tuberculosis. ANTIGEN–ANTIBODY REACTIONS 115 Immunoelectronmicroscopic Tests ◗ These are the types of antigen–antibody reactions that are visualized directly by electron microscope. These are of the following types: This test is used to detect antigen directly in tissue specimens, in which tissue sections are treated with peroxidase-labeled antisera to detect corresponding antigen. The peroxidase bound to the antigen is visualized under the electron microscope. ◗ Immunoelectronmicroscopy ◗ Immunoferritin test Electron-dense substances, such as ferritin are conjugated with antibody and such labeled antibodies reacting with antigen can be visualized under the electron microscope. Section II This is a test used to detect rotavirus and hepatitis A virus directly in feces. In this test, viral particles are mixed with specific antisera and are demonstrated as clumps of virion particles under the electron microscope. Immunoenzyme test Chapter 14 43 15 Mycobacterium Complement System Leprae Introduction The term complement refers to the ability of a system of some nonspecific proteins in normal human serum to complement, i.e., augment the effects of other components of immune system, such as antibody. The complement system, which is an important component of the human innate host defense system, consists of approximately 20 proteins that are present in normal human serum. is designated “b”, and the small fragment “a”. But for historical reasons, with respect to the fragments of C2, the large fragment is designated C2a and the small one is designated C2b. Key Points Effects of complement There are four main effects of complement: ■ ■ The Complement System ■ The complement system is an extremely powerful system comprising of rapidly acting glycoproteins, several proenzymes, and components, and it exists in an inactive state in the plasma. All normal individuals always have complement components in their blood. Properties of Complement Complement shows the following properties: 1. It is present in sera of all mammals including humans and in lower animals including birds, amphibians, and fishes. 2. These are heat-labile substances that are inactivated by heating serum at 56°C for 30 minutes. 3. These are glycoproteins and are synthesized primarily by liver cells and to a very less extent by macrophages and many other cell types. The rate of synthesis of the various complement glycoproteins increase when complement is activated and consumed. 4. The complement usually does not bind to the antigen or antibody but only to antigen–antibody complex. 5. The importance of the complement lies in the fact that it contributes to both the acquired and innate immunity of an individual. ■ It causes lysis of cells (such as bacteria, viruses, allografts, and tumor cells). It generates mediators that participate in triggering specific cell functions, inflammation, and secretion of immunoregulatory molecules. It facilitates opsonization, the process by which bacteria are more readily and more efficiently engulfed by phagocytes. It causes immune clearance, in which immune complexes from the circulation are removed and are transported to spleen and liver. Activation of Complement Complement activation takes place through any of the following three pathways: 1. The classical pathway 2. The alternative pathway 3. The lectin pathway Of these, alternative and lectin pathways are important in the innate immunity of the host. These two are also more important when the human host is infected by a microorganism for the first time, because the antibody required to trigger the classical pathway is not present. All the three activation pathways lead to activation of C3, resulting in the production of C3b. Hence, C3b is considered as the central molecule in the activation of the complement cascade. Nomenclature of Complement Key Points Complement components are designated by numerals, viz., C1–9. These components circulate in plasma in the form of proenzymes that are functionally inactive. Activation involves cleavage by proteolysis into peptide fragments. The fragments are designated with lowercase suffixes—for example, C3 is cleaved into two fragments, C3a and C3b. Normally, the large fragment The C3b has two important functions to perform: ■ ■ First, it combines with other components of the complement to produce C5 convertase, the enzyme that leads to the production of membrane attack complex; and Second, it opsonizes bacteria due to the presence of receptors for C3b on the surface of the phagocytes. 117 COMPLEMENT SYSTEM The final steps that lead to the formation of a membrane attack complex are same in all the pathways. When these complement components are activated, a sequential, rapid cascading pattern ensues. This is because once a complement component is activated, it is either cleaved or becomes bound to a previously activated component or complex of complement components. Also, each component or complex of components, once activated, generally amplifies the cascading process by activating many molecules of the next component in the series. C1q, r, s Activated C1 Ag-Ab complex C3 C14b C4 C4a Mg++ C3a C14b2a C2 C2b C3 convertase The classical pathway is a chain of events in which complement components react in specific sequences as a cascade resulting in cell lysis. It is activated by antibody bound to antigen but never by native or free antibody. C5 C5a Classical pathway of activation of the complement. Key Points ■ Immunoglobulins IgM and IgG. The IgG subclasses vary with regard to their efficiency in activating the complement; IgG3 immunoglobulins are the most efficient, followed by IgG1 and IgG2. IgG4 immunoglobulins do not activate the classical pathway. ● ■ ■ ■ Native, free IgG or IgM do not activate the complement system. A single, native IgG molecule will not bind and activate the complement pathway. However, if antibodies of the IgG class are aggregated by antigen binding, this will result in complement fixation and activation. The formation of an antigen–antibody complex induces conformational changes in the Fc portion of the IgM molecule that expose a binding site for the C1 component of the complement system Staphylococcal protein A, C-reactive protein, and DNA Steps of activation of classical pathway: The classical pathway of complement activation usually begins with the formation of soluble antigen–antibody complexes (immune complexes) or with the binding of antibody to antigen on a suitable target, such as a bacterial cell (Fig. 15-1). Following are the sequential steps in the activation of classical pathway: 1. Activation of C1 is the first step in the cascade of classical pathway activation. The C1 actually is a complex of three different types of molecules: C1q, C1r, and C1s. C1q first combines with the Fc portion of the bound antibody, IgM or IgG. This results in the sequential activation of C4, C2, and C3. For C1 to be activated, it must bind to at least two adjacent Fc regions. This means that the concentration of antibody of the IgG class must be relatively high and that the specific antigenic determinants recognized by the IgG antibody must be in close proximity. When pentameric IgM is bound to antigen on a target surface, it assumes the so-called stable configuration, in which at least three binding sites for the C1q are exposed. Since IgG molecules have a lower valency, about 1000 of them are needed to ensure the initiation of the complement pathway as against only one IgM molecule. 2. C1q binding in the presence of calcium ions leads to activation of C1r and C1s. Activated C1s is an esterase that splits C4 into two fragments: a small soluble fragment (C4a) and a larger fragment (C4b). C4a has anaphylatoxin activity, and C4b binds to cell membrane along with C1. C4b in the presence of Mg2⫹ splits C2 into C2a and C2b. The smaller fragment (C2b) diffuses away, while the larger fragment (C2a) remains attached to C4b. The resulting C4b2a complex possesses enzymatic activity and is called C3 convertase, which converts C3 into an active form. 3. The C3 convertase activate thousands of C3 molecules and splits these molecules into C3a and C3b. A single C3 convertase molecule can generate over 200 molecules of C3b, resulting in tremendous amplification at this step of the sequence. The biological importance of activated C3b as well as C4b is that they are able to bind to C3b/C4b receptors (currently designated as CR1 receptors) present on almost all host cells, most notably phagocytes. The increased affinity of phagocytic cells for C3b (or iC3b)/C4b-coated particles is known as immune adherence. The latter is responsible for a significant enhancement of phagocytosis, which is one of the main defense mechanisms of the body. 4. Some of the C3b binds to C4b2a to form a trimolecular complex C4b2a3b called C5 convertase. The C5 convertase splits C5 into C5a and C5b. C5a diffuses away, while C5b attaches to C6 and initiates formation of C5b–9 complex otherwise known as membrane attack complex (MAC). Chapter 15 FIG. 15-1. Activators of the classical pathway: Activators of classical pathways include the following: Section II C14b2a3b C5 convertase Classical Pathway of Complement Activation 118 IMMUNOLOGY C5a C5 C5b inserted into lipid bilayer C6 insertion C5b C6 C7 Insertion of C7 C8 C8 insertion Section II C9 (multiple) Insertion of C9 Pore formation by MAC Pore formation by multiple C9 Influx and efflux of fluids and molecules Lysis Chapter 15 Formation of MAC Cell lysis FIG. 15-2. Formation of membrane attack complex. Microbe Key Points FIG. 15-3. Membrane attack complex The formation of the MAC (Fig. 15-2) is the terminal sequence of all three different pathways including the classical pathway. All the three pathways converge at the step involving the formation of the MAC. The formation of the MAC involves the participation of the complement components C5b, C6, C7, C8, and C9. A complex of C5b, C6, and C7 is first formed in the soluble phase and then attaches to the cell membrane through the hydrophobic amino acid groups of C7. The C7 becomes exposed as a consequence of the binding of C7 to the C5b–C6 complex. Lysed microbe Action of membrane attack complex. Factor H + Factor I − Free C3b Inactivated C3b C3 Microbe C3a Factor B Mg++ Bound C3b C3bB Factor D Ba Released C5b67 complexes can insert into the membrane of nearby cells and mediate “innocent-bystander” lysis. Regulator proteins in human sera normally prevent this from occurring, but in certain diseases cell and tissue damage may occur due to this process of innocent-bystander lysis. The membrane-bound C5b–6–7 complex acts as a receptor for C8 and C9. C8, on binding to the complex, stabilizes the attachment of the complex to the foreign cell membrane. The C5b–8 complex acts as a catalyst for C9, which is a single chain glycoprotein with a tendency to polymerize spontaneously. 5. The C5b–8 complex on binding to C9 molecules undergoes polymerization, which finally ends in the formation of C5b–9 complex also known as MAC. The MAC forms a transmembrane channel of 100 Å diameter in the cell. This transmembrane channel allows the free exchange of ions between the cell and the surrounding medium. Due to the rapid influx of ions into the cell and their association with cytoplasmic proteins, the osmotic pressure rapidly increases inside the cell. This results in an influx of water, swelling of the cell, and, for certain cell types, rupture of the cell membrane and finally lysis (Fig. 15-3). C3a C3bBbP3b C5 convertase C3bBb C3 C3 convertase C3bBbP Stabilized C3 convertase Properdin C5 C5a FIG. 15-4. Alternative pathway of activation of the complement. Alternative Pathway of Complement Activation The alternative pathway was first described by Pillemer in 1954. It differs from the classical pathway in (a) the nature of activating substances and (b) the sequence of events itself. The alternative pathway is unique in not requiring antigen–antibody complexes to activate the complement. This pathway does not depend on antibody and does not involve the early complement components (C1, C2, and C4) for activation of the complement. It, therefore, can be activated before the establishment of an immune response to the infecting pathogen (Fig. 15-4). COMPLEMENT SYSTEM Key Points Activators of the alternative pathway: Activators of the alternative pathway of complement activation include (a) IgA, (b) IgD, (c) bacterial endotoxin, (d) cobra venom factor, and (e) nephritic factor. Regulation of Complement System Since the complement system involves the formation of many biologically active substances, there are many regulatory systems to prevent unwarranted damage to the human host. The activities of the different complement components activated at each stage of the cascade are regulated by several MBL MBL-MASP complex Glycoproteins on microbes MASP C4 C2 C4a C2a C4b2b C3 convertase C4b2b3b C5 convertase C3 Lectin Pathway of Complement Activation The lectin pathway, as the name suggests, is triggered by lectins. Lectins are the proteins that recognize and bind to specific carbohydrate targets. The mannose-binding lectin (MBL) is one such protein that takes part in the lectin pathway of TABLE 15-1 C3a C5 C5a FIG. 15-5. Lectin pathway of activation of the complement. Comparison of classical, alternative, and lectin pathways Classical pathway Alternative pathway Lectin pathway Chain of events in which components react in specific sequence following activation of C1 Activation of C3 without prior participation of C1,4,2 Activated by binding of mannose-binding lectin to mannose residues on surface of microorganisms Requires binding of C1 to antigen–antibody complex Activators are bacterial endotoxins, IgA and IgD, cobra venom factor, and nephritic factor No role for antibodies; similar to alternate pathway Cannot be considered as a component of innate immune mechanism It is a component of the innate immune mechanism Can be considered as a component of innate immune mechanism Chapter 15 1. The C3b binds with factor B to form C3bB complex. The interaction between C3b and factor B is stabilized by Mg2⫹, which is the only ion required for functional activation of the alternative pathway. Therefore, tests to discriminate between the two complement activation pathways are often based on the selective chelation of Ca2⫹ (to disrupt C1q, C1r2, and C1s2) and the addition of sufficient Mg2⫹ to allow activation of the alternative pathway. 2. The C3bB is split into two fragments, Ba and Bb, by another serum protein called factor D or C3 proactive convertase. Since factor D has never been isolated in its proenzyme form, it is generally believed to be activated immediately upon leaving the hepatocyte where it is synthesized. The Ba is released into the medium and the Bb binds to C3b forming the C3bBb complex, which possesses the C3 convertase activity. 3. The C3bBb complex activates more C3, leading to the formation of more C3bBb, which in turn is capable of activating C5 and the MAC. The C3bBb complex has a half-life of only 5 minutes, but by binding with properdin it forms PC3bBb complex, which is relatively heat stable. 4. The alternative pathway then proceeds from C3 to produce finally the MAC, in the same way as occurs in the classical pathway. complement activation. MBL is a large serum protein that binds to nonreduced mannose, fructose, and glucosamine on bacterial and other cell surfaces with mannose-containing polysaccharides (mannans) (Fig. 15-5). The binding of MBL to a pathogen results in the secretion of two MBL-associated serine proteases: MASP-1 and MASP-2. MASP-1 and MASP-2 are similar to C1r and C1s, respectively, and MBL is similar to C1q. Formation of the MBL/MASP-1/ MASP-2 trimolecular complex results in activation of MASPs and subsequent cleavage of C4 into C4a and C4b. Subsequently, it proceeds to produce MAC in the same way as that occurs in the classical and alternative pathways. Differences between classical, alternative, and lectin pathways are summarized in Table 15-1. Section II Steps of activation of alternative pathway: The initial component of the alternative pathway involves four serum proteins: C3b, factor B, factor D, and properdin. 119 Chapter 15 Section II 120 IMMUNOLOGY mechanisms. The following are regulators of the complement system: 1. Level of antibody: The level of antibody itself is the first regulatory step in the classical pathway. If antigen is not bound to the antibodies, the complement-binding sites on the heavy chains of IgG and IgM are unavailable to the C1 component of the complement. This means that complement is not activated even if IgM and IgG are present in the blood at all times. However, when antigen binds with specific antibodies, a conformational shift occurs and that allows the C1 component to bind and initiate the cascade reaction. 2. C1 inhibitors: These inhibitors play a critical role in limiting unnecessary complement activation. These prevent the formation and function of C1qrs complex by causing C1s to dissociate from C1qrs. The C1 inhibitors may also aid in the removal of the entire C1 complex from the antigen–antibody complexes. 3. Other inhibitory substances: Multiple substances have inhibitory effects over different steps of the activation sequence of the classical pathway. These are considered to be host cell protective mechanisms. These mechanisms probably help to protect the host cells from the possible bystander damage initiated by activated complement fragments (C3b and C4b) being formed on and near its surface. 4. Decay-accelerating factor (DAF): It is another inhibitory substance located in a large variety of host cell membranes. It is so termed because it can accelerate the dissociation of active C4b2a complexes, turning off their ability to activate native C3. In addition, DAF remains attached to membranebound C4b and C3b, and prevents the subsequent interaction with C2a and factor B, respectively. As a consequence, the two types of C3 convertases, C4b2a and C3bBb, are not formed; hence, the rate of C3 breakdown is reduced and the host cells are spared from complement-mediated membrane damage. 5. Regulation of alternative pathway: The alternative pathway has its own set of regulatory proteins and mechanisms. It is mediated by the binding of factor H to C3b and cleavage of this complex-by specific plasma inhibitor factor I, a protease. This reduces the amount of C5 convertase available. Biological Effects of Complement The main role of complement is to amplify the humoral immune response. The complement through its various products participates in the inflammatory response, opsonization of antigen, viral neutralization, and clearance of immune complexes as follows: Chemotaxis ■ C5a is a chemotactic molecule specifically recognized by polymorphonuclear leukocytes or phagocytic cells. This substance causes leukocytes to migrate to a tissue in ■ ■ which an antigen–antibody reaction is taking place. At that site, a phagocytic cell recognizes opsonized particles and ingests them. C5a not only has a chemotactic effect on neutrophils, but also activates these cells causing their reversible aggregation and release of stored enzymes, including proteases. C5a also enhances the adhesiveness of neutrophils to the endothelium. Opsonization Complement plays an important role in opsonization of pathogenic bacteria and viruses. Bacteria and viruses are easily phagocytosed by phagocytic cells in the presence of complement component C3b. This is because the receptors for the C3b component are present on the surface of many phagocytes. Hypersensitivity Reactions Complement participates in type II (cytotoxic) and type III (immune-complex) hypersensitivity reactions. The C3a, C4a, and C5a components stimulate degranulation of mast cells with release of mediators, such as histamine. The C3a fragments bind to receptors on basophils and mast cells and induce the release of stored vasoactive amines (e.g., histamine) and heparin. The release of histamine into the tissues results in increased capillary permeability and smooth muscle contraction. Fluid is released into the tissue, thereby causing edema and swelling. There is some evidence that C3a and C5a may also act directly on endothelial cells, causing increased vascular permeability. The end result is very similar to the classical anaphylactic reaction that takes place when IgE antibodies bound to the membranes of mast cells and basophils react with the corresponding antigens. For this reason, C3a and C5a are called as anaphylatoxins. Cytolysis Complement mediates cytolysis. Insertion of C5b–9 complex (MAC) into the cell membrane leads to killing or lysis of erythrocytes, bacteria, and tumor cells. The insertion of the MAC complex results in disruption of the membrane, thereby leading to entry of water and electrolytes into the cell. Enhancement of Antibody Production The binding of C3b to the surface receptors on the activated B cells markedly enhances the production of antibodies in comparison to that of B cells activated by antigen alone. Hence, deficiency of C3b leads to reduced production of antibodies. Therefore, low concentration of both C3b and antibodies affects host defense, resulting in severe pyogenic infections. COMPLEMENT SYSTEM TABLE 15-2 121 Diseases associated with complement deficiencies Disorder Salient features C1 esterase inhibitor Hereditary angioedema Transient but recurrent localized edema in the skin, gastrointestinal tract, and respiratory tract C1q Associated with hypogammaglobulinemia and severe combined immunodeficiency disease Repeated infections C2 and C4 Similar to SLE Due to failure in clearance immune-mediated complexes C3 Severe recurrent pyogenic infections Streptococcus pneumoniae infections C5 Impaired chemotaxis Increased susceptibility to bacterial infection C5–C8 Bacteremia Gram-negative diplococci and toxoplasmosis Complement plays an important role in the well-being of humans. Deficiency of various components may result in many diseases as follows: 1. Inherited deficiency of C1 esterase inhibitors causes angioedema. The low level of C1 esterase inhibitors leads to overproduction of esterase. This leads to an increase in release of anaphylatoxins, which cause capillary permeability and edema. 2. Acquired deficiency of DAF results in an increase in complement-mediated hemolysis. The condition manifests clinically as paroxysmal nocturnal hemoglobinuria. 3. Inherited or acquired deficiency of C5–8 components greatly enhances susceptibility to Neisseria bacteremia and other infections. Deficiency of C3 leads to severe recurrent pyogenic sinusitis and respiratory infections. 4. The synthesis of sufficient quantities of complement is reduced in the patients with severe liver disease, such as chronic hepatitis or alcoholic cirrhosis. These patients, therefore, are highly susceptible to infections caused by pyogenic bacteria. Deficiency of complement components and diseases associated with these complement deficiency are summarized in Table 15-2. Biosynthesis of Complement Various components of the complement are synthesized at various sites of the body. For example, C1 is synthesized in the intestinal epithelium, both C2 and C4 in the macrophages, C5 and C8 in the spleen, and C3, C6, and C9 in the liver. There is an increase in the level of C3, C4, C5, and C6 in the acute phase of inflammation. Complement along with some other plasma proteins known as acute phase reactants show a rise in acute inflammation. Quantitation of Complement Estimation of the highest dilutions of serum lysing sheep erythrocytes sensitized by antierythrocytic antibody complement activity of the serum is a method frequently used to measure complement activities of the serum. Radial immunodiffusion in agar is employed to measure complement components in the serum. Chapter 15 Deficiency of Complement Section II Deficiency 16 43 Structure and Function of Immune System Mycobacterium Leprae Introduction The lymphoreticular system is a complex organization of cells of diverse morphology, distributed widely in different organs and tissues of the human body, and is responsible for immunity. It consists of lymphoid and reticuloendothelial components and is responsible for immune response of the host. The lymphoid cells, which include lymphocytes and plasma cells, are responsible for conferring specific immunity. On the other hand, the reticuloendothelial system, which consists of phagocytic cells and plasma cells, is responsible for nonspecific immunity. These cells kill microbial pathogens and other foreign agents, and remove them from blood and tissues. ◗ Thymus is the first lymphoid organ to develop. It reaches its maximal size at puberty and then atrophies, with a significant decrease in both cortical and medullary cells and an increase in the total fat content of the organ. The thymus is a flat, bilobed organ situated above the heart. Each lobe is surrounded by a capsule and is divided into lobules, which are separated from each other by strands of connective tissue called trabeculae. Each lobule is organized into two compartments: cortex and medulla. The stroma of the organ is composed of dendritic cells, epithelial cells, and macrophages (Fig. 16-1, Color Photo 9). ■ Lymphoid Tissues and Organs ■ The specific immune response to antigen is of two types: (a) humoral or antibody-mediated immunity, mediated by antibodies produced by plasma cells; and (b) cell-mediated immunity, mediated by sensitized lymphocytes. The immune system is organized into several special tissues, which are collectively termed lymphoid or immune tissues. The tissues that have evolved to a high degree of specificity of function are termed lymphoid organs. Lymphoid organs include the gut-associated lymphoid tissues—tonsils, Peyer’s patches, and appendix—as well as aggregates of lymphoid tissue in the submucosal spaces of the respiratory and genitourinary tracts. The lymphoid organs, based on their function, are classified into central (primary) and peripheral (secondary) lymphoid organs. Central (Primary) Lymphoid Organs Central or primary lymphoid organs are the major sites for lymphopoiesis. These organs have the ability to produce progenitor cells of the lymphocytic lineage. These are the organs in which precursor lymphocytes proliferate, develop, and differentiate from lymphoid stem cells to become immunologically competent cells. The primary lymphoid organs include thymus and bone marrow. In mammals, T cells mature in thymus and B cells in fetal liver and bone marrow. After acquiring immunological competency, the lymphocytes migrate to secondary lymphoid organs to induce appropriate immune response on exposure to antigens. Thymus Cortex: It consists mainly of (a) cortical thymocytes, the immunologically immature T lymphocytes, and (b) a small number of macrophages and plasma cells. In addition, the cortex contains two subpopulations of epithelial cells, the epithelial nurse cells and the cortical epithelial cells, which form a network within the cortex. Medulla: It contains predominantly mature T lymphocytes and has a larger epithelial cell-to-lymphocyte ratio than the cortex. The concentric rings of squamous epithelial cells known as Hassall’s corpuscles are found exclusively in the medulla. Thymus is the site where a large diversity of T cells is produced and so they can recognize and act against a myriad number of antigen–MHCs (major histocompatibility complexes). The thymus induces the death of those T cells that cannot recognize antigen–MHCs. It also induces death of those T cells that react with self-antigen MHC and pose a danger of causing autoimmune disease. More than 95% of all thymocytes die by apoptosis in the thymus without ever reaching maturity. Subcapsular (marginal) capsule Primary follicle Germinal center of secondary follicle High endothelial venule Afferent lymphatic vessel FIG. 16-1. A schematic diagram of the thymus. Collagenous capsule Cortex Paracortex Medulla Efferent lymphatic vessels Lymphatic artery and vein Hilus Medullary cords Trabeculum STRUCTURE AND FUNCTION OF IMMUNE SYSTEM Key Points Functions of the thymus: The thymus is the only clearly individualized primary lymphoid organ in mammals. It has many functions: ■ ■ Bone marrow Some lymphoid cells develop and mature within the bone marrow and are referred to as B cells (B for bursa of Fabricius, or bone marrow). The function of bursa of Fabricius in birds is played by bone marrow in humans. Bone marrow is the site for proliferation of stem cells and for the origin of pre-B cells and their maturation to become immunoglobulin-producing lymphocytes. Immature B cells proliferate and differentiate within the bone marrow. Stromal cells within the bone marrow interact directly with the B cells and secrete various cytokines that are required for the development of B cells. Like thymic selection during T-cell maturation, a selection process within the bone marrow eliminates B cells with self-reactive antibody receptors. B lymphocytes develop their B-cell receptors (BCRs) by DNA rearrangement. They express auxiliary molecules, such as Ig␣ and Ig␤, and begin to express IgM on their surfaces before leaving the bone marrow. Subsequently, mature B lymphocytes also acquire C3 and Fc receptors on their surfaces. B lymphocytes on their surfaces either bear IgM alone or in association with IgG or IgA depending upon the production of particular class of immunoglobulin. The B lymphocytes are transformed into plasma cells and secrete antibodies. B lymphocytes are primarily responsible for antibody-mediated immunity. Peripheral (Secondary) Lymphoid Organs Peripheral or secondary lymphoid organs consist of (a) lymph nodes, (b) spleen, and (c) nonencapsulated structures, such as mucosa-associated lymphoid tissues (MALT). These organs serve as the sites for interaction of mature lymphocytes with antigens. ◗ Lymph nodes The lymph nodes are extremely numerous and disseminated all over the body. They play a very important and dynamic role in Key Points Functions of the lymph nodes: Lymph nodes serve the following functions: ■ ■ ■ They act as filter for the lymph, the fluid, and cellular content of the lymphocytic circulatory system. They also provide sites for mingling of lymphocytes, monocytes, and dendritic cells for initiation of immune responses. Most antigen-activated B cells divide and differentiate into antibody-producing plasma cells in lymphoid follicles, but only a few B cells in the antigen-activated population find their way into germinal centers. Those that do, undergo one or more rounds of cell division during which the genes that encode their antibodies mutate at an unusually high rate. They phagocytose microbial pathogens and other foreign substances. Chapter 16 ◗ the initial or inductive states of the immune response. Lymph nodes measure 1–25 mm in diameter and are surrounded by a connective tissue capsule. The lymph node has two main parts: cortex and medulla. The reticulum or framework of the lymph node is composed of phagocytes and specialized types of reticular or dendritic cells (Color Photo 10). Cortex: The cortex and the deep cortex, also known as paracortical area, are densely populated by lymphocytes. Roughly spherical areas containing densely packed lymphocytes, termed primary lymphoid follicles or nodules, are found in the cortex. B and T lymphocytes are found in different areas in the cortex. The primary lymphoid follicles predominately contain B lymphocytes. They also contain macrophages, dendritic cells, and some T lymphocytes. The primary follicles are very densely packed with small lymphocytes, not actively involved in an immune response. The larger, less dense follicles, termed secondary follicles, are found in the cortex of a lymph node draining an area in which an infection has taken place. The secondary follicles contain clear germinal centers where B lymphocytes actively divide as a result of antigenic stimulation. T lymphocytes are found predominantly in the deep cortex or paracortical area; for this reason, the paracortical area is designated as T-dependent. Interdigitating cells are also present in this area, where they present antigen to T lymphocytes. Medulla: It is less densely populated and is composed mainly of medullary cords. These cords are elongated branching bands of the lymphocytes, plasma cells, and macrophages. They drain into the hilar efferent lymphatic vessels. Plasma cells are also found in the medullary cords. Following the period of division, there is a rigorous selection process in which more than 90% of these B cells die by apoptosis or cell death. As antigen is carried into a regional node by the lymph, it is trapped, processed, and presented together with class II MHC molecules by interdigitating dendritic cells in the paracortex, resulting in the activation of TH cells. The initial activation of B cells is also thought to take place within the T-cell-rich paracortex. Once activated, TH and B cells form small foci consisting largely of proliferating B cells at the edges of the paracortex. Some B cells within the foci differentiate into plasma cells secreting IgM and IgG. Section II ■ Production of thymic lymphocytes is the primary function of the thymus. It is a major organ for proliferation of lymphocytes in the body. It is believed to play a key role in determining the differentiation of T lymphocytes. The lymphocytes during maturation acquire new surface antigens (Thy antigens) and are called as T lymphocytes or T cells (thymus dependent). The thymus confers immunological competence on these cells during their stay in the organ. Lymphocyte proliferation in thymus, unlike in the peripheral lymphoid organs, is not dependent on antigenic stimulus. The T lymphocytes are primarily responsible for cellmediated immunity (CMI). The absence of thymus in neonatally thymectomized mice is associated with gross deficiency of CMI, resulting in lymphopenia, deficient graft rejection, and runting disease. Congenital aplasia of thymus in man in Di-George syndrome is another example of deficiency of CMI due to absence of thymus. 123 124 Spleen The spleen is the largest lymphoid organ. It is a large, ovoid secondary lymphoid organ situated high in the left abdominal cavity. The spleen parenchyma is heterogeneous and is composed of the white and the red pulp. It is surrounded by a capsule made up of connective tissue (Color Photo 11). The spleen unlike the lymph nodes is not supplied by lymphatic vessels. Instead, blood-borne antigens and lymphocytes are carried into the spleen through the splenic artery. The narrow central arterioles, which are derived from the splenic artery after multiple branchings, are surrounded by lymphoid tissue (periarteriolar lymphatic sheath). In the white pulp, T lymphocytes are found in the lymphatic sheath immediately surrounding the arteriole. B lymphocytes are primarily found in perifollicular area, germinal center, and mantle layer, which lie more peripherally relative to the arterioles. Chapter 16 Section II ◗ IMMUNOLOGY The follicles of the Peyer’s patches are extremely rich in B cells, which differentiate into IgA-producing plasma cells. Specialized epithelial cells, known as M cells, are found in abundance in the dome epithelia of Peyer’s patches, particularly at the ileum. These cells take up small particles, virus, bacteria, etc., and deliver them to submucosal macrophages, where the engulfed material is processed and presented to T and B lymphocytes. Key Points MALTs play an important role in defense system of the human host. This is demonstrated by large population of antibody-producing plasma cells in MALT, whose number far exceeds that of plasma cells in the spleen, lymph nodes, and bone marrow, when combined together. In addition to spleen and lymph nodes, MALTs facilitate interaction among circulating leukocytes. Key Points Functions of the spleen: The spleen plays a major role in: ■ ■ Mounting immune responses to antigens in the blood stream. The circulating antigens are trapped by the macrophages present in the marginal zone. These macrophages then process the antigen, migrate deeper into the white pulp, and initiate the immune response by interacting with T and B lymphocytes. Filtering or clearing of (a) infectious organisms; (b) aged or defectively formed elements (e.g., spherocytes, ovalocytes); and (c) particulate matter from the peripheral blood. In addition, the spleen traps blood-borne antigens and microbes. The main filtering function is performed by the macrophages lining up the splenic cords. The effect of splenectomy on the immune response depends on the age at which the spleen is removed: ■ ■ ◗ In children, splenectomy often leads to an increased incidence of bacterial sepsis caused primarily by Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. In adults, the adverse effects are less; although in some, it makes the host more susceptible to blood-borne bacterial infections. Mucosa-associated lymphoid tissues Mucosa-associated lymphoid tissues (MALT) consist of the lymphoid tissues of the intestinal tract, genitourinary tract, tracheobronchial tree, and mammary glands. All of the MALT are noncapsulated and contain both T and B lymphocytes, and the latter predominate. Structurally, these tissues include clusters of lymphoid cells in the lamina propria of intestinal villi, tonsils, appendix, and Peyer’s patches. Tonsils: These are present in the oropharynx and are predominantly populated by B lymphocytes. These are the sites of intense antigenic stimulation, as shown by the presence of numerous secondary follicles with germinal centers in the tonsillar crypts. Peyer’s patches: These are lymphoid structures that are found within the submucosal layer of the intestinal lining. Lymphatic Circulatory System Leukocytes and their products use two circulatory systems: cardiovascular system and the lymphatic circulatory system. The cardiovascular system is responsible for circulation of blood throughout the body. Peripheral blood is “filtered” by the spleen and liver. Organisms and antigens that enter directly into the systemic circulation are trapped in these two organs, of which the spleen plays the most important role as a lymphoid organ. The lymphatic circulatory system, on the other hand, is an extensive capillary network that collects lymph, a clear watery fluid containing leukocytes and cellular debris, from various organs and tissues. Cleared lymph originating from below the diaphragm and the upper left half of the body drains via efferent lymphatics into the thoracic duct for subsequent drainage into the left innominate vein. Cleared lymph originating from the right side above the diaphragm drains into the right lymphatic duct, which subsequently drains into the origin of the right innominate vein. The same routes are followed by the lymphocytes stimulated and produced in the lymph nodes, or peripheral lymphoid tissues, which eventually reach the systemic circulation. Cells of the Lymphoreticular System It is essential for the immune system to distinguish its own molecules, cells, and organs (self) from those of foreign origin (nonself). The innate immunity does this by expressing germline-encoded pattern recognition receptors on the surface of its cells, receptors that recognize structures on potentially invasive microorganisms. The adaptive immunity, on the other hand, makes use of somatically generated epitope-specific T-cell receptors (TCRs) and B-cell receptors (BCRs). These receptors are produced randomly and fresh within each individual T and B lymphocytes by gene 125 STRUCTURE AND FUNCTION OF IMMUNE SYSTEM Natural killer cell Self-renewing stem cell Pluripotent stem cell Lymphoid progenitor Thymus T lymphocytes Myeloid progenitor Section II B lymphocytes Megakaryocyte Basophil CFU Eosinophil CFU Erythrocytes Platelets Basophils Eosinophils Lymphocytes The lymphocytes occupy a very special place among the leukocytes. ■ Granulocyte – Monocyte CFU Neutrophils Monocytes Proliferation and development of cells of lymphoreticular system. recombination prior to encounter with antigens (Fig. 16-2). The cells involved in the adaptive immune responses are (a) lymphocytes, (b) antigen-presenting cells (APCs), and (c) effector cells that function to eliminate antigens. ■ Chapter 16 FIG. 16-2. Erythroid CFU They participate in immune reactions due to their ability to interact specifically with antigenic substances and to react to nonself antigenic determinants. They also contribute to the memory of the immune system. The lymphocytes consist of heterogeneous populations of cells that differ greatly from each other in terms of origin, lifespan, preferred areas of settlement within the lymphoid organs, surface structure, and function. They differentiate from stem cells in the fetal liver, bone marrow, and thymus into two main functional classes: B cells and T cells. They are found in the peripheral blood and in all lymphoid tissues. The lymphocytes are classified depending upon where they undergo their development and proliferation: (a) T lymphocytes or T cells undergoing development in the thymus or (b) B lymphocytes, or B cells undergoing development in the bone marrow. Differences between T cells and B cells are summarized in Table 16-1. TABLE 16-1 Differences between T cells and B cells Property T cell B cell Antigen recognition receptors T-cell receptors Membranebound Ig Surface glycoprotein antigens CD3 CD19 IgM on surface ⫺ ⫹ Immunoglobulin synthesis ⫺ ⫹ IL-2, IL-4, IL-5, and gamma interferon synthesis ⫹ ⫺ Receptor for Fc fragment of IgG ⫺ ⫹ EAC rosette (receptors for C3 component of complement) ⫺ ⫹ Sheep red blood cells (SRBC) rosette (E rosette) ⫹ ⫺ Effect of cell-mediated immunity ⫹ ⫺ Thymus-specific antigens ⫹ ⫺ Maturation in bursa or its equivalent ⫺ ⫹ Antigen receptor recognizes processed peptides with MHC ⫹ ⫺ ◗ Thymus-derived cells T lymphocytes, or T cells, are so designated because the thymus plays a key role in their differentiation. They are the key players in adaptive immunity. They participate directly in immune 126 IMMUNOLOGY responses as well as in orchestrating and regulating activities of other cells. ■ ■ ■ Chapter 16 Section II ■ ■ T cells constitute 65–80% of the circulating pool of small lymphocytes. They are found in the inner subcortical regions but not in the germinal centers of the lymph nodes. They have a longer lifespan (months or years) than B lymphocytes. They are stimulated to divide on exposure to certain mitogens, such as phytohemagglutinin or concavalin A, the T cells can be stimulated to divide. Most human T cells have receptors for sheep erythrocytes on their surface and have the ability to form rosettes with them; this property is made use of for identifying T cells in a mixed population of cells. The T lymphocytes perform two important groups of functions as follows: Regulation of immune responses: Regulatory function is mediated primarily by helper (CD4⫹) T cells, which produce interleukins. Various effector functions: Effector functions are mediated primarily by cytotoxic (CD8⫹) T cells, which kill allografts, tumor cells, and virus-infected cells. Depending on whether they have CD4 or CD8 proteins on their surface, T cells are subdivided into two major groups: CD4⫹ T cells and CD8⫹ T cells. Mature T cells have either CD4 or CD8 proteins, but never both. TABLE 16-2 Comparison of Th-1 cells and Th-2 cells Features Th-1 cells Th-2 cells Enhances cell-mediated immunity and delayed hypersensitivity Yes No Enhances antibody production No Yes Activation of cytotoxic T lymphocytes Yes No Stimulated by IL-12 Yes No Stimulated by IL-4 No Yes Produces IL-2 and gamma interferon Yes No Produces IL-4, IL-5, IL-6, and IL-10 No Yes All these functions are mediated by Th-1 cells and Th-2 cells— the two subpopulations of CD4⫹ T cells: ■ ■ The Th-1 cells activate cytotoxic T cells by producing IL-2. They help in the development of hypersensitivity responses by producing primarily IL-2 and gamma interferon. The Th-2 cells perform B-cell helper function by producing primarily IL-4 and IL-5. The balance between Th-1 and Th-2 cells is regulated by gamma interferon and IL-12. Gamma interferon inhibits the production of Th-2 cells, whereas IL-12 increases the number of Th-1 cells, thereby increasing host defense against microorganisms that are controlled by a delayed hypersensitivity reaction. Table 16-2 shows a comparison of Th-1 and Th-2 cells. CD4⫹ T cells CD8⫹ T cells CD4 cells are also known as helper T (Th) cells. They constitute about 65% of peripheral T cells and are found mainly in the thymic medulla, tonsils, and blood. CD4 displayed on the surfaces of these T cells recognize a nonpeptide-binding portion of MHC class II molecules. Hence, CD4⫹ T cells are restricted to the recognition of pMHC class II complexes. Helper T lymphocytes are involved in the induction and regulation of immune responses. CD4⫹ T cells perform following helper functions: CD8⫹ T cells are also known as cytotoxic T (Tc) and suppressor T (Ts) cells. They account for approximately one-third of all mature CD3⫹ cells. They are found mainly in the human bone marrow and gut lymphoid tissue. CD8⫹ T glycoprotein displayed on the surfaces of these T cells recognize a nonpeptide-binding portion of MHC class I molecules. Hence, CD8⫹ T cells are restricted to the recognition of pMHC class I complexes. CD8⫹ T cells perform mainly cytotoxic functions. They kill (a) virus-infected cells, (b) allograft cells, and (c) tumor cells. T-cell mediated cytotoxicity is an apoptotic process that appears to be mediated by two different pathways: ■ ■ ■ They help B cells to be transformed into plasma cells. They help CD8⫹ T cells to become activated cytotoxic T cells. They help macrophages to mediate delayed type hypersensitivity reactions. The main functions of helper T cells are summarized in Box 16-1. Box 16-1 Main function of helper T cells 1. Help in the antigen-specific activation of B cells and effector T cells. 2. Th-1 cytokines activate cytotoxic inflammatory and delayed hypersensitivity reactions. 3. Th-2 cells help in the production of interleukins which encourage production of antibodies especially IgE. 4. Th-2 cytokines are associated with regulation of strong antibody and allergic responses. (i) One pathway involves the release of proteins known as perforins, which insert themselves in the target cell membranes forming channels. These channels allow the diffusion of enzymes (granzymes, which are serine esterases) into the cytoplasm. The exact way in which granzymes induce apoptosis has not been established, but granzyme-induced apoptosis is Ca2⫹-dependent. (ii) The other pathway depends on signals delivered by the cytotoxic cell to the target cell, which require cell-to-cell contact. This pathway is Ca2⫹ independent. The ratio of CD4⫹ and CD8⫹ T cells is approximately 2:1 in normal human peripheral blood. This may be significantly STRUCTURE AND FUNCTION OF IMMUNE SYSTEM TABLE 16-3 Differences between helper T cells (CD4) and cytotoxic T (CD8) cells Helper T cells Cytotoxic T cells Carries CD4 marker Carries CD8 cells Helps or induces immune responses Predominantly cytotoxic Recognize antigen in association with class II MHC Recognize antigen in association with class I MHC altered in immunodeficiency diseases, autoimmune diseases, and other disorders. Differences between CD4 and CD8 T cells are summarized in Table 16-3. Recognition of complex on the surface of APCs, such as macrophages and dendritic cells, consisting of both the antigen and a class II MHC protein by TCR present on T cells, is most important for activation of helper T cells. Two signals are required to activate T cells: ■ ■ The interaction of the antigen and the MHC protein with the T-cell-receptor-specific antigen is the first signal required in the activation of process. IL-1 secreted by the macrophages is also necessary for efficient helper T-cell activation. A costimulatory signal is the second signal required for activation of T cells. In this signal, B7 protein present on the APC must interact with CD28 protein on the helper T cells. Following the costimulatory signal, IL-2 is produced by helper T cells, which is most crucial in producing a helper T cell capable of performing their regulatory, effector, and memory functions. After activation of the T cells, a new different protein called CTLA-4 appears on the cell surface of T cells and binds to B7 by displacing CD28. The interaction of CTLA-4 with B7 inhibits T-cell activation by blocking IL-2 synthesis. This makes T cells to remain in a quiescent state and thereby plays an important role in T-cell homeostasis. On the other hand, mutant T cells that lack CTLA-4 and hence cannot be deactivated participate more frequently in autoimmune diseases. Memory T cells Memory T cells, as the name suggests, confer host immunity with the ability to respond rapidly and vigorously for many years after the initial exposure to a microbe or other foreign substances. The memory produced against a specific antigen shows the following characteristics: 1. Memory cells live for many years or have the capacity to reproduce them. 2. A large number of memory cells are produced, and so secondary response is enhanced and is greater than the primary response. T-cell receptor T- cell receptor (TCR) for antigen consists of two polypeptides: alpha and beta. These two peptides are associated with CD3 proteins. Each T cell has a unique TCR on its surface, thereby implying that hundreds of millions of different T cells occur in each person. Activated T cells as well as activated B cells produce large number of cells specific for those antigens. T-cell alpha and beta polypeptides show many similarities to immunoglobulin heavy chain in the following ways: ■ ■ ■ The genes coding for T-cell polypeptides are formed by rearrangement of multiple regions of DNA. There are V (variable), D (diversity), J (joining), and C (constant) segments that rearrange to provide diversity, thereby resulting in more than 107 different receptor proteins. RAG-1 and RAG-2 are the two genes that encode the recombinase enzymes that catalyze these gene rearrangements and are similar in T cells and B cells. T cells, however, differ from immunoglobulins in the following ways: ■ ■ T cells have two chains rather than four chains in immunoglobulins. T cells recognize antigen only in conjunction with MHC proteins, whereas immunoglobulins recognize free antigens. Effect of superantigens on T cells Certain proteins such as staphylococcal enterotoxins and toxic shock syndrome toxins, and certain viral proteins, such as mouse mammary tumor virus, are called superantigens. These are called “super” because they activate a large number of helper T cells unlike “antigens”, which activate one or a few helper cells. The superantigens play a very important role in pathogenesis of staphylococcal toxic shock syndrome caused by Staphylococcus aureus. In this condition, toxic shock syndrome toxin produced by S. aureus binds directly to class II MHC proteins without internal processing of the toxin. Subsequently, this toxin interacts with variable component of the beta chain (V␤) of the T-cell receptor of many T cells. The activation of T cells results in release of the interleukins, IL-2 from the T cells and tumor necrosis factor (TNF) from macrophages. These interleukins are responsible for many of the clinical presentations observed in toxin-mediated staphylococcal diseases. Effector functions of T cells T cells perform two important functions: (a) cytotoxicity and (b) delayed hypersensitivity. Chapter 16 Activation of T cells 3. Memory cells are activated by small quantity of antigens and require less costimulation than do the naïve and unactivated T cells. 4. Activated memory cells produce greater amounts of interleukins than do naïve T cells when they are first activated. Section II Destroy virus-infected and Macrophages are activated to kill intracellular microorganisms tumor cells directly by secreting cytokines 127 128 IMMUNOLOGY Cytotoxicity: Cytotoxicity activity of T cells is required primarily to destroy virus-infected cells and tumor cells. It also plays an important role in graft rejection. The cytotoxic T cells kill the virus-infected cells: Chapter 16 Section II (a) By inserting perforins and granzymes (degrading enzymes) into the infected cell, (b) By the Fas–Fas ligand (FasL) interaction, and (c) By antibody-dependent cellular cytotoxicity (ADCC mechanism. A. By inserting perforins and granzymes: Perforins are inserted into the cells, leading to formation of a channel through the membrane. This results in the loss of cell contents and finally death of the cell. Granzymes are proteins that degrade proteins in the cell membrane, which also results in loss of cell contents. These enzymes also activate caspases that causes apoptosis, resulting in cell death. B. By the Fas–Fas ligand (FasL) interaction: Cytotoxic T cells kill virus-infected cells by the FasL interaction. FasL is a protein which is expressed on the surface of many cells. When a cytotoxic TCR recognizes an epitope on the surface of virusinfected cells, FasL appears on the cytotoxic T cells. When Fas and FasL interact, it results in death or apoptosis of target cells. NK cells can also kill target cells by FasL interaction. C. By antibody-dependent cellular cytotoxicity (ADCC): Virusinfected cells can also be killed by ADCC. In this process, target cells are killed by a combination of IgG and phagocytic cells. The antibody bound to the surface of the infected cells is recognized by IgG receptor on the surface of phagocytic cells (e.g., macrophages, NK cells) and the infected cell is killed. After killing of the virus-infected cells, the cytotoxic T cells are not damaged and can continue to kill other cells infected with the same virus. However, the cytotoxic T cells do not have any effect on free virus; they have effect only on virus-infected cells. The cytotoxic T cells kill the tumor cells by a phenomenon called immune surveillance. New antigens are usually developed on surface of many tumor cells. These antigens bound to class I proteins are recognized by cytotoxic T cells, which are activated to proliferate by IL-2. The resultant clone of cytotoxic T cells can kill the tumor cells. The cytotoxic T cells also play an important role in graft rejection. In this process, cytotoxic CD8 cells recognize the class I MHC molecules on the surface of the foreign cells. Helper CD4 cells recognize the foreign class II molecules on certain cells, such macrophages and lymphocytes in the graft. The activated helper cells secrete IL-2, which stimulates the cytotoxic cells to produce a clone of cells, which kills the cells in the allograft. Delayed hypersensitivity: The CD4 cells particularly the Th-1 subset cells and macrophages mediate the delayed hypersensitivity reactions against antigens of many intracellular pathogens. The CD4 cells produce interleukins, such as gamma interferon, macrophage activation factor, and macrophage inhibition factor, which mediate delayed hypersensitivity reactions. Th-1 cells produce IL-12-gamma interferon, which activates macrophages and thereby enhances the ability of the macrophages to kill Mycobacterium tuberculosis. The gamma interferon, therefore, plays an important role in the ability of host immunity to control infections caused by M. tuberculosis, Listeria monocytogenes, and other intracellular microbes. A deficiency of CMI makes the person highly susceptible to infection by these microorganisms. Regulatory functions of T cells T cells play key role in regulating antibody production and in suppression of certain immune responses. 1. Regulation of antibody production: Production of antibodies by B cells may be (a) T-cell dependent, requiring the participation of helper T cells (T-cell-dependent response), or (b) non-T-cell dependent (T-cell-independent response). In the T-cell-dependent response, all the classes of immunoglobulins, such as IgG, IgM, IgA, IgE, and IgD, are synthesized. The T-cell-dependent response produces memory B cells. In the non-T-cell dependent response (T-cell-independent response), only IgM antibody is synthesized. This response does not produce any memory cells. Hence, a secondary antibody response does not occur. In this response, the multivalent macromolecules, such as bacterial capsule polysaccharide are not effectively processed and presented by APCs; hence these do not activate helper T cells. This is because polysaccharides do not bind to class II MHC proteins, whereas peptide antigens do. 2. Stimulation of helper and cytotoxic T cells to participate in the CMI: In CMI, the antigen is processed by macrophages and is presented in conjunction with class II MHC molecules on the surface. These interact with the receptor on the helper T cells, which is then activated to produce IL-2, a T-cell growth factor that stimulates the specific helper and cytotoxic T cells to grow and participate in the CMI. 3. Suppression of certain immune responses: T cells have been shown to inhibit several immune-mediated diseases in animals. Regulatory T cells (TR), also called suppressor T cells, is a subset of T cells and are associated with the suppression of certain immune responses. TR cells also called suppressor T cells are characterized by possessing CD25 marker and comprise 5–10% of the CD4⫹ cells. The exact mechanism by which the regulatory cells suppress the immune response is not known. Imbalance in numbers or activity between CD4 and CD8 cells also leads to impairment of the cellular immune response of the host. ◗ Bone marrow-derived cells The bone marrow-derived lymphocytes are known as B lymphocytes or B cells. Plasma cells are derived from mature B cells. Both B cells and plasma cells synthesize and secrete immunoglobulin. B cells B lymphocytes or B cells are so designated because the bursa of Fabricius, a lymphoid organ located close to the caudal end of the gut in birds, plays a key role in their differentiation. A mammalian equivalent of the bursa is yet to be found. Here the early stages of maturation of these lymphocytes occur in the bone marrow. STRUCTURE AND FUNCTION OF IMMUNE SYSTEM Key Points ■ ■ ■ ■ ■ ■ Antigen-independent phase, which consists of stem cells and pre-B cells Antigen-dependent phase, which consists of the cells, such as activated B cells and plasma cells that proliferate on interactions of antigen with B cells. B cells possess surface IgM, which acts as a receptor for antigen. Some B cells may also carry on their surface IgD as receptor for the antigen. There are many other molecules expressed on the surface of the B cells, which serve different functions. A few of them are B220, class II MHC molecules, CR1 and CR2, CD40, etc. Activation of B cells: Activation of B cells to produce the full range of antibodies first requires recognition of the epitope by the T-cell-antigen receptor and the production of IL-4 and IL-5 by the helper T cells. In addition, it also requires other costimulatory interactions of CD28 on the T cells with B7 on the B cells. The CD28–B7 interaction is essential to produce IL-2. It also includes CD40L on the T cells, which must interact with CD40 on the B cells. The CD40L–CD40 interaction is essential for class switching from IgM to IgG and for switching between other immunoglobulin classes to take place. Effector functions of B cells: Production of many plasma cells is the end result of activation of B cells. The plasma cells in turn produce large amounts of immunoglobulins specific for the epitope of the antigen. Some activated B cells also produce memory cells, which remain in a stage of quiescence for months ◗ Antigen-presenting cells Antigen presenting cells (APCs) include (a) macrophages and (b) dendritic cells. Macrophages The mononuclear phagocytic system consists of monocytes circulating in the blood and macrophages in the tissues. The monocyte is considered a leukocyte in transit through the blood, which becomes a macrophage when fixed in a tissue. Monocytes and macrophages as well as granulocytes are able to ingest particulate matter (microorganisms, cells, inert particles) and for this reason are said to have phagocytic functions. The phagocytic activity is greater in macrophages, particularly after activation by soluble mediators released during immune responses, than in monocytes. Differentiation of a monocyte into a tissue macrophage involves a number of changes as follows: 1. 2. 3. 4. 5. The cell enlarges 5–10 folds. Its intracellular organelles increase in number and complexity. It acquires increased phagocytic ability. It produces higher levels of hydrolytic enzymes. It begins to secrete a variety of soluble factors. Macrophage-like cells serve different functions in different tissues and are named according to their tissue location. Examples include (a) alveolar macrophages in the lung, (b) histiocytes in connective tissues, (c) Kupffer cells in the liver, (d) mesangial cells in the kidney, (e) microglial cells in the brain, and (f) osteoclasts in the bone. For their participation in the immune reaction, the macrophages need to be stimulated and reach an “activated state.” ■ ■ ■ Macrophages can be activated by various cytokines, components of the bacterial cell wall, and mediators of the inflammatory response. Gamma interferon produced by helper T cells is a potent activator of macrophages and is secreted by various cells in response to appropriate stimuli. Bacterial lipopolysaccharides (endotoxin), bacterial peptidoglycan, and bacterial DNA are the substances that also activate macrophages. Activated macrophages are more potent than normal macrophages in many ways, such as having greater phagocytic ability and increased ability to kill ingested microbes. They are better APCs, and they activate T-cell response in a more effective manner. By secreting various cytotoxic proteins, they help in eliminating a broad range of pathogens, including virus-infected cells, tumor cells, and intracellular bacteria. Functions of macrophages: Macrophages perform three main functions: (a) phagocytosis, (b) antigen presentation, and (c) cytokine production (Table 16-4). Chapter 16 Origin of B cells: The clonal selection theory explains the origin of antibody formation. According to this postulation, each immunologically competent B cell possesses receptor for either IgM or IgD that can combine with one antigen or closely related antigens. After binding of the antigen, the B cell is activated to proliferate and form a clone of cells. Selected B cells are transformed to plasma cells that secrete antibodies specific for the antigen. Plasma cells synthesize the immunoglobulin with the same antigenic specificity as those carried by activated B cells. The same clonal selection also occurs with T cells. B cell precursors, during embryogenesis, first proliferate and develop in the fetal liver. From there, they migrate to the bone marrow, the main site of B-cell maturation in the adults. Unlike T cells, they do not require the thymus for maturation. The Pre-B cells have only ␮ heavy chains in the cytoplasm but do not have surface immunoglobulins and light chains. Pre-B cells are found in the bone marrow, while B cells are found in the circulation. B cells mature in two phases: or years. Most memory B cells have surface IgG that acts as the antigen receptor, but some even have surface IgM. These quiescent memory cells are activated rapidly on reexposure to antigen. Memory T cells produce interleukins that facilitate antibody production by the memory B cells. The presence of these cells is responsible for the rapid appearance of antibody in the secondary immune responses. Section II Nearly 30% of the recirculating small lymphocytes are composed of B cells. The B cells have a short lifespan of days or weeks. Nearly 109 B cells are produced every day. B cells are found in the germinal centers of the lymph nodes, in the white pulp of the spleen, and in the MALT. B cells perform two important functions. First, they differentiate into plasma cells and produce antibodies. Second, they can present antigen to helper T cells. 129 130 IMMUNOLOGY Important features of macrophages TABLE 16-4 Features Mechanism Phagocytosis Ingestion and killing of microbes in phagolysosome Antimicrobial and cytotoxic activities Oxygen-dependent killing: by superoxides, nitric oxide, and hydrogen peroxide Chapter 16 Section II Oxygen-independent killing: by tumor necrosis factor, lysozyme, and hydrolytic enzymes Antigen processing Phagocytic antigen 1. Phagocytosis: Phagocytosis of bacteria, viruses, and other foreign particles is the most important function of macrophages. The macrophages on their cell surfaces have Fc receptors that interact with Fc component of the IgG, thereby facilitating ingestion of the opsonized organisms. They also have receptors for C3b, another important opsonin. After ingestion, the phagosome containing the microbe fuses with a lysosome. The microbe within the phagolysosome is killed by reactive oxygen, reactive nitrogen compounds, and lysosomal enzymes. 2. Antigen presentation: After ingestion and degradation of foreign materials, the fragments of antigen are presented on the macrophage cell surface in conjunction with class II MHC proteins for interaction with the TCR of CD4⫹ helper T cells. Degradation of the foreign protein is stopped following the association of antigen with the class II MHC proteins in the cytoplasm. This is followed by transportation of the complex to the cell surface by transporter proteins. 3. Cytokine production: Macrophages produce several cytokines including the IL-1, TNF, and IL-8. IL-1 plays an important role in activation of helper T cells, while TNF plays as important mediator in inflammatory reactions. IL-8 attracts neutrophils and T cells to the site of infection. Dendritic cells Dendritic cells are so named because of their many long, narrow processes that resemble neuronal dendrites, which make them very efficient at making contacts with foreign materials. They are primarily present in the skin (e.g., Langerhans cells) and the mucosa, from where they migrate to local lymph nodes for presentation of antigen to helper T cells. Key Points ■ ■ ■ ■ Dendritic cells are very important for presentation of the antigen to T cells during primary immune response. They are bone marrow-derived cells that express class II MHC proteins and present antigen to CD4+ T cells. They have little or no phagocytic activity. They also serve as professional APCs, although macrophages and B cells are the major APCs. Four types of dendritic cells are known: (i) Langerhans cells, (ii) interstitial dendritic cells, (iii) myeloid cells, and (iv) lymphoid dendritic cells. All these cells constitutively express high levels of both class II MHC molecules and members of the costimulatory B7 family. Following microbial invasion or during inflammation, mature and immature forms of Langerhans cells and interstitial dendritic cells migrate into draining lymph nodes, where they make the critical presentation of antigen to TH cells, which is required for the initiation of responses by those key cells. Follicular dendritic cells Follicular dendritic cells are similar to the dendritic cells except for their sites of presence and functions. These cells are present in B-cell-containing germinal centers of the follicles in the spleen and lymph nodes. These cells do not present antigen to helper T cells, but combine with antigen–antibody complexes by Fc receptors found on their surfaces. ◗ Effector cells that function to eliminate antigens Plasma cells Plasma cells originate from terminally differentiated B cells. Plasma cells are oval or egg-shaped structures characterized by a stellate (star-like pattern) nucleus, nonstaining Golgi, and basophilic cytoplasm. ■ ■ ■ ■ ■ The main function of the plasma cells is to produce and secrete all the classes of immunoglobulins into the fluids around the cells. They secrete thousands of antibody molecules per second, which are specific for the epitope of the antigen for a few days and then die. They, however, do not express membrane immunoglobulins. They divide very poorly, if at all, and are usually found in the bone marrow and in the perimucosal lymphoid tissues. They have a short lifespan of 30 days during which they produce large quantities of immunoglobulins. Natural killer cells Natural killer (NK) cells are morphologically described as large granular lymphocytes. These cells are called natural killer cells due to their ability to kill certain virally infected cells and tumor cells without prior sensitization. Their activities are not enhanced by exposure and are not specific for any virus. NK cells comprise approximately 5–10% of peripheral lymphocytes and are found in spleen and peripheral blood. Key Points ■ ■ ■ ■ They lack both T cell (CD3) and B cell (surface immunoglobulin) markers. They lack immunologic memory and unlike cytotoxic T cells do not have any TCRs, and killing of target cells does not require recognition of MHC proteins. These cells do not carry antigen receptors of any kind, but can recognize antibody molecules bound to target cells and destroy those cells using the same general mechanisms involved on T-lymphocyte cytotoxicity (ADCC). They also have a recognition mechanism that allows them to destroy tumor cells and virus-infected cells. 131 STRUCTURE AND FUNCTION OF IMMUNE SYSTEM Box 16-2 1. 2. 3. 4. 5. Properties of natural killer cells Large granular lymphocytes. Lack T-cell receptor, CD3 proteins, and surface IgM and IgD. Prior exposure does not increase the activity. Thymus is not required for development. Number remains normal in severe combined immunodeficiency disease. Granulocytes are a collection of white blood cells with segmented or lobulated nuclei and granules in their cytoplasm, which are visible with special stains. The granulocytes are classified as neutrophils, eosinophils, or basophils on the basis of cellular morphology and cytoplasmic-staining characteristics. Both neutrophils and eosinophils are phagocytic, whereas basophils are not. Eosinophils play an important role in defense against parasitic infections, though their phagocytic role is significantly lower than neutrophils. Basophils, on the other hand, are nonphagocytic granulocytes that function by releasing pharmacologically active substances from their cytoplasmic granules. These substances play a major role in certain allergic responses. Mast cells are the other granulocytic cells that have a role in the immune system. These cells are found in a wide variety of tissues, including the skin, connective tissues of various organs, and mucosal epithelial tissue of the respiratory, genitourinary, and digestive tracts. Like circulating basophils, these cells have large numbers of cytoplasmic granules that contain histamine and other pharmacologically active substances. Mast cells, together with blood basophils, play an important role in the development of allergies. 1. 2. 3. 4. Kill virus-infected cells and tumor cells. Nonspecific killing of virus-infected cells and tumor cells. Killing is independent of antigen presentation by MHC proteins. Mechanism of killing is by perforins and granzymes, which cause apoptosis of target cell. 5. Killing is activated by failure of a cell to present antigen with class I MHC or by reduction of class I MHC proteins on the cell surface molecules (transplantation antigens) responsible for the rapid rejection of tissue grafts transplanted between genetically nonidentical individuals. Gorer in 1930 was the first to identify the antigens responsible for allograft rejection that led to the discovery of major histocompatible complex. He demonstrated two blood group antigens (antigen 1 and antigen 2) in mice. Antigen 1 was found in all strains of mice, while antigen 2 was found in certain strains of mice and was responsible for allograft rejection. This was named H2 antigen and was found to be the major histocompatible antigen. This antigen was coded for by a closely linked multiallelic cluster of genes called the MHC, named as H-2 complex. Histocompatible antigen denotes the cell surface antigens that induce immune responses to an incompatible host, resulting in allograft rejection. The MHC in humans is known as human leukocyte antigens (HLA) complex. In humans, these alloantigens are present on the surface of leukocytes and are called HLA and the set of genes encoding for them is named the HLA complex. Carbohydrate antigens of erythrocytes (blood groups) and glycoprotein antigens of cell membranes are the two major transplantation antigens of humans. Snell, Dauseset, and Benacerraf (1980) were awarded the Nobel Prize for their work on MHC and genetic control of immune responses. HLA Complex In humans, the HLA complex of genes is located on short arm of chromosome 6 containing several genes that are critical to immune function (Fig. 16-3). The HLA complex of genes is classified into three classes as follows: Short arm of chromosome 6 Major Histocompatibility Complex The major histocompatibility complex (MHC) was first detected as the genetic locus encoding the glycoprotein FIG. 16-3. DP DQ DR C4a C4b BF C2 Hsp70 TNF Class II Class III A schematic diagram of HLA complex. B C Class I A Chapter 16 Granulocytes Functions of natural killer cells Section II Properties of NK cells are summarized in Box 16-2. NK cells develop within the bone marrow and lack TCR, but possess another set of receptors called killer activation receptors and killer inhibition receptors. They also posses NK T cells, another subset of T cells, which share some functional characteristics with NK cells. These NK T cells unlike NK cells are stimulated by lipids, glycolipids, and hydrophobic peptides presented by a nonclassical class I molecule CD1D and secrete large amounts of cytokines, especially IL-4. The main functions of the NK cells are to kill virus-infected cells and tumors. They do so by secreting cytotoxins, such as perforins and granzymes similar to those of cytotoxic T lymphocytes and also by FasL-mediated apoptosis. They kill the viruses without presence of specific antibodies but by a mechanism called ADCC. Both IL-12 and gamma interferons are potent activators of NK cells (Box 16-3). Box 16-3 132 IMMUNOLOGY Chapter 16 Section II 1. Class I: HLA-A, HLA-B, and HLA-C. 2. Class II: HLA-DR, HLA-DQ, and HLA-DP. All of these are present within HLA-D region of HLA complex. 3. Class III: Complement loci that encode for C2, C4, and factor B of complement system and TNFs alpha and beta. A locus means the position where a particular gene is located on the chromosome. HLA loci are usually multiallelic, meaning that genes present on the locus can be any one of several alternative forms. There are 24 alleles at HLA-A locus and 50 at HLA-B locus. Each allele expresses for a distinct antigen. HLA system is pleomorphic and every person inherits one set of HLA genes from each parent. The official Committee of the WHO has recommended a set of guidelines for nomenclature of the HLA system. Recognized alleles and their corresponding antigens are indicated by alphabet(s) and a number (e.g., HLA-A1, HLA-DR7, etc.). The HLA genes encode a variety of enzymes and structural molecules essential for the activation and function of B and T cells. The genes encode MHC proteins that are classified into three groups or classes known as the class I, class II, and class III molecules. ◗ MHC class I molecules MHC class II molecules These are glycoproteins and unlike class I proteins, they have a restricted tissue distribution. They are chiefly found on macrophages, B cells, and other APCs, such as dendritic TABLE 16-5 ◗ MHC class III molecules The class III MHC locus encodes complement proteins (C2, C4) of the classical pathways, properdin and factor B of the alternative pathway, and several cytokines. Biologic Importance of MHC These are glycoproteins found on the surface of virtually all nucleated cells in the body. Class I proteins are encoded by the HLA-A, -B, and -C loci. Approximately 20 different proteins are encoded by allelic genes at the A locus, 40 at the B locus, and 8 at the C locus (Table 16-5). The complete class I is composed of a transmembrane glycoprotein of 45,000 Da, heavy chain, noncovalently associated with a ␤2-microglobulin (a non-HCencoded polypeptide of MW 12,000 Da). The heavy chain like that of an immunoglobulin molecule has a variable and constant region. The variable region is highly pleomorphic. The polymorphism of these molecules is important in the recognition of self and nonself. The constant region of the heavy chain binds with the CD8 proteins of the cytotoxic T cells. Class I proteins are involved in graft rejection and cell-mediated cytolysis. ◗ cells of the spleen and Langerhans cells of the skin. Their expression on other cells (e.g., endothelial cells) can be induced by gamma interferon. They are highly polymorphic glycoproteins composed of two noncovalently associated transmembrane glycoproteins of MW of about 33,000 and 29,000 Da. Class II proteins are encoded by the genes on the HLA-D locus. This locus retains control of immune responsiveness, and different allelic forms of these genes confer striking differences in the ability to mount an immune response against a given antigen. The class II MHC locus also includes genes encoding proteins involved in antigen processing, e.g., transporter associated with antigen processing (TAP). Class II proteins are primarily responsible for the graft-versus-host response and the mixed leukocyte response. It is now known that MHC molecules bind peptide antigens and present them to T cells. Thus, these transplantation antigens are responsible for antigen recognition by the TCR. In this respect, the TCR is different from antibody. ■ ■ Antibody molecules interact with antigen directly; the TCR only recognizes antigen presented by MHC molecules on another cell, the APC. The TCR is specific for antigen, but the antigen must be presented on a self-MHC molecule. The TCR is also specific for the MHC molecule. If the antigen is presented by another allelic form of the MHC molecule in vitro (usually in an experimental situation), there is no recognition by the TCR. This phenomenon is known as MHC restriction. Peptide antigens associated with class I MHC molecules are recognized by CD8⫹ cytotoxic T lymphocytes, whereas class II-associated peptide antigens are recognized by CD4⫹ helper T cells. Important features of some human MHC gene products Class I Class II Genetic loci (partial list) HLA-A, -B, and -C HLA-DP, -DQ, and -DR Polypeptide composition MW 45,000 Da⫹ MW 12,000 Da Light chain (MW 33,000 Da), light chain (MW 29,000 Da), light chain (MW 30,000 Da) Cell distribution All nucleated somatic cells and platelets Antigen-presenting cells (macrophages, B cells, etc.) and activated human T cells Present peptide antigens to CD8 T cells CD4 T cells Size of peptide bound 8–11 residues 10–30 or more residues STRUCTURE AND FUNCTION OF IMMUNE SYSTEM HLA Typing which are first killed by irradiation. Then it is mixed with live responder lymphocytes from the recipient. The resultant mixture is then incubated in cell culture to allow DNA synthesis, which is detected by adding tritiated thymidine. The more the amount of DNA synthesis in the responder cells, the more foreign are the class II MHC protein of donor cells. Therefore, a large amount of DNA synthesis indicates that class II (HLA-D) MHC proteins of donor and recipients are not similar. This shows an unsatisfactory match between donor and recipients, thereby graft is likely to be rejected. Least production of DNA suggests the best donor and a good match between the donor and recipients. HLA typing is carried out: ■ ■ ■ Chapter 16 usually before tissue transplantation, for determination of paternity in case of dispute, and for finding association of HLA with diseases, such as association of HLA-B27 with ankylosing spondylitis and HLA-DR4 with rheumatoid arthritis. Section II HLA typing or tissue typing are usually performed to determine the closest MCH match between the donors and recipients before performing transplantation surgery. The methods commonly used in the laboratory include (a) molecular methods using DNA sequence, (b) serological assays, and (c) mixed lymphocyte culture (MLC) techniques. All these methods are used to determine the haplotype, i.e., the class I and class II alleles on both chromosomes of both the donor and recipient. DNA probe and PCR are highly specific and sensitive methods used to detect the different alleles. Serological assays using a battery of antibodies specific for a different class I and class II proteins are also used to demonstrate the alleles. If these two methods fail to provide sufficient data, then additional information can be obtained by performing the MLC technique, also known as mixed lymphocyte reaction. Mixed lymphocyte reaction: This test is performed by using stimulator lymphocytes from a potential donor, 133 43 17 Mycobacterium Immune Response Leprae Introduction The adoptive immune system is developed in a host primarily to protect the host from harmful effects of pathogens and other foreign substances. The adoptive response can be antibody-mediated (humoral), cell-mediated (cellular), or both. An encounter with a microbial or viral agent usually elicits a complex variety of responses. There are two main sites where pathogens may reside in an infected host—extracellularly in tissue spaces or intracellularly within a host cell; the immune system has different ways of dealing with pathogens at these sites. Humoral immunity acts mainly against extracellular pathogens, while cell-mediated immunity (CMI) acts against intracellular pathogens. Humoral Immunity Humoral immunity is based on the action of antibodies and complement. It is directed primarily against: ■ ■ ■ Extracellular bacteria, in particular exotoxin-producing bacteria, such as Corynebacterium diphtheriae, Clostridium tetani, etc., Bacteria whose virulence is due to polysaccharide capsules (e.g., Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, etc.), and Certain viruses that cause infection through respiratory or intestinal tract. The humoral immunity also participates in the pathogenesis of hypersensitivity reactions and certain autoimmune diseases. Production of antibodies is the main feature of humoral immune responses. The production of antibodies follows a characteristic pattern as follows: 1. Lag phase: This is the immediate phase following exposure to antigen. During this phase, no antibodies are detected in circulation. 2. Log phase: This is the next phase characterized by a steady rise in antibody titers in the circulation. 3. Plateau: This is a phase of equilibrium between antibody synthesis and catabolism. 4. Phase of decline: This phase is characterized by an increase in the catabolism of antibodies compared to the production of antibodies, leading to a fall in antibody titer in the circulation. Humoral immune response is of two types: primary and secondary. Primary Response During the primary response, when an individual encounters an antigen for the first time, antibody response to that antigen is detectable in the serum after a longer lag period than occurs in the secondary response. The serum antibody concentration continues to rise for several weeks and then declines; it may drop to very low levels. During this primary response, a small clone of B cells and plasma cells specific for the antigen are formed. The lag period is typically of 7–10 days duration but can be longer, even for weeks, depending on the nature of the antigen. For example, the lag phase may be as long as 2–3 weeks with some antigens, such as diphtheria toxoid, while it may be a short as a few hours with pneumococcal polysaccharide. The lag period also depends on dose of the antigen and the route of administration whether oral or parenteral. IgM is the first antibody to be formed, followed by IgG, IgA, or both. IgM levels tend to decline sooner as compared to IgG levels (Fig. 17-1). Secondary Response The antibody response is typically more rapid in the secondary response, due to second encounter with the same antigen, or a closely related “cross-reacting” antigen, months or years after the primary response. The lag period is typically very short (only 3–5 days). The level of antibody is also much higher than that during the primary response. These changes in secondary response are attributed to the persistence of antigen-specific “memory cells” following the first contact with the antigen. These memory cells proliferate in large numbers to produce large clones of specific B cells and plasma cells that mediate the secondary response. In the secondary response: ■ ■ The amount of IgM produced is qualitatively similar to that produced after the first contact with the antigen; however, much more IgG is produced and the level of IgG tends to persist much longer than in the primary response. Furthermore, such antibody tends to bind antigen more firmly (i.e., to have higher affinity) and thus to dissociate less easily. Improved antibody binding is due to mutations that occur in the DNA that encodes the antigen-binding site. This process is called somatic hypermutation. Fate of Antigen in Tissues Route of administration of antigen affects the site of localization of these antigens in the body. For example, most of the IMMUNE RESPONSE Primary immune response Secondary immune response IgG IgM 14 21 28 35 IgG appears earlier than in primary immune response IgM 0 7 14 Days First exposure to antigen FIG. 17-1. 21 28 35 Days Second exposure to antigen Section II 7 Final concentration of IgG is higher than in primary response IgG Amount of antibody Amount of antibody IgM is the first antibody to appear in the primary immune response 0 135 A schematic diagram showing primary and secondary response. Production of Antibodies Synthesis and production of antibodies typically is dependent on complex interaction of three cells: (a) macrophages, (b) helper T cells, and (c) B cells. Antigens are presented to immunocompetent cells by antigen presenting cells (APCs), such as macrophages and dendritic cells. Processing by macrophages appears to be a prerequisite for formation of antibodies against many T-cell-dependent antigens, such as proteins and erythrocytes. However, antibody production does not require macrophage participation in case of T-cell-independent antigens. Both the macrophages and dendritic cells present the antigen either native or processed at the cell surface. Macrophages play a key role by modulating the optimum dose of antigen presented to lymphocytes to induce the immune responses. After processing of antigens by a macrophage, fragments of antigen appear on surfaces of macrophages in association with class II MHC proteins. The antigen-class II MHC protein complex binds to specific receptors present on the surface of helper T cells. Subsequently, these helper T cells produce cytokines that activate B cells, producing antibodies that are specific for that antigen. The activated cytokines are interleukin-2 (T-cell growth factor), interleukin-4 (B-cell growth factor), and interleukin-5 (B-cell differentiation factor). The activated B cells undergo clonal proliferation and differentiate to form plasma cells, which then produce specific immunoglobulins (antibodies). Major host defense functions of antibodies include neutralization of toxins and viruses and opsonization (coating) of the pathogen, which aids its uptake by phagocytic cells. Although helper T cells play a key role in the formation of antibodies, certain substances (e.g., polysaccharides) can activate B cells directly without the help of T cells. Such substances are called T-cell-independent antigens. These antigens, however, induce only the production of IgM antibodies but not other antibodies by B cells. This is because B cells require interleukins 4 and 5 to switch classes to produce IgG, IgA, and IgE. These interleukins 4 and 5 are produced by T helper cells only. B cells perform two important functions: First, they recognize antigens with their surface IgM that acts as an antigen receptor; second, they present epitopes to helper T cells in association with class II MHC proteins. IgM antigen receptor on the B cells recognizes foreign proteins as well as lipids, carbohydrates, DNA, RNA, etc. On the other hand, class II MHC proteins present protein fragments to the helper T cells. The IgM antigen receptor binds with this wide variety of molecules that stimulate B cells to produce antibodies against all the molecules possible. ◗ Theories of antibody formation There are two sets of theories of antibody formation. These are instructive theory and selective theories. Instructive theory Instructive theory suggests that an immunocompetent cell is capable of synthesizing antibodies of all specificity. The antigen directs the immunocompetent cell to produce complementary antibodies. Two instructive theories are postulated as follows: Direct template theory: This theory was first postulated by Breinl and Haurowitz (1930). They suggested that a particular antigen or antigenic determinants would serve as a template against which antibodies would fold. The antibody molecule would thereby assume a configuration complementary to antigen template. Indirect template theory: This theory was first postulated by Burnet and Fenner (1949). They suggested that the entry of antigenic determinants into the antibody-producing cells induced a heritable change in these cells. A genocopy of the antigenic Chapter 17 antigens introduced subcutaneously are localized mainly in the draining lymph nodes and only a small amount is there in the spleen. On the other hand, most of the antigens introduced intravenously are localized in the spleen, liver, bone marrow, kidney, and lungs but not in lymph nodes. Approximately, three-fourths of these antigens are broken down by reticuloepithelial cells and are excreted out in the urine. 136 IMMUNOLOGY determinant was incorporated in genome and transmitted to the progeny cells. However, this theory that tried to explain specificity and secondary responses is no longer accepted. Chapter 17 Section II Selective theories Three selective theories were postulated as follows: Side chain theory: This theory was proposed by Ehrlich (1898). According to this theory, immunocompetent cells have surface receptors that are capable of reacting with antigens, which have complementary side chains. When antigens are introduced into host, they combine with those cell receptors that have a complementary fit. This inactivates the receptors. There is an overproduction of the same type of receptors that circulate as antibodies, as a compensatory mechanism. Natural selection theory: This theory was proposed by Jerne (1955). According to this theory, during the embryonic life, millions of globulin molecules were formed against all possible range of antigens. The antigen when introduced to the host combines selectively with the globulin molecule that has the nearest complementary fit. The globulin with the combined antigen stimulates antibody-forming cells to produce the same type of antibody. Clonal selection theory: Burnet (1957) suggested that immunological specificity existed in the cell but not in the serum and proposed the most acceptable clonal selection theory. According to this theory, a large number of clones of immunological competent cells bearing specific antibody patterns are produced during fetal development by a process of somatic mutations of immunological competent cells (ICCs) against all possible antigens. This theory suggests that an individual ICC expresses membrane receptors that are specific for a distinct antigen. This unique receptor specificity is determined before the lymphocyte is exposed to antigen. Binding of antigen to its specific receptor activates the cell and leads to cellular proliferation to form clones, synthesizing the antibody. The clonal selection theory is most widely accepted and provides a framework for better understanding of the specificity, immunological memory, and the property of recognition of self and nonself by adoptive immunity. ◗ Factors affecting production of antibodies Many factors affect the production of antibodies. These factors are discussed below: Genetic factors Genetic factors influence the response of the host to antigen. Persons responding to antigens are called responders, while persons not responding are called nonresponders. These differences are controlled genetically and are being controlled by immune response (Ir) gene located in the short arm of the 6th chromosome. of 5–7 years for IgG and 10–15 years for IgA by the development of lymphoid organs. Nutritional status Malnutrition affects both the humoral and cell-mediated immunities. Deficiencies of amino acid and vitamins have shown to decrease the production of antibodies. Route of antigen Induction of immune response in a host depends on the route of administration of the antigen. Parenteral administration of the antigen induces a better immune response than the oral or nasal routes. Dose of antigen A minimum critical dose of antigen is essential to elicit an optimum immunological response. A very high or small dose fails to stimulate the immune system. This phenomenon is referred to as immunological paralysis. Multiple antigens Antibody responses vary when two or more antigens are administered simultaneously. Antibody responses to one or more of them may be diminished due to antigenic competition, or enhanced as seen after vaccination with triple vaccine (diphtheria, pertussis, and tetanus), or may be similar. Hence, the nature and relative proportions of different antigens should be carefully adjusted for optimal effect. Adjuvants Adjuvants are the substances that enhance the immunogenicity of an antigen. The adjuvants delay the release of an antigen from the site of injection and prolong the antigenic stimulus. The substances that are used as adjuvants include: (a) Freund’s incomplete adjuvant (protein antigen incorporated in water phase of water in oil emulsion); (b) Freund’s complete adjuvant (incomplete adjuvant along with suspension of killed tubercle bacilli); (c) Aluminum salts both phosphate and hydroxide; and (d) Others, such as silica particles, beryllium sulfate, endotoxin, etc. Key Points An adjuvant functions in the following ways: ■ ■ ■ Age The embryo and the infant, at birth, are not fully immunologically competent. Full competence is achieved by about the age ■ ■ ■ It causes sustained release of antigen from depot. It enhances immunogenicity of nonantigenic substances. It increases the concentration and persistence of antibodies. It induces and enhances CMI. It activates macrophages. It stimulates lymphocytes nonspecifically. IMMUNE RESPONSE Immunosuppressive agents Antilymphocyte serum: Antilymphocyte serum (ALS) is a heterogeneous antiserum raised against T lymphocytes. The ALS acts mainly against circulating lymphocytes but not against lymphocytes in lymphoid organs. It is mainly used to prevent graft rejection in transplantation surgery. ■ ■ ■ ■ ■ First, an animal (e.g., mouse) is immunized with the antigen of interest. Spleen cells (lymphocytes) are then fused with mouse myeloma cells and grown in culture, which are deficient in the enzyme hypoxanthine phosphoribosyl transferase (HPRT). Fusion of the cells is facilitated by addition of certain chemicals, such as polyethylene glycol. The fused cells are grown in a special culture medium (HAT medium) that supports the growth of the fused hybrid cells but not of the parent cells. Finally, the resulting clones of cells are screened for the production of antibody to the antigen of interest. These clones are then selected for continuous cultivation. The hybridomas can be maintained indefinitely and will continue to produce monoclonal antibodies. Human monoclonal antibodies, such as chimeric antibodies, have been produced with modification of the original technique for therapeutic use, since mouse monoclonal antibodies are not suitable. The chimeric antibodies consisting of human constant regions and mouse variable regions are being prepared for use in treatment of leukemia. Chimeric antibodies are also used to kill tumor cells either by delivering toxins, such as diphtheria to tumor cells, or by killing tumor cells through complement-mediated cytotoxicity. Key Points ■ ■ Monoclonal Antibodies Antibodies that arise from a single clone of cells (e.g., myeloma) are homogenous and are called monoclonal antibodies. For example, in multiple myeloma, antibodies are produced by a single clone of plasma cells against a single antigenic determinant, and hence antibodies are monoclonal. The monoclonal antibodies differ from polyclonal antibodies, which are heterologous and are formed by several different clones of plasma cells in response to antigen. ◗ Method of production of monoclonal antibodies Kohler and Milstein (1975) were the first to describe a method for production of monoclonal antibodies against a desired antigen for which they were awarded Nobel Prize in 1984. Monoclonal antibodies are produced by fusion of myeloma cells with antibody-producing cells, resulting in production of hybridomas. Such hybridomas produce virtually unlimited quantities of antibodies that are useful in research and diagnostics. In this procedure, mouse splenic lymphocytes are first fused with mouse myeloma cells to produce hybrid cells or hybridomas. Myeloma cell provides the hybrid cell immortality, ■ Monoclonal antibodies are now used widely in research and diagnostics. They are used in various clinical situations, such as treatment of cancer and autoimmune diseases. They are used in inducing immune suppression in transplant surgery, and in the prevention of infectious diseases. Function of Antibodies Antibodies are the primary defense against infectious pathogens or their products. Antibodies can be induced in the host actively by use of vaccines or acquired passively for conferring immediate protection against the pathogen. For example, hyperimmunized sera containing readymade antitoxins against toxins of tetanus, botulism, or diphtheria are given to neutralize the actions of these toxins immediately in the body. Also, hyperimmune sera containing high titer of specific antibodies are given to inhibit attachment and replication of rabies and hepatitis A and B viruses early during the period of incubation. The functions of the antibodies can be summarized as follows: Neutralization: By binding to the pathogen or foreign substance, antibodies can block the binding of the pathogen with their targets. For example, antibodies to bacterial toxins can prevent the binding of the toxin to host cells, thereby rendering the toxin ineffective. Similarly, antibody binding to a Chapter 17 Antimetabolites: These include folic acid antagonists (such as methotrexate); analogs of purine (6-mercaptopurine and azathioprine); and analogs of cytosine (cytosine arabinose); and uracil (5-fluorouracil). These substances inhibit DNA and RNA synthesis, thereby inhibiting the cell division and differentiation, which is essential for cellular and humoral immune responses. These are usually used for prevention of graft rejection. whereas splenic plasma cell provides the antibody-producing capacity. These hybridomas can be maintained indefinitely in culture and continue to produce monoclonal antibodies. Hybridoma cells are prepared in following ways (Fig. 17-2): Section II Immunosuppressive agents are those that suppress immune response. They are used in transplantation surgery and in situations that require suppression of host immunity. The agents are as follows: X-irradiation: Sublethal dose of irradiation is toxic to replicating cells and is used to suppress antibody formation. Antibody production ceases after 24 hours of receiving irradiation. Radiometric drugs: These include alkylating agents (such as cyclophosphamide, nitrogen mustard, etc.), which suppress antibody production. Cyclophosphamide, given for 3 days, completely suppresses the antibody response. It selectively prevents replication of B cells. Corticosteroids: Corticosteroids are anti-inflammatory drugs that diminish the responsiveness of both B and T cells. They alter maturation of activated cells by suppressing the production of interleukins. They suppress delayed hypersensitivity, but in therapeutic doses for a short period, they have little effect on the production of antibodies. 137 138 IMMUNOLOGY Mouse injected with specific antigen to raise antibody Cultured myeloma cells (Cancerous B cells) Suspension of myeloma cells Spleen Section II Unfused myeloma cells Unfused spleen cells Fused hybrid cells Suspension of spleen cells Chapter 17 Mixture of cells placed in selective medium that allows only fused hybrid cells to grow The hybrid cells proliferate into clones called hybridomas followed by screening of hybridomas for desired antibodies Hybridomas The selected hybridomas are then cultured to produce desired monoclonal antibodies on large scale FIG. 17-2. Monoclonal antibodies of interest A schematic diagram showing the production of monoclonal antibodies. virus or bacterial pathogen can block the attachment of the pathogen to its target cell, thereby preventing infection or colonization. Opsonization: Antibody binding to a pathogen or foreign substance can opsonize the material and facilitate its uptake and destruction by phagocytic cells. The Fc region of other antibody interacts with Fc receptors on phagocytic cells, rendering the pathogen more readily phagocytosed. Complement activation: Activation of the complement cascade by antibody can result in lysis of certain bacteria and viruses. In addition, some components of the complement cascade (e.g., C3b) opsonize pathogens and facilitate their uptake via complement receptors on phagocytic cells. Tests for Detection of Humoral Immunity The measurement of IgG, IgM, and IgA in the patient’s serum is the primary method for detection of humoral immunity. Radial immunodiffusion and immunoelectrophoresis are the methods frequently employed for measurement of antibodies. Cell-Mediated Immunity Cell-mediated immunity (CMI) is a specific type of acquired immune response not mediated by antibodies but by sensitized T cells. This form of immunity is transferred from donor to recipient, not with antisera but with intact lymphocytes; hence it is called cell-mediated immune reaction. CMI performs the following immunological functions: 1. It confers immunity in diseases caused by obligate intracellular bacteria (Mycobacterium tuberculosis, Mycobacterium leprae, Brucella, etc.), viruses (small pox, measles, mumps, etc.), fungi (Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, etc.), and parasites (Toxoplasma gondii, Leishmania donovani, etc.). 2. It participates in immunological surveillance and immunity against cancer. 3. It plays an important role in pathogenesis of delayed hypersensitivity reactions and in pathogenesis of certain autoimmune diseases, such as autoimmune thyroiditis, encephalitis, etc. IMMUNE RESPONSE 139 Induction of CMI ◗ Antigen processing and presentation are the means by which antigens become associated with self-MHC molecules for presentation to T cells with appropriate receptors. Proteins from exogenous antigens, such as bacteria, are internalized via endocytic vesicles into APCs, such as macrophages. Then, they are exposed to cellular proteases in intracellular vesicles. Peptides, approximately 10–30 amino acid residues in length, are generated in endosomal vesicles. The endosomal vesicles can then fuse with exocytic vesicles containing class II MHC molecules. Induction of CMI involves sequence of events, which is explained below. This stimulates Tc lymphocytes to release cytokines, resulting in the lysis of the target cells. The T cells then detach from the target cells and attach with other target cells, and the same process is repeated. Interferon-gamma synthesized and secreted by Tc lymphocytes possibly also contributes for macrophage activation in some way. Presentation of foreign antigen by APCs to T lymphocytes ◗ Recognition of antigen by T cells T cells recognize antigens only when presented with MHC molecules. The combination of foreign antigen and class I MHC molecule is recognized by CD8⫹ cells. These CD8⫹ cells after recognition differentiate into Tc and Ts lymphocytes. On the other hand, CD4⫹ cells recognize the combination of antigen and class II MHC antigen, after which they are differentiated into Th and Td cells. The class II MHC molecules are synthesized, as for other membrane glycoprotein, in the rough endoplasmic reticulum and then proceed out through the Golgi apparatus. A third polypeptide, the invariant chain (Ii), protects the binding site of the class II dimer until the lowered pH of the compartment created after fusion with an endosomal vesicle causes a dissociation of the Ii chain. The MHC class II peptide antigen complex is then transported to the cell surface for display and recognition by a TCR of a CD4 T cell. The lymphocyte recognizes antigen and class I MHC molecule and gets attached to the target cells. Endogenous antigens such as cytosolic viral proteins synthesized in an infected cell are processed for presentation by class I MHC molecule. In brief, cytosolic proteins are broken down by a peptidase complex known as the proteasome. The cytosolic peptides gain access to nascent MHC class I molecules in the rough endoplasmic reticulum via peptide transporter systems (transporters associated with antigen processing; TAPs). The TAP genes are also encoded in the MHC. The binding groove of the class I molecule is more constrained than that of the class II molecule; for that reason, shorter peptides are found in class I than in class II MHC molecules. Cytokines are biologically active substances secreted by monocytes, lymphocytes, and other cells and are actively involved in innate immunity, adoptive immunity, and inflammation. They actively take part in a wide range of biological activities varying from chemotaxis to activation of specific cells. Cytokines were initially identified as products of immune cells that act as mediators and regulators of immune processes. Many cytokines are now known to be produced by cells other than immune cells, and they can have effects on nonimmune cells as well. Cytokines are currently being used clinically as biological response modifiers for the treatment of various disorders. Cytokines are not typically stored as preformed proteins. Rather their synthesis is initiated by gene transcription and their mRNAs are short-lived. They are produced as needed in immune responses. Many individual cytokines are produced by many cell types and act on many cell types (i.e., they are pleiotropic), and in many cases cytokines have similar actions (i.e., they are redundant). Redundancy is due to the nature of the cytokine receptors. ◗ Categories of cytokines Cytokines can be grouped into different categories based on their functions or their source, but it is noteworthy that because they can be produced by many different cells and act on many different cells (Table 17-1), any attempt to categorize them will be subject to limitations. Cytokines may be categorized as follows: 1. 2. 3. 4. 5. Mediators affecting lymphocytes. Mediators affecting macrophages and monocytes. Mediators affecting polymorphonuclear leukocytes. Mediators affecting stem cells. Mediators produced by macrophages that affect other cells. Mediators affecting lymphocytes Interleukin-1 (IL-1): It is a protein produced mainly by activated macrophages and monocytes. Its production is stimulated by antigens, toxins, and inflammatory processes but inhibited by cyclosporine and corticosteroids. It is an important interleukin, which mediates a wide range of metabolic, physiological, inflammatory, and hematological activities. It has many important functions, which are given below: ■ ■ It activates a wide range of target cells including T and B lymphocytes, neutrophils, epithelial cells, and fibroblasts to proliferate, differentiate, or synthesize specific products. For example, it stimulates helper T cells to produce IL-2, and stimulates B cells to proliferate and synthesize antibodies, etc. It acts on the hypothalamus to cause fever associated with infections and other inflammatory reactions. Chapter 17 Induction of CMI begins with presentation of foreign antigen by APCs to T lymphocytes. T-cell receptors (TCRs), which are antigen recognition receptors, are present on T lymphocytes, and recognize foreign antigen and a self-MHC molecule on the surface of APCs. Subsequently, the sensitized T lymphocytes undergo blast transformation, clonal proliferation, and differentiation into memory cells and effector cells, such as Th, Tc, Td, and Ts. Finally, the lymphokines, which are biologically active products responsible for various manifestations of CMI, are released by the activated lymphocytes. Cytokines Section II ◗ Release of cytokines by Tc lymphocytes 140 IMMUNOLOGY TABLE 17-1 Chapter 17 Section II Cytokine Important functions of the main cytokines Source Functions IL-1 Macrophages Activates helper T cells, causes fever IL-2 Th-1 cells Activates helper, cytotoxic T cells and B cells IL-3 Th cells, NK, and mast cells Supports growth and differentiation, stimulates histamine release IL-4 Th-2 cells Stimulates B-cell growth, increases isotype switching and IgE, up-regulates class II MHC expression IL-5 Th-2 cells Stimulates B-cell differentiation, increases eosinophils and IgA Interferon-␣ Leukocytes Inhibits viral replication Interferon-␤ Fibroblasts Inhibits viral replication Interferon-␥ Th-1, Tc, and NK cells Inhibits viral replication, increases expression of class I and II MHC, stimulates phagocytosis and killing by macrophages and NK cells Tumor necrosis factor Macrophages Activates neutrophils and increases their adhesion to endothelial cells, mediates septic shock, causes necrosis of tumors, lipolysis, wasting, antiviral and antiparasitic effects Transforming growth factor-␤ Platelets, mast cells, and lymphocytes Induces increased IL-1 production, induces class switch to IgA, limits inflammatory response, and promotes wound healing Interleukin-2 (IL-2): IL-2 is a protein produced mainly by helper T cells. It is a major T-cell growth factor. It stimulates both helper and cytotoxic T cells to grow. It also promotes the growth of B cells and can activate natural killer (NK) cells and monocytes. IL-2 acts on T cells in an autocrine fashion. Activation of T cells results in expression of IL-2R and the production of IL-2. The IL-2 binds to the IL-R and promotes cell division. When the T cells are no longer being stimulated by antigen, the IL-2R will eventually decay and the proliferative phase ends. Interleukin-4 (IL-4): It is a protein produced mainly by helper T cells and macrophages. It stimulates the development of Th-2 cells, the subset of helper T cells that produces IL-4 and IL-5 and enhances humoral immunity by producing antibodies. It is also required for class (isotype) switching from one class of antibodies to another within antibody-producing cells. Interleukin-5 (IL-5): It is a protein produced by helper T cells. It promotes the growth and differentiation of B cells and eosinophils. It enhances the synthesis of IgA and also stimulates the production and activation of eosinophils. Interleukin-6 (IL-6): It is a protein produced by helper T cells and macrophages. It stimulates the production of acute phase proteins by the liver. It also acts on the hypothalamus to cause fever. Other interleukins: IL-10, IL-12, and IL-13 are the other interleukins that affect lymphocytes. IL-10 is produced by activated macrophages and Th-2 cells. It is predominantly an inhibitory cytokine. It inhibits production of type I interferon. It inhibits production of interferon-gamma by Th-1 cells, which shifts immune responses toward a Th-2 type. It also inhibits cytokine production by activated macrophages and the expression of class II MHC and costimulatory molecules on macrophages, resulting in a depression of immune responses. IL-12 is produced by activated macrophages and dendritic cells. It stimulates the production of interferon-gamma and induces the differentiation of Th cells to become Th-1 cells. In addition, it enhances the cytolytic functions of Tc and NK cells. IL-13 is produced by Th-2 cells. It is associated with pathogenesis of allergic airway disease (asthma). It is involved in the occurrence of hyper-responsiveness seen in asthma. Transforming growth factor-beta (TGF-␤): It is produced by T cells and many other cell types. It is primarily an inhibitory cytokine. It inhibits the proliferation of T cells and the activation of macrophages. It also acts on polymorphonuclear leukocytes and endothelial cells to block the effects of proinflammatory cytokines. In essence, it suppresses the immune response when it is not required after an infection, and thereby it promotes the healing process. Mediators affecting macrophages and monocytes Chemokines are a subtype of cytokines of low molecular weight and with a characteristic structural pattern. More than 50 chemokines varying in size from 68 to 120 amino acids have been identified. The alpha-chemokines, such as IL-8 are produced by activated mononuclear cells, which attract neutrophils. The beta-chemokines, such as RANTES (regulated upon activation, normal T-cell expressed and secreted) and MCAF (monocyte chemotactic and activating factor), are produced by activated T cells and attract macrophages and monocytes. Chemokines are produced by endothelial cells, resident macrophages and various cells present at the site of infection: ■ ■ They attract either macrophages or neutrophils to the site of infection, hence are involved in chemical-induced migration of leukocytes—a process called chemotaxis. Specific receptors for chemokines are present on the surface of monocytes and neutrophils. They also facilitate migration of white cells into the tissue to reach the infected area. They do so by activating integrins IMMUNE RESPONSE on the surface of neutrophils and macrophages that bind to the intercellular adhesion molecule (ICAM) proteins on the surface of the endothelium. Mediators affecting polymorphonuclear leukocytes Leukocyte-inhibiting factor: It inhibits migration of neutrophils, thereby retaining the cells at the site of infection. Mediators affecting stem cells Mediators affecting stem cells include (a) IL-3, (b) granulocyte macrophage colony-stimulating factor (GM-CSF), and (c) granulocyte colony-stimulating factor (G-CSF). ■ ■ ■ IL-3 produced by helper T cells suppresses the growth and differentiation of bone marrow stem cells. GM-CSF produced by macrophages and T lymphocytes stimulates the growth of granulocytes. G-CSF is produced by macrophages, fibroblasts, and endothelial cells. It facilitates development of neutrophils from stem cells, and hence used to prevent infection in patients receiving cancer chemotherapy. Mediators produced by macrophages that affect other cells Tumor necrosis factor (TNF-␣): TNF-␣, as the name suggests, causes death and necrosis of certain tumor cells in experimental animals. It is also called cachectin because it inhibits lipoprotein lipase in adipose tissues, thereby reducing the utilization of fatty acids, leading to cachexia. TNF-␣ performs many functions: ■ ■ ■ It activates respiratory burst within neutrophils, thereby enhancing killing activities of phagocytes. It facilitates adhesion of neutrophils to endothelial cells of blood vessels. It also stimulates growth of B cells and increases synthesis of lymphokines by helper T cells. Macrophage migration inhibition factor (MIF): It is produced by macrophages in response to action by endotoxin. It retains the macrophages at the site of infection. It plays an important role in the induction of septic shock. Tests for Detection of CMI The CMI can be detected by in vivo and in vitro tests as follows: ◗ CMI in vivo tests Skin tests are useful to detect delayed hypersensitivity reaction to common antigens that come in contact with the body. Purified protein derivative in tuberculin test, dinitrochlorobenzene, or dinitrofluorobenzene are the antigens used for the skin testing. Most of the normal people respond with delayed type reactions to these skin antigens. Absence of reactions to these skin tests suggests impairment of the CMI. ◗ CMI in vitro tests Many in vitro tests are available for detection of CMI. These are: 1. Migration inhibition factor (MIF) test: This test is performed to determine the CMI by making a semiquantitative assessment of the migration inhibition of leukocytes. This test depends on the principle that cultured T cells produce macrophage migration inhibition factor on exposure to the antigen to which they are sensitized. In this test, human peripheral leukocytes are incubated in capillary tubes in culture chamber containing culture fluid. The leukocytes, in the absence of antigen, migrate out to the open end of the tube to form a fan-like pattern. In the presence of antigen, the leukocytes are prevented from migrating. 2. Lymphocyte blast transformation: A large number of T cells undergo blast transformation when exposed to certain mitogens, such as the phytohemagglutinin and concavalin. Sensitized T lymphocytes are transformed into large blast cells with great increase in the synthesis of DNA, on exposure to the specific antigen. An increase in DNA synthesis is then measured by incorporation of tritiated thymidine. 3. Enumeration of T cells, B cells, and subpopulation: Fluorescence-activated cell sorter is used to count the number of each type of cells, whether T or B cells. In this method, cells are labeled with monoclonal antibody tagged with a fluorescent dye, such as fluorescent or rhodamine. The number of cells that fluoresce is registered by passing those single cells through a laser light beam. The total number of B cells can be counted by using fluorescein-labeled antibodies against all immunoglobulin classes. Specific monoclonal antibodies directed against T-cell marker allows the counting of T cells, CD4 helper cells, CD8 suppressor cells, and other cells. The ratio of CD4 to CD8 in normal person is 1.5 or more but becomes less than one in persons with AIDS. 4. Rosette formation: Rosette is a lymphocyte to which three or more sheep erythrocytes are attached. Most T cells form rosettes when mixed with sheep erythrocytes. T-cell rosette is Chapter 17 Chemokines and other chemotactic factors: Chemokines and other chemotactic factors attract selectively neutrophils, basophils, and eosinophils to the site of infection. For example, IL-8 and C5a component of the complement attract specifically neutrophils. Nitric oxide (NO): It is produced by macrophages in response to action by endotoxin. NO causes vasodilatation, thereby inducing hypotension in septic shock. Section II Tumor necrosis factor (TNF-␣): It is produced by activated macrophages in response to microbes, especially the lipopolysaccharide of Gram-negative bacteria. It is an important mediator of acute inflammation. It mediates the recruitment of neutrophils and macrophages to sites of infection by stimulating endothelial cells to produce adhesion molecules. It also produces chemokines, which are chemotactic cytokines. TNF-␣ also acts on the hypothalamus to produce fever, and it promotes the production of acute phase proteins. 141 142 IMMUNOLOGY referred to as E-rosette. T cells can be determined by counting E-rosettes. Rosette formation is useful to detect T cells, hence the CMI of the host. preserved. However, expression of immune responses following antigen recognition might be inhibited by active suppression. Types of Immune Tolerance Chapter 17 Section II Transfer Factor Lawrence in 1954 first reported transfer of CMI in humans by injection of an extract from the leukocytes from the immunized host. The extract from leukocytes contained soluble substance known as transfer factor (TF). ■ ■ ■ The transferred immunity is specific in that CMI can be transferred only to those antigens to which the donor is specific. Transferred immunity is systemic and not local. TF does not transfer humoral immunity. The immune tolerance may be of two types: natural or acquired. Natural tolerance: Natural tolerance is nonresponsiveness to self-antigens. It develops during the embryonic life, and any antigen that comes in contact with the immune system during its embryonic life is recognized as self-antigen. The selfantigen would not induce any immune response. Burnet and Fenner (1949) also postulated that foreign antigens would not induce immune response if they were administered during the embryonic life. Key Points Acquired tolerance: Acquired tolerance develops when a potential immunogen induces a state of unresponsiveness to itself. The antigen needs to be repeatedly or persistently administered to maintain the acquired tolerance. This is probably necessary because of the continuous production of new B and T cells that must be rendered tolerant. Induction of immune tolerance depends on a number of factors. These include (a) species and immune competence of the host and (b) physical nature, dose, and route of administration of antigens. TF has following clinical applications: ■ TF is a nucleopeptide with a low molecular weight of 2000– 4000 Da. It is resistant to trypsin but gets inactivated by heating at 56°C for 30 minutes. It is nonantigenic. The exact mode of action of TF is not known. It is believed to stimulate the release of lymphokines from sensitized T lymphocytes. ■ ■ ■ It is used in treatment of disseminated infections associated with deficient CMI, such as tuberculosis and lepromatous leprosy. It is used in treatment of malignant melanoma and other types of cancer. It is used in treatment of Wiskott–Aldrich syndrome and other T-cell-deficient syndromes. Immunological Tolerance Immunological tolerance is a state of specific immunologic unresponsiveness to a particular antigen to which a person has been exposed earlier. The immune tolerance prevents the body to mount immune response against the self-antigen. Mechanisms of Tolerance Suggested mechanism of tolerance includes (a) clonal deletion, (b) clonal anergy, and (c) suppression. 1. Clonal deletion: Clones of B and T lymphocytes that recognize self-antigens are selectively deleted in embryonic life, hence are not available to respond on subsequent exposure to antigen. This is known as clonal deletion. 2. Clonal anergy: Clonal anergy means a condition in which clones of B and T lymphocytes that recognize self-antigens might be present but cannot be activated. 3. Suppression: In this mechanism, clones of B and T lymphocytes expressing receptors that recognize self-antigens are ■ Species and immune competence of the host: Tolerance depends on the immunological maturity of the host. Embryos and neonatal animals are immunologically immature, hence are more susceptible for induction of tolerance. Rabbits and mice can be made tolerant more rapidly than guinea pigs and chickens. Physical nature, dose, and route of administration of antigens: Soluble antigens and haptens can induce more immune tolerance than the aggregated antigens. For example, heat aggregated human gamma globulin is more tolerogenic than deaggregated gamma globulin in mice. It may possibly be due to enhanced phagocytosis of aggregated proteins than soluble antigens by macrophages, in which they can be presented to antibody-forming cells, thus inducing antibody synthesis. The induction of tolerance is dose dependent also. For example, a very simple molecule induces tolerance more readily than a complex one. Repeated minute doses as well as high doses of antigen induce B-cell tolerance, whereas a moderate degree of same antigen may be immunogenic. The route of administration is also important. In guinea pigs, intravenous or oral administration of certain haptens causes tolerance, whereas intradermal administration causes induction of immunity. T cells become tolerant more readily than B cells and also remain tolerant longer than B cells. Tolerance is overcome spontaneously or by injection of cross-reacting antigens. For example, in rabbits, tolerance to bovine serum albumin can be abolished by immunization with cross-reacting human serum albumin. Tolerance can be enhanced by administration of immunosuppressive drugs. For example, tolerance is enhanced in patients who have received organ transplants. 18 43 Mycobacterium Leprae Immunodeficiency Introduction When a system errs by failing to protect the host from diseasecausing agents or from malignant cells, the result is immunodeficiency. Immunodeficiency diseases and syndromes are the causes of significant mortality and morbidity, as well as a source of extremely valuable information about the physiology of the human immune system. Immunodeficiency can occur in T cells, B cells, complement, and phagocytes—the major components of the immune system. A functional defect of the immune system is suspected when a patient: ■ ■ ■ Has unusual frequency of infections with common or opportunistic microorganisms; Has unusually severe infections; and Is unable to eradicate infections with antibiotics to which the microorganisms are sensitive. Recurrent infections with certain viruses, protozoa, and fungi indicate a T-cell deficiency, whereas recurrent infections with pyogenic bacteria (such as staphylococci) indicate a B-cell deficiency. ◗ X-linked hypogammaglobulinemia, or infantile agammaglobulinemia or X-linked agammaglobulinemia (XLA), is the prototype of “pure” B-cell deficiency. In the majority of cases, the disease is transmitted as a sex-linked trait. The defective gene is located on Xq21.2–22, the locus coding for the B-cell progenitor kinase or Bruton’s tyrosine kinase (Btk). Btk plays an important role in B-cell differentiation and maturation, and is also part of the group of tyrosine kinases involved in B-cell signaling in adult life. Most cases of infantile agammaglobulinemia are associated with mutations affecting Btk. X-linked hypogammaglobulinemia shows the follow ing features: ■ ■ Immunodeficiency disorders can be classified as (a) primary immunodeficiencies or (b) secondary immunodeficiencies. Primary Immunodeficiencies A condition resulting from a genetic or developmental defect in the immune system is called a primary immunodeficiency. In such a condition, the defect is present at birth, although it may not manifest itself until later in life. Most of the primary immunodeficiencies are inherited from parents to offsprings. Primary immunodeficiency may affect either adaptive or innate immune functions. Most defects that lead to immunodeficiencies affect either myeloid or lymphoid cell lineages. The lymphoid cell disorders may affect T cells, B cells, or both B and T cells, whereas the myeloid cell disorders may affect phagocytic function. Primary immunodeficiency diseases can be classified as: (a) B-cell immunodeficiencies, (b) T-cell immunodeficiencies, (c) combined B-cell and T-cell deficiencies, (d) complement immunodeficiencies, and (e) phagocyte deficiencies. B-Cell Immunodeficiencies B-cell deficiencies include (a) X-linked hypogammaglobulinemia, (b) selective immunoglobulin deficiencies, (c) hyperIgM syndrome, and (d) interleukin-12 receptor deficiency. X-linked hypogammaglobulinemia It is characterized by extremely low IgG levels and by the absence of other immunoglobulin classes. Individuals with XLA have no peripheral B cells and suffer from recurrent bacterial infections, beginning at about 9 months of age. Patients suffer from repeated infections caused by common pyogenic organisms (Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Staphylococcus aureus, etc.) causing pyoderma, purulent conjunctivitis, pharyngitis, otitis media, sinusitis, bronchitis, pneumonia, empyema, purulent arthritis, meningitis, and septicemia. Chronic obstructive lung disease and bronchiectasis develop as a consequence of repeated bronchopulmonary infections. Infections with Giardia lamblia are diagnosed with increased frequency in these patients and may lead to chronic diarrhea and malabsorption. Agammaglobulinemic patients are at risk of developing paralytic polio after vaccination with the attenuated virus; they also are at risk of developing chronic viral meningoencephalitis, usually caused by an echovirus. Arthritis of the large joints develops in about 30–35% of the cases and is believed to be infectious, caused by Ureaplasma urealyticum. This condition is best treated with replacement therapy using gamma globulin (a plasma fraction containing predominantly IgG, obtained from normal healthy donors) administered intravenously. ◗ Selective immunoglobulin deficiencies In this condition, only one or more of the immunoglobulins are deficient in serum, while the others remain normal or elevated. IgA deficiency is the most common example of selective immunoglobulin deficiencies. IgA deficiency is characterized by nearly absent serum and secretory IgA. The IgA level is less than 5 ng/dL, but the remaining immunoglobulin class levels are normal or elevated. The disorder is either familial or it may Chapter 18 Section II 144 IMMUNOLOGY be acquired in association with measles or other types of viral infection, or toxoplasmosis. The etiology of IgA deficiency is unknown, but is believed to be due to arrested B-cell development. The principal defect appears to be in IgA B-cell differentiation. The adult patients with selective IgA deficiency usually express the immature phenotype, only a few of which can transform into IgA-synthesizing plasma cells. Although IgA cells are produced, these cells fail to secrete IgA. IgA is the principal immunoglobulin in secretions and is an important part of the defense of mucosal surfaces. Thus, IgA-deficient individuals have an increased incidence of respiratory, gastrointestinal, and urogenital infections. They also have an increased incidence of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. There is an increased incidence of the disorder in certain atopic individuals. Some selective IgA-deficiency patients form significant titers of antibody against IgA. They may develop anaphylactic reactions upon receiving IgA-containing blood transfusions. Selective IgA deficiency is diagnosed by the demonstration of less than 5 mg/dL of IgA in serum. They, however, have normal levels of IgG and IgM antibodies. Some individuals develop antibodies against IgG, IgM, and IgA. clefts at 6–8 weeks of fetal life, leading to deficient development of the thymus and parathyroids. Tetany and hypocalcemia, both characteristics of hypoparathyroidism, are observed in DiGeorge syndrome in addition to the defects in T-cell immunity. Peripheral lymphoid tissues exhibit a deficiency of lymphocytes in thymic-dependent areas. A defect in delayed-type hypersensitivity is demonstrated by the failure of affected patients to develop positive skin tests to commonly employed antigens, such as candidin or streptokinase and the inability to develop an allograft response. There is also minimal or absent in vitro responsiveness to T-cell antigens or mitogens. Defective cell-mediated immunity may increase susceptibility of the patient to opportunistic infections and render the individual vulnerable to a graft-versus-host reaction in blood transfusion recipients. However, the B or bursa equivalent-dependent areas, such as lymphoid follicles, show normal numbers of B cells and plasma cells in patients with DiGeorge syndrome. Serum immunoglobulin levels are within normal limits, and there is a normal immune response following immunization with commonly employed immunogens. Considerable success in treatment of DiGeorge syndrome has been achieved with fetal thymic transplants and by the passive administration of thymic humoral factors. ◗ ◗ Hyper-IgM syndrome This condition is characterized by high concentration of serum IgM but very low concentration of serum IgG, IgA, and IgE. They have normal numbers of T cells and B cells. Some of these immunodeficiencies are X-linked and some are inherited as autosomal recessives. Patients with this condition are susceptible to recurrent microbial infections and many autoimmune disorders, such as thrombocytopenia, neutropenia, and hemolytic anemia. ◗ Interleukin-12 receptor deficiency Patients with interleukin-12 receptor deficiency are highly susceptible to disseminated mycobacterial infections. Lack of interleukin-12 receptor prevents IL-12 initiating a Th-1 response, which is essential to prevent mycobacterial infections. T-Cell Immunodeficiencies T-cell deficiencies include (a) DiGeorge syndrome, (b) chronic mucocutaneous candidiasis, (c) transient hypogammaglobulinemia of infancy, and (d) common, variable, unclassified immunodeficiency. ◗ Thymic aplasia (DiGeorge syndrome) The DiGeorge syndrome or thymic aplasia is a classic example of a pure T-cell deficiency. Although the DiGeorge syndrome is a congenital immunodeficiency, it is not hereditarily transmitted. The condition is believed to be caused by an intrauterine infection prior to the eighth week of life, possibly of viral etiology. It is associated with microdeletions of chromosomal region 22q11. From the immunological point of view, it results due to defective embryogenesis of the third and fourth pharyngeal Chronic mucocutaneous candidiasis Some patients with chronic infection of skin and mucosa with Candida albicans have exhibited a selective deficiency of cell-mediated immunity. Affected individuals develop severe and widespread forms of candidal infections. Skin tests with Candida antigens and in vitro lymphocyte proliferation responses to C. albicans reveal a selective lack of reactivity. T-lymphocyte functions are normal when tested with other antigens and mitogens. The humoral response to C. albicans is also normal. Symptomatic therapy with antimycotic agents is often unsuccessful. ◗ Transient hypogammaglobulinemia of infancy This condition is characterized by the hypogammaglobulinemia in infants that occurs due to progressive catabolism of maternal IgG during the second and third months of life. This condition may persist until 2–3 years of age and become progressively more accentuated. In most cases, a deficiency of helper T-cell function appears to be responsible for the delay in immunoglobulin synthesis. The diagnostic hallmark for this disease is low-for-age circulating immunoglobulin levels. Peripheral blood B lymphocytes are usually normal. Intravenous gamma globulin is indicated until the child’s immunoglobulin levels normalize. With time, most children develop normal immune function. ◗ Common, variable, unclassified immunodeficiency This condition, also known as late onset hypogammaglobulinemia, includes a large number of cases of primary immunodeficiency with heterogeneous presentations. These conditions IMMUNODEFICIENCY Combined B-Cell and T-Cell Deficiencies ◗ Severe combined immunodeficiency Severe combined immunodeficiency (SCID) includes many syndromes with severe deficiency of both humoral and cellmediated immune responses. All these are inherited diseases with a defect in the differentiation of early stem cells. These are of two types: X-linked and autosomal. X-linked SCID: The sex-linked SCID is associated with a defect of the gene that codes for a polypeptide chain common to several interleukin receptors (IL-2, IL-4, IL-7, IL-11, and IL-15). This chain is involved in signaling of second messages, thus in its absence, T-cell precursors fail to receive the signals necessary for their proliferation and differentiation. There is T- and B-cell lymphopenia and decreased production of IL-2. There is an absence of delayed-type hypersensitivity, cellular immunity, and of normal antibody synthesis following immunogenic challenge. Autosomal SCID: This is due to a mutation in the gene encoding a tyrosine kinase called ZAP-70, which plays an important role in signal transduction in T cells. Other SCID patients show mutations in other genes, such as RAG-1 or RAG-2, that are essential to produce T-cell antigen receptor and the IgM monomer on the B cell that acts as antigen carrier. SCID is a disease of infancy, presenting with failure to thrive. Affected individuals frequently die during the first 2 years of life. Clinically, they may develop a measles-like rash, show hyperpigmentation, and develop severe recurrent (especially pulmonary) infections. These patients have heightened susceptibility to Pneumocystis carinii, C. albicans, and other pathogens. Even attenuated microorganisms, such as those used for immunization, e.g., attenuated poliomyelitis viruses, may induce infection in SCID patients. All these forms of SCID can be corrected with a bone marrow graft from HLA-DR matched siblings. The graft is usually successful, but there is a great risk for the development of graft-versus-host disease. Graft-versus-host disease (GVHD): It is a problem in SCID patients receiving unirradiated blood transfusions. It can also develop after transfusion of any fresh blood component ◗ Wiskott–Aldrich syndrome Wiskott–Aldrich syndrome is an X-linked recessive immunodeficiency disease of infants. It is characterized by thrombocytopenia, eczema, and increased IgA and IgE levels. There is decreased cell-mediated immunity. The inability to mount an IgM response to capsular polysaccharide of bacteria is the most important defect. IgA and IgE are increased, but IgM is diminished, although IgG serum concentrations are usually normal. By electron microscopy, T cells appear to lack the markedly fimbriated surface of normal T cells. T cells have abnormal sialophorin. The defect appears to be caused by the inability of T cells to provide help to B cells. Bone marrow transplantation corrects the deficiency. ◗ Ataxia telangiectasia It is an autosomal recessive disease caused by mutations in the gene that encodes DNA repair enzyme. This condition is characterized by ataxia, telangiectasia, and recurrent infections in babies by 2 years of age. IgA deficiency and lymphopenia commonly occur. ◗ MHC class II deficiency It is an autosomal recessive disease failing to express MHC molecules on the surface of antigen presenting cells, such as macrophages and B cells. This results in a deficiency of CD4 T cells. The lack of these helper T cells results in production of deficient antibodies. Complement Deficiencies Complement deficiencies include the following conditions: ◗ Recurrent infections This is a condition caused by a deficiency of C1, C3, or C5, or even C6, C7, or C8 components of the complement. Patients with C3 deficiency are highly susceptible to infection with S. aureus and other pyogenic bacteria. Similarly, patients with C6, C7, or C8 deficiency are more susceptible to bacteremia with N. meningitidis or Neisseria gonorrhoeae. ◗ Autoimmune diseases Patients with deficiencies in C2 and C4 components have disease resembling systemic lupus erythematosus or other Chapter 18 Combined B-cell and T-cell deficiencies include (a) severe combined immunodeficiency, (b) Wiskott–Aldrich syndrome, (c) ataxia telangiectasia, and (d) MHC class II deficiency. contaminated with viable T lymphocytes. It is characterized by fever, maculopapular rash involving the volar surfaces, diarrhea and protein-losing enteropathy, Coombs’ positive hemolytic anemia, thrombocytopenia, and splenomegaly. In full-blown cases, the outcome is generally poor, with death occurring within 10–14 days from the onset of symptomatology. The reaction may be prevented in the case of transfusion by using frozen or irradiated blood products. Current attempts at eliminating all cells except stem cells from bone marrow grafts appear promising. Section II show a variable age of onset and patterns of inheritance, whose clinical picture is similar to that of XLA, but with a less severe course of clinical manifestations. T-cell function appears to be deficient in most cases, with abnormally low proliferative responses to T-cell mitogens. Sinusitis and bacterial pneumonia are the predominant infections in patients with common, variable, unclassified immunodeficiency. Intestinal giardiasis is common. Opportunistic infections caused by Pneumocystis jiroveci, mycobacteria, viruses, and other fungi are also more frequent in these patients. Treatment usually involves administration of intravenous gamma globulin. 145 146 IMMUNOLOGY autoimmune diseases. Patients with C2 deficiency are usually asymptomatic, and C2 deficiency is the most common complement defect. Chapter 18 Section II ◗ Paroxysmal nocturnal hemoglobinuria It is a disease characterized by hemoglobinuria during night when patient is asleep. The hemoglobinuria occurs due to a complement-mediated hemolysis, especially at night. This is because the lower concentration of oxygen in the blood during sleep increases the susceptibility of the red blood cells to lysis. This occurs in the patients with a defect in the gene for the molecules that attach decay-accelerating factor (DAF) and other proteins to the cell membrane. This results in a deficiency of DAF on the surface of blood cell precursors, leading to an increased activation of complement and increased hemolysis. ◗ Hereditary angioedema Hereditary angioedema is a disease caused by a deficiency of C1 inhibitor, a component of the complement. This deficiency results in the continual action of C1 on C4 to produce more C4a and subsequently more C3a and C5a complement components. An increased production of the vasoactive components, such as C3a and C5a, results in the production of capillary permeability and edema in larynx and several other organs. Steroids (such as oxymetholone and danazol) are used to treat the condition, because they increase the concentration of C1 inhibitors, thereby preventing increased production of more C3a and C5a. Phagocyte Deficiencies Phagocyte deficiencies include (a) chronic granulomatous disease, (b) Chediak–Higashi syndrome, (c) Job’s syndrome, (d) leukocyte adhesion deficiency, (e) myeloperoxidase deficiency, and ( f ) cyclic neutropenia. ◗ Chronic granulomatous disease Chronic granulomatous disease (CGD) is a disorder that is inherited as an X-linked trait in two-thirds of the cases and as an autosomal recessive trait in the remaining one-third. Clinical manifestations are usually apparent before the end of the 2nd year of life. This is a condition associated with deficiency of an enzyme NADPH oxidase. This enzyme deficiency causes neutrophils and monocytes to have decreased consumption of oxygen and diminished glucose utilization by the hexose monophosphate shunt. Although neutrophils phagocytose microorganisms, they do not form superoxide and other oxygen intermediates that usually constitute the respiratory burst. All of these lead to decreased intracellular killing of bacteria and fungi. Thus, these individuals have an increased susceptibility to infection with microorganisms that normally are of relatively low virulence. These include Aspergillus, Serratia marcescens, and Staphylococcus epidermidis. Patients with CGD may have hepatosplenomegaly, pneumonia, osteomyelitis, abscesses, and draining lymph nodes. The quantitative nitroblue tetrazolium (NBT) test and the quantitative killing curve are both employed to confirm the diagnosis of CGD. Therapy includes interferon gamma, antibiotics, and surgical drainage of abscesses. ◗ Chediak–Higashi syndrome It is a childhood disorder with an autosomal recessive mode of inheritance. The condition is identified by the presence of large lysozomal granules in leukocytes that are very stable and undergo slow degranulation. The large cytoplasmic granular inclusions that appear in white blood cells may also be observed in blood platelets and can be seen by regular light microscopy in peripheral blood smears. The condition is characterized by a defective neutrophil chemotaxis and an altered ability of the cells to kill ingested microorganisms. The majority of affected individuals die during childhood, although occasional cases may live longer. There is no effective therapy other than the administration of antibiotics for treatment of the infecting microorganisms. High doses of ascorbic acid have been shown to restore normal chemotaxis, bactericidal activity, and degranulation. ◗ Job’s syndrome Job’s syndrome is caused by failure of helper T cells to produce gamma interferon, which in turn reduces the ability of macrophages to kill bacteria. This results in an increased production of Th-2 and consequently an increased production of IgE. All these in turn cause more production of histamine that prevents certain components of inflammatory reaction and also inhibits chemotaxis. Therefore, the patient with this syndrome suffers repeatedly from staphylococcal abscesses as well as eczema with a high level of IgE. ◗ Leukocyte adhesion deficiency It is an autosomal recessive disease caused by mutation in the gene encoding the B chain of an integrin that mediates adhesion of leukocytes to microbes. This causes poor adhesion of neutrophils to endothelial surfaces; hence phagocytosis of bacteria is inadequate. ◗ Cyclic neutropenia It is an autosomal dominant disease in which there is a mutation in the gene encoding neutrophil esterase, an enzyme produced by neutrophils. The disease is characterized by a very low neutrophil count, less than 200/␮L for 3–6 days of a 21-day cycle. The patients are susceptible to life-threatening bacterial infections during these 3–6 days of low neutrophil count but not when neutrophil counts are normal. ◗ Myeloperoxidase deficiency It is a disease associated with deficiency of an enzyme myeloperoxidase, which is essential for the production of hypochlorite, a microbicidal agent. The deficiency of this enzyme is frequently seen but has little clinical importance. This is because other intracellular killing mechanisms of leukocytes are intact. 147 IMMUNODEFICIENCY T-Cell Deficiencies Secondary Immunodeficiencies ◗ Common variable hypogammaglobulinemia This condition is caused due to a defective T-cell signaling resulting in failure to produce IgG in the body. This occurs in persons between the ages of 13 and 35 years. In this condition, the number of B cells is normal, but the ability to produce IgG and other immunoglobulins is greatly reduced. The cell-mediated immunity is normal. Patients with this condition are highly susceptible to infections caused by S. pneumoniae, H. influenzae, and other pyogenic bacteria. Administration of intravenous gamma globulin reduces the infections caused by these bacteria. ◗ Malnutrition In malnutrition, the synthesis of IgG is reduced due to low supply of amino acids. People with malnutrition, hence, are susceptible to infection by pyogenic bacteria. TABLE 18-1 Acquired immunodeficiency syndrome (AIDS) Patients with AIDS caused by HIV are highly susceptible to infection by many opportunistic pathogens including bacteria, viruses, fungi, and parasites. This is attributed to the loss of helper T-cell activity. The virus specifically infects and kills the cells bearing CD4 surface receptors. The immunity is highly suppressed, and failure of immune surveillance leads to a high incidence of tumors. For detail, refer Chapter 68. ◗ Measles T-cell function is altered, but immunoglobulins are normal in patients suffering from measles. Patients show a temporary suppression of delayed hypersensitivity. Chapter 18 B-Cell Deficiencies ◗ Complement Deficiencies ◗ Liver failure The synthesis of complement proteins is very much reduced in chronic hepatitis B or C and in liver failure caused by alcoholic cirrhosis. Hence, these patients are highly susceptible to infection by pyogenic bacteria. ◗ Malnutrition In severe malnutrition, the synthesis of complement proteins by liver is reduced due to low supply of amino acids. Therefore, people with malnutrition are susceptible to infection by pyogenic bacteria. Phagocyte Deficiencies ◗ Neutropenia The condition is characterized by a low neutrophil count (less than 500/␮L), caused commonly by cytotoxic drugs, such as those used in cancer therapy. The patients are susceptible to severe bacterial infections caused by pyogenic bacteria, such as S. aureus and S. pneumoniae. Immunodeficiency diseases have been summarized in Table 18-1. Immunodeficiency syndromes Disease Specific deficiency Molecular defect Absence of B cells, very low IgG levels Mutant tyrosine kinase Selective IgA deficiency Very low IgA levels Failure of heavy-chain gene switching Transient hypogammaglobulinemia of infants Delay in initiation of IgG synthesis Common variable immunodeficiency Total Ig is less, inability of B cells to differentiate Immunodeficiency with hyper-IgM Low IgA and IgG, elevated IgM Transcobalamin II deficiency Metabolic defects of vitamin B12 deficiency B-cell defects X-linked agammaglobulinemia Section II Secondary immunodeficiencies occur secondary to numerous diseases or conditions, or as a consequence of therapeutic measures that depress the immune system. Most immunodeficient patients have secondary forms of immunodeficiency, caused by either pathological conditions that affect the immune system or the administration of therapeutic compounds with immunosuppressive effects. By far, the most common secondary immunodeficiency is acquired immunodeficiency syndrome (or AIDS), which results from infection with the human immunodeficiency virus (HIV). Secondary immunodeficiencies are more common than primary immunodeficiencies and include AIDS, chemotherapy by immunosuppressive drugs (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs), psychological depression, burns, radiation, Alzheimer’s disease, celiac disease, sarcoidosis, lymphoproliferative disease, Waldenstrom’s macroglobulinemia, multiple myeloma, aplastic anemia, sickle cell disease, malnutrition, aging, neoplasia, diabetes mellitus, and numerous other conditions. Secondary immunodeficiencies may be categorized as (a) B-cell deficiencies, (b) T-cell deficiencies, (c) complement deficiencies, and (d) phagocytic deficiencies as follows: (Continued) 148 IMMUNOLOGY TABLE 18-1 Immunodeficiency syndromes Disease (Continued) Specific deficiency Molecular defect Chapter 18 Section II T-cell defects Thymic aplasia (DiGeorge syndrome) Absence of T cells Defective development of pharyngeal pouches Chronic mucocutaneous candidiasis Deficient T-cell response to Candida Unknown Purine nucleoside phosphorylase (PNP) deficiency PNP deficiency Autosomal recessive Both B- and T-cell defects Nezelof syndrome Deficient T-cell and B-cell immunity Ataxia telangiectasia Lack of serum and secretory IgA, IgE Autosomal recessive Wiskott–Aldrich syndrome Depressed cell-mediated immunity, serum IgM X-linked disease Severe combined immunodeficiency Deficiency of both T cell and B cell Defective IL-2 receptor, kinases, recombinases Immunodeficiency with thymoma Impaired cell-mediated immunity, thymic tumor, agammaglobulinemia Complement disorders Hereditary angioedema Deficiency of C1 protease inhibitor Excess C3a, C4a, and C5a generated Complement component deficiencies Insufficient C3, C6, C7, C8 Unknown Chronic granulomatous disease Defective bactericidal activity Deficient NADPH oxidase activity Myeloperoxidase deficiency Leukocytes have reduced myeloperoxidase Chediak–Higashi syndrome Inclusions in leukocytes, diminished phagocytic activity Leukocyte G6PD deficiency Deficient G6PD in leukocytes Disorders of phagocytosis 43 19 Mycobacterium Leprae Hypersensitivity Introduction Hypersensitivity reaction denotes an immune response resulting in exaggerated or inappropriate reactions harmful to host. It is a harmful immune response in which tissue damage is induced by exaggerated or inappropriate immune responses in a sensitized individual on re-exposure to the same antigen. Both the humoral and cell-mediated arms of the immune response may participate in hypersensitivity reactions. Hypersensitivity essentially has two components. First priming dose (first dose) of antigen is essential, which is required to prime the immune system, followed by a shocking dose (second dose) of the same antigen that results in the injurious consequences. Depending on the time taken for the reactions and the mechanisms that cause the tissue damage, hypersensitivity has been broadly classified into immediate type and delayed type. In the former, the response is seen within minutes or hours after exposure to the antigen and in the latter, the process takes days together to manifest as symptoms. Prince of Monaco first observed the deleterious effects of jellyfish on bathers. Subsequently, Portier and Richet (1906) suggested a toxin to be responsible for these effects and coined the term “anaphylaxis”. Gell and Coombs (1963) classified hypersensitivity reactions into four categories based on the time elapsed from exposure of antigen to the reaction and the arm of immune system involved. Types I, II, and III are antibody-mediated and are known as immediate hypersensitivity reactions, while type IV is cell-mediated (i.e., mediated by cell-mediated immunity) and is known as delayed hypersensitivity reactions. Type V hypersensitivity reaction has been described later. It is called stimulatory type reaction and is a modification of type II hypersensitivity reaction. Differences between immediate and delayed hypersensitivities have been summarized in Table 19-1. Type I (Anaphylactic) Hypersensitivity Type I hypersensitivity reaction is commonly called allergic or immediate hypersensitivity reaction. This reaction is always rapid, occurring within minutes of exposure to an antigen, and always involves IgE-mediated degranulation of basophils or mast cells. Type I reactions are also known as IgE-mediated hypersensitivity reactions. IgE is responsible for sensitizing mast cells and providing recognition of antigen for immediate hypersensitivity reactions. The short time lag between exposure to antigen and onset of clinical symptoms is due to the presence of preformed mediators in the mast cells. Thus, the time taken for these reactions to initiate is minimal, so the onset of symptoms seems to be immediate. Type I reaction can occur in two forms: anaphylaxis and atopy. Anaphylaxis Anaphylaxis is an acute, potentially fatal, and systemic manifestation of immediate hypersensitivity reaction. It occurs when an antigen (allergen) binds to IgE on the surface of mast cells with the consequent release of several mediators of anaphylaxis. On exposure to the antigen, TH2 cells specific to the antigen are activated, leading to the stimulation of B cells to produce IgE antibody (Fig. 19-1). The IgE then binds to Fc portion of mast cells and basophils with high affinity. On reexposure to the antigen, the allergen cross-links the bound IgE, followed by activation of IgE and degranulation of basophils and mast cells to release pharmacologically active mediators within minutes. Binding of IgE to the mast cells is also known as sensitization, because IgE-coated mast cells are ready to be activated on repeat antigen encounter. ◗ Initiator cells in anaphylaxis The initiator of type I reaction is otherwise known as allergen. Typical allergens include: ■ ■ ■ Plant pollen, proteins (e.g., foreign serum and vaccines), Certain food items (e.g., eggs, milk, seafood, and nuts), Drugs (e.g., penicillin and local anesthetics), TABLE 19-1 Differences between immediate and delayed hypersensitivities Properties Immediate Delayed Type I, II, III IV Time to manifest Minutes to hours Days Mediators Antibodies T cells Route of sensitization Any route Intradermal Passive transfer with serum Possible Not possible Desensitization Easy but short lived Difficult but long lasting 150 IMMUNOLOGY Mast cell IgE antibodies Binding of IgE on mast cell Chapter 19 Section II Allergen Release of inflammatory mediators Degranulation Anaphylaxis FIG. 19-1. A schematic diagram showing type I hypersensitivity reaction. ■ ■ ■ Insect products (venom from bees, wasps, and ants), Dust mites, mould spores, and Animal hair and dander. The exact reason for these substances to act as allergens is not known, although they show some common characteristics. Because these reactions are T-cell dependent, T-cell-independent antigens like polysaccharides cannot elicit type I reactions. ◗ Effector cells in anaphylaxis The effector cells in anaphylaxis include (a) mast cells, (b) basophils, and (c) eosinophils. All these three cells contain cytoplasmic granules whose contents are the major mediators of allergic reactions. Also, all these three cell types produce lipid mediators and cytokines that induce inflammation. Mast cells: Mast cells are the prime mediators of anaphylaxis. These cells are found throughout connective tissue, particularly near blood and lymphatic vessels. IgE-mediated degranulation of mast cells occurs when an allergen causes cross-linkage of the membrane-bound IgE. The importance of cross-linkage in the process can be understood by the fact that monovalent molecules, which cannot cause cross-linkage, are unable to cause degranulation. Key Points Activation of mast cells results in three types of biologic responses: ■ ■ ■ ◗ secretion of preformed contents of their granules by a regulated process of exocytosis; synthesis and secretion of lipid mediators; and synthesis and secretion of cytokines. Mediators of anaphylaxis Many substances instead of a single substance are responsible for all manifestations of anaphylaxis. Important mediators include (a) histamine, (b) slow-reacting substances of anaphylaxis (SRS-A), (c) serotonin, (d) eosinophilic chemotactic factors of anaphylaxis, and (e) prostaglandins and thromboxanes. Histamine: It is the most important mediator of anaphylaxis. It is found in a preformed state in granules of mast cells and basophils. It causes vasodilatation, increased capillary permeability, and smooth muscle contraction. It is the principal mediator of allergic rhinitis (hay fever), urticaria, and angioedema. Antihistamines that block histamine receptors are relatively effective against allergic rhinitis but not against asthma. Slow-reacting substances of anaphylaxis: These are produced by leukocytes. These consist of several leukotrienes, which do not occur in preformed state but are produced during reactions of anaphylaxis. Leukotrienes are principal mediators of bronchoconstriction in asthma and are not inhibited by antihistamines. They cause increased vascular permeability and smooth muscle contraction. Serotonin: Serotonin is found in preformed state in mast cells and platelets. It causes vasoconstriction, increased capillary permeability, and smooth muscle contraction. Eosinophilic chemotactic factors of anaphylaxis: It is found in preformed state in granules of mast cells. It attracts eosinophils to the site of action. The role of eosinophils, however, is not clear in type I hypersensitivity reaction. Nevertheless, it is believed to reduce severity of type I hypersensitivity by releasing the enzymes histaminase and arylsulfatase that degrade histamine and SRS-A, respectively. Prostaglandins and thromboxanes: Prostaglandins cause bronchoconstriction as well as dilatation and increased permeability of capillaries. Thromboxanes cause aggregation of platelets. All these mediators are inactivated by enzymatic reactions very rapidly, hence are active only for a few minutes after their release. ◗ Phases of anaphylaxis The spectrum of changes seen in type I hypersensitivity can be considered under immediate and late phases. Immediate phase: This phase is characterized by degranulation and release of pharmacologically active mediators within minutes of re-exposure to the same antigen. Histamine is the principal biogenic amine that causes rapid vascular and smooth muscle reactions, such as vascular leakage, vasodilatation, and bronchoconstriction. It is responsible for the “wheal and flare” response seen in cutaneous anaphylaxis and also for the increased peristalsis and bronchospasm associated with ingested allergens and asthma, respectively. Other lipid mediators, such as prostaglandins (PGD2) and leukotrienes (LTC4)—which are derived from arachidonic acid by the cyclooxygenase pathway and lipoxygenase pathway, respectively, also cause similar reactions. Prostaglandins and leukotrienes promote bronchoconstriction, neutrophil chemotaxis, and aggregation at inflammatory sites. Late phase: This phase begins to develop 4–6 hours after the immediate phase reaction and persists for 1–2 days. It is characterized by the infiltration of neutrophils, macrophages, HYPERSENSITIVITY eosinophils, and lymphocytes to the site of reaction. This leads to an amplification of the various inflammatory symptoms seen as a part of the early reaction like bronchoconstriction and vasodilatation. The cells remain viable after degranulation and proceed to synthesize other substances that are released at a later time, causing the late phase of type I reactions. The mediators are not detectable until after some hours of the immediate reaction. The important mediators involved during the late phase are: ■ ■ ◗ slow-reacting substances of anaphylaxis (SRS-A) that contain several leukotrienes (e.g., LTC4, LTD4, and LTE4); platelet-aggregating factor; and cytokines released from the mast cells. Clinical manifestations of anaphylaxis ◗ Management and prevention of anaphylaxis Desensitization is an effective way for prevention of systemic anaphylaxis. It is of two types: acute desensitization and chronic desensitization. Acute desensitization involves the administration of small amounts of antigen to which the person is sensitive, at an interval of 15 minutes. The complex of antigen–IgE is produced in TABLE 19-2 Atopy The term atopy was first coined by Coca (1923) to denote a condition of familial hypersensitivities that occur spontaneously in humans. Atopy is recurrent, nonfatal, and local manifestation of immediate hypersensitivity reaction. The reaction shows a high degree of familial predisposition and is associated with a high level of IgE. It is localized to a specific tissue, often involving epithelial surfaces at the site of antigen entry. It is mediated by IgE antibodies, which are homocytotropic (i.e., species specific). Only human IgE can fix to surface of mast cells. Common manifestations of atopy are asthma, rhinitis, urticaria, and atopic dermatitis. The commonest of atopic reactions is bronchial asthma. Atopy is associated with mutations in certain genes encoding the alpha chain of the IL-4 receptor. These mutations facilitate the effectiveness of IL-4, resulting in an increased production of IgE synthesis by B cells. Atopic individuals produce high levels of IgE in response to allergens as against the normal individuals who do not. This depends on the propensity of an individual to mount a TH2 response, because it is only TH2 cell-derived cytokines that stimulates the heavy-chain isotype switching to the IgE class in B cells. Stimulation of heavy chain isotype switching to the IgE class may be influenced by a variety of factors including inherited genes, the nature of the antigens, and the history of antigen exposure. Mechanism and manifestations of hypersensitivity reactions Type Syndromes caused Mechanism Type I Hay fever, asthma, hives, and eczema IgE mediated Type II Blood transfusion reactions, erythroblastosis fetalis, and autoimmune hemolytic anemias Antibodies against cell surface antigen, causing damage by ADCC or complement activation Type III Arthus reaction, Farmer’s lung, Serum sickness, rheumatoid arthritis, necrotizing vasculitis, glomerulonephritis, SLE, immune complex in hepatitis B, and malaria Mediated by immune complexes containing complement-fixing antibodies Type IV Contact dermatitis, tubercular lesions, and graft rejection TH cells cause the release of cytokines which stimulate macrophages or cytotoxic T cells to mediate direct cellular damage Chapter 19 Anaphylaxis is an acute, life-threatening reaction usually affecting multiple organs. The time of onset of symptoms depends on the level of hypersensitivity and the amount, diffusibility, and site of exposure to the antigen. Multiple organ systems are usually affected, including the skin (pruritus, flushing, urticaria, and angioedema), respiratory tract (bronchospasm and laryngeal edema), and cardiovascular system (hypotension and cardiac arrhythmias). When death occurs, it is usually due to laryngeal edema, intractable bronchospasm, hypotensive shock, or cardiac arrhythmias developing within the first 2 hours (Table 19.2). Anaphylactoid reaction: This appears to be clinically similar to anaphylactic reaction but differs from it in many ways. First, it is not IgE mediated. Second, the inciting agents (such as drugs or iodinated contrast media) stimulate directly basophils and mast cells to release mediators without any involvement of the IgE. small quantities; hence enough mediators are not released to produce a major reaction. However, this action is short lived because of the return of hypersensitivity reaction due to continued production of IgE. Chronic desensitization involves the long-term administration of antigen to which the person is sensitive, at an interval of weeks. This stimulates the production of IgA- and IgG-blocking antibodies that prevent subsequent antigen to binding to mast cells, therefore, preventing the reaction. Administration of drugs to inhibit the action of mediators, maintenance of airways, and support of respiratory and cardiac functions form the mainstay of treatment of anaphylactic reactions. Section II ■ 151 152 IMMUNOLOGY Atopic hypersensitivity is non-transferable by lymphoid cells, but by serum. This observation was used in the past for diagnosis of passive cutaneous anaphylaxis reaction by Prausnitz–Kustner reaction. Chapter 19 Section II Key Points Prausnitz–Kustner reaction: This is based on the special affinity of IgE antibody for cells of the skin. In this experiment, serum was collected from Kustner who suffered from gastrointestinal allergy to certain cooked fish. The same serum was given intradermally to Prausnitz, who was then given another intradermal injection of small quantity of cooked fish into the same site, 24 hours’ later. This resulted in a wheal and flare at the site of injection within minutes. The test, however, is not done nowadays due to risk of transmission of certain bloodborne viral infections, such as hepatitis B, hepatitis C, and HIV. Radioallergosorbent test (RAST), enzyme linked immunosorbent assay (ELISA), and passive agglutination tests are the frequently used tests for detection of IgE in the serum for diagnosis of atopy. Type II cytotoxic reaction is mediated by antibodies directed against antigens on the cell membrane that activates complement thereby causing antibody-mediated destruction of cells (Fig. 19-2). The cell membrane is damaged by a membrane attack complex during activation of the complement. The reactions involve combination of IgG or IgM antibodies with the cell-fixed antigens or alternately circulating antigens absorbed onto cells. Antigen–antibody reaction leads to complement activation, resulting in the formation of membrane attack complex. This complex then acts on the cells, causing damage to the cells, as seen in complement-mediated lysis in Rh hemolytic disease, transfusion reaction, or hemolytic anemia. Similarly, the antibodies combining with tissue antigens Target cell Surface antigen A large number of proteins and glycoproteins are present on the surface of RBCs, of which A, B, and O antigens are of particular importance. Antibodies to these antigens are called isohemagglutinins and are of IgM class. When transfusion with mismatched blood occurs, a transfusion reaction takes place due to the destruction of the donor RBCs through the isohemagglutinins against the foreign antigen. The clinical manifestations result from the massive intravascular hemolysis of the donor cells by antibody and complement. This condition develops when maternal antibodies specific for fetal blood group antigens cross the placenta and destroy fetal RBCs. This condition is seen in cases where a presensitized Rh-negative mother mounts an immune response against Rh-positive RBCs of the fetus. This results in severe hemolysis, leading to anemia and hyperbilirubinemia, which can even be fatal. Drug-Induced Hemolysis Certain drugs (such as penicillin, quinidine, phenacetin, etc.) may induce hemolysis of red blood cells. They attach to the surface of red blood cells and induce formation of IgG antibodies. These autoantibodies then react with red blood cell surface, causing hemolysis. Similarly, quinacrines attach to surface of platelets and induce autoantibodies that lyse the platelets, causing thrombocytopenia. Goodpasture’s Syndrome Antibody Binding of antibody to surface antigen Cytotoxic action by NK cell Complement mediated reactions C3b Phagocyte Membrane attack complex Transfusion Reactions Erythroblastosis Fetalis Type II (Cytotoxic) Hypersensitivity Antibody dependent cell-mediated cytotoxicity contribute to the pathogenesis of Goodpasture’s syndrome, pemphigus, and myasthenia gravis. Antibody-dependent cell-mediated cytotoxicity (ADCC): It is another mechanism, which involves the binding of cytotoxic cells with Fc receptors in the Fc binding part of the antibodies coating the target cells. The antibody coating the target cell can also cause its destruction by acting as an opsonin. This mechanism is important in immunity against large-sized pathogens, such as the helminths. Autoantibodies of IgG class are produced against basement membrane of the lungs and kidneys in Goodpasture’s syndrome. Such autoantibodies bind to tissues of the lungs and kidneys and activate the complement that leads to an increased production of C5a, a component of the complement. The C5a causes attraction of leukocytes, which produce enzyme proteases that act on lung and kidney tissues, causing damage of those tissues. Rheumatic Fever Osmotic lysis Opsonization by C3b and phagocytosis FIG. 19-2. A schematic diagram showing type II hypersensitivity reaction. In this condition, antibodies are produced against group A streptococci that cross-react with cardiac tissues and activate complement and release of components of complement, which in turn causes damage of cardiac tissues. HYPERSENSITIVITY Type III (Immune-Complex) Hypersensitivity Type III reaction is mediated by antigen–antibody immune complexes, which induce an inflammatory reaction in tissues. Mechanism of Immune-Complex Hypersensitivity Manifestations of Immune-Complex Hypersensitivity Arthus reactions and serum sickness reactions are two typical manifestations of type III hypersensitivity. ◗ Arthus reactions Arthus reaction is an inflammatory reaction caused by deposition of immune complexes at a localized site. This reaction is named after Dr. Arthus who first described this reaction. This reaction is edematous in the early stages, but later can become hemorrhagic and, eventually, necrotic. The lag time between antigen challenge and the reaction is usually 6 hours. This is considerably longer than the lag time of an immediate hypersensitivity reaction, but shorter than that of a delayed hypersensitivity reaction. Tissue damage is caused by deposition of antigen–antibody immune complexes and complement. The activation of complement through its product of activation causes vascular occlusion and necrosis. Key Points ■ B cell ■ Hypersensitivity pneumonitis is the clinical manifestation of Arthus reaction. Farmer’s lung, cheese-washer’s lung, wood-worker’s lung, and wheat-miller’s lung are the examples of hypersensitivity pneumonitis associated with different occupations. All these conditions are caused by inhalation of fungi or bacteria present in different products handled by the infected people. Arthus reaction can also occur locally at the site of tetanus immunization, if toxoids are given at the same site within a very short period of 5 years. Production of antibodies ◗ Plasma cell Immune complex formation Complement Neutrophil Complement activation Neutrophil activation Tissue damage FIG. 19-3. A schematic diagram showing type III hypersensitivity reaction. Serum sickness Serum sickness is a systemic inflammatory reaction caused by deposition of immune complexes at many sites of the body. The condition manifests after a single injection of a high concentration of foreign serum. It appears a few days to 2 weeks after injection of foreign serum or certain drugs, such as penicillin. However, serum sickness is considered as an immediate hypersensitivity reaction, because symptoms appear immediately after formation of immune complex. Unlike type I hypersensitivity reaction, a single injection acts as both priming and shocking doses. Fever, lymphadenopathy, rashes, arthritis, splenomegaly, and eosinophilia are the typical manifestations. Disease is self-limited and clears without sequelae. Chapter 19 Antigen The proteolytic enzymes (including neutral proteinases and collagenase), kinin-forming enzymes, polycationic proteins, and reactive oxygen and nitrogen intermediates cause damage in the local tissues and enhance the inflammatory responses. Platelets aggregated by intravascular complexes provide yet another source of vasoactive amines and may also form microthrombi, which can lead to local ischemia. Section II In many situations, reactions between the various antigens and antibodies in the body give rise to formation of immune complexes (Fig. 19-3). In the normal course, these immune complexes are normally removed by mononuclear-phagocyte system through participation of RBC. However, the body may be exposed to an excess of antigen in many conditions, such as persistent infection with a microbial organism, autoimmunity to self-components, and repeated contact with environmental agents. When the clearance capacity of this system is exceeded, deposition of the complexes takes place in various tissues. Immune complexes are deposited (a) on blood vessel walls, (b) in the synovial membrane of joints, (c) on the glomerular basement membrane of the kidneys, and (d) on the choroid plexus of the brain. Sometimes, immune complexes are formed at the site of inflammation itself. These in situ immune complexes, in certain cases, may be beyond the reach of phagocytic clearance and hence aggregate and cause disease. Immune complexes fix complement and are potent activators of the complement system. Activation of the complement results in the formation of complement components, such as C3a- and C5a-anaphylatoxins that stimulate release of vasoactive amines. The C5a attracts neutrophils to the site, but these neutrophils fail to phagocytose large aggregated mass of immune complexes, and instead release lysosomal enzymes and lytic substances that damage host tissue. 153 154 IMMUNOLOGY Immune-Complex Diseases T lymphocyte Chapter 19 Section II Formation of circulating immune complexes contributes to the pathogenesis of a number of conditions other than serum sickness. These include the following: 1. Autoimmune diseases ■ Systemic lupus erythematosus (SLE) ■ Rheumatoid arthritis 2. Drug reactions ■ Allergies to penicillin and sulfonamides 3. Infectious diseases ■ Poststreptococcal glomerulonephritis ■ Meningitis ■ Hepatitis ■ Infectious mononucleosis ■ Malaria ■ Trypanosomiasis Antigen presenting cell Sensitized T lymphocyte Cytokines Macrophage Granuloma formation FIG. 19-4. A schematic diagram showing type IV hypersensitivity reaction. Type IV Delayed (Cell-Mediated) Hypersensitivity Type IV hypersensitivity reaction is called delayed type hypersensitivity (DTH), because the response is delayed. It starts hours or days after primary contact with the antigen and often lasts for days. The reaction is characterized by large influxes of nonspecific inflammatory cells, in particular, macrophages. It differs from the other types of hypersensitivity by being mediated through cell-mediated immunity. This reaction occurs due to the activation of specifically sensitized T lymphocytes rather than the antibodies. Initially described by Robert Koch in tuberculosis as a localized reaction, this form of hypersensitivity was known as tuberculin reaction. Later, on realization that the reaction can be elicited in various pathologic conditions, it was renamed as delayed type hypersensitivity. Activated macrophage ■ ■ MHC class II alleles, human leukocyte antigen (HLA)-DR, HLA-DP, and HLA-DQ. Specific MHC class II alleles are recognized to produce excessive immune activation to antigens. On subsequent exposure, the effector phase is stimulated. The TH1 cells are responsible in secreting a variety of cytokines that recruit and activate macrophages and other nonspecific inflammatory cells. The response is marked only after 2–3 days of the second exposure. Generally, the pathogen is cleared rapidly with little tissue damage. However, in some cases, especially if the antigen is not easily cleared, a prolonged DTH response can itself become destructive to the host, as the intense inflammatory response develops into a visible granulomatous reaction. Types of DTH Reactions Mechanism of DTH The DTH response begins with an initial sensitization phase of 1–2 weeks after primary contact with an antigen (Fig. 19-4): ■ ■ ■ ■ TH1 subtypes CD4 are the cells activated during the sensitization phase. A variety of antigen-presenting cells (APCs) including Langerhans cells and macrophages have been shown to be involved in the activation of a DTH response. These cells are believed to pick up the antigen that enters through the skin and transport it to regional lymph nodes, where T cells are activated by the antigen. The APCs present antigens complexed in the groove of major histocompatibility complex (MHC) molecules expressed on the cell surface of the APCs. For most protein antigens or haptens associated with skin DTH, CD4⫹ T cells are presented with antigens bound to DTH reactions are of two types: contact hypersensitivity and tuberculin-type hypersensitivity reactions. ◗ Contact hypersensitivity Contact hypersensitivity is a manifestation of DTH occurring after sensitization with certain substances. These include drugs, such as sulfonamides and neomycin; plant products, such as poison ivy and poison oak; chemicals, such as formaldehyde and nickel; and cosmetics, soaps and other substances. This reaction manifests when these substances acting as haptens enter the skin and combine with body proteins to become complete antigens to which a person becomes sensitized. On second exposure to the same antigen, the immune system responds by attack of cytotoxic T cells that cause damage, mostly in the skin. HYPERSENSITIVITY The condition manifests as itching, erythema, vesicle, eczema, or necrosis of skin within 12–48 hours of the second exposure. ◗ Tuberculin-type hypersensitivity reaction ■ Infected persons receiving therapy with immunosuppressive drugs (such as corticosteroids and anticancer drugs) and In those suffering from the diseases associated with suppressed cell-mediated immunity (such as AIDS, sarcoidosis, lymphoma, post measles vaccination, etc.). The response to M. tuberculosis illustrates that while on one hand mechanisms involved in DTH are required for defense against the organism; on the other hand, these are also responsible for tissue damage in the longer run. Cytokines (like TNF and IFN-␥), which have been produced to activate the macrophages and thus contain the infection, also trigger other cascades that lead finally to extensive tissue damage. Various other skin tests are used to detect DTH. These include many skin tests in bacterial, fungal, viral, and helminthic infections. Lepromin test is a useful test for leprosy. A positive lepromin test indicates the presence of tuberculoid leprosy with intact Characteristics Contact hypersensitivity Tuberculin-type hypersensitivity Site Epidermal Intradermal Antigen Organic chemicals, poison ivy, metals, etc. Tuberculin, lepromin, leishmanin skin tests, etc. Reaction time 48–72 hours 48–72 hours cell-mediated immunity. On the other hand, a negative lepromin test indicates the presence of lepromatous leprosy with impaired cell-mediated immunity. Positive skin tests in coccidioidomycosis, paracoccidioidomycosis and other fungal infections suggest exposure to the fungi. In both viral and parasitic infections, skin tests are less specific and less useful than the serological tests for diagnosis. Differences between contact hypersensitivity and tuberculintype hypersensitivity reaction are summarized in Table 19-3. Type V (Stimulatory Type) Hypersensitivity In this type of hypersensitivity reaction, antibodies combine with antigens on cell surface, which induces cells to proliferate and differentiate and enhances activity of effector cells. Type V hypersensitivity reaction plays an important role in pathogenesis of Graves’ disease, in which thyroid hormones are produced in excess quantity. It is postulated that long-acting thyroid-stimulating antibody, which is an autoantibody to thyroid membrane antigen, combines with thyroid-stimulating hormone (TSH) receptors on a thyroid cell surface. Interaction with TSH receptor produces an effect similar to the TSH, resulting in an excess production and secretion of thyroid hormone, which is responsible for Graves’ disease. Table 19-4 summarizes important features of various types of hypersensitivity. Comparison of different types of hypersensitivity reactions TABLE 19-4 Type I Type II Type III Type IV Antigen Exogenous Cell surface Soluble Tissue and organ Antibody IgE IgG, IgM IgG, IgM None Reaction time 15–30 minutes Minutes to hours 3–8 hours 48–72 hours Transfer Antibody Antibody Antibody T cells Conditions Hay fever, allergy, and asthma Erythroblastosis fetalis and Goodpasture’s syndrome SLE, serum sickness Tuberculin test, poison ivy, etc. Chapter 19 ■ Differences between contact and tuberculin-type hypersensitivity TABLE 19-3 Section II Tuberculin reaction is a typical example of delayed hypersensitivity to antigens of microorganisms, which is being used for diagnosis of the disease. Tuberculin skin test: This test is carried out to determine whether an individual has been exposed previously to Mycobacterium tuberculosis or not. In this test, a small amount of tuberculin (PPD), a protein derived from the cell wall of M. tuberculosis, is injected intradermally. Development of a red, slightly swollen, firm lesion at the site of injection after 48–72 hours indicates a positive test. A positive test indicates that the person has been infected with the bacteria but does not confirm the presence of the disease, tuberculosis. However, if a person with a tuberculin-negative skin test becomes positive, then it indicates that the patient has been recently infected. The skin test, however, can even become negative in: 155 43 20 Mycobacterium Leprae Autoimmunity Introduction The immune system is a finely tuned system, which functions round-the-clock whole life to protect the body against various foreign cells, be it microbes or abnormal cells. Though the immune system is up and running all the time surrounded by self-antigens, it does not mount a response against them. At times, these mechanisms go awry, and this results in injury to various tissues. Autoimmunity is a condition when the body produces autoantibodies and immunologically competent T lymphocytes against its own tissues. Conditions where the mechanisms of self-tolerance fail are termed as autoimmune disorders and the phenomenon is called autoimmunity. Autoimmunity literally means “protection against self”; however, in practice it leads to “injury to self.” At the clinical level, autoimmunity is apparently involved in a variety of apparently unrelated diseases, such as systemic lupus erythematosus (SLE), insulin-dependent diabetes mellitus, myasthenia gravis, rheumatoid arthritis, multiple sclerosis, and hemolytic anemias. Ehrlich in 1901 first postulated the existence of tolerance to self-antigens as also those situations where this mechanism would fail, leading to “horror autotoxicus”. More recently, an understanding of the various immunological mechanisms and disorders has led to the same conclusions. Tolerance Tolerance is a state of specific immunological unresponsiveness to a certain antigen or epitope, although the immune system is otherwise functioning normally. The antigens that are present during embryonic life are usually considered self and do not stimulate an immunologic response, hence the host remains tolerant to those antigens. The absence of an immune response in the fetus is due to the deletion of self-reactive T-cell precursors in the thymus. On the other hand, the antigens that are not present during the process of maturation are considered nonself and usually elicit an immunologic response against those antigens. Mechanisms of Tolerance Both T cells and B cells participate in tolerance, but it is T-cell tolerance that plays a major role. ◗ T-cell tolerance T-cell tolerance is explained by theories of (a) clonal deletion, (b) clonal anergy, and (c) clonal ignorance. 1. Clonal deletion: The theory of clonal deletion described by Burnet, Fenner, and Medawar based on their studies on mice was the first theory of tolerance. The recent studies suggest that T lymphocytes acquire the ability to distinguish self from nonself by the process of clonal deletion during the early phases of life. This process involves the killing of T cells (negative selection) that acts against antigens, mainly self-MHC (major histocompatibility complex) molecules present in the fetus during that time. The self-reactive cells die by apoptosis, a process of programed cell death. 2. Clonal anergy: Clonal anergy is a process that leads to the incapacitation of the self-reactive T cells. These cells become incapable of mounting an immune response due to lack of proper costimulation and are called as anergic. 3. Clonal ignorance: This is the term used to describe selfreactive T cells that ignore self-antigens. These self-reactive T cells ignore self-antigens because the antigens are present in very small quantities. Also, these self-reactive cells are kept ignorant by physical separation from the target antigens, such as blood–brain barrier. ◗ B-cell tolerance B cells become tolerant to self-antigens also by (a) clonal deletion of B-cell precursors while they are in the bone marrow and (b) clonal anergy of B cells in the periphery. Pathogenesis of Autoimmunity Mechanisms The following mechanisms have been proposed for pathogenesis of autoimmunity: 1. 2. 3. 4. Release of sequestrated antigens Antigen alteration Epitope spreading Molecular mimicry AUTOIMMUNITY ◗ Release of sequestrated antigens Binding of IgG antibodies to the antigens on cardiac cells that resemble M protein Destruction of the cardiac valves and adjacent areas by these antibodies FIG. 20-1. T cells, resulting in autoimmune diseases. For example, in experimental animal infection, a multiple sclerosis-like disease is caused by an infection with an encephalomyelitis virus. In this condition, the self-reactive T cells are directed against cellular antigens but not against the virus that cause the sclerosislike disease. ◗ Molecular mimicry Molecular mimicry is a process in which infection by particular microbial pathogen is associated with the subsequent development of specific autoimmune diseases. Key Points ■ Antigen alteration Certain physical, chemical, or biological factors may alter tissue antigens, resulting in formation of new cell surface antigens called neoantigens. These neoantigens are no longer recognized as self, therefore, appear foreign to immune system, thereby eliciting an immune response. Procainamide-induced SLE is an example of an autoimmune disease caused by this mechanism. Pathogenesis of acute rheumatic fever. ■ ■ Sharing of M protein of Streptococcus pyogenes and myosin of cardiac muscle is one of many examples of molecular mimicry. Repeated infections with S. pyogenes induce the production of antibodies against certain M proteins that crossreact with myosin of cardiac muscle, resulting in damage to cardiac tissue, leading to rheumatic fever (Fig. 20-1). Similarly, infection with Shigella spp. or Chlamydia spp. may result in Reiter’s syndrome and infection with Campylobacter spp. may lead to Guillain–Barre syndrome. Development of encephalitis in certain cases following vaccination with rabies Semple vaccine is due to molecular mimicry of sheep brain antigens, used in the vaccine, with that of neural tissues in the brain. Autoimmune Pathological Process ◗ Epitope spreading Epitope spreading is the term used to describe the new exposure of sequestrated autoantigens as a result of damage to cells caused by viral infections. It is another process that is believed to contribute to pathogenesis of autoimmunity. These newly exposed autoantigens or epitopes stimulate autoreactive The autoimmune pathological process may be initiated and maintained by (a) autoantibodies, (b) immune complexes containing autoantigens, and (c) autoreactive T lymphocytes. Each of these immune processes plays a major role in certain diseases or may be synergistically associated, particularly in multiorgan, systemic autoimmune diseases. Chapter 20 ◗ Production of enormous amounts of IgG antibodies against M protein M protein expressed by group A Streptococcus infecting the throat Section II Certain tissues, such as sperm, central nervous system, and the lens and uveal system of the eye, are sequestrated or hidden. These sites are normally never exposed to the immune system for various reasons. These are called immunologically privileged sites. When these hidden or sequestrated antigens are released, exposed to as a result of injury, the host immune system—both cellular and humoral—does not consider them as self but as foreign, and hence attacks them. For example, lens protein is enclosed within its capsule and has no contact with circulation. Therefore, immunological tolerance against lens protein is not developed during fetal life. Following injury or cataract surgery, when this antigen is leaked into circulation, it elicits an immune response, which results in damage to the lens of other eye. Similarly, developing sperms are found within the lumen of the testicular tubules, which are sealed off early in embryonic development, prior to the development of the immune system. These developing sperms are enclosed within a sheath of tightly joined Sertoli cells, hence are never accessed to immune cells. If these are exposed by surgery or vasectomy and injury, an immune response occurs against the sperm, producing aspermatogenesis that may lead to male sterility. DNA, histones, and mitochondrial enzymes are the intracellular antigens that are normally sequestrated from the immune system. However, certain viral or bacterial infections and exposure to radiation and chemicals can damage these cells and release sequestrated intracellular antigens into circulation. These antigens then elicit a strong immune response. The autoantibodies are produced against these antigens, which combine with subsequently released sequestrated antigens. This results in the formation of immune complexes, which causes damage to tissues. For example, following an infection by mumps, the virus causes damage to the basement membrane of seminiferous tubules, thereby eliciting an immune response and resulting in orchitis. 157 Chapter 20 Section II 158 IMMUNOLOGY 1. Autoantibodies: Autoantibody associated diseases are characterized by the presence of autoantibodies in the individual’s serum and by the deposition of autoantibodies in tissues. Autoantibodies may be directly involved in the pathogenesis of certain diseases, while in others they may serve simply as disease markers without a known pathogenic role. They may also be instrumental in triggering various pathogenic mechanisms leading to tissue injury and cell death. Autoantibodies play a key role in the pathogenesis of (a) myasthenia gravis, (b) pemphigus vulgaris, and (c) various autoimmune cytopenias. 2. Immune complexes containing autoantigens: The formation of immune complexes between self-antigens and autoantibodies, leading to end organ damage, is another pathogenic mechanism seen in autoimmune disorders. Only those immune complexes that are of adequate size manage to activate the complement system and are involved in the pathogenesis of autoimmune diseases. Systemic lupus erythematosus and polyarteritis nodosa are two classic examples of autoimmune diseases in which immune complexes play a major pathogenic role. 3. Autoreactive T lymphocytes: Antigens that are sequestered from the circulation, and are therefore not seen by the developing T cells in the thymus, do not induce self-tolerance. Exposure of mature T cells to such normally sequestered antigens at a later time might result in their activation. Induction of autoantibodies to sperms after vasectomy, sympathetic ophthalmitis, and the presence of antibodies to myocardial cells after myocardial infarction are the examples. Inappropriate expression of class II MHC molecules can also sensitize self-reactive T cells in certain other situations. This is supported by the clinical observations showing increased frequency of autoimmune diseases in families and by increased rates of clinical concordance in monozygotic twins. Polyclonal B-cell activation may also lead to initiation of autoimmune disease process. Animal Models of Autoimmunity Better understanding of autoimmune disease has been facilitated by many experimental studies in animal models. Several animal models have been developed, each sharing some characteristics of a human disease of autoimmune etiology. These animal models often provide only the experimental approaches to the study of pathogenesis of autoimmune diseases. In some animal models, autoimmune diseases are induced by injecting normal animals with antigens extracted from the human target tissues, resulting in an autoimmune disease with a rapid onset and an acute course. The usual models used are mice and rats. Experimentally induced autoimmune diseases in these animals include (a) myasthenia gravis, (b) multiple sclerosis, (c) rheumatoid arthritis, and (d) Hashimoto’s thyroiditis. Autoimmune Diseases Different molecules, cells, and tissues are affected in autoimmune diseases. Table 20-1 summarizes affected tissue, target antigens, and resultant autoimmune diseases. The autoimmune diseases can be broadly classified as (a) organ-specific autoimmune disease and (b) systemic autoimmune diseases Organ-Specific Autoimmune Diseases These are diseases in which autoantibodies are produced targeting only the tissue of a single organ, thus affecting it solely. A few examples of such disorders are Addison’s disease, autoimmune hemolytic anemia, Goodpasture’s syndrome, Graves’ disease, Hashimoto’s thyroiditis, idiopathic thrombocytopenic purpura, insulin-dependent diabetes mellitus, myasthenia gravis, pernicious anemia, poststreptococcal glomerulonephritis, etc. These diseases can be further subgrouped on the basis of tissue damage as: (a) diseases mediated by the action of cellmediated immunity and (b) diseases mediated by the action of autoantibodies. ◗ Diseases mediated by the action of cell-mediated immunity Some of the diseases where the main mechanism of cell damage is directly mediated by lymphocytes are listed below: Hashimoto’s thyroiditis: Hashimoto’s thyroiditis primarily is a subclinical disease in which no thyroid dysfunction is evident and no therapy is needed until the later stages of disease. A cell-mediated autoimmune reaction triggered by unknown factors is believed to be responsible for the development of this disease. The disease occurs most often in middle-aged women producing both autoantibodies and TH1 cells specific for thyroid antigens. It is the most common form of thyroiditis, and it usually has a chronic evolution. It occurs most commonly during the third to fifth decades, with a female to male ratio of 10:1. The disease is functionally characterized by a slow progression to hypothyroidism with an insidious onset of symptoms. Most patients become hypothyroid with symptoms of malaise, fatigue, cold intolerance, and constipation. The diagnosis is usually confirmed by the detection of antithyroglobulin antibodies. Addison’s disease (chronic primary hypoadrenalism): This disease can either be caused by exogenous agents (e.g., infection of the adrenals by Mycobacterium tuberculosis) or may be idiopathic. The idiopathic form is believed to have an immune basis, since 50% of patients have been found to have autoantibodies to the microsomes of adrenal cells (as compared to 5% in the general population). The autoantibodies directed against the adrenals are believed to play the main role in pathogenesis of the disease. Symptoms of Addison’s disease include weakness, fatigability, anorexia, nausea, vomiting, weight loss, and diarrhea. Signs include increased skin pigmentation, vascular collapse, and hypotension. The disease finally ends in atrophy and loss of function of the adrenal cortex. The diagnosis is confirmed by demonstration of antiadrenal antibodies by indirect immunofluorescence test. Addison’s disease is found frequently in association with other autoimmune diseases, such as thyroiditis, pernicious anemia, and diabetes mellitus. AUTOIMMUNITY TABLE 20-1 159 Autoimmune disorders Affected tissue Disease Self-antigen Immune response Organ-specific autoimmune diseases Adrenal cells Autoantibodies Erythrocytes Autoimmune hemolytic anemia RBC membrane protein Autoantibodies Kidneys and lungs Goodpasture’s syndrome Renal and lung basement membrane Autoantibodies Thyroid gland Graves’ disease Thyroid stimulation hormone receptor Autoantibodies Thyroid gland Hashimoto’s thyroiditis Thyroid stimulation hormone receptor Antibodies to thyroglobulin. “Long acting thyroid stimulator”-IgG antibody to thyroid membrane. Stimulating T cells and autoantibodies Platelets Idiopathic thrombocytopenic purpura Platelet membrane protein Autoantibodies Pancreas Insulin-dependent diabetes mellitus Pancreatic beta cells TH cells and autoantibodies Skeleton muscle Myasthenia gravis Acetylcholine receptors Autoantibodies Stomach Pernicious anemia Gastric parietal cells, intrinsic factor Autoantibodies Kidney Poststreptococcal glomerulonephritis Basement membrane Antigen–antibody complex Sperm Male sterility Spermatogonia Autoantibodies Lower spine Ankylosing spondylitis Vertebrae Immune complex Myelin of CNS Multiple sclerosis Brain or white matter TH, TC cells, and autoantibodies Synovial membranes and joints Rheumatoid arthritis Connective tissue, IgG Autoantibodies, and immune complex Connective tissue Scleroderma Nuclei, heart, lungs, GI tract and kidney Autoantibodies Tear ducts Sjögren’s syndrome Salivary gland, liver, kidney, thyroid Autoantibodies Skin, joints, vasculature, muscle, and kidney Systemic lupus erythematosus DNA, nuclear protein, RBC, and platelets Autoantibodies, and immune complex Systemic autoimmune diseases ◗ Diseases mediated by the action of autoantibodies In some autoimmune diseases, antibodies act as agonists, binding to hormone receptors in lieu of the normal ligand and stimulating inappropriate activity. This usually leads to an overproduction of mediators or an increase in cell growth. Conversely, autoantibodies may act as antagonists, binding hormone receptors but blocking receptor function. This generally causes impaired secretion of mediators and gradual atrophy of the affected organ. Some important representative disorders in this group are mentioned below: Myasthenia gravis: Myasthenia gravis is the prototype autoimmune disease mediated by blocking antibodies. It is a disorder of neuromuscular transmission. A patient with this disease produces autoantibodies that bind the acetylcholine receptors on the motor end-plates of muscles. These antibodies block the normal binding of acetylcholine and also induce complement-mediated lysis of the cells. Increased muscular fatigue and weakness, especially becoming more pronounced following exercise, are the usual symptoms of myasthenia gravis. Weakness is usually first detected in extraocular muscles resulting in diplopia or ptosis. The face, tongue, and upper extremities are also frequently involved. Skeletal muscle involvement is usually proximal. The disease is usually marked by spontaneous remission periods. Thymic abnormalities are frequent in myasthenia gravis. About 10% of the patients develop malignant tumors of the thymus (thymomas). The diagnosis is confirmed by the finding of antiacetylcholine receptor antibodies. Graves’ disease: Graves’ disease, also known as thyrotoxicosis, diffuse toxic goiter, and exophthalmic goiter, occurs as a result of the production of autoantibodies against the thyrotrophic hormone (thyroid-stimulating hormone [TSH]) receptor (TSH receptor antibodies). The TSH receptor antibodies (also known as long-acting thyroid stimulator, thyroid-stimulating immunoglobulin, and thyroid-stimulating antibodies) stimulate the activity of thyroid gland in Graves’ disease. These antibodies have the Chapter 20 Addison’s disease Section II Adrenal cortex Chapter 20 Section II 160 IMMUNOLOGY capacity to stimulate the production of thyroid hormones by activating the adenylate cyclase system after binding to the TSH receptor. These antibodies are detected in 80–90% of patients with Graves’ disease and are usually of the IgG isotype. Protruding eyeballs, also known as exophthalmos, is the classical presentation of the condition. Other symptoms of hyperthyroidism include increased metabolic rate with weight loss, nervousness, weakness, sweating, heat intolerance, and loose stools. This disease is more prevalent in women in their 30s. Biopsy of the thyroid gland shows diffuse lymphoplasmacytic interstitial infiltration. Laboratory tests show increased levels of thyroid hormones (triiodothyronine, or T3, and thyroxine, or T4), increased uptake of T3, and antithyroid receptor antibodies. Systemic Autoimmune Diseases The autoantibodies are produced against a wide range of host tissues in systemic autoimmune diseases. These diseases reflect a general defect in immune regulation that results in hyperactive T cells and B cells. Tissue damage is widespread, much more generalized and usually leads to multisystem disorders. Tissue damage is caused by cell-mediated immune responses, by autoantibodies, or by accumulation of immune complexes. A few of the representative systemic diseases are discussed below: ◗ Systemic lupus erythematosus Systemic lupus erythematosus (SLE) is a generalized autoimmune disorder associated with multiple cellular and humoral immune abnormalities and protean clinical manifestations. It is most common in females of childbearing age. It is 10 times more common in females than males. The clinical manifestations of SLE vary among different patients. The kind of organ (vital versus nonvital) that becomes involved determines the seriousness and the overall prognosis of the disease. A wide range of autoantibodies to a vast range of tissue antigens, such as DNA, histones, RBCs, platelets, leukocytes, and clotting factors are produced in patients with SLE. Combination of these autoantibodies with their specific antigens produces a variety of symptoms. Demonstration of lupus erythematosus (LE) cell on incubation of normal neutrophils with damaged leukocytes preincubated with sera obtained from SLE patients is typical of SLE. The cell is a peculiar-looking polymorphonuclear leukocyte that has ingested nuclear material. Laboratory diagnosis of SLE is made by demonstration of antinuclear antibodies (ANAs) in a variety of tissues and cell lines as substrates by an indirect immunofluorescence test. Demonstration of nuclear fluorescence after incubating the cells with the patient’s serum indicates a positive test. Four patterns of fluorescence can be seen, indicating different types of ANAs. The test for ANAs is not very specific but is very sensitive. A negative result virtually excludes the diagnosis of SLE (95% of patients with SLE are ANA positive), while high titers are strongly suggestive of SLE but not confirmatory. False positives can occur in other systemic autoimmune/collagen diseases and chronic infections. Anti-dsDNA antibodies are found almost exclusively in SLE (60–70% of the patients). Levels of serum anti-DNA antibodies may vary with disease activity, but they are poor predictors of disease activity. Marked elevations in the levels of circulating immune complexes can be detected in patients with SLE sera during acute episodes of the disease by a variety of techniques. Sunlight exposure is the first environmental factor to be identified that influences the clinical evolution of SLE in a patient. Exposure to sunlight may precede the clinical expression of the disease or disease relapse. This is due to the Langerhans cells of the skin and keratinocytes that release significant amounts of interleukin-1 upon exposure to UV light. Infections and also drugs are responsible for setting off the disease process in certain susceptible individuals. Treatment of the condition is carried out by administration of corticosteroids and other anti-inflammatory drugs. Other examples of systemic autoimmune diseases include multiple sclerosis and rheumatoid arthritis. HLA Association with Autoimmune Diseases The risk for many autoimmune diseases appears to be associated with the presence of particular human leukocyte antigen (HLA) genes. Some autoimmune diseases have increased frequencies in persons carrying certain HLA genes. Table 20-2 summarizes MHC association with some important autoimmune diseases. TABLE 20-2 MHC associations with autoimmune diseases HLA gene Disease B8 Myasthenia gravis B27 Acute uveitis Ankylosing spondylitis Reiter’s disease Cw6 Psoriasis vulgaris DR2 Goodpasture’s syndrome Multiple sclerosis DR3 Graves’ disease Multiple sclerosis Myasthenia gravis Systemic lupus erythematosus DR4 Pemphigus vulgaris Rheumatoid arthritis DR3/DR4 heterozygote Type I insulin-dependent diabetes mellitus DR5 Hashimoto’s thyroiditis Immunology of Transplantation and Malignancy Introduction Modern medicine continues to offer many miracles that lengthen the lifespan of humans, as well as greatly increase the quality of life they enjoy. The replacement of defective organs by transplantation was one of the impossible dreams of medicine for many centuries. The dream of health professionals have been to replace or restore damaged tissues or organs that are irreparably damaged. The successful transplantation requires a multitude of important steps: surgical asepsis; development of surgical techniques of vascular anastomosis; genetic matching of donors with hosts; use of agents that could suppress the immune system; and prevention of infection in both recipient and donor. The development of strict antiseptic techniques contributes immensely to control the infection, while the proper use of immunosuppressive drugs and tissue typing increases the rate of success of transplantation. Transplant Immunology Transplantation can be defined as the transfer of cells, tissues, or organs from one site in an individual to another, or between two individuals. In the latter case, the individual who provides the transplant organ is termed a donor and the individual receiving the transplant is known as the recipient. 21 3. Allograft: An allograft is the transfer of tissue or an organ between genetically different members of the same species, i.e., from one human to another. This is the predominant form of transplantation today, and allografts have dominated transplant research for many years. 4. Xenograft: A xenograft is the transfer of tissues or organs between members of different species. It represents the most disparate of genetic relationships and is always rejected by an immunocompetent recipient. A major limitation in the success of transplantation is the immune response of the recipient to the donor tissue. Problem of rejection with autografts is usually minimal or absent. It is only when tissues from ”others” are used, as in allografts and xenografts, the problem of rejection arises. Transplantation immunology is the study of the events that occur after an allograft or a xenograft is removed from a donor and then transplanted into a recipient. Allograft Rejection Allografts are rejected by a process called allograft reaction. Graft rejection is the consequence of an immune response mounted by the recipient against the graft as a consequence of Autograft Same individual Types of Transplants Syngeneic graft There are four different basic types of transplants. These reflect the genetic relationship of the donor to the recipient. The degree of immune response to a graft varies with the type of graft (Fig. 21-1). 1. Autograft: An autograft is the transfer of individual’s own tissue or organ from one site to another site in the body. In other words, the recipient is also the donor. Common examples of autografts include skin transplants in burn patients and bypass surgery in patients suffering from coronary heart disease. 2. Syngraft: A syngraft is a transfer of tissue between two genetically identical individuals, i.e., identical twins. The first successful human kidney transplant was a syngraft, carried out in 1954 between identical twins. Identical twins Allograft Nonidentical individuals Xenograft Different species FIG. 21-1. Grafts in transplantation. Chapter 21 Section II 162 IMMUNOLOGY the incompatibility between tissue antigens of the donor and recipient. The problem of rejection was first recognized when attempts to replace damaged skin on burn patients with skin from unrelated donors were found to be relatively unsuccessful. During a period of 1–2 weeks, the skin would undergo necrosis and peel off. The failure of such grafts led scientists like Peter Medawar and many others to study skin transplantation in animal models. These experiments established that the failure of skin grafting was caused by an inflammatory reaction, now called as rejection. Results of several experimental studies imply that adaptive immune response is responsible for rejection. Histocompatibility antigens: Cells expressing class II MHC (major histocompatibility complex) antigens play a major role in sensitizing the immune system of the recipient. The sensitization of alloreactive helper T lymphocytes from the recipient is followed by their clonal expansion. This in turn causes multiple immunological and inflammatory phenomena. Some of these phenomena are mediated by activated T lymphocytes and also by antibodies, which eventually result in graft rejection. Recognition of transplanted cells as self or foreign is determined by polymorphic genes that are inherited from both parents and are expressed codominantly. MHC molecules are responsible for almost all strong rejection reactions. The rejection reactions are mediated by T cells. Both CD4 and CD8 cells coordinate to bring about an effective and pronounced rejection reaction. Nude mice, which lack a thymus, are incapable of launching an allogeneic immune response. Histocompatibility is tissue compatibility as demonstrated in the transplantation of tissues or organs from one member to another of the same species (an allograft), or from one species to another (a xenograft). Key Points ■ ■ The major histocompatibility genes include class I and class II MHC genes that are important in tissue transplantation. These are the genes that encode antigens that should match if a tissue or organ graft is to survive in the recipient and are located in the MHC region. These are located on the short arm of chromosome 6 in humans and of chromosome 17 in the mouse. Minor histocompatibility antigens are the molecules expressed on cell surfaces that are encoded by the minor histocompatibility loci, not the major histocompatibility locus. They represent weak transplantation antigens than the major histocompatibility antigens. However, they are multiple and their cumulative effect may contribute considerably to organ or tissue graft rejection. The greater the match between donor and recipient, the more likely the transplant is to survive. For example, a six-antigen match implies sharing of two HLA-A antigens, two HLA-B antigens, and two HLA-DR antigens between donor and recipient. Even though antigenically dissimilar grafts may survive when a strong immunosuppressive drug, such as cyclosporine, is used; the longevity of the graft is still improved by having as many antigenic match as possible. ◗ Mechanisms of graft rejection Allogeneic MHC molecules are presented for recognition by the T cells of a graft recipient in two distinctly different ways: (a) direct presentation and (b) indirect presentation. Direct presentation: Direct presentation involves recognition of an intact MHC molecule displayed by donor antigen-presenting cells (APCs) in the graft. It depends on the similarity in the structure of an intact foreign (allogeneic) molecule and self-MHC molecules. Direct recognition of foreign MHC molecules is a crossreaction of a normal T-cell receptor, which is selected to recognize a self-MHC molecule and foreign peptide, with an allogeneic MHC molecule and peptide. This is because an allogeneic MHC molecule with a bound peptide can mimic the determinant formed by a self-MHC molecule and a particular foreign peptide. As many as 2% of an individual’s T cells are capable of recognizing and responding to a single foreign MHC molecule, and this high frequency of T cells reactive with allogeneic MHC molecules is one reason that allografts elicit strong immune responses in vivo. Indirect presentation: The “indirect presentation” involves the recognition of processed allogeneic MHC molecules but not an intact MHC molecule. It involves processing of donor MHC molecules by recipient APCs and presentation of derived peptides from the allogeneic MHC molecules in association with self-MHC molecules. Here the processed MHC molecules are recognized by T cells like conventional foreign protein antigens. Indirect presentation may result in allorecognition by CD4 T cells. This is because alloantigen is acquired primarily through the endosomal vesicular pathway and is therefore presented by class II MHC molecules. Some antigens of phagocytosed graft cells appear to enter the class I MHC pathway of antigen presentation and are indirectly recognized by CD8 T cells. ◗ Stages of cell-mediated graft rejection Cell-mediated graft rejection could occur in two stages: (a) A sensitization phase, in which antigen-reactive lymphocytes of the recipient proliferate in response to alloantigens on the graft and (b) An effector stage, in which immune destruction of the graft takes place. Sensitization phase: During the sensitization phase, CD4 and CD8 T cells recognize alloantigens expressed on the cells of foreign graft and proliferate in response. The response to major histocompatibility antigens involves recognition of both the donor MHC molecule and an associated peptide ligand in the cleft of the MHC molecule. The peptides present in the groove of allogeneic class I MHC molecules are derived from proteins synthesized within the allogeneic cell. The peptides present in the groove of allogeneic class II MHC molecules are generally proteins that are taken up and processed through the endocytic pathway of the allogeneic APC. Recognition of the alloantigens expressed on the cells of a graft induces vigorous T-cell proliferation in the host. This proliferation can be demonstrated in vitro in a mixed lymphocyte reaction. Both dendritic cells and vascular endothelial IMMUNOLOGY OF TRANSPLANTATION AND MALIGNANCY cells from an allogeneic graft induce host T-cell proliferation. The CD4 T cell is the major proliferating cell that recognizes class II alloantigens directly or alloantigen peptides presented by host APCs. This amplified population of activated TH cells is believed to play a key role in inducing the various effector mechanisms of allograft rejection. Effector mechanisms in allograft rejection: A variety of effector mechanisms participate in allograft rejection: ■ ◗ Clinical features of graft rejection Rejection episodes, based primarily on the time elapsed between transplantation and the rejection episode, are traditionally classified as (a) hyperacute, (b) acute, and (c) chronic rejections. Hyperacute rejection: Hyperacute rejection occurs usually within the first few hours post-transplantation and is mediated by preformed antibodies against ABO or MHC antigens of the graft. Possibly, antibodies directed against other alloantigens, such as vascular endothelial antigens, also play a role in this type of rejection. Once the antibodies bind to the transplanted tissues, rejection can be caused either (a) by activation of the complement system, which results in the chemotactic attraction of granulocytes and the triggering of inflammatory circuits, or (b) by ADCC. Pathological features of hyperacute rejection are following: ■ ■ ■ This is associated with the formation of massive intravascular platelet aggregates leading to thrombosis, ischemia, and necrosis. The hyperacute rejection episodes are irreversible and invariably results in graft loss. With proper cross-matching techniques, this type of rejection is almost 100% avoidable. The hyperacute rejection by antibodies to all human cellular antigens is the major limitation of xenogeneic transplantation (e.g., pig to human). Acute rejection: Acute rejection occurs mostly in the first few days or weeks after transplantation: ■ When acute rejection takes place in the first few days after grafting, it may correspond to a secondary (second set) immune response. This indicates that the patient had been Acute rejection is predominantly mediated by T lymphocytes. CD4 helper T lymphocytes are believed to play the key role in acute rejection of the graft. This is because they release growth factors like IL-2 and IL-4 for the promotion of clonal expansion of CD8 lymphocytes and B cells. In rejected organs, the cellular infiltrates contain mostly monocytes and T lymphocytes of both helper and cytotoxic phenotypes, and lesser numbers of B lymphocytes, NK (natural killer) cells, neutrophils, and eosinophils. All these cells have the potential to play significant roles in the rejection process. The initial diagnosis of acute rejection is usually based on clinical suspicion: ■ ■ ■ ■ Functional deterioration of the grafted organ is the main basis for considering the diagnosis of acute rejection. Confirmation usually requires a biopsy of the grafted organ. Mononuclear cell infiltration in tissues of rejected graft tissue is characteristic finding. The measurement of cytokines (such as IL-2) in serum and in urine (in the case of renal transplants) is another diagnostic approach. In most cases, acute rejection, if detected early, can be reversed by increasing the dose of immunosuppressive agents or by briefly administering additional immunosuppressants. Delayed or chronic rejection: This is characterized by an insidiously progressive loss of function of the grafted organ. The functional deterioration associated with chronic rejection appears to be due to both immune and nonimmune processes. Vascular endothelial injury is the most common feature. Granulocytes, monocytes, and platelets are found to increasingly adhere to injured vascular endothelium. The damaged endothelium is covered by a layer of platelets and fibrin, and eventually by proliferating fibroblasts and smooth muscle cells. The end result is a proliferative lesion in the vessels, which progresses toward fibrosis and occlusion. ◗ Prevention of graft rejection Immunosuppression of the host prevents graft rejection (Table 21-1). It is achieved by treatment with radiation, corticosteroids, and antilymphocyte serum. Cyclosporin A and rapamycin are also used, which cause immunosuppression by specific inhibition of T cells. Graft-Versus-Host Reaction Whenever a patient with a profound immunodeficiency (primary, secondary, or iatrogenic) receives a graft of an organ rich in immunocompetent cells, there is a considerable risk Chapter 21 An influx of T cells and macrophages into the graft is the hallmark of graft rejection involving cell-mediated reactions. Histologically, the infiltration in many cases resembles that seen during a delayed-type hypersensitive response, in which cytokines produced by TD and TH cells promote macrophage infiltration. Recognition of foreign class I alloantigens on the graft by host CD8 cells results in CTL-mediated killing. In some cases, CD4 T cells that function as class II MHC-restrict cytotoxic cells mediate graft rejection. previously sensitized to the HLA antigens present in the organ donor (as a consequence of a previous transplant, pregnancy, or blood transfusions). When graft rejection occurs first week after grafting, it usually corresponds to a first-set (primary) response. Up to 70% of graft recipients experience one or more acute rejection episodes. Section II ■ The most common are cell-mediated reactions involving delayed-type hypersensitivity and cytotoxic T lymphocyte (CTL)-mediated cytotoxicity. Less common mechanisms are antibody plus complement lysis and destruction by antibody-dependent cell-mediated cytotoxicity (ADCC). ■ 163 164 IMMUNOLOGY Chapter 21 Section II TABLE 21-1 Immunosuppressive Agents used in Transplantation Agent Mode of action Azathioprine Inhibition of nucleotide synthesis of multiple cells Cyclophosphamide Inhibition of nucleotide synthesis of multiple cells Cyclosporine Inhibition of transcription of cytokines in lymphocytes Corticosteroids Inhibition of transcription for cytokines and products involved in inflammation in multiple cells Sirolimus Inhibition of transduction induced by cytokines in T cells Irradiation DNA damage in all rapidly proliferating cells Anti-CD4 and CD8 antibodies Interference with T-cell receptor binding of CD4 and CD8 T cells that a graft-versus-host (GVH) reaction may develop. The probability of developing a GVH reaction is greatest in the 2-month period immediately following transplantation. GVH reactions require three important components, which are: ■ ■ ■ the donor graft must contain immunocompetent T cells, the host must be immunocompromised, and the recipient should express antigens, such as MHC proteins, which will be identified as foreign to the donor. For example, donor T cells recognize the recipient cells as foreign. Key Points GVH reactions are a significant problem in the following conditions: ■ ■ ■ Transplantation of bone marrow or thymus in infants and children with primary immunodeficiencies. Transplantation of bone marrow in adults. Transplantations of small bowel, lung, and even liver—the organs that have substantial amount of lymphoid tissue. successfully for the control of chronic GVH unresponsive to traditional immunosuppressants. Tumor Immunology Tumor immunology has been defined as part of immunology that deals with the antigens on tumor cells and the immune response to them. As a consequence of their loss of differentiation, tumor cells may express developmental antigens that are usually seen only in the prenatal period. Examples of these antigens are alpha-fetoprotein and carcinoembryonic antigen. Tumors or neoplasia is said to develop when the balance between cell death and renewal is disturbed in a way that numerous clones of a single cell group are produced in an uncontrolled fashion. Key Points ■ Transplantation of organs, such as the heart and kidneys—poor in endogenous lymphoid tissue—very rarely results in a GVH reaction. GVH reactions occur because the donor T lymphocytes become activated, proliferate, and differentiate into helper and effector cells in the irradiated, immunocompromised host. These activated T cells attack the host cells and tissues, producing the signs and symptoms of GVH disease. The donor’s cytotoxic T cells play a key role in destroying recipient’s cells. The crucial role played by the donor T cells is demonstrated by the fact that removal of these T cells from a bone marrow graft prevents GVH reactions. The initial proliferation of donor T cells takes place in lymphoid tissues, particularly in the liver and spleen leading to hepatomegaly and splenomegaly. Later, at the peak of the proliferative reaction, the skin and intestinal walls are heavily infiltrated leading to severe skin rashes or exfoliative dermatitis and severe diarrhea. Finally, many GVH reactions end in overwhelming infections and death. All immunosuppressive drugs used in the prevention and treatment of rejection have been used for treatment of the GVH reaction. Thalidomide, the tranquilizer drug that achieved notoriety due to its teratogenic effects, has been used ■ ■ A tumor that is not capable of indefinite growth and does not invade the healthy surrounding tissue extensively is benign. A tumor that continues to grow and becomes progressively invasive is malignant; the term cancer refers specifically to a malignant tumor. In addition to uncontrolled growth, malignant tumors exhibit metastasis; in this process, small clusters of cancerous cells dislodge from a tumor, invade the blood or lymphatic vessels, and are carried to other tissues, where they continue to proliferate. In this way a primary tumor at one site can give rise to a secondary tumor at another site. Features of Malignant Cells Malignant cells show the following features: 1. Once cells become malignant, they stop functioning normally and add to the burden on the body by competing with the normal cells for space and nutrition. 2. The more “undifferentiated” a cell is, the lesser its functionality and more its malignant nature. 3. They undergo rapid and uncontrolled division. 4. They lose their homing instinct and start invading the basement membrane and enter the vasculature to spread to dissimilar tissues, leading to metastasis and spread of cancer. IMMUNOLOGY OF TRANSPLANTATION AND MALIGNANCY It has been postulated that the immune system is responsible in part for the protection of the body against the development of malignancies. At the same time, the prevalence of numerous cancers in immunocompetent individuals indicates that immune system has only a partial role in protecting against malignancies and also that it is not very efficient at it. Tumor Antigens 1. Tumor-specific antigens 2. Tumor-associated transplantation antigens ◗ Tumor-specific antigens ◗ Tumor-associated transplantation antigens Tumor-associated transplantation antigens (TATAs) are the other class of tumor antigens. These antigens are expressed by (a) tumor cells and also by (b) normal cells at low levels or only during the process of differentiation. The expression of these antigens is considerably derepressed or enhanced after the process of malignant transformation. TATAs can be of the following types: 1. Tumor-associated carbohydrate antigens: They represent abnormal form of mucin-associated antigen detected in breast and pancreatic cancers. 2. Differential antigens: These include CD10 and prostatespecific antigens (PSA). The latter is used as a diagnostic indicator in prostatic cancer. ■ ■ ■ First, these antigens are uniquely expressed by tumor cells alone. Also, there are the products of genes that have been mutated during the process of transformation, leading to the expression of abnormal products. Second, certain antigens expressed by tumors are present only when normal cells are undergoing the process of differentiation and these are also readily recognized by the immune system. Finally, the antigens that are overexpressed by the tumor cells elicit a good immune response. Immune Reactions against Tumors Tumor antigens are capable of eliciting a comprehensive immune response involving both the cellular and humoral immune responses. ◗ Cellular immune responses T lymphocytes play an important role in tumor immunity. They act both as cytotoxic effector cells and as central modulating cells. Through these effector cells, they control the specific cellmediated antitumor immune responses and upregulate nonspecific killing mechanisms. The activation of T lymphocytes by tumor cell products as a consequence of antigen recognition may result in the secretion of nonspecific immunoregulatory factors. ■ ■ ■ These factors are capable of “upregulating” the tumorkilling function of mononuclear phagocytes, NK cells, and granulocytes. These factors also enhance the ability of NK cells and monocytes to participate in ADCC against tumor cells. Macrophages also play an important role in tumor response. Clustering of macrophages around tumor cells is associated with tumor regression and seen in the case of numerous cancers. Chapter 21 The tumor-specific antigens (TSAs), also called tumor-specific transplantation antigens, are unique to tumors. They are not found on other cells of the body. They are usually the products of mutated genes seen in the cancer cells. Cytosolic processing of the abnormal proteins yields peptides that are unique and when presented by the appropriate MHC class I molecules elicit a cell-mediated immune response. Various physical and chemical carcinogens cause malignancies by inducing mutation in key genes involved in modulating cell growth. Ras proto-oncogene products including the p21 Ras proteins and other related gene products are an example of TSAs. Ras proteins bind guanine nucleotides (GTP and GDP) and possess intrinsic GTPase activity. The mutations associated with Ras genes in malignant cells appear to cause a single amino acid substitutions at specific positions (12, 13, or 61), which results in increased enzymatic activity of the gene product. As a consequence, the cells acquire transforming capacity. Moreover, these products are also recognized as foreign antigens by the cellular immune response. Another mode through which the tumor cells may express unique and novel antigens “is by” integration with proviral genomes. These virus-induced tumors usually have their genome integrated with proviral genome, hence the proteins encoded and expressed are sometimes novel and recognized by the cellular immune response. Viruses that have been implicated in tumorigenesis include Epstein–Barr virus (EBV), hepatitis B virus (HBV), hepatitis C virus (HCV), etc. 3. Oncofetal antigens: These antigens are found in embryonic and malignant cells but are absent in normal adult cells. Alphafetoprotein and carcinoembryonic antigens are examples of this antigen, which are found in hepatomas and colonic cancers, respectively. Silent tumor-associated genes are not expressed in normal cells but are actively transcribed in tumor cells. Tissuespecific genes or differentiation genes are present in the surface of normal cells or may be shed to the circulation, but the levels of expression are usually very low in normal cells. This finds practical application in the diagnosis of malignancies as illustrated by the assay of PSA for the diagnosis of carcinoma of prostate. PSA: It is a kallikrein-like serine protease produced exclusively by the epithelial cells in the prostate gland. The antigen is detectable at relatively high levels in seminal plasma and at very low levels in the serum of healthy men. The assay of serum PSA levels is a very useful marker of prostate carcinoma, perhaps the most meaningful serum marker for neoplasia. In healthy men, the levels of PSA vary between 0.65  0.66 ng/mL at ages 21–30 and 1.15  0.68 ng/mL at ages 61–70. Significantly elevated levels are demonstrated in 63–86% of patients with prostatic carcinoma, depending on the stage. In essence, antigens of tumors that are capable of eliciting an immune response may be one of the following nature: Section II Tumor cells also express unique molecules that can be classified into two groups: 165 166 ◗ IMMUNOLOGY Humoral immune responses Chapter 21 Section II B lymphocytes produce tumor-specific antibodies, which may induce complement-dependent cytotoxicity of tumor cells or may mediate ADCC. ADCC can be mediated by a variety of cells expressing Fc receptors (NK cells, monocytes or macrophages, and granulocytes) by recognizing and destroying IgG-coated tumor cells. ◗ This includes treatment with various nonspecific immune modulators. ■ Immunosurveillance The emergence of cancer cells within the body may not be a rare or unusual event at all. Of the trillions of normal cells found in the body, several hundred per day may be undergoing malignant degeneration in response to the cancer-promoting stimuli. The immune system may possibly play a significant role in halting the growth of these cells and preventing the development of overt malignancy. The concept of immune surveillance was initially put forward by Ehrlich, and later on modified by Thomas and Burnet. Ehrlich first suggested that though cancer cells frequently arise in the body, they are recognized as foreign and eliminated. Later, Burnet postulated the immunosurveillance theory. He suggested that the immune system routinely patrols the cells of the body and upon recognition of a cell or a group of cells that has become cancerous attempts to destroy them, thus preventing the growth of some tumors. ■ ■ ◗ Immunotherapy of Cancer Immunotherapy of cancer can be considered as the following two broad groups: 1. Antigen-nonspecific treatment 2. Antigen-specific treatment Antigen-nonspecific treatment Bacillus Calmette–Guérin (BCG) vaccine has been shown to possess antitumor activity. The vaccine when injected directly into certain solid tumors may cause regression of tumor. Antitumor effect of tumor is believed to be due to activation of macrophages and NK cells. The BCG therapy has been reported to be beneficial in treatment of bladder cancer, malignant melanomas, stage I lung cancer, and certain leukemias. Corynebacterium parvum also possesses antitumor activities. Its antitumor effect is due to its ability to stimulate macrophages and B cells. It shows a synergistic effect when used in conjunction with cyclophosphamide. It is found to be useful in treatment of metastatic breast cancer and various types of lung cancer. Other nonspecific immune modulators include (i) dinitrochlorobenzene (DNCB), evaluated in squamous and basal carcinoma, (ii) levamisole for stimulating cell-mediated immunity and macrophage function, (iii) interferon to stimulate NK cell function, (iv) cytokine IL-2 to stimulate killing of cancer cells by cytotoxic T cells, (v) NK cells, and macrophages, thymic hormones to restore T cell function, and (vi) tuftsin to stimulate phagocytic cells. Antigen-specific treatment Antigen-specific treatment includes (a) vaccination with tumor antigen, (b) treatment with transfer factor, (c) treatment with immune RNA, (d) treatment with monoclonal antibodies raised against tumor-associated antigens (TAAs) given alone or in conjunction with cytotoxic drug, and (e) modification of tumor antigenicity by treatment with neuraminidase. 22 Immunohematology ABO Blood Group Antigens Introduction Immunohematology is the study of blood group antigens and antibodies, and their interactions in health and disease. Ehrlich and Morgenroth first described blood groups in goats based on antigens of their red cells in an article published in the Berliner klinische Wochenschrift in 1900. Subsequently, Karl Landsteiner, a Viennese pathologist, successfully identified the human ABO blood groups for which he was awarded the Nobel Prize 30 years later. After this initial discovery, blood grouping was developed as a science, and many different systems of grouping were designed on the basis of many isoantigens on the surface of erythrocytes. The ABO and Rh systems are among the wellknown human blood groups described in the literature. ABO Blood Group System ABO blood group system was the first human red-cell antigen system to be characterized. The ABO blood group substances are glycopeptides with oligosaccharide side chains (Fig. 22-1). The ABO blood group specificity is determined by the presence of terminal sugar in an oligosaccharide structure. The terminal sugars of the oligosaccharides are specific for blood groups A and B. They are also immunogenic. The red cells express either A, B, both A and B, or neither, and antibodies are found in serum to antigens not expressed by the red cells. The blood group of an individual is determined by presence or absence of two antigens, A and B, on the surface of the red cell membrane. Red cells of blood group A carry antigen A, cells of blood group B carry antigen B, and cells of blood group AB have both A and B antigens. On the other hand, blood group O cells have neither A nor B antigens. The blood groups are also differentiated by the presence or absence of two distinct isoantibodies in the serum. Serum of blood group A individuals have anti-B antibodies, blood group B have anti-A antibodies, and blood group O have both anti-A and anti-B antibodies. The blood group AB does not contain any anti-A and anti-B antibodies in the serum. Soluble ABO blood group substances may be found in mucous secretions of humans, such as saliva, gastric juice, ovarian cyst fluid, etc. Such persons are termed secretors, while those without the blood group substances in their secretions are nonsecretors. The ABO group of a given individual is determined by testing both cells and serum. In this method, the subject’s red cells are mixed with serum containing known antibody and the subject’s serum is tested against cells possessing known antigen. For example, the cells of a group A individual are agglutinated by anti-A serum but not by anti-B serum, and his or her serum agglutinates type B cells but not type A cells. The typing of cells as group O is done by exclusion (a cell not reacting with anti-A or anti-B is considered to be of blood group O). It is A blood group R Erythrocyte A enzyme R H enzyme + B enzyme R R B blood group Fucose H substance Galactose N-acetylglucosamine N-acetylgalactosamine FIG. 22-1. ABO blood group system. O enzyme (inactive) R O blood group Chapter 22 Section II 168 IMMUNOLOGY noteworthy that these antibodies to the isoantigens are found in all individuals including those that have had no transfusions. It is believed that the anti-A and anti-B isoagglutinins are synthesized as a consequence of cross-immunization with bacteria of the family Enterobacteriaceae that colonize the infants’ gut. These bacteria have outer membrane oligosaccharides strikingly similar to those that define the A and B antigens in the human body. For example, a newborn with group A will not have anti-B in his or her serum, since there has been no opportunity to undergo cross-immunization. When the intestine is eventually colonized by the normal microbial flora, the infant will start to develop anti-B, but will not produce anti-A because of tolerance to his or her own blood group antigens. The inheritance of the ABO groups follows simple Mendelian rules, with three common allelic genes: A, B, and O (A can be subdivided into A1 and A2), and any individual will carry two alleles, one inherited from the mother and one from the father. ◗ ◗ Individuals either have or do not have the RhD antigen on the surface of their red blood cells. This is usually indicated by “RhD positive” (does have the RhD antigen) or “RhD negative” (does not have the antigen) suffix to the ABO blood type. This suffix is often shortened to “D pos”/“D neg,” “RhD pos”/“RhD neg,” or ⫹/⫺. The latter symbol is generally not preferred in research or medical situations, because it can be altered or obscured accidentally. There are several alloantigenic determinants within the Rh system. ■ ■ H antigen Red cells of all ABO groups possess a common antigen, the H antigen, or H substance. H antigen is a glycoprotein and structurally is an L-fucose. It is a precursor for the production of A and B antigens. A and B antigens are formed by addition of N-acetylgalactosamine and galactose, respectively, to L-fucose of H antigen. The H antigen, due to its universal distribution, is not that important in blood grouping or transfusion. However, in rare instances, such as in “Bombay,” or OH blood, both A and B antigens as well as H antigens are absent in the blood. Individuals with “Bombay” blood group have anti-A, anti-B, and anti-H antibodies, hence are not compatible with most of the red cells. Rh Blood Group System Philip Levine, in 1939, discovered that the sera of most women who gave birth to infants with hemolytic disease contained an antibody that reacted with the red cells of the infant and with the red cells of 85% of Caucasians. In 1940, Landsteiner and Wiener injected blood from the monkey Macacus rhesus into rabbits and guinea pigs, and discovered the resulting antibody agglutinated rhesus (Rh) red cells, which appeared to have the same specificity as the neonatal antibody. The donors whose cells were agglutinated by the antibody to Rh red cells were termed Rh positive; those whose cells were not agglutinated were termed Rh negative. It is now known that the antibody obtained by Landsteiner and Wiener reacts with an antigen (LW) is different but is closely related to the one that is recognized in human hemolytic disease, but nevertheless the Rh nomenclature is still retained. Rh Blood Group Antigens The term Rh blood group system refers to the five main Rh antigens (C, c, D, E, and e) as well as many other less frequent Rh antigens. The terms Rh factor and Rh antigen are similar, and both refer to the RhD antigen only. Of all the Rh antigens, antigen D (RhD) is most important. D antigen Clinically, the D antigen has a lot of medical importance. This is because RhD negative individuals who receive RhD positive erythrocytes by transfusion can develop alloantibodies that may lead to severe reactions with further transfusions of RhD-positive blood. The D antigen also poses a problem in RhD-negative mothers who bear a child with RhD-positive red cells inherited from the father. The entry of fetal erythrocytes into the maternal circulation at parturition or trauma during the pregnancy (such as in amniocentesis) can lead to alloimmunization against the RhD antigen. This may cause hemolytic disease of the newborn in subsequent pregnancies. This can now be prevented by the administration of Rh (D) immunoglobulin to these women within 72 hours of parturition. Unlike ABO system, there are no natural antibodies against Rh antigens. Antibodies against Rh antigens develop only in certain situations, such as in Rh incompatible pregnancy or transfusion. Most of these antibodies are IgG antibodies, and few IgM antibodies. These are incomplete antibodies and can be detected in newborn blood by direct Coombs’ test and in mother blood by indirect Coombs’ test. Blood Transfusion Blood transfusion is the process of transferring blood or bloodbased products from one person into the circulatory system of another. Blood transfusions have many indications as mentioned below: ■ ■ ■ Blood transfusions can be life-saving in some situations, such as massive blood loss due to trauma, or can be used to replace blood lost during surgery. Blood transfusions may also be used to treat a severe anemia or thrombocytopenia caused by a blood disease. People suffering from hemophilia or sickle-cell disease may require frequent blood transfusions. Before a blood transfusion, a series of procedures need to be done to establish the proper selection of blood for the patient. Basically, those procedures try: ■ ■ to establish ABO and Rh compatibility between donor and recipient and to rule out the existence of antibodies in the recipient’s serum, which could react with transfused red cells. IMMUNOHEMATOLOGY Rh−/Rh− Rh−/Rh+ Rh−/Rh+ fetus Rh−/Rh− fetus Complications of Blood Transfusion Transfusion reaction is the major immunological complication following incompatible blood transfusion. Other transfusion reactions may be caused due to factors other than incompatibility, such as a person being hypersensitive to some allergens present in the blood. Transmission of infectious agents through blood is the most important complication. These include: ■ ■ ■ ■ Viruses, such as human immunodeficiency viruses I and II (HIV I and II), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and cytomegalovirus (CMV); Bacteria, such as Treponema pallidum and Leptospira interrogans; and Protozoa, such as Toxoplasma gondii, Leishmania donovani, and Plasmodium species. Transmission of HIV I and II, HBV, and HCV, which is a major concern. Hemolytic Disease of Newborn (Erythroblastosis Fetalis) Hemolytic disease of the newborn, also known as HDN, is an alloimmune condition. It develops in a fetus, which contains the IgG antibodies that have been produced by the mother and have passed through the placenta. These antibodies subsequently Mother sensitized by Rh−/Rh+ fetal RBCs Mother not sensitized by Rh−/Rh− fetal RBCs Production of IgG against Rh+ antigens on fetal RBCs Maternal anti-Rh IgG crosses placenta and attacks fetal RBCs FIG. 22-2. Hemolytic diseases of newborn. Chapter 22 attack and lyse the red blood cells in the fetal circulation, resulting in reticulocytosis and anemia. This condition in fetus varies from mild to very severe, and fetal death may occur due to heart failure (hydrops fetalis). When the disease is moderate or severe, many erythroblasts are present in the fetal blood, and this form of the disease is called erythroblastosis fetalis. Immunological destruction of fetal and/or newborn erythrocytes is likely to occur when IgG antibodies are present in the maternal circulation directed against the antigen(s) present on the fetal red blood cells. This is because only IgG antibodies can cross the placenta and reach the fetal circulation. Anti-D and anti-A or anti-B are the two types of antibodies most usually involved in hemolytic disease of the newborn. Anti-A or antiB antibodies are usually IgM, but, in some circumstances, IgG antibodies may develop (usually in group O mothers). This can be secondary to immune stimulation (some vaccines contain blood group substances or cross-reactive polysaccharides), or may occur without apparent cause for unknown reasons. Antibodies are produced when the body is exposed to an antigen foreign to the make-up of the body. If a mother is exposed to an alien antigen and produces IgG (as opposed to IgM which does not cross the placenta), the IgG will combine with the antigen, if present in the fetus, and may affect it in utero and persist after delivery (Fig. 22-2). Section II To establish the ABO and Rh compatibility between donor and recipient, both the recipient and the blood to be transfused are typed. The most direct way to detect antibodies in the recipient’s serum that could cause hemolysis of the transfused red cells is to test the patient’s serum with the donor’s cells (major crossmatch). The minor cross-match, which consists of testing a patient’s cells with donor serum is of little significance and rarely performed, since any donor antibodies would be greatly diluted in the recipient’s plasma, and rarely, it causes clinical problems. Universal recipient: It is an ABO blood group individual whose red blood cells express antigens A and B, but whose serum does not contain anti-A and anti-B antibodies. Thus, red blood cells containing any of the ABO antigens, i.e., from an individual with type A, B, AB, or O, may be transfused to the universal recipient without inducing a hemolytic transfusion reaction. It is best if the universal recipient is Rh positive, i.e., has the RhD antigen on the erythrocytes to avoid developing a hemolytic transfusion reaction. However, blood group systems other than ABO may induce hemolytic reactions in a universal recipient. Thus, it is best to use type-specific blood for transfusions. Universal donor: It is a blood group O RhD-negative individual whose erythrocytes express neither A nor B surface antigens. This type of red blood cell fails to elicit a hemolytic transfusion reaction in recipients with blood group A, B, AB, or O. However, group O individuals serving as universal donors may express other blood group antigens on their erythrocytes that may induce hemolysis. It is preferable to use type-specific blood for transfusions, except in cases of disaster or emergency. 169 170 IMMUNOLOGY Chapter 22 Section II The three most common ways in which a woman becomes sensitized (i.e., produces IgG antibodies against) toward particular blood types are as follows: 1. Fetal–maternal hemorrhage can occur due to trauma, abortion, childbirth, ruptures in the placenta during pregnancy, or medical procedures carried out during pregnancy that breach the uterine wall. In subsequent pregnancies, if there is a similar incompatibility in the fetus, these antibodies then cross the placenta into the fetal bloodstream, combine with the red blood cells, and finally cause hemolysis. In other words, if a mother has anti-RhD (D being the major Rh antigen) IgG antibodies as a result of previously carrying an RhD-positive fetus, these antibodies will only affect a fetus with RhD-positive blood. 2. The woman may receive a therapeutic blood transfusion with an incompatible blood type. ABO blood group system and Rh blood group system typing are routine prior to transfusion. Suggestions have been made that women of childbearing age or young girls should not be given a transfusion with Rhc-positive blood or Kell-positive blood to avoid possible sensitization. However, it is considered uneconomical to screen for these blood groups. 3. The third sensitization model can occur in women of blood type O. The immune response to A and B antigens, which are widespread in the environment, usually leads to the production of IgM anti-A and IgM anti-B antibodies early in life. On rare occasions, IgG antibodies are produced. In contrast, Rhesus antibodies are generally not produced from exposure to environmental antigens. A positive direct Coombs’ (antiglobulin) test with cord RBC is invariably positive in cases of Rh incompatibility. In ABO incompatibility, the direct antiglobulin test is usually weakly positive and may be confirmed by eluting antibodies from the infant’s red cells and testing the elute with A and B cells. Before birth, treatment of the condition include intrauterine transfusion or early induction of labor when (a) pulmonary maturity has been attained, (b) fetal distress is present, or (c) 35–37 weeks of gestation have passed. The mother is also administered with plasma to reduce the circulating levels of antibody by as much as 75%. After birth, treatment depends on the severity of the condition. These include temperature stabilization, phototherapy, transfusion with compatible packed red blood cells, administration of sodium bicarbonate for correction of acidosis, and/or assisted ventilation and exchange transfusion with a blood cells, type compatible with both the infant and the mother. Rh-negative mothers who have had a pregnancy with or are pregnant with an Rh-positive infant are given Rh immunoglobulin (RhIG) during pregnancy and after delivery to prevent sensitization to the D antigen. The RhIG acts by binding any fetal red cells with the D antigen before the mother is able to produce an immune response and form anti-D IgG. All the offsprings of Rh-incompatible marriages, however, do not suffer from hemolytic diseases of newborn. This may be due to either of the following causes: 1. Mother–fetus ABO incompatibility: When the mother and fetus possess the same ABO group, Rh immunization is more likely to occur. However, when Rh and ABO incompatibility coexist, Rh sensitization from the mother is very rare. In this condition, the fetal cells entering the maternal circulation are destroyed rapidly by the ABO antibodies before they can form the Rh antibodies. 2. Immune unresponsiveness to Rh antigen: Some Rh-negative individuals even after repeated injections of Rh-positive cells fail to form Rh antibodies. Such individuals are known as nonresponders. The exact reason for such immunological unresponsiveness, however, is not known. 3. Number of pregnancies: The risk of hemolytic disease of new born is more in second and successive child, but not in first child. This is because sensitization occurs only during the delivery; hence the first child escapes. ABO Hemolytic Diseases ABO hemolytic diseases occur in a very less number of cases, even though materno-fetal ABO incompatibility is very common. The condition is usually seen in O group mothers bearing blood group A or B fetus. It occurs largely in O group mothers because the isoantibodies are largely IgG in nature, which can cross the placenta. It does not occur in mothers with blood groups A or B because natural antibodies are mainly IgM in nature, which does not cross the placenta and sensitize the fetus. Unlike hemolytic disease of the newborn, the ABO hemolytic diseases can occur even in first child even without prior immunization. This is because the ABO disease is caused by naturally occurring maternal isoantibodies. ABO hemolytic disease is much milder condition than that of the Rh disease. The diagnosis of ABO incompatibility is made by a positive indirect Coombs’ test but a negative direct Coombs’ test. Peripheral blood smear characteristically shows spherocytosis. “This page intentionally left blank" “This page intentionally left blank" 23 43 Mycobacterium Leprae Staphylococcus ■ Introduction ■ Family Micrococcaceae consists of Gram-positive cocci, which are aerobic and anaerobic, and are arranged in tetrads or clusters. Micrococcaceae consists of four genera, Staphylococcus, Micrococcus, Planococcus, and Stomatococcus. Differences between these genera are summarized in Table 23-1. Human infections caused by these genera are summarized in Table 23-2. Among these, Staphylococcus is the only genus of medical importance. Staphylococcus aureus Staphylococcus The genus Staphylococcus consists of 32 species, most of which are animal pathogens or commensals. The bacteria belonging to this genus are aerobic and facultative anaerobic, catalase positive, oxidase negative, and are arranged in clusters, pairs, or tetrads. Features distinguishing Staphylococcus, Micrococcus, and Planococcus TABLE 23-1 Characters Staphylococcus Micrococcus Planococcus S. aureus is an important human pathogen that causes a spectrum of clinical diseases. These range from superficial skin lesions like folliculitis to deep-seated abscess and various pyogenic infections like endocarditis, osteomyelitis, etc. The bacterium also causes toxin-mediated diseases, such as food poisoning, toxic shock syndrome (TSS), and staphylococcal scalded skin syndrome (SSSS). Properties of the Bacteria ◗ Clusters Clusters/ Tetrads Tetrads Presence of teichoic acid ⫹ ⫺ ⫺ ■ Production of brown pigment ⫺ ⫺ ⫹ ■ Glucose fermentation ⫹ ⫹ ⫺ Arrangement of bacteria TABLE 23-2 Staphylococcus aureus is the most important human pathogen. The other important human pathogens are coagulasenegative staphylococci (CONS), which include Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus warneri, Staphylococcus saccharolyticus, Staphylococcus schleiferi, and Staphylococcus lugdunensis. Staphylococci are capable of acquiring resistance to many antibiotics and therefore can cause major clinical and epidemiological problems in hospitals. Morphology Staphylococci show following features: ■ They are Gram-positive cocci, measuring around 1 ␮m in diameter. They are nonmotile, nonsporing. They are noncapsulated. They, however, contain a microcapsule, which can be visualized by electron microscope only, but not by a light microscope. Human infections caused by Staphylococcus, Micrococcus, and Stomatococcus Bacteria Diseases Staphylococcus aureus Skin infections—impetigo, folliculitis, furuncle, carbuncle, paronychia, wound infection Systemic infections—bacteremia, osteomyelitis, septic arthritis, endocarditis, pneumonia, meningitis, deep-seated abscess Toxin-mediated infections—food poisoning, toxic shock syndrome, staphylococcal scalded skin syndrome Staphylococcus epidermidis Opportunistic infections—intravenous catheter infections, CSF shunt infections, and catheter-associated peritonitis endocarditis in “immunocompromised” patients Staphylococcus saprophyticus Urinary tract infection particularly in sexually active young women Micrococcus spp. Opportunistic infections Stomatococcus spp. Opportunistic infections, bacteremia, endocarditis 174 BACTERIOLOGY The cocci are typically arranged in irregular grape-like clusters. This appearance is due to incomplete separation of daughter cells during successive divisions of bacteria, which takes place in perpendicular planes. The grape-like clustering is seen when the bacteria are grown in solid media, but usually short chains are seen when grown in liquid media. In smears taken from pus, the cocci are present either singly or in pairs, in clusters, or in short chains of three or four cells. ◗ Culture Section III Staphylococci are aerobes and facultative anaerobes but can grow in the absence of oxygen also. They grow at a temperature range of 10–42°C (optimum temperature 37°C) and a pH range of 7.4–7.6 (optimum pH 7). Culture on solid media: Staphylococci can grow on a wide range of media including Mueller–Hinton agar, nutrient agar, blood agar, and MacConkey agar. Primary isolation can be made on nutrient agar and blood agar. Chapter 23 Color Photo 12). Hemolysis is well marked on sheep or rabbit blood agar, especially when incubated in an atmosphere of 20–25% CO2. Sheep blood agar is used for primary isolation. Hemolysis is weak on horse blood agar. Human blood is not used, as it may contain antibiotics or other inhibitors. Other species of Staphylococcus do not produce hemolysis. 3. MacConkey agar: S. aureus produces small pink colonies due to fermentation of lactose. 4. Selective media: Mannitol salt agar, milk agar, and glycerol monoacetate agar are the commonly used selective media for isolation of S. aureus from clinical specimens containing normal bacterial flora (e.g., stools). Mannitol salt agar contains 1% mannitol, 7.5% sodium chloride, and 0.0025% phenol red indicator. Most strains of S. aureus ferment mannitol with acid production, which gives rise to yellow zone formation around the colonies. 1. Nutrient agar: S. aureus produces round, convex, welldefined colonies measuring 2–4 mm in diameter. The colonies show a butyrous consistency with a smooth glistening surface. S. aureus produces characteristic golden-yellow colonies due to production of a nondiffusible golden-yellow pigment. The pigment is believed to be a lipoprotein allied to carotene. The production of the pigment is enhanced by incubation at 22°C in the presence of oxygen. Milk agar and 1% glycerol monoacetate agar are other media that facilitate the production of pigment. On nutrient agar slopes, the growth gives a characteristic “oil paint” appearance. 2. Blood agar: S. aureus produces a clear zone of hemolysis (beta-hemolysis) surrounding the colonies (Fig. 23-1, Culture in liquid media: S. aureus produces turbidity in liquid media and there is no production of pigment. ◗ S. aureus shows following reactions: ■ ■ ■ ■ ■ ■ ◗ FIG. 23-1. Blood agar plate showing beta-hemolysis surrounding the colonies of Staphylococcus aureus. Biochemical reactions It is coagulase positive. The production of coagulase is used as a test to differentiate S. aureus from S. epidermidis and other CONS. It is phosphatase positive. Phosphatase production can also be used to differentiate S. aureus from S. epidermidis, as the latter either does not produce or has very weak phosphatase activity. It is catalase positive. It produces enzyme catalase (unlike Streptococcus), which degrades H2O2 into nascent oxygen and water. It is oxidase negative. S. aureus ferments mannitol, sucrose, maltose, and trehalose under aerobic conditions, with the production of acid but no gas. Fermentation of mannitol is of diagnostic importance, because most strains of S. aureus ferment mannitol while those of S. epidermidis and S. saprophyticus do not ferment mannitol. It liquefies gelatin, hydrolyzes urea, reduces nitrate to nitrite, and is “Voges-Proskauer (VP)” and “methyl red (MR)” positive but indole negative. Other properties Susceptibility to physical and chemical agents: The cocci withstand moist heat at 60°C for 30 minutes but are killed after 30 minutes. They are also killed rapidly by disinfectants, such as phenol, chlorhexidine, and hexachlorophene. The cocci are very sensitive to aniline dyes, such as crystal violet. The dye at a concentration of 1:500,000 inhibits the growth of the cocci on blood agar medium but permits the growth of streptococci. STAPHYLOCOCCUS Cell Wall Components and Antigenic Structure ◗ Cell wall associated proteins and polymers include the following (Fig. 23-2): ◗ Cell wall peptidoglycan Teichoic acid Teichoic acid is the major antigenic determinant of the cell wall of S. aureus. It is a polymer of ribitol phosphate. Antibodies to teichoic acids develop in endocarditis and in certain other staphylococcal infections. ◗ S. aureus cell wall is rich in peptidoglycans. Peptidoglycan is a polymer of the polysaccharide, which provides rigidity to the cell wall of the bacteria. It has the characteristic penta glycine bridges that link tetrapeptides to the muramic acid residues. 175 Protein A It is the major protein in the cell wall and has a molecular weight of 13,000 Da. It is present in large quantities in the cell wall of certain strains of S. aureus, such as the Cowan’s strain of S. aureus (SAPA). This is a group specific antigen. The antigen is present in more than 90% strains of S. aureus. Protein A is absent in both the coagulase-negative staphylococci (CONS) and micrococci. Capsule or polysaccharide slime layer Pathogenesis and Immunity TTeichoic acid Cytoplasmic membrane S. aureus causes disease by multiplying in tissues and causing inflammation, and also by liberating toxin. Peptidoglycan layer ◗ Cytoplasm Staphylococcal cell wall structure Cell wall structure of Staphylococcus aureus. TABLE 23-3 S. aureus produces several virulence factors (Table 23-3), which include the following: (a) Cell wall associated proteins and polymers (b) Extracellular enzymes (c) Toxins Virulence factors of Staphylococcus aureus Virulence factors Biological functions Cell wall associated polymers and proteins Peptidoglycan Inhibits chemotaxis of inflammatory cells Capsular polysaccharide Inhibits phagocytosis and chemotaxis Teichoic acid Mediates attachment of staphylococci to mucosal cell Protein A Chemotactic, anticomplementary, and antiphagocytic; causes platelet injury; and elicits hypersensitivity reactions Enzymes Coagulase The enzyme coats the bacterial cells with fibrin, rendering them resistant to opsonization and phagocytosis Catalase Produces nascent oxygen which causes oxidative damage to host tissue Hyaluronidase Hydrolyzes hyaluronic acids present in the matrix of the connective tissues, thereby facilitating the spread of bacteria in the tissues Penicillinase Inactivates penicillins Nuclease Hydrolyzes DNA Lipases Hydrolyzes lipids Toxins Toxic shock syndrome toxin Superantigen, stimulates the release of large amount of interleukins (IL-1 and IL-2) Enterotoxin Superantigen, acts by producing large amounts of interleukins (IL-1 and IL-2) Exfoliative toxin Splits intercellular bridges in the stratum granulosum of epidermis of the skin Leukocidin toxin Leukolysin is thermostable and causes lysis of leukocytes Hemolysin Causes lysis of erythrocytes Chapter 23 FIG. 23-2. Virulence factors Section III Protein A Clumping factor 176 BACTERIOLOGY Cell wall associated proteins and polymers These include capsular polysaccharide, protein A, peptidoglycan, and teichoic acid that contribute to pathogenesis of staphylococcal diseases. Capsular polysaccharide: Few strains of S. aureus are capsulated. These strains are more virulent than the noncapsulated ones. ■ ■ The capsule protects the bacteria from phagocytosis. The capsule also facilitates adherence of the cocci to host cells and to prosthetic implants. Protein A: Protein A is an important virulence factor since it has non-specific interaction with Fc portion of the immunoglobulin G (IgG) leaving the Fab portions free to combine with specific antigen. ■ Chapter 23 Section III ■ It is chemotactic, anticomplementary, and antiphagocytic. It causes platelet injury and elicits hypersensitivity reactions. Peptidoglycan: It activates the complement, stimulates production of the antibodies, and inhibits chemotaxis by inflammatory cells. TABLE 23-4 Differentiating features of coagulase and clumping factors Coagulase Clumping factor Produced extracellularly Present on the surface Detected by tube test Detected by slide test Heat labile Heat stable Eight serotypes One serotype Needs CRF Does not need CRF Is a virulence factor Is not a virulence factor Catalase: The enzyme catalase reduces H2O2 to nascent oxygen and water. This nascent oxygen causes oxidative damage of host tissue. This enzyme is produced after phagocytosis or during metabolism of the bacteria. All strains of staphylococci produce catalase unlike streptococci. Hyaluronidase: The enzyme hyaluronidase hydrolyzes the acidic mucopolysaccharides present in the matrix of the connective tissues, thereby facilitating the spread of bacteria in tissues. Extracellular enzymes Penicillinase: More than 90% of S. aureus produce enzyme penicillinase. The enzyme inactivates penicillin group of antibiotics, hence is responsible for widespread occurrence of penicillin-resistant staphylococci. The gene for this enzyme is acquired through plasmids. The enzymes include (a) coagulase, (b) catalase, (c) hyaluronidase, (d) penicillinase, and (e) other enzymes. Other enzymes: These include phosphatase, deoxyribonucleases, nucleases, proteases, phospholipase, and lipases. Coagulase: S. aureus has a unique ability to clot a variety of mammalian plasma. Clotting of plasma is brought about by the action of the enzyme coagulase secreted by the pathogenic strains of S. aureus. The enzyme coagulase is of two types: (a) free coagulase and (b) bound coagulase. Toxins Teichoic acid: It mediates attachment of staphycocci to mucosal cell. A. Free coagulase: Free coagulase is a heat-labile and filterable enzyme. It has eight antigenic types (A, B, C, D, E, F, G, and H). Antigenic type A is produced by most human S. aureus strains. The enzyme coagulase in association with coagulasereacting factor (CRF) present in plasma converts fibrinogen to fibrin. In the absence of CRF, coagulase cannot bring about clotting like in case of the guinea pig plasma. This fibrin coats the bacterial cells, rendering them resistant to opsonization and phagocytosis and hence making bacteria more virulent. All coagulase-producing staphylococci are, by definition, S. aureus. Coagulase production is demonstrated by tube coagulase test, which is an important test for the identification of S. aureus. B. Bound coagulase: Bound coagulase is otherwise known as clumping factor. It is a heat-stable protein and is present in the cell wall. This enzyme brings about clumping of the staphylococci when mixed with plasma by directly acting on fibrinogen. Lysis of the cell releases the enzyme. Unlike free coagulase, clumping factor does not need CRF for its action; till date only one type has been identified. Bound coagulase is not a virulence factor. Differences between coagulase and clumping factors are summarized in Table 23-4. Toxins include (a) toxic shock syndrome toxin, (b) enterotoxin, (c) exfoliative toxin, (d) leukocidins, and (e) hemolysins. Toxic shock syndrome toxin: Toxic shock syndrome toxin (TSST) is a protein with a molecular weight of 22,000 Da and resembles enterotoxin F and exotoxin C. It is antigenic. Production of toxin is pH dependent and occurs at pH 7–8. The toxin causes toxic shock syndrome (TSS). ■ ■ S. aureus strains responsible for menstruation-associated TSS and half of the strains responsible for non-menstruation associated TSS produce TSST-1. The strains producing TSST-1 belong to the bacteriophage group I. TSST is a superantigen and hence a potent stimulant of T lymphocytes, resulting in release of large amount of interleukins (IL-1 and IL-2) and tumor necrosis factor, ultimately manifesting in TSS. Enterotoxin: Enterotoxin is a heat-stable protein, capable of resisting boiling for about 30 minutes. It is also gut-enzyme resistant. The toxin is produced by nearly one-third of all the strains of S. aureus, and these strains belong to bacteriophage group III (6/47). Nine antigenic types (A, B, C1,2,&3, D, E, G, H, I, and J) of enterotoxins have been described, out of which type A and B are most important. These proteins are of molecular weights ranging from 26,000 to 30,000 Da. STAPHYLOCOCCUS ■ ■ The toxins are superantigens and act by producing large amounts of interleukins, IL-1 and IL-2. The enterotoxins are responsible for clinical conditions like staphylococcal food poisoning and pseudomembranous enterocolitis postantibiotic therapy. Exfoliative toxin: Exfoliative toxin is of two types: (a) toxin A (molecular weight of 30,000 Da) and (b) toxin B (molecular weight of 29,500 Da). Toxin A is heat stable, while toxin B is heat labile. The toxin is antigenic, and specific antibodies against the toxin are protective. The strains producing this toxin belong to bacteriophage group II. ■ ■ The toxin breaks intercellular bridges in the stratum granulosum of epidermis and causes its separation from the underlying tissue, resulting in a blistering and exfoliating disease of the skin. Toxin in localized form causes bullous impetigo and in generalized form causes staphylococcal scalded skin syndrome (SSSS) in children below 4 years of age. ■ ■ ■ ◗ The diseases caused by S. aureus can be divided into two groups: (a) inflammatory and (b) toxin-mediated staphylococcal diseases. ◗ These include the following conditions: ■ ■ ■ ■ ■ Hemolysins: S. aureus produces four hemolysins: alpha (␣), beta (␤), gamma (␥), and delta (␦) hemolysins. ■ ■ ■ ■ ◗ Alpha-hemolysin is a protein with a molecular weight of 33 kDa. It has lethal effects on a wide variety of cell types and lyses erythrocytes of several animal species. Beta-hemolysin is a sphingomyelinase that is active on a variety of cells. It is a protein with a molecular weight of 35 kDa. It is a hot–cold hemolysin; i.e., its hemolytic properties are increased by exposure of the RBCs to cold temperature. Delta-hemolysin is a protein with a molecular weight of 8 kDa. It acts primarily as a surfactant. Gamma-hemolysin actually consists of three proteins. The three delta-hemolysin proteins interact with one of the two PV–leukocidin proteins. Pathogenesis of staphylococcal infections S. aureus are pyogenic bacteria that cause localized lesions in contrast to streptococci that are spreading in nature. Staphylococci adhere to the damaged skin, mucosa, or tissue surfaces. At these sites, they evade defense mechanisms of the host, colonize, and cause tissue damage. They produce disease by: ■ ◗ Staphylococcal skin infections include impetigo, folliculitis, furuncles, carbuncles, paronychia, surgical wound infection, blepharitis, and postpartum breast infection. S. aureus is the most common cause of boils. The infection is acquired either by self-inoculation from a carrier site, such as the nose or through contact with another person harboring the bacteria. Bacteremia and septicemia may occur from any localized lesion, especially wound infection or as a result of intravenous drug abuse. S. aureus is an important cause of acute bacterial endocarditis, of normal or prosthetic heart valves, which is associated with high mortality. S. aureus is the most common cause of osteomyelitis in children. The bacteria reach bone through blood stream or by direct implantation following trauma. S. aureus causes pneumonia in postoperative patients following viral respiratory infection, leading to empyema; it also leads to chronic sinusitis. S. aureus causes deep-seated abscesses in any organ after bacteremia. Toxin-mediated staphylococcal diseases These include (a) staphylococcal food poisoning, (b) staphylococcal toxic shock syndrome, and (c) staphylococcal scalded skin syndrome. Staphylococcal food poisoning: Staphylococcal food poisoning is caused by enterotoxin. The enterotoxin is a preformed toxin, already present in the contaminated food before consumption. Milk and milk products and animal products like fish and meat kept at room temperature after cooking are mainly incriminated. When kept at room temperature, the contaminating staphylococci multiply and produce toxin adequate enough (as little as 25 ␮g of toxin B can lead to illness) to cause food poisoning. The toxin acts by stimulating the release of large amounts of interleukins IL-1 and IL-2. It is fairly heat resistant and so is not inactivated by brief cooking. Often a food handler, who either is a carrier of S. aureus (nose, skin) or is suffering from staphylococcal skin infection, Chapter 23 ■ Inflammatory staphylococcal diseases Section III ■ Host immunity Clinical Syndromes ■ The alpha-lysin is the most important leukocidin. It causes marked necrosis of the skin and hemolysis by damaging the cell membrane, leading to release of low-molecular-weight substances from the damaged cells. PV–leukocidins are six in number, each consisting of two components. The molecular weight is around 32 kDa. These toxins cause death of human leukocytes and macrophages without causing any lysis. Leukolysin is thermostable and causes lysis of leukocytes and necrosis of tissues in vivo. Multiplying in tissues, Liberating toxins, and Stimulating inflammation. S. aureus infection does not cause any life-long immunity. It causes repeated infections in a susceptible host. Leukocidins: Leukocidins include (a) alpha-lysin, (b) PantonValentine–leukocidin (PV–leukocidin), and (c) leukolysin. ■ 177 Chapter 23 Section III 178 BACTERIOLOGY is the source of infection. The onset of symptoms is sudden, appearing within 2–6 hours of ingestion of food. It is a selflimiting condition characterized by nausea, vomiting, abdominal cramps, and watery, nonbloody diarrhea. Staphylococcal toxic shock syndrome: Staphylococcal toxic shock syndrome (STSS) is caused by TSST. The toxin is a superantigen, which causes STSS by stimulating the release of large amounts of interleukins IL-1 and IL-2 in the body. The STSS is an acute and potentially life-threatening condition similar to Gram-negative sepsis and septic shock. STSS is a multisystem disease characterized by fever, hypotension, myalgia, vomiting, diarrhea, mucosal hyperemia, and an erythematous rash followed by desquamation of the skin, particularly on palms and soles. This condition was first documented in 1980 in the United States among the menstruating women who used highly absorbent vaginal tampons; the vaginal swab from these women showed a heavy growth of S. aureus. This condition can also occur in other individuals, who have a local site of staphylococcal infection on skin or mucosa or on any other extragenital site. Staphylococcal scalded skin syndrome: Staphylococcal scalded skin syndrome (SSSS) is caused by the exfoliative toxin, exfoliatin. The condition is seen commonly in infants and children. It is associated with extensive exfoliation of the skin, in which outer layer of the epidermis is separated from the underlying tissue and is characterized by the appearance of extensive bullae. These bullae when ruptured may leave behind scalded, red, tender skin. The lesion typically starts periorificially or in skin folds. It usually resolves within 10 days’ time. Pemphigus neonatorum and bullous impetigo are the milder forms, whereas Ritter’s disease in the newborn and toxic epidermal necrolysis in the older persons are the severe forms of the SSSS. Complications of Staphylococcal Diseases Complications of staphylococcal diseases include bacterial pneumonia, septicemia, arthritis, meningitis, etc. These complications are frequently seen in persons with extreme of age, debilitated persons, and immunosuppressed hosts. Epidemiology ◗ Reservoir, source, and transmission of infection Human cases and carriers are the important reservoir of infection. ■ ■ ■ ■ Human cases of cutaneous and respiratory infections shed large numbers of staphylococci into the environment for a prolonged period of time. Staphylococci colonize the skin very early in life (in neonates on the umbilical stump). Staphylococci shed by the patients and carriers contaminate handkerchiefs, bed linens, blankets, and other inanimate fomites and persist in them for weeks. S. aureus found in the nose and sometimes on the skin, especially in hospital staff and patients is the main source of infection in hospitals. Domestic animals, such as cows, can also be reservoirs of staphylococcal infection. Key Points Staphylococcal infections may be acquired through: ■ ■ Self-inoculation from nose or other sites in patients who harbor staphylococci (endogenous infection) or Direct contact with infected humans, carriers, and less frequently, animals (exogenous infection). Exogenous infection can also be acquired by close contact with infected fomites or inhalation of air droplets in heavily contaminated environment. Hospital-acquired S. aureus infections: This is the most common cause of hospital-acquired infections. Certain strains of S. aureus causing hospital infections are known as hospital strains. They exhibit certain properties, which are presented in Box 23-1. ◗ Bacteriophage typing Strains of staphylococci can be typed by bacteriophage typing, which is useful in epidemiological studies (Fig. 23-3). Bacteriophage typing is based on the susceptibility of cocci to bacteriophages. This is carried out by pattern method, where a set of 23 standard typing phages of S. aureus is used to type staphylococcal isolates and distinguish them from one another by their patterns of susceptibility to lysis. In this method, the Geographical distribution Staphylococcal infections are found throughout the world. Nearly one-third of the adult population is asymptomatic carrier of staphylococci. Hospital infections caused by S. aureus are worldwide in distribution. ◗ ◗ Habitat Staphylococci are primary pathogens of humans and animals. They are present as commensals on skin, in the glands of the skin, and on mucous membranes. The cocci are commonly found in the intertriginous skin folds, perineum, axillae, and vagina. Approximately, 35–50% of normal adults carry S. aureus in the anterior nares, 10% in the perineum, and 5–10% in the vagina. Box 23-1 Hospital strains of Staphylococcus aureus Certain strains of staphylococci are the common causes of postoperative wound infections and other infections in the hospital environment. These strains are known as “hospital strains”. These hospital strains show following characteristics: 1. They are usually resistant to penicillin, methicillin, and other routinely used antibiotics. 2. They belong to certain bacteriophage types. 3. Some of the strains (e.g., phage type 80/81) are known to cause hospital infections throughout the world. Such strains are known as “epidemic strains”. STAPHYLOCOCCUS ◗ 179 Specimens Specimens to be collected for demonstration of staphylococci depend on the nature of lesion (Table 23-6). ◗ Microscopy Demonstration of Gram-positive cocci arranged in clusters and pus cells in the Gram-stained smears of pus (Fig. 23-4, Color Photo 14), wound exudate, etc. are the characteristic features of pyogenic infection caused by S. aureus. It is noteworthy that microscopy: ■ FIG. 23-3. Bacteriophage typing of staphylococci. TABLE 23-5 Group Phage typing of human isolates of Staphylococcus aureus Phage 29, 52, 52A, 79, 80 3A, 3C, 55, 71 III 6, 42E, 47, 53, 54, 75, 77, 83A, 84, 85 IV — V 94, 96 Not allocated 81, 95 ◗ Culture The identification of staphylococci is confirmed by culture and other identification tests comprising a range of biochemical and enzymatic tests followed by antibiotic sensitivity. The specimens are inoculated onto nutrient agar and blood agar and incubated at 37°C for 24 hours. On nutrient agar, large, circular, smooth, convex, and glistening colonies showing goldenyellow pigments can be observed. On blood agar, the colonies show a zone of beta-hemolysis, which is not shown by any other species of staphylococci. Specimens from heavily contaminated sources, such as vomitus and feces, are inoculated on selective media (e.g., mannitol salt agar or salt milk agar). These media inhibit growth of Gram-negative bacteria but allow the growth of staphylococci and certain other Gram-positive cocci. ◗ Identification of bacteria The identifying features of S. aureus are summarized in Box 23-2. Coagulase test Coagulase test is an important test carried out to detect S. aureus. The test is done in two ways: tube coagulase test and slide coagulase test. Other typing methods S. aureus has been classified into six biotypes (A, B, C, D, E, and F). Most human pathogenic strains belong to biotype A. Other typing methods include (a) plasmid profile, (b) DNA fingerprinting, (c) ribotyping, and (d) PCR-based analysis of genetic pleomorphism and (e) serotyping. Laboratory Diagnosis Laboratory diagnosis of staphylococcal infections is based on the demonstration of staphylococci, in appropriate clinical specimens, by microscopy and culture. TABLE 23-6 Various specimens collected in staphylococcal infections Specimen Condition Pus Suppurative lesions and osteomyelitis Sputum Respiratory infections Blood Bacteremia Feces and vomitus Food poisoning Urine Urinary tract infections Nasal and perineal swab Suspected carriers Chapter 23 strain of S. aureus to be typed is inoculated on a nutrient agar plate to produce a lawn culture. After drying the plate, various phages at their routine test dilution (RTD) are applied over marked squares on plate. Such plates are then incubated overnight at 30°C and observed for the presence or absence of lysis of the colonies by the phages. The phage type of a strain is known by designation of the phages that lyse it. Thus, if a strain is lysed by phages 83A, 84, and 85, it is called phage type 83A/84/85. By this method, most of the strains of staphylococci can be classified and are divided into five lytic groups, while there are a few which cannot be classified and constitute the unclassified group (Table 23-5). The national reference centre for staphylococcal phage typing in India is located in the Department of Microbiology, Maulana Azad Medical College, New Delhi. ◗ Section III I II ■ Alone is not adequate to differentiate various species of staphylococci or micrococci from one another. Is also of no value for sputum and other specimens where mixed bacterial flora is present. 180 BACTERIOLOGY ■ ■ Human or rabbit plasma, which is rich in CRF, is used in the test. The plasma is collected in vials containing anticoagulants, such as oxalate, heparin, or EDTA. Citrated plasma is not used because if the specimen is contaminated with Gram-negative bacilli, the latter may utilize the citrate and produce false positive reaction. Slide coagulase test: Slide coagulase test detects the bound coagulase or the clumping factor. The test is performed by mixing a dense suspension of the staphylococci with a loopful of undiluted rabbit plasma on a slide. In a positive test, clumping takes place within 10 seconds. Phosphatase test FIG. 23-4. Gram-stained pus smear showing staphylococci (⫻1000). Chapter 23 Section III Box 23-2 Identifying features of Staphylococcus aureus 1. S. aureus are Gram-positive cocci arranged in irregular grape-like clusters. 2. On nutrient agar, S. aureus colonies produce characteristic goldenyellow colonies. 3. On blood agar, S. aureus produces a clear zone of hemolysis (beta-hemolysis). 4. S. aureus are coagulase positive. All coagulase-producing staphylococci are, by definition, S. aureus. 5. S. aureus are phosphatase positive, DNAase positive, and mannitol positive. 6. S. aureus are novobiocin and polymyxin B sensitive. Coagulase test The production of phosphatase can be demonstrated by culturing a mixed specimen on phenolphthalein phosphate agar and exposing the colonies to ammonium vapors. S. aureus colonies turn bright pink due to the release of phenolphthalein. Novobiocin sensitivity Novobiocin sensitivity is a simple disk diffusion test to differentiate S. aureus from other staphylococci. This test is carried out by using a 5-␮g novobiocin disk on an overnight culture of staphylococci on Mueller–Hinton agar. Novobiocin sensitivity is shown by an inhibition zone of ⱖ16 mm. S. aureus is novobiocin sensitive, while S. saprophyticus is novobiocin resistant. Polymyxin B resistance Polymyxin B sensitivity is again a simple disk diffusion test to differentiate S. aureus from other staphylococci. This is carried out by using a 300-U polymyxin B disk on an overnight culture of staphylococci on Mueller–Hinton agar. Polymyxin resistance is shown by an inhibition zone of ⬍10 mm. S. aureus is usually polymyxin resistant. Treatment Positive Negative Skin and soft tissue infections are treated best with local wound care with or without topical antibiotics (e.g., neomycin). Spontaneous or surgical drainage of pus and debridement of necrotic tissue is an effective mode for treatment of staphylococcal abscess. Systemic antibiotics are necessary for deep-seated and systemic infections. Key Points FIG. 23-5. ■ Tube coagulase test. ■ Tube coagulase test: Tube coagulase test is carried out to detect free coagulase. In this test, 0.1 mL of an overnight broth culture is mixed with 0.5 mL of a 1:10 dilution of human or rabbit plasma. The plasma-broth culture mixture is incubated in a water bath at 37°C for 3–6 hours. In a positive test, the plasma is coagulated and does not flow (Fig. 23-5, Color Photo 13). ◗ Benzyl penicillin is the drug of choice for penicillinsensitive strains of S. aureus. Erythromycin, vancomycin, or first-generation cephalosporins are recommended for patients with allergy to penicillin. Penicillin resistance in staphylococci Penicillin resistance in the bacteria is increasingly recognized since 1945. Nearly 80% or more strains of S. aureus are resistant to penicillin. It is of three types: STAPHYLOCOCCUS 1. Plasmid-mediated resistance: This type of resistance may be due to the production of enzyme penicillinase (beta-lactamase), which is plasmid mediated. This enzyme inactivates penicillin by splitting the beta-lactam rings. Staphylococci produce four types of penicillinases (A, B, C, and D). Penicillinase plasmids are transmitted to the staphylococci by both transduction and conjugation. The plasmids also carry markers of resistance to heavy metals, such as arsenic, cadmium, mercury, lead, and bismuth as well as to other antibiotics, such as erythromycin and fusidic acid. 2. Chromosomal-mediated resistance: This type of resistance has also been documented. Reduction in the affinity of the penicillin-binding proteins (PBPs; present on the cell wall of the staphylococci) to the beta-lactam antibiotics also contributes to the resistance of the bacteria to penicillins and other beta-lactam antibiotics. 3. Tolerance to penicillin: Staphylococci developing tolerance to penicillin are only inhibited but not killed. Penicillin-resistant strains can be treated with beta-lactamase-resistant penicillins, e.g., oxacillin, flucloxacillin, cloxacillin, methicillin, or vancomycin. Methicillin-resistant staphylococci Prevention and Control There is no effective immunization with toxoids or bacterial vaccines against staphylococcal infection. Cleanliness, frequent hand-washing, and aseptic management of lesions help in the Coagulase-Negative Staphylococci Coagulase-negative staphylococci (CONS) are the normal flora of the skin. CONS are opportunistic bacteria. They cause infections in debilitated or immunocompromised patients and in patients fitted with urinary catheters, cardiac valves, pacemakers, and artificial joints. ■ ■ They form white nonpigmented colonies, morphologically similar to those of S. aureus. They are differentiated from S. aureus by their failure to coagulate the plasma due to the absence of the enzyme coagulase. CONS of medical importance include (a) S. epidermidis, (b) S. saprophyticus, (c) S. haemolyticus, (d ) S. saccharolyticus, (e) S. hominis, ( f ) S. schleiferi, ( g) S. lugdunensis, and (h) Staphylococcus simulans. Staphylococcus epidermidis S. epidermidis forms white colonies on blood agar. It is catalase positive, coagulase negative, and does not ferment mannitol. It tolerates salt, survives drying, and is highly antibiotic resistant. It is a normal skin commensal. Carriage rate is as high as 100%. This bacterium is transmitted by self-inoculation or by contact with infected patients and hospital personnel. Ability to produce slime is an important virulence factor of the bacterium. S. epidermidis causes infection by adhering itself to the surface of the intravenous plastic catheters and prosthetic devices. The adherence is believed to be facilitated by polysaccharide glycocalyx known as slime, produced in large quantities by the bacteria. Slime also inhibits the action of lymphocytes and neutrophils. S. epidermidis is an important agent of hospital-acquired infection. It causes: ■ ■ ■ infection in compromised hosts, such as neutropenic patients, particularly in association with intravenous catheters and other prosthetic devices, such as heart valves. endocarditis in patients with prosthetic valves, intravenous catheter infections, CSF shunt infections, catheter-associated peritonitis and endocarditis. sepsis in neonates, osteomyelitis, wound infections, vascular graft infections, and mediastinitis. Vancomycin is the drug of choice for treatment of infection caused by S. epidermidis. Staphylococcus saprophyticus S. saprophyticus forms white colonies on blood agar. It is catalase positive, coagulase negative, and does not ferment mannitol. It normally inhabits the skin and genital mucosa. The bacterium causes: Chapter 23 Methicillin-resistant S. aureus (MRSA) denotes resistance of S. aureus to penicillin, as well as to all other beta-lactam antibiotics including the third-generation cephalosporins and carbapenems. Resistance to methicillin is due to the production of a novel PBP, designated as PBP 2a. PBPs are the targets of beta-lactam antibiotics. Infections caused by MRSA are being increasingly reported worldwide since 1980. The infection is also being increasingly reported now, from different hospitals. MRSA usually colonizes the broken skin and can cause a wide range of local and systemic staphylococcal infections. Hospital staffs harboring MRSA are the chief source of infection for the patients. These strains can cause a wide range of infections including bacteremia, endocarditis, and pneumonia. These strains are increasingly recognized as important agents of hospital-acquired infection in hospitalized patients undergoing prosthetic heart valve surgery. MRSA strains can be treated with glycopeptide antibiotics, such as vancomycin and teicoplanin in serious systemic infections, such as pneumonia, bacteremia, and endocarditis. MRSA are sensitive to one or more of the second-line drugs, which include erythromycin, clindamycin, quinolones, fusidic acid, trimethoprim, chloramphenicol, tetracycline, and rifampicin. However, ciprofloxacin, rifampicin, and fusidic acid are not used simply because of the possibility of emergence of resistance. Proper hand-washing and use of topical agents, such as mupirocin and chlorhexidine on skin and nose to eradicate the agents are effective to prevent and control nosocomial infections caused by MRSA. control of S. aureus infection. Treating with nasal creams containing neomycin or bacitracin prevents recurrent infections in cases of nasal carriers of S. aureus. Topical application of antimicrobial agents prevents dissemination of infection from the abscesses. Section III ◗ 181 182 BACTERIOLOGY Differences between Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus TABLE 23-7 S. aureus S. epidermidis S. saprophyticus Coagulase ⫹ ⫺ ⫺ Clumping factor ⫹ ⫺ ⫺ Heat-stable nuclease ⫹ ⫺ ⫺ Variable ⫺ ⫹ ␤-galactosidase ⫺ ⫺ ⫹ Alkaline phosphatase ⫹ ⫹ ⫺ Polymyxin B Resistant Resistant Sensitive Novobiocin Sensitive Sensitive Resistant Acid from mannitol ⫹ ⫺ ⫺ Acid from trehalose ⫹ ⫺ Acid from mannose ⫹ ⫹ ⫺ PYR test ⫺ ⫺ ⫹ Test Chapter 23 Section III Urease ■ ■ Urinary tract infection by endogenous spread in colonized women. It adheres to the epithelial cells lining the urogenital tract. It causes dysuria, pyuria, and hematuria. Urethritis, catheter-associated urinary tract infections, prostatitis in elderly men, and rarely, sepsis and endocarditis. Urinary tract infection caused by S. saprophyticus can be treated with quinolones (such as norfloxacin) or with trimethoprim–sulfamethoxazole. S. epidermidis and S. saprophyticus are distinguished from each other by their reaction to antibiotic novobiocin—S. epidermidis is sensitive, while S. saprophyticus is resistant. The differences between S. aureus, S. epidermidis, and S. saprophyticus are summarized in Table 23-7. Other Coagulase-Negative Staphylococci There are many other coagulase-negative staphylococci that have been reported recently to cause human infections. These include the following: 1. S. haemolyticus causes bacteremia, endocarditis, urinary tract infection, and wound infection. 2. S. saccharolyticus causes endocarditis. 3. S. hominis causes bacteremia in cancer patients. 4. S. schleiferi causes wound infections, bacteremia, and indwelling catheter infections. 5. S. lugdunensis causes endocarditis, peritonitis, osteomyelitis, and breast abscesses. 6. S. simulans causes septicemia, osteomyelitis, and septic arthritis. Micrococcus Micrococci in comparison to staphylococci are larger and measure up to 2 ␮m in diameter. In smears, they appear as Gram-positive cocci arranged in tetrads. On culture, they produce colonies with yellow, pink, or red pigments. It is doubtful that they are human pathogens. Planococcus They are Gram-positive cocci most commonly found in seawater, prawns, and shrimp. They are distinguished from other Grampositive cocci by their ability to grow in a higher salt concentration of 12% sodium chloride. They are nonpathogenic to humans. Stomatococcus They are capsulated Gram-positive cocci arranged in pairs or clusters. On culture, they produce white and mucoid colonies. It is doubtful that they are human pathogens. CASE STUDY A group of 25 students of 11–12 years of age studying in a higher secondary school in Haripur were admitted to a hospital with complaints of severe vomiting and diarrhea within 3 hours of consuming the food prepared in their school. History revealed that a new cook appointed a few days back in the school prepared the food. All the students consumed the same food. Children were treated and were discharged after observing them overnight in the hospital. ■ ■ ■ ■ What is the possible cause of this food poisoning? What is the possible reservoir for this organism that was responsible for this outbreak? What are the tests you will perform to establish the etiological diagnosis of the condition? What steps you will take to prevent this infection? 24 Streptococcus and Enterococcus The streptococci based on their oxygen requirement are classified into aerobes and obligate anaerobes. Obligate anaerobes are designated as peptostreptococci, which will be described in detail in Chapter 30. The aerobes and facultative anaerobes are further classified as follows: agar. Brown (1919) classified these aerobic streptococci into three groups on the basis of their growth in 5% horse blood agar. Alpha-hemolytic streptococci: These cocci produce colonies surrounded by a narrow zone (greenish zone) of hemolysis with persistence of some partially lysed RBCs. The greenish discoloration is due to the formation of a reduced product of hemoglobin. Alpha-hemolytic streptococci are known as viridans streptococci. These are found as commensals in the upper respiratory tract of humans, and these may cause opportunistic infections. Streptococcus salivarius is an important opportunistic pathogen belonging to this group (Streptococcus pneumoniae also belongs to alpha-hemolytic group). Beta-hemolytic streptococci: These cocci produce a welldefined, clear, colorless zone of hemolysis (2–4 mm wide) around the colonies. RBCs in the zone of hemolysis are completely lysed. This lysis is due to the liberation of enzymes streptolysin O (SLO) and streptolysin S (SLS). The term hemolytic streptococci is applicable only to beta-hemolytic streptococci. Most of the pathogenic streptococci belong to this group, and among them Streptococcus pyogenes is the most important one. Gamma-hemolytic streptococci: These streptococci do not produce any hemolysis or discoloration on blood agar. These nonhemolytic streptococci are generally found as commensals. Streptococcus faecalis (Enterococcus faecalis) belongs to this group. ◗ ◗ Introduction Streptococci are aerobic and facultatively anaerobic Grampositive cocci, arranged in pairs, or chains. The enterococci are facultative anaerobes. They require complex nutrients for their growth. Streptococci and enterococci causing human infections are summarized in Table 24-1. Streptococcus Streptococci are part of the normal flora in humans and animals. ■ ■ ■ They are nonmotile, nonsporing, spherical or ovoid cocci, and have hyaluronic acid capsules. They are catalase negative by which they are distinguished from staphylococci. They are relatively fastidious bacteria requiring enriched medium, such as blood agar for their growth. Classification Classification based on hemolysis in blood agar The aerobes and facultative anaerobes are further classified on the basis of their hemolytic properties on blood TABLE 24-1 Classification based on antigenic structure Lancefield classification is a serological classification of the beta-hemolytic streptococci. This serological classification is Human infections caused by Streptococcus and Enterococcus Bacteria Diseases Streptococcus pyogenes Pharyngitis, pyoderma (impetigo, erysipelas, cellulites), necrotizing fasciitis, scarlet fever, streptococcal toxic shock syndrome, acute glomerulonephritis (AGN), and acute rheumatic fever Streptococcus agalactiae Neonatal infections (septicemia, meningitis, or pneumonia); urinary tract infection in pregnant women; osteomyelitis, arthritis, peritonitis, and skin infections in nonpregnant women and in men Other hemolytic streptococci Pharyngitis, bacteremia, abscess formation Viridans streptococci Dental caries, subacute endocarditis, and intra-abdominal suppurative infections Enterococcus Urinary tract infection especially in hospitalized patients; bacteremia, infection of the bile duct, and endocarditis 184 BACTERIOLOGY based on the detection of group-specific carbohydrate antigen (C antigen) on the cell wall of the streptococci. The beta-hemolytic streptococci are classified into 21 serological groups known as Lancefield groups, designated from A to W (with exception of I and J). The majority of hemolytic streptococci that cause human infections belong to group A. Group A streptococci are also known as S. pyogenes, while group B streptococci are known as Streptococcus agalactiae. Based on the M, T, and R protein antigens present on the cell surface, S. pyogenes have been further classified into 80 serotypes. This classification is known as Griffith typing. M protein is the most important type-specific antigen. This serotyping is important for epidemiological studies. Hyaluronic acid capsule Protein lipoteichoic acid fimbria Group specific carbohydrate Peptidoglycan Cytoplasm Cytoplasmic membrane FIG. 24-1. Pili covered with lipoteichoic acid Antigenic structure of Streptococcus pyogenes. Chapter 24 Section III Streptococcus pyogenes S. pyogenes is the species classified under group A streptococci. It is the most important human pathogen causing: 1. Pyogenic infections, such as bacterial pharyngitis and cellulitis. 2. Toxin-mediated diseases, such as scarlet fever and toxic shock syndrome. 3. Immunologic diseases, such as acute glomerulonephritis (AGN) and rheumatic fever. Properties of the Bacteria ◗ Morphology S. pyogenes shows following features: ■ ■ ■ ◗ They are Gram-positive cocci measuring 0.6–1.0 ␮m in diameter and are arranged in long chains. Streptococci are nonmotile and nonsporing. Streptococci divide in one plane and thus occur in pairs or in chains of varying lengths, especially in liquid media and clinical specimens. They are motile and nonsporing. Some strains of S. pyogenes and some strains of group C streptococci produce capsule during the first 2–4 hours of growth. The capsule is composed of hyaluronic acid containing repeating molecules of glucuronic acid and N-acetylglucosamine. It is chemically similar to that of host connective tissue and is therefore nonantigenic. Capsulated strains produce mucoid colonies on the blood agar (Fig. 24-1). Culture S. pyogenes is an aerobe and facultative anaerobe. It grows at 37°C and at a pH of 7.2–7.4 on enriched medium, such as blood agar. 1. Blood agar: S. pyogenes produces small white to gray colonies, measuring 0.5–1 mm in diameter with a clear zone FIG. 24-2. Blood agar plate showing beta-hemolysis surrounding the colonies of Streptococcus pyogenes. of beta-hemolysis (Fig. 24-2, Color Photo 15). Presence of 10% CO2 enhances the growth and hemolysis of colonies. The virulent strains on fresh isolation produce matt colonies, while avirulent strains produce glossy colonies. Colonies that produce large amounts of hyaluronic acid appear mucoid on the culture plate. 2. Selective media: Crystal violet blood agar is a selective medium for culture of S. pyogenes. Addition of 0.0001% crystal violet to blood agar makes the medium highly selective for S. pyogenes. This inhibits all other Gram-positive cocci while allowing selective growth of S. pyogenes. PNF medium (horse blood agar containing polymyxin B sulfate, neomycin sulfate, and fusidic acid) is another selective medium used for isolation of S. pyogenes. 3. Transport medium: Pikes transport medium containing (1:1,000,000) crystal violet and (1:16,000) sodium azide is a frequently used transport medium for transporting throat swab for culture of S. pyogenes. STREPTOCOCCUS AND ENTEROCOCCUS 4. Liquid medium: S. pyogenes produces granular turbidity with powdery deposit when grown in the liquid media, such as serum or glucose broth. ◗ Biochemical reactions S. pyogenes shows following biochemical reactions: ■ ■ ■ ■ The bacteria are catalase negative; by this property, they are distinguished from staphylococci. They ferment many sugars, producing acid but no gas; howevr, these are of little diagnostic value in the identification of cocci. They do not ferment ribose They are not soluble in bile. They cause hydrolysis of pyrrolidonyl naphthylamide (PYR). M protein: M protein is the most important protein. It is acid- and heat-stable and trypsin-sensitive. It is the chief virulence factor of the cocci. It inhibits phagocytosis and facilitates the attachment of cocci to epithelial cells. M protein, a complex alpha-helix, consists of carboxyl terminus and an amino terminus. The carboxyl terminus is bound to the cytoplasmic membrane and is a highly conserved structure. The amino terminus is present through the cell wall to the cell surface and is highly variable. This variable component is responsible for antigenic differences in the M protein and based on that S. pyogenes is divided into more than 80 (l–60) serotypes. M proteins are further subdivided into class I and class II protein molecules: ■ ■ ◗ 185 Other properties Antibodies develop against the exposed constant (C) region of the class I M proteins and are responsible for the pathogenesis of rheumatic fever. In contrast, antibodies do not develop against class II M proteins. Cell Wall Components and Antigenic Structure ◗ Other cell surface components The cell wall of S. pyogenes consists of the following components (Fig. 24-1): Peptidoglycan, lipoteichoic acid, and F proteins are other components of the cell wall of S. pyogenes. Peptidoglycan confers rigidity to the cell wall. It is also responsible for producing fever, dermal and cardiac necrosis in animals, and lysis of erythrocytes. ◗ Pathogenesis and Immunity Group-specific carbohydrate The cell wall contains a group-specific polysaccharide that forms approximately 10% of the dry weight of the cell. It is a polymer of N-acetylglucosamine and rhamnose. It is nontoxic and hapten in rabbits. On the basis of group-specific carbohydrate (C) antigen, S. pyogenes strains have been divided into 21 groups (A–W) except I and J by Lancefield, hence are known as Lancefield groups. The “C” antigen can be extracted by the following methods: ■ ■ ■ ■ Acid extraction with hydrochloric acid (Lancefield method ), Formamide extraction at 150°C (Fuller’s method ), Autoclaving (Rantz and Randall’s method ), and Enzyme extraction (Maxted’s method ). After the extraction, the carbohydrate component is treated with type-specific antiserum by a precipitation reaction or by immunofluorescence for grouping of the isolates of S. pyogenes. ◗ Type-specific proteins The cell wall of S. pyogenes has three major proteins, M, T, and R proteins. These proteins are useful for serologic typing (Griffith typing) of S. pyogenes. Streptococci have more than 20 soluble antigens, enzymes, and toxins that contribute in the pathogenesis of various stages of streptococcal diseases. ◗ Virulence factors Streptococci produce a wide range of virulence factors responsible for the disease, which include the following (Table 24-2): (a) Cell wall associated proteins and polymers (b) Enzymes (c) Toxins Cell wall associated proteins and polymers These include capsule, teichoic acid, M protein, and F protein that contribute to pathogenesis of diseases in various ways as mentioned in Table 24-2. Capsule: The cell wall of the Streptococcus is surrounded by a capsule. The capsule is nonantigenic and weakly antiphagocytic. It acts like a barrier between the complement proteins bound to the bacteria and the phagocytic cells, thereby preventing phagocytosis of the bacteria. Capsulated strains that are rich in M proteins are highly pathogenic. Chapter 24 T proteins: These are trypsin-resistant (T) proteins and are acid and heat labile. T typing of strains of S. pyogenes is useful in the epidemiological surveillance of the infection caused by cocci. R proteins: These are pepsin sensitive but trypsin resistant. These proteins are not important for typing the strains. Section III Susceptibility to physical and chemical agents: Streptococci are killed by heating at 54°C for 30 minutes and by usual strengths of disinfectants. They are sensitive to bacitracin. Sensitivity to bacitracin is an important diagnostic feature by which S. pyogenes can be differentiated from other hemolytic streptococci. They are resistant to crystal violet. 186 BACTERIOLOGY TABLE 24-2 Virulence factors of Streptococcus pyogenes Virulence factors Biological functions Cell wall associated polymers and proteins Capsule Prevents phagocytosis Teichoic acid Binds to epithelial cells M protein Adhesin and antiphagocytic; inactivates C3b—an important complement factor responsible for phagocytosis. Strains that are rich in M protein are resistant to phagocytosis and intracellular killing by PMNs. Interferes with opsonisation via the alternative complement pathway F protein Mediates attachment to epithelial cells Enzymes Streptokinase Breaks down the fibrin barrier around the infected site, thereby facilitating spread of the infection Deoxyribonucleases Depolymerizes free DNA present in the pus Hyaluronidase Hydrolyzes hyaluronic acids in the matrix of the connective tissues Chapter 24 Section III Toxins Streptococcal pyrogenic exotoxins (SPEs) Dissolves the clot, thrombi, and emboli; thereby facilitates spread of the bacteria in tissues Streptolysin O and Streptolysin S Lyse erythrocytes, leukocytes, and platelets; and stimulate production of lysosomal enzymes Pyrogenic exotoxins Release large amounts of cytokines from helper T cells and macrophages; rapidly destroy tissues Teichoic acid, M protein, and F protein: Lipoteichoic acid and F proteins mediate the binding of the cocci with fibronectin present on the host cell surface. M protein is an adhesin and antiphagocytic, which inactivates C3b—an important complement factor responsible for phagocytosis. Enzymes Serum opacity factor: This is a lipoproteinase produced by some M types of S. pyogenes. This enzyme produces opacity when applied to agar gel containing swine or horse serum, hence is known as serum opacity factor (SOP). SOP is antigenic. Other enzymes: These include neuraminidase, amylase, esterase, lipase, and beta-glucuronidase. Streptokinase: This enzyme is produced by S. pyogenes as well as by group C and G streptococci. Two types of streptokinase have been described—streptokinase A and B. Streptokinase activates plasminogen to form plasmin, which breaks down the fibrin barrier around the infected site, thereby facilitating the spread of the infection. This thrombolytic property is made good use of in medical management of myocardial infarction. Antibodies appear against streptokinase (A and B) during the course of infection and are diagnostic. Toxins Deoxyribonucleases: Four types of deoxyribonucleases have been described—deoxyribonucleases A, B, C, and D. Most strains of S. pyogenes produce these enzymes. The enzyme depolymerizes free DNA present in the pus, thereby reduces viscosity of pus and helps in spread of the infection. The enzymes are antigenic, and the demonstration of antideoxyribonuclease B antibody in serum is diagnostic of S. pyogenes infections, particularly of skin infections. Dick test: The susceptibility of a person to these toxins is determined by performing a skin test known as Dick test. In this test, 0.2 mL of the diluted toxin is injected intradermally. In a positive test, a localized erythematous reaction, around 1 cm in diameter, develops within 12–24 hours. A positive test indicates absence of antibodies against the toxin and shows susceptibility to the toxin and thus to scarlet fever. No reaction takes place in a negative test, which indicates the presence of specific antibodies against erythrogenic toxin in the serum. Hyaluronidase: The enzyme is produced by S. pyogenes as well as by other groups like B, C, G streptococci, Streptococcus suis, Streptococcus anginosus, and S. pneumoniae. The enzyme splits hyaluronic acid present in host connective tissue, thereby facilitating spread of the bacteria through tissues. The enzyme is antigenic. Streptococcal pyrogenic exotoxins (SPEs): Streptococcal pyogenic exotoxin, otherwise called erythrogenic toxins, are of three types: Spes A, Spes B, and Spes C. ■ ■ These toxins are antigenic, and their production is regulated by a temperate phage in their genome. These toxins act as superantigen and are responsible for scarlet fever and streptococcal toxic shock syndrome. Schultz Charlton test: Intradermal injection of antitoxin in a patient with scarlet fever causes local blanching of the rash. This is due to neutralization of erythrogenic toxin. This test is known as Schultz Charlton test. STREPTOCOCCUS AND ENTEROCOCCUS 187 Hemolysins: Two types of hemolysins, oxygen-labile streptolysin O (SLO) and oxygen-stable and serum-soluble streptolysin S (SLS), are produced by S. pyogenes. Hyaluronic acid capsule, M protein, and C5a peptidase of S. pyogenes are antiphagocytic. These factors prevent opsonization and phagocytosis of bacteria in many ways. Streptolysin O: SLO is an oxygen-labile and heat-labile protein with a molecular weight of 50,000–75,000 Da. It causes betahemolysis only when colonies are grown under the surface of blood agar plate. ■ ■ ■ Is responsible for hemolysis around the colonies grown on surface of the blood agar. Inhibits chemotaxis and is antiphagocytic. ■ ■ ◗ Pyrogenic exotoxin A is similar to that of staphylococcal toxic shock syndrome toxin (TSST). It has the same mode of action as staphylococcal TSST. It acts by releasing large amounts of cytokines from helper T cells and macrophages. Pyrogenic exotoxin B is a protease that rapidly destroys tissues and is produced in large amount by S. pyogenes. Pathogenesis of streptococcal infections S. pyogenes produces suppurative as well as nonsuppurative streptococcal diseases by following mechanisms: 1. Adherence: Adherence of S. pyogenes to surface of host cells is the first stage in pathogenesis of the disease. The cocci adhere to the epithelium of the pharynx with the help of pili, lipoteichoic acid, F proteins, and M proteins. Initially, adherence is mediated by a weak binding between lipoteichoic acid and fatty acid of the cocci with fibronectin of epithelial cells of the host. Subsequently, a strong binding is established by M protein, F protein, and other adhesins of the cocci. 2. Invasion by the cocci: S. pyogenes invades into epithelial cells mediated by M protein, F protein, and other antigens of the cocci. Invasion of the cocci is suggested to be responsible for the persistence of infection, such as streptococcal pharyngitis, as well as for invasion into deep tissues. 3. Production of toxins and enzymes: S. pyogenes then produces a wide variety of toxins and enzymes that contribute to pathogenesis of many streptococcal diseases. ◗ Host immunity Acquired immunity to streptococcal infection is based on the development of specific antibodies against the antiphagocytic epitopes of M protein. The acquired immunity against a particular M type of streptococci lasts longer in untreated persons than in treated persons. Although such antibodies protect from infection against a homologous M protein type, they confer no immunity against other M serotypes. S. pyogenes is a highly communicable bacterium. It can cause disease in people of all ages who do not have type-specific immunity against the specific serotype. Clinical Syndromes S. pyogenes produces a variety of clinical manifestations. These infections can be classified broadly as: (a) suppurative streptococcal diseases, (b) toxin-mediated disease, and (c) nonsuppurative streptococcal diseases. ◗ Suppurative streptococcal diseases Respiratory infections Pharyngitis: S. pyogenes is the most common bacterium causing pharyngitis or sore throat. Pharyngitis is characterized by inflammation of pharyngeal mucosa with exudate formation, tender enlarged cervical lymph nodes, fever, and leukocytosis. The condition is commonly seen in children and it spreads by droplet nuclei. The incubation period is 1–4 days. Uncomplicated pharyngitis resolves within 3–5 days. Skin and soft tissue infections Pyoderma: Pyoderma or impetigo is a localized infection of the skin, primarily affecting face, arms, legs, and other exposed parts of the body. The infection is acquired by direct contact with an infected person or fomites. The condition is caused by a limited number of serotypes (49, 53–55, 59–61, etc.) of S. pyogenes. The condition is seen mainly in young children. In tropics, impetigo is one of the important causes of acute glomerulonephritis (AGN) in children. Chapter 24 Pyrogenic exotoxins: There are two types of exotoxins— exotoxin A and B. ■ Section III Streptolysin S: SLS is a serum-soluble (hence named as S) and oxygen-stable protein. It is a small polypeptide of 20,000 Da and is nonantigenic. Hence, no antibodies against this toxin are demonstrated in serum. This toxin: ■ ■ It is antigenic, and antibodies (ASLO) against it develop in group A streptococcal infection. Demonstration of ASLO antibodies is important for the determination of a recent group A streptococcal infection and also the late complications of streptococcal infections after the organisms have been eliminated from the host. The SLO cross-reacts with similar hemolysins produced by streptococci of groups C and G, pneumolysins of S. pneumoniae, tetanolysin of Clostridium tetani, theta toxin of Clostridium perfringens, cereolysin of Bacillus cereus, and listeriolysin of Listeria monocytogenes. ■ ■ The capsule prevents phagocytosis of the bacteria by acting as a barrier between bacteria and cell. The M protein inactivates C3b, an important complement factor that mediates phagocytosis of bacteria. The M protein also binds to the fibrinogen and blocks the activation of complement by alternate pathway, thereby reducing the amount of C3b production. The C5a peptidase inactivates the complement component C5a, which mediates chemotaxis of neutrophils and phagocytes. 188 BACTERIOLOGY Erysipelas: Erysipelas is an acute and diffused infection of the skin, affecting the superficial lymphatics. It is characterized by red, swollen, and indurated skin with well-marked and raised borders. The affected skin is clearly demarcated from the surrounding healthy area. Commonly, face and legs are affected. Erysipelas occurs most commonly in young children or in older adults. Chapter 24 Section III Cellulitis: Cellulitis is the infection of skin and subcutaneous tissues characterized by local inflammation like edema, erythema, tenderness, fever, headache, malaise, and other systemic manifestations. It is spreading in nature, often without any apparent focus of infection. The entry of the pathogen may be at a location distant to the lesion. Necrotizing fasciitis: Necrotizing fasciitis occurs as a rapidly spreading streptococcal infection of superficial and deep fascia. The infection is caused by certain M strains of S. pyogenes (M types 1 and 3), which produce pyrogenic exotoxins. These strains are also called as “flesh-eating strains” due to the extensive destruction of muscle and fascia caused by them. The condition is also associated with a toxic shock-like syndrome, leading to disseminated intravascular coagulation and multisystem failure. by certain pharyngeal (M types 1, 12) and pyodermal strains (M types 49, 53–55, 59–61) of S. pyogenes. M protein type 49 skin infection is most frequently implicated. The disease occurs as a result of deposition of antigen–antibody complexes on the glomerular basement membrane, initiating inflammation. This leads to the manifestation of disease with hypertension, generalized edema, hematuria, and proteinuria. It has an excellent prognosis, and most young patients usually recover completely. Rheumatic fever: Rheumatic fever is an immunologically mediated disease, which affects the heart, joints, skin, and brain. It has a latent period of 2–4 weeks. It is characterized by fever, migrating polyarthritis, and carditis, and is frequently associated with subcutaneous nodules. Damage to heart valves may occur during the course of infection. Key Points ■ ■ ■ ■ Toxin-mediated diseases Scarlet fever: Scarlet fever is a complication of streptococcal pharyngitis caused by certain strains of S. pyogenes producing pyrogenic exotoxins. It manifests as fever, pharyngitis, and by a characteristic rash. The rash is followed by desquamation. However, with the use of penicillin and other antibiotics, the suppurative complications of pharyngitis including scarlet fever have become rare. Streptococcal toxic shock syndrome: Streptococcal toxic shock syndrome is caused by certain strains of S. pyogenes (M serotypes 1 or 3) that have prominent hyaluronic acid capsule. This is a condition similar to staphylococcal toxic shock syndrome. The condition manifests initially as pain at the site of inflammation and nonspecific systemic complaints, such as nausea, vomiting, diarrhea, fever, and chills. The condition progresses subsequently to multiorgan failure and shock. S. pyogenes is always isolated from the blood in toxic shock syndrome. The condition occurs in people of all ages. However, patients with HIV infection, varicella-zoster infection, diabetes, heart diseases, and intravenous drug and alcohol abusers are at high risk for streptococcal toxic shock syndrome. Other suppurative streptococcal diseases Other pyogenic infections caused by S. pyogenes include lymphangitis, puerperal sepsis, abscesses of the internal organs (liver, lungs, kidneys, brain, etc.), and bacteremia. ◗ Nonsuppurative streptococcal diseases Acute glomerulonephritis: AGN is a nonsuppurative complication of S. pyogenes infection. The onset of infection typically occurs 2–3 weeks following skin infection or pharyngitis caused Rheumatic fever occurs mainly due to antigenic crossreaction between streptococcal proteins and the connective tissue antigens of the heart and joints. It is an autoimmune disease exacerbated by recurrence of streptococcal infection. The diagnosis of this condition is mainly clinical, supplemented by relevant laboratory investigations. If streptococcal infections are treated within 8 days after onset, rheumatic fever is usually prevented. The comparison of AGN and rheumatic fever is presented in Table 24-3. Epidemiology ◗ Geographical distribution S. pyogenes infections are worldwide in distribution. ■ ■ ◗ Prevalence of streptococcal pyoderma is higher in tropics with no seasonal variation, whereas it is more common in winter months in temperate countries. Rheumatic fever is most frequently observed in children aged 5–15 years, the age group most susceptible to S. pyogenes infections. The attack rate of rheumatic fever following upper respiratory tract infection is approximately 3% for persons with untreated or inadequately treated infections. Habitat Streptococci are normal flora of the oral cavity, nasopharynx, skin, fingernails, perianal region, intestine, and upper respiratory tract of humans. ◗ Reservoir, source, and transmission of infection Infected human cases are the reservoirs of infection. Respiratory and salivary secretions in the form of droplets and contaminated fomites are the sources of S. pyogenes infection. Streptococcal carrier rate as high as 20–40% has been reported. However, these carriers with chronic asymptomatic pharyngeal and nasopharyngeal colonization are not usually STREPTOCOCCUS AND ENTEROCOCCUS TABLE 24-3 189 Differences between acute glomerulonephritis and acute rheumatic fever Particulars Acute rheumatic fever Acute glomerulonephritis Hereditary factors Contribute Not known Initial site of infection Throat Skin/throat Prior exposure to Streptococcus pyogenes Essential Not required Serotypes involved All serotypes Pyodermal strains: M types 42, 49, 53–55, 59–61; throat infection strains: 1, 2, and 4 Immune response Prominent Lower Complement level Unaffected Decreased Repeated attacks Common Absent Penicillin prophylaxis Essential Not required Course of the disease Static/progressive Complete recovery Prognosis Variable Excellent at risk of spreading disease, as they mostly inhabit avirulent organisms. Overcrowding (crowded homes and class rooms) is an important factor in transmission of S. pyogenes infection. Both impetigo and pharyngitis are more likely to occur in children living in crowded homes and under poor hygienic conditions. Bacteriocin and phage typing of streptococci are employed in research and epidemiologic studies. Laboratory Diagnosis ◗ Specimens The nature of specimens to be collected for bacteriological investigations of S. pyogenes infections depends upon the disease manifestations. The frequently used specimens include: ■ ■ ◗ Throat swab, nasal swabs, high vaginal swabs (puerperal sepsis), pus or pus swabs, pharyngeal secretions, blood, cerebrospinal fluid, joint aspirate, edge aspirate of cellulitis, skin biopsy specimen, epiglottic secretions, bronchoalveolar lavage fluid, thoracocentesis fluid, or abscess fluid. A frozen section biopsy obtained in the operating room may be used in cases of suspected necrotizing fasciitis. Microscopy Gram staining of pus or exudate is a rapid and presumptive diagnostic procedure for S. pyogenes infection of skin and soft tissues. The presence of Gram-positive cocci in pairs FIG. 24-3. Gram-stained pus smear showing streptococci in chains (X1000). and chains (Fig. 24-3) in association with leukocytes is suggestive of streptococcal infection. This is because S. pyogenes are not found as normal flora on the skin surface. However, demonstration of streptococci in respiratory specimens from a patient with pharyngitis by Gram staining is of no value, because the streptococci are found as part of the normal flora in the oropharynx. ◗ Culture Throat swab culture is the most specific method for diagnosis of streptococcal pharyngitis. Ideally, the throat swab specimen should be collected from tonsils and posterior part of the oropharynx because more number of bacteria are present at this site than in the anterior part of mouth. After collection of the specimen, they are plated immediately on the blood agar plate, and in case of delay they are sent to the laboratory in Pikes transport medium. In laboratory, the specimens are inoculated on a 5% sheep blood agar and incubated at 37°C aerobically in the presence of 5–10% CO2 for 2–3 days. Chapter 24 ■ Person-to-person transmission is the main route of transmission. The infection is transmitted from person to person through respiratory droplets. The infection is also transmitted through breaks in the skin by direct contact with infected patient, fomites, or arthropod vectors. Children with untreated acute infections spread organisms by their salivary droplet and nasal discharge. Occasional food-borne and waterborne outbreaks have also been documented. Section III ■ 190 BACTERIOLOGY Box 24-1 1. 2. 3. 4. 5. Chapter 24 Section III ◗ Identifying features of Streptococcus pyogenes Gram-positive cocci arranged in short chains or pairs. On blood agar, produces a clear zone of hemolysis (beta-hemolysis). Positive for bacitracin susceptibility test. Positive for L-pyrrolidonyl-alpha-naphthylamide (PYR) test. Positive for group-specific C antigen by direct antigen detection tests. Identification of bacteria Culture of the swabs on blood agar shows a clear zone of beta-hemolysis (Fig. 24-2) surrounding the small translucent to opaque colonies. The identifying features of S. pyogenes are summarized in Box 24-1. Bacitracin sensitivity test: S. pyogenes can be distinguished from other streptococcal groups by their sensitivity to bacitracin. In this method, a filter paper disc containing 0.04 U of bacitracin is applied on the surface of an inoculated blood agar and is incubated overnight. Any zone of inhibition around the colonies confirms the presence of S. pyogenes (Fig. 24-4, Color Photo 16). Bacitracin test is simple to perform and is useful for presumptive identification of S. pyogenes. It is positive in more than 95% of S. pyogenes strains and negative in nongroup A streptococci. L-pyrrolidonyl-beta-naphthylamide test: This is a test performed to differentiate S. pyogenes from other beta-hemolytic streptococci. S. pyogenes produces the enzyme L-pyrrolidonylbeta-naphthylamidase (PYRase), which hydrolyzes L-pyrrolidonyl-beta-naphthylamide to produce a substance called beta-naphthylamine. This substance can be detected in the presence of p-dimethylamino cinnamaldehyde by formation of a characteristic red color after applying a disc containing p-dimethylamino cinnamaldehyde on an inoculated agar plate followed by overnight incubation. This makes the presumptive identification of a strain as group A streptococci. Direct antigen detection test: Detection of group-specific carbohydrate antigen A directly in the throat swabs by direct fluorescent antibody test is a very rapid and specific method. The result for this test is obtained within 4 hours. The test is as specific as culture but is less sensitive. ◗ Serodiagnosis Serological tests are of value in the diagnosis of AGN and rheumatic fever. These tests detect high level of antibodies produced against many streptococcal antigens. The tests detecting antibodies against SLO (anti-SLO, or ASO antibodies) are most frequently used for confirming rheumatic fever and AGN. The ASO antibodies appear in serum 3–4 weeks after initial infection by S. pyogenes. A titer of more than 200 indicates streptococcal infections. Higher antibody titers are found in acute rheumatic fever, whereas they are not raised in patients with glomerulonephritis and streptococcal pyoderma. Antibodies against other streptococcal enzymes, such as DNAase B (anti-DNase B antibodies), hyaluronidase (anti-hyaluronidase antibodies), and streptokinase (anti-streptokinase antibodies) are also demonstrated in S. pyogenes infections. The demonstration of antibodies against these antigens may prove useful in the diagnosis of streptococcal pharyngitis and pyoderma. Treatment Treatment of S. pyogenes infections by antibiotics varies depending upon the clinical conditions. Penicillin is highly effective against S. pyogenes. As of now, no penicillin-resistant strains of S. pyogenes have been documented in clinical practice. Penicillin, therefore, remains the drug of choice, except in penicillin-allergic individuals (with pharyngeal infections) and in complicated or invasive diseases. Failures of penicillin therapy: In uncomplicated cases, penicillin is given orally in a dosage of 250–500 mg twice daily for at least 10 days. Noncompliance is the most common reason for the failure to respond to therapy. The drug is often discontinued before the 10-day course is completed, because children usually appear to have recovered in 3–4 days. And the presence of betalactamase-producing flora (particularly organisms, such as mouth anaerobes), which could inactivate penicillin, has also been proposed. However, this theory is yet to be proved conclusively. Most of the failures of penicillin therapy have been thought to occur in patients where streptococcal pharyngitis has not been well defined and some of these patients may in fact be streptococcal carriers who actually had viral pharyngitis. ■ ■ Erythromycin and clindamycin are given to patients allergic to penicillins. Recently, strains resistant to erythromycin have been reported. Sulfonamides and tetracycline are not used for streptococcal infections. Prevention and Control FIG. 24-4. Bacitracin sensitivity of Streptococcus pyogenes. Chemoprophylaxis is most important in prevention of AGN or rheumatic fever. STREPTOCOCCUS AND ENTEROCOCCUS ◗ 191 Chemoprophylaxis Long-term chemoprophylaxis using penicillins to prevent streptococcal infection is recommended for patients with a history of acute rheumatic fever (up to age 21 years) or rheumatic heart disease (lifelong). Antibiotic prophylaxis prevents streptococcal reinfection and further damage to the heart. The role of chemoprophylaxis for household contacts of patients with either acute streptococcal disease or nonsuppurative complications is yet to be ascertained. Vaccines Multivalent streptococcal vaccine containing multiple M protein epitopes has been evaluated; its efficacy is being proved in animal models. The vaccine is still in experimental stage and yet to be used in clinical practice. FIG. 24-5. ■ ■ ■ S. agalactiae in neonates can cause either early-onset or lateonset infections. Early-onset infection is acquired either in utero or from mother’s vagina during delivery. The clinical symptoms develop during the first week of life. The condition is characterized by septicemia, meningitis, or pneumonia. Late-onset infection is acquired from mother or from another infant (environment) during 2–12 weeks of life. The condition manifests as septicemia and meningitis. S. agalactiae in pregnant women causes urinary tract infection particularly immediately after delivery. In nonpregnant women and in men, S. agalactiae can cause infections, such as osteomyelitis, arthritis, peritonitis, and skin infections. The condition is diagnosed by culturing the specimen in the blood agar and identifying the colonies by various tests as mentioned below: 1. CAMP test: CAMP (Christie, Atkins, Munch-Peterson) test was first described in 1944 by Christie, Atkins, and MunchPeterson. The basis for the test is that S. agalactiae produces a diffusible and heat-stable protein, known as CAMP factor, which accentuates hemolysis of RBCs. The staphylococci Other Hemolytic Streptococci Streptococci belonging to groups C, F, and G, and rarely H, K, O, and R can also be associated with human infections. Group C Streptococci Group C streptococci are usually pathogens of animals. Streptococcus equisimilis is a group C Streptococcus, which can Chapter 24 S. agalactiae is the only species belonging to group B streptococci. This is a pathogen of the cattle causing bovine mastitis, hence named “agalactiae”. S. agalactiae are Gram-positive cocci arranged in pairs and short chains in clinical specimens and are morphologically similar to S. pyogenes. The cocci grow readily on enriched medium, such as blood agar and produce large colonies after overnight incubation. S. agalactiae are found as commensals in the genitourinary tract and lower gastrointestinal tract. Vaginal carriage rate as high as 40–50% has been observed in some pregnant women. More than 50% of infants born to these mothers through vaginal delivery are colonized with S. agalactiae. produce an enzyme sphingomyelinase C, which binds to the RBCs present in the blood agar. On exposure to CAMP factor liberated by S. agalactiae, RBCs undergo hemolysis, producing a butterfly appearance (Fig. 24-5, Color Photo 17). In this test, S. aureus is streaked from top to bottom on a blood agar plate. Then perpendicular streaks of S. agalactiae are made on either side, leaving at least 1 cm space from S. aureus. The plate is incubated overnight at 37°C in 20% CO2. Haemolysis showing a typical butterfly appearance indicates a positive test. S. agalactiae is CAMP positive. 2. Hippurate hydrolysis test: S. agalactiae are hippurate positive. They hydrolyze the hippurate to produce hippuric acid. 3. Demonstration of group-specific cell wall antigen: The bacteria are identified by the group-specific cell wall polysaccharide antigen or B antigen. This antigen is composed of rhamnose, N-acetylglucosamine, and galactose. 4. Demonstration of type-specific capsular antigen: Depending upon type-specific capsular polysaccharide antigens, S. agalactiae strains have been classified into 11 distinct serotypes (Ia, Ia/c, Ib/c, II, IIc, III, IV, V, VI, VII, and VIII). Serum antibodies confer specific protection against these serotypes. Penicillin is the drug of choice for treatment of S. agalactiae infection. Vancomycin is given to persons allergic to penicillins. Recently, strains resistant to erythromycin and tetracycline have been documented. Section III Streptococcus agalactiae CAMP test for Streptococcus agalactiae. 192 BACTERIOLOGY cause occasional infections in humans. S. equisimilis resembles S. pyogenes in fermenting trehalose but differs from it by not fermenting ribose. Like S. pyogenes, it also produces SLO, streptokinase, and other proteins. It causes upper respiratory tract infections and also pneumonia, osteomyelitis, endocarditis, brain abscess, and puerperal sepsis. S. equisimilis shows tolerance to treatment with penicillin; therefore patients may not respond to treatment with penicillin. Group F Streptococci These cocci are called “minute streptococci”. They grow on blood agar well in the presence of CO2. Streptococcus MG is a member of this group that can cause primary atypical pneumonia in humans. Demonstration of cold agglutinins in serum is diagnostic of primary atypical pneumonia. Characters Streptococcus pyogenes Streptococcus agalactiae Viridans streptococci CAMP test ⫺ ⫹ ⫺ Hippurate hydrolysis ⫺ ⫹ ⫺ Sensitive Resistant Resistant Bacitracin susceptibility ■ Chapter 24 Section III Group G Streptococci Group G streptococci are the commensals of humans and of animals, such as monkeys and dogs. They may occasionally cause infections, such as tonsillitis, urinary tract infection, and endocarditis in humans. Nonenterococcal Group D Streptococci Streptococcus bovis and Streptococcus equinus are the nonenterococcal group D streptococci, which are associated with human infections, such as urinary tract infections and rarely endocarditis. They are susceptible to penicillins. Viridans Streptococci Viridans streptococci are a heterogenous group of alphahemolytic and nonhemolytic streptococci. These are found as commensal flora in the oral cavity, oropharynx, gastrointestinal tract, and genitourinary tract. These bacteria produce a green pigment on the blood agar and hence are called viridans (Latin for “green”). Most isolates of viridans streptococci do not possess a group-specific carbohydrate; hence, they cannot be classified under Lancefield classification of streptococci. These cocci, however, have been classified into different species, such as Streptococcus mutans, Streptococcus sanguis, Streptococcus mitis, S. salivarius, etc. based on various properties, such as (a) cell wall composition, (b) production of dextrans and levans, and (c) fermentation of sugars. Viridans streptococci are nutritionally fastidious, requiring complex media supported with blood for their growth. Growth of the colonies is facilitated by the presence of 5–10% carbon dioxide. Some strains are nutritionally deficient, requiring supplementation of pyridoxal, the active form of vitamin B6 for their growth. Viridans streptococci can cause a variety of infections. They are commonly implicated in dental caries, subacute bacterial endocarditis, and intra-abdominal suppurative infections. Important biochemical characters of common streptococci TABLE 24-4 ■ S. sanguis is the most common causative agent of bacterial endocarditis in individuals with preexisting heart lesions. There is a transient bacteremia following tooth extraction or other dental procedures after which bacteria adhere to the damaged heart valves or prosthetic heart valves. Prophylactic use of antibiotics before dental procedures prevents such complications. S. mutans is an important causative agent of dental caries. It splits dietary sucrose, producing acid and a dextran. The acid damages the dentine. The dextran binds together exfoliative epithelial cells, mucus, food debris, and bacteria to form dental plaques. Earlier, most strains of viridans streptococci were sensitive to penicillins. However, recently moderately resistant and highly resistant strains have been reported particularly in the S. mitis group. Broad-spectrum cephalosporins or vancomycin are recommended for treatment of these penicillin-resistant strains. Table 24-4 summarizes the differences between important biochemical characteristics of common streptococci. Enterococcus The enterococci were classified earlier as group D streptococci, because they possess the group D cell wall antigen. These enterococci, however, showed several distinctive features (Table 24-5) by which they were separated from the streptococci. Thiercelin proposed the term Enterococcus in 1899. The term enterococcal group was used by Sherman to describe the streptococci that grew at 10–45°C, with pH 9.6, in broth containing 6.5% sodium chloride and survived heating to 60°C for 30 minutes. Based on acid formation from mannitol, sorbitol, and sorbose TABLE 24-5 Features for distinguishing Streptococcus and Enterococcus Streptococcus Enterococcus Arrangement of bacteria Pairs or in short chains Pairs of oval cocci Growth in the presence of 40% bile ⫺ ⫹ Growth in the presence of 6.5% sodium chloride ⫺ ⫹ Growth at 45°C ⫺ ⫹ Growth at pH 9.6 ⫺ ⫹ Characters STREPTOCOCCUS AND ENTEROCOCCUS broth and hydrolysis of arginine, genus Enterococcus is classified into five groups (Table 24-6). The genus Enterococcus has 16 species. Enterococcus faecalis and Enterococcus faecium are two important species known to cause human infections. The enterococci are Gram-positive, spherical, oval, or coccobacillary and are arranged in pairs and short chains. Most of the species are nonmotile and noncapsulated. They grow at a temperature range of 35–37°C. Colonies on blood agar media are 1–2 mm in diameter and alpha-hemolytic (actually nonhemolytic; appearance of alpha-hemolysis is due to the production of the enzyme peroxidase rather than hemolysins). Some cultures are beta-hemolytic on agar containing rabbit, horse, or human blood but not on agar containing sheep blood. Enterococcus durans is beta-hemolytic on agar containing sheep blood as well. On MacConkey agar they produce tiny and magenta-colored colonies. On potassium tellurite agar they produce black colonies. Bile-esculin-azide Classification of Enterococcus Species Group I Enterococcus avium Group II Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum Group III Enterococcus durans Group IV Enterococcus sulfurous and Enterococcus cecorum Group V Variants of Enterococcus faecalis, Enterococcus gallinarum CASE STUDY A 32-year-old male was admitted to a hospital with fever, chills, and generalized body pain. The patient had received multiple courses of antibiotics prescribed by private practitioners. Serum of the patient was tested positive for HIV by ELISA. Blood culture showed growth with Gram-positive cocci. The colonies were positive with group D antisera. These were PYRase test positive, CAMP test negative, resistant to bacitracin, and they did not hydrolyze hippurate. The bacterial isolate was identified as enterococci. The isolate showed resistance to penicillin and vancomycin. ■ ■ ■ Which is the most likely bacteria species to cause this condition? What are the other tests you will perform to identify the bacteria? What antibiotics you will use to treat this infection? Chapter 24 Groups medium and Columbia colistin-nalidixic acid agar are used as selective media. They are catalase negative and are resistant to optochin and bile. They ferment sucrose, sorbitol, mannitol, and esculin. They are PYRase test positive, CAMP test negative, resistant to bacitracin, and they do not hydrolyze hippurate. They possess two important virulence factors: (a) aggregation substances and (b) carbohydrate adhesions. Aggregation substances are hair-like proteins that facilitate binding of bacteria to the epithelial cells. Carbohydrate adhesins facilitate binding of cocci to host cells gelatinase. Cytolysin and pheromone are the other virulence factors. The cell wall of the bacteria possesses group-specific antigen, which is group D glycerol teichoic acid. The enterococci are commonly found in gastrointestinal and genital tract of humans and animals. Enterococci are nonpathogenic but now are emerging as important agents of nosocomial infection. They cause urinary tract infection especially in hospitalized patients. Indwelling catheters and urinary tract instrumentation are important predisposing factors. They are frequently isolated from cases of wound infections particularly intra-abdominal. They also cause bacteremia, infection of the bile duct, and endocarditis. The antimicrobial therapy includes combination of aminoglycosides with penicillin, ampicillin, or vancomycin. Enterococci are less sensitive to penicillin and gentamicin and are resistant to cephalosporins. Plasmid-mediated resistance is a major cause of resistance among drug-resistant strains of Enterococcus. Vancomycin-resistant Enterococcus (VRE) has been emerging in the recent years. Section III TABLE 24-6 193 43 25 Pneumococcus Mycobacterium Leprae Introduction Pneumococcus was earlier classified as Diplococcus pneumoniae. The bacterium has now been reclassified as Streptococcus pneumoniae due to its genetic similarities to Streptococcus. S. pneumoniae, however, differs from streptococci in its morphology (by having a specific polysaccharide capsule), bile solubility, and optochin sensitivity. or noncapsulated), the type of incubation (whether aerobic or anaerobic), and the time of incubation: ■ ■ Streptococcus pneumoniae ■ A lot of advances have been made toward the better understanding of the pathogenesis, antibiotic resistance, and use of vaccines in pneumococcal infections caused by S. pneumoniae. Properties of the Bacteria ◗ Morphology S. pneumoniae shows the following morphological features: ■ ■ ■ ◗ They are Gram-positive cocci measuring 0.5–1.25 m in diameter. Older cells decolorize rapidly and might appear Gram negative. In clinical specimens, they appear typically “lanceolate shaped” with one end pointed and the other end round. They are arranged in pairs (diplococci) with the broad ends in apposition to each other. In cultures, they usually appear more rounded and are arranged in short chains (Color Photo 18). They are capsulated. A polysaccharide capsule completely envelops each pair of cocci. The capsule is visualized by staining it directly with specific stains or by Indian ink negative staining or by Quellung reaction (Color Photo 19). They are nonsporing and nonmotile. Colonies on blood agar in anaerobic incubation show beta-hemolysis (greenish discoloration), but show alphahemolysis in aerobic incubation (Color Photo 20). Capsulated strains after overnight incubation produce round and mucoid colonies measuring 1–3 mm in diameter. Some strains, e.g., type 3 S. pneumoniae (most virulent), produce copious quantities of capsular material and hence produce large mucoid colonies. Noncapsulated strains produce small and flat colonies. On prolonged incubation, the colonies undergo autolysis and the centers become flattened or depressed (umbonation) and edges become raised, giving the colonies a typical draughtsman appearance (Fig. 25-1). The central flattening or depression is due to the production of intracellular enzymes, such as amidase, which lyses the bacteria. Bile salts, sodium lauryl sulfate, and other surface active agents enhance the process of autolysis of the bacterial colonies. The capsules are present in strains isolated from clinical specimens but are lost on repeated cultivation, which is called smooth to rough variation. Smooth to rough variation: The capsules are present in strains isolated from clinical specimens but are lost on repeated cultivation. This is called smooth to rough variation. Noncapsulated rough (R) strains are avirulent; these forms arise as spontaneous mutants and outgrow the parental smooth (S) strains in artificial culture. In tissues R forms are eliminated by phagocytosis. Transformation of a rough strain to a smooth one is also possible by treatment with smooth capsular substance containing DNA. Culture S. pneumoniae is an aerobe and a facultative anaerobe. It grows at an optimum temperature of 37°C (range 25–42°C) and pH 7.8 (range 6.5–8.3). The growth is enhanced by the presence of 5–10% CO2. Pneumococci are fastidious. They grow only on an enriched media, such as blood agar or chocolate agar (supplemented with blood products), which will supply nutrients, pH buffers, etc. Blood agar: On blood agar, morphology of the colonies varies depending on the nature of the strain (whether capsulated Draughtsman colony FIG. 25-1. Draughtsman colonies of Streptococcus pneumoniae. PNEUMOCOCCUS ◗ Biochemical reactions S. pneumoniae shows following reactions: ■ ■ ■ ■ ■ ◗ S. pneumoniae ferments many sugars, producing acid only but no gas. Fermentation of sugars is carried out in Hiss’s serum water or in serum agar slopes. S. pneumoniae ferments inulin, and this is an important test to differentiate it from those streptococci that do not ferment inulin. S. pneumoniae produces an autolytic enzyme amidase, which solubilizes the peptidoglycan of the cell wall; hence in old cultures, typical draughtsman colonies are formed. This autolytic activity can be augmented by surface active agents, such as bile and bile salts. Bile solubility is a constant feature of pneumococci, and is positive in all the capsulated and some noncapsulated variants. Pneumococci are catalase and oxidase negative. Other properties differentiate these from streptococci. Optochin sensitivity is usually performed by a paper disk containing 5 g of the compound; an inhibition zone of 14 mm or more indicates sensitivity to optochin. In recent times, occasional optochinresistant pneumococci have also been documented. The target of optochin in pneumococci is an H  ATPase, and resistance is thought to be due to a point mutation in one of subunit a or c of the H  ATPase. This resistance to optochin is not related to the virulence of the bacteria. Cell Wall Components and Antigenic Structure The cell wall of S. pneumoniae consists of the following components (Fig. 25-2): ■ ■ ■ ■ Capsule: The polysaccharide capsule surrounds the cell wall. The capsule is essential for virulence, its role being to protect the cocci from phagocytosis. Cell wall: It consists of peptidoglycan, teichoic acid, and proteins. Peptidoglycan: Peptidoglycan confers rigidity to the cell wall. Teichoic acid: The teichoic acid present in cell wall is of two types: (a) C polysaccharide and (b) Forssman or F antigen. ● C polysaccharide: It is present on the surface of cell wall. The exposed part of teichoic acid, which is linked Penicillin-binding proteins Polysaccharide coat Peptidoglycan Teichoic acid Lipoteichoic acid Cell membrane TAs are linked to the peptidoglycan via a phosphodiester linkage, whereas LTAs are linked to the cell membrane via a C-terminal fatty acylgroup. Choline-binding proteins are linked to cell-wall TAs or LTAs via choline-binding domains (CBDs). Penicillin-binding proteins (PBPs) are located in the periplasmic space and interact with the peptidoglycan. FIG. 25-2. Schematic diagram of antigenic structure of Streptococcus pneumoniae. Chapter 25 Choline-binding proteins Section III Susceptibility to physical and chemical agents: Pneumococci are delicate bacteria. They are killed by heating at 52°C for 15 minutes and by usual strengths of disinfectants. Pneumococcal colonies die on prolonged incubation. Optochin sensitivity test: Pneumococci are sensitive to optochin (ethyl hydrocupreine)—a useful property to 195 Chapter 25 Section III 196 ■ ◗ BACTERIOLOGY to peptidoglycan layer and extends through the capsule, is known as C polysaccharide or C antigen. This is a species-specific antigen and in no way related to the groupspecific C carbohydrate antigen found in beta-hemolytic streptococci. The C polysaccharide present in the cell wall of pneumococci precipitates with C reactive protein (CRP), a serum globulin. ❍ C-reactive protein: The CRP is an acute-phase substance synthesized in the liver. It is not produced specifically against C antigen of pneumococci. It is present in low concentrations in healthy individuals, but the concentration increases in inflammation, malignancies, and bacterial infections. CRP increases during pneumonia and disappears during convalescence, hence can be used as a prognostic tool. CRP is used as an index of the treatment in rheumatic fever and certain other conditions. ● F antigen: F antigen is the other type of teichoic acid, which is covalently bound to the lipids in cytoplasmic membrane. It is so called because it cross-reacts with the Forssman surface antigen of the mammalian cells. F proteins mediate the binding of pneumococci to the host cell surface. M protein: M protein is a type-specific protein similar to the M protein of Streptococcus pyogenes, but is immunologically distinct. Antibodies against M proteins are not protective, as they do not inhibit phagocytosis of pneumococci. Serotyping of pneumococci Capsular polysaccharide is antigenic in humans and rabbits. The capsular polysaccharide is also called specific soluble substance (SSS) as it diffuses into the culture medium or infective pus or host tissues. S. pneumoniae are classified into more than 90 different serotypes (1–90) based on the antigenic structure of the capsular polysaccharide. Of these, only 23 serotypes are associated with pneumococcal diseases. Serotyping of S. pneumoniae is not carried out routinely and is done only for epidemiological studies. There are three typing methods: ■ ■ ■ Agglutination of the bacteria with type-specific antiserum by co-agglutination (Co-A) test. The Co-A test is a rapid slide agglutination test, uses less antiserum, and is in complete agreement with Quellung test. Precipitation of the specific soluble substances with specific serum by counter-current immunoelectrophoresis (CIEP). This is also a rapid test used to serotype pneumococci. Capsular swelling reaction with type-specific antiserum by “Quellung reaction”. This is called capsular swelling reaction due to swelling (Latin quellung: swelling) of the capsule observed in this test. This reaction was first described by Neufeld in the year 1902. In this test, a drop of type-specific antiserum is added to a drop of suspension of pneumococci on a glass slide along with a drop of methylene blue solution. The capsule, in the presence of the specific homologous antiserum, becomes apparently swollen, clearly delineated, and refractile. Pathogenesis and Immunity Pneumococci cause disease primarily by their capacity to multiply in host tissues by avoiding the host defense mechanisms (Table 25-1). ◗ Virulence factors Capsule: Virulent strains of S. pneumoniae have a complex polysaccharide capsule. The acidic and hydrophilic nature of the capsule allows the bacteria to escape phagocytosis by macrophages. Pneumolysin: Pneumolysin is a toxin produced by pneumococci. The toxin alters the mucociliary clearance function of respiratory epithelium and inhibits phagocytic cell oxidative burst essential for intracellular killing of the bacteria. It activates the classical complement pathway resulting in the production of C3a and C5a, thereby contributing to the disease process. IgA protease: The enzyme produced by pneumococci disrupts secretory IgA-mediated clearance of the bacteria and thereby enhances the ability of the bacteria to colonize mucosa of the upper respiratory tract. Cell wall associated polymers and proteins: These include teichoic acid, peptidoglycan, protein adhesin, phosphorylcholine, F protein, etc. that contribute to pathogenesis of pneumococcal diseases in various ways as mentioned in Table 25-1. TABLE 25-1 Virulence factors Virulence factors of Streptococcus pneumoniae Biological functions Cell wall associated polymers and proteins Capsule Prevents phagocytosis Teichoic acid Binds to epithelial cells and activates alternative complement pathway Peptidoglycan Activates alternative complement pathway Protein adhesin Binds to epithelial cells Phosphorylcholine Mediates invasion of host cell by cocci F protein Mediates attachment to epithelial cells Enzymes Secretory IgA protease Destroys secretory IgA Toxins Pneumolysin Alters mucociliary clearance function of respiratory epithelium and inhibits phagocytic cell oxidative burst essential for intracellular killing of the bacteria Autolysin N-acetyl muramoyl-L-alanine amidase that along with a glycosidase enzyme function during cell division to separate daughter cells and to break down the organism after exponential growth PNEUMOCOCCUS ◗ Pathogenesis of pneumococcal diseases Host immunity Host immunity is type specific with production of anticapsular antibodies. These antibodies appearing in serum 5–8 days after the onset of infection are protective against the pneumococcal serotype causing the infection. Natural immunity follows infections as well as colonization. Clinical Syndromes Ninety serotypes of S. pneumoniae have been identified with varying degrees of pathogenicity, out of which 23 serotypes are known to cause disease in humans. S. pneumoniae serotypes 3, 4, 6B, 9V, 14, 18C, 19F, and 23F cause the majority of invasive disease. S. pneumoniae causes (a) pneumonia, ( b) meningitis, (c) sinusitis and otitis media, (d ) bacteremia, and (e) other infections. Pneumonia S. pneumoniae is the leading cause of bacterial pneumonia, both lobar and bronchopneumonia. Pneumonia develops when bacterium multiplies in the alveoli. Since the disease is associated with aspiration and is localized in the lower lobes of the lungs, it is called lobar pneumonia. Pneumonia is common at the extreme of ages, in children and in elderly, who have a more generalized bronchopneumonia. S. pneumoniae is the most common bacterial cause of childhood pneumonia, especially in children younger than 5 years. Serotypes 6, 14, 18, 19, and 23 are responsible for most cases of pneumonia in children, while serotypes 1, 3, 4, 7, 8, and 12 cause pneumonia in adults leading to mortality in more than 5–10%. Haemophilus influenzae and Moraxella catarrhalis are the other causes of acute pneumonia. Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella spp. are the causative agents of atypical pneumonia. ◗ Meningitis Pneumococcus is the most common cause of pyogenic meningitis in children, although the condition can occur in all age groups. Meningitis is always secondary to other pneumococcal infections, such as pneumonia, bacteremia, infections of the ear or sinuses. The bacteria reach the brain through blood stream or from nasopharynx (following head trauma or dural tear particularly with cerebrospinal fluid leak). Pneumococcal meningitis is now emerging as a common cause of death in children and in adults. Meningitis caused by S. pneumoniae is associated with a higher mortality and more neurological complications than the meningitis caused by any other bacteria. Even with antibacterial therapy, the mortality due to pneumococcal meningitis is nearly 25%. Streptococcus agalactiae, Escherichia coli, Neisseria meningitidis, H. influenzae type B, Listeria monocytogenes, Pseudomonas spp., Flavobacterium meningosepticum, and Staphylococcus aureus are the other bacteria causing meningitis. ◗ Sinusitis and otitis media Sinusitis and otitis media occur in patients with prior viral infections. The viral infection lowers the mucosal immunity, facilitating the invasion by S. pneumoniae. Sinusitis caused by the pneumococci occurs in patients of all ages, but otitis media caused by the bacteria is seen only in young children. Pneumococci cause approximately 40% of otitis media cases. H. influenzae is another causative agent. ◗ Bacteremia This condition is more frequent in children than in adults. Bacteremia occurs in more than two-thirds of patients with meningitis and in one-fourth of the patients with pneumococcal pneumonia. This does not occur in patients with sinusitis or otitis media. Chapter 25 ◗ ◗ Section III S. pneumoniae causes disease through the following stages: (i ) colonization and invasion, (ii ) tissue destruction, and (iii ) avoidance of opsonization and phagocytosis. Colonization and invasion: S. pneumoniae colonizes the oropharynx by adhering to the epithelial cells of pharynx. This adhesion is mediated by pneumococcal neuraminidase or by pneumococcal cell-surface ligands of the cocci called adhesins. The cocci also release an enzyme, secretory IgA protease, which destroys the secretory IgA and thereby enhances the ability of the cocci to colonize mucosa of the upper respiratory tract. Pneumolysin liberated by the cocci destroys the ciliated epithelial cells and phagocytic cells by binding to cholesterol in the epithelial cell membrane. This adversely affects mucociliary clearance function of respiratory epithelium. Tissue destruction: The process of tissue destruction is mediated by factors, such as cell wall teichoic acid, peptidoglycan, and phosphorylcholine. The destructive action is further supplemented by hydrogen peroxide produced by the bacteria. The peptidoglycan–teichoic acid complex of the pneumococcus is highly inflammatory. This complex activates the alternate complement pathway producing C5a, which being chemotaxic attracts neutrophils to the site of inflammation. Migration of inflammatory cells to the site of infection is the key feature of pneumococcal infection. The activated leukocytes produce cytokines, such as IL-1 and TNF-, which further contribute to the migration of inflammatory cells to the site of infection, tissue damage, fever, and other manifestations of pneumococcal disease. Phosphorylcholine present in the bacterial cell wall binds with phosphodiesterase-activating factors of the host endothelial cells and helps the cocci to enter the host cells. Avoidance of opsonization and phagocytosis: The ability to evade phagocytosis allows S. pneumoniae to survive, multiply, and spread to various organs. The antiphagocytic action of capsule is further supplemented by pneumolysin, which causes inhibition of phagocytic cell oxidative burst, required for intracellular killing of bacteria. 197 198 ◗ BACTERIOLOGY Other infections These include spontaneous bacterial peritonitis, postsplenectomy sepsis, endocarditis associated with rapid destruction of heart valves, bone and joint infections (prosthetic or natural joint septic arthritis, occasionally as a complication of rheumatoid arthritis), myositis, and brain and epidural abscesses. All these infections result from seeding of tissues during bacteremia. Epidemiology Chapter 25 Section III ◗ Geographical distribution S. pneumoniae is worldwide in distribution. It is the major cause of community-acquired bacterial pneumonia. S. pneumoniae serotypes 6, 14, 18, 19, and 23 are usually associated with infections in children, whereas serotypes 1, 3, 4, 7, 8, and 12 predominate in infections in adults. S. pneumoniae serotypes 6, 14, 18, and 23 cause 60–80% of lower respiratory tract infections. Pneumococcal infection accounts for more deaths than any other vaccine preventable disease. Children between 6 months and 4 years of age and adults over 60 years of age are most commonly at risk for pneumococcal infection. In developing countries like India, the incidence of pneumococcal diseases in children is many times higher than that in the developed countries. ◗ FIG. 25-3. Gram-stained smear of CSF showing Streptococcus pneumoniae in pairs (1000). ◗ Microscopy Gram staining of sputum: It is a rapid method for diagnosis of acute pneumonia. Stained smears showing lanceolateshaped, Gram-positive cocci in pairs surrounded by a capsule is good evidence for pneumococcal infection. The morphology of the pneumococci may be altered in the patient receiving antibacterial therapy. Key Points Gram staining of CSF: It is a rapid method for demonstration of Gram-positive diplococci—inside the polymorphs as well as outside in a CSF smear. Gram staining is positive in 90% of these cases (Fig. 25-3, Color Photo 21). Habitat S. pneumoniae is a normal inhabitant of throat and nasopharynx. Nasopharyngeal colonization occurs in approximately 5–75% of the population. Colonization is more common in children than in adults. The colonization occurs at about 6 months of age. Gram staining of a buffy coat or blood smear: It is frequently positive in cases of overwhelming pneumococcal sepsis and is useful for rapid presumptive diagnosis of this condition. In acute pneumococcal otitis media, Gram stain of an aspirated fluid smear from middle ear is useful to demonstrate the bacteria. ◗ ◗ Reservoir, source, and transmission of infection Pneumococci are strict parasites, and cause infection only under specific predisposing conditions like prior viral or other infections, aspiration, immune suppression, anatomical deformity, etc. S. pneumoniae infection occurs exclusively in human beings, and no animal reservoir is found in nature. Respiratory and pharyngeal secretions of carriers and patients are the sources of infection. Horizontal transmission requires close person-to-person contact, hence overcrowding facilitates spread of infection. Infection is acquired by inhalation of droplets nuclei and by coming in contact with contaminated fomites. Laboratory Diagnosis ◗ Specimens Sputum, endotracheal aspirate, bronchoalveolar lavage fluid, cerebrospinal fluid (CSF), pleural fluid, joint fluid, abscess fluid, bones, and other biopsy material are the specimens collected for Gram staining and culture. Culture Sputum is plated on blood agar and incubated in the presence of 5–10% carbon dioxide. Gray colonies with alpha-hemolysis are observed after overnight incubation. Sputum culture may be negative due to normal flora outgrowing pneumococci or due to rapid autolysis. Diagnosis of pneumococcal meningitis is confirmed by CSF culture and is positive in 90% of untreated cases. However, the culture is negative in more than 50% of cases who have received treatment even with a single dose of antibiotics. In the acute phase of pneumonia, the blood can be cultured in glucose broth. Demonstration of the pneumococci in the blood shows bad prognosis. Culture of aspirated fluid from the middle ear or from the sinus is a definitive method for diagnosis of otitis media or sinusitis. However, culture is not recommended for specimens collected from the nasopharynx or from external ear. ◗ Identification of bacteria The identifying features of S. pneumoniae are summarized in Box 25-1. S. pneumoniae colonies are identified by the following tests: PNEUMOCOCCUS Box 25-1 1. 2. 3. 4. 5. Identifying features of Streptococcus pneumoniae Gram-positive capsulated lanceolate cocci arranged in pairs. On blood agar, produces alpha hemolysis. Bile solubility test positive. Optochin sensitivity test positive. Inulin fermentation test positive. Optochin sensitivity test: S. pneumoniae is identified by its sensitivity to optochin. In this method, a filter paper disc containing optochin (ethylhydrocupreine dihydrochloride) is applied on the middle of blood agar plate streaked with pneumococci and is incubated overnight. A zone of inhibition of 14 mm or more is observed around the disk after overnight incubation. Animal inoculation: S. pneumoniae can be isolated from clinical specimens containing few pneumococci by intraperitoneal inoculation in mice. Pneumococci are demonstrated in the peritoneal exudate and heart blood of the mice, which die 1–3 days after inoculation. Table 25-2 summarizes important biochemical tests used to differentiate pneumococci from viridans streptococci. ◗ Serodiagnosis Pneumococcal antigen detection: The CIEP is a useful test to detect pneumococcal capsular polysaccharide antigen in the CSF for diagnosis of meningitis, and in the blood or urine for diagnosis of bacteremia and pneumonia. Latex agglutination test using the latex particles coated with anti-CRP antibody is employed to detect C reactive protein. The CRP is used as a prognostic marker in acute cases of acute pneumococcal pneumonia, acute rheumatic fever, and other infectious diseases. CRP is found in sera from cases of acute pneumonia but is absent during the convalescent phase of the disease. Pneumococcal antibody detection: The indirect hemagglutination, indirect fluorescent antibody test, and ELISA are used to demonstrate specific pneumococcal antibodies in invasive pneumococcal diseases. Treatment Most pneumococci are susceptible to penicillin, amoxicillin, and erythromycin. Characters Streptococcus pneumoniae Streptococcus viridans Arrangement of bacteria Pairs of lanceolate diplococci Pairs of oval cocci, short chains Polysaccharide capsule Present Absent Colonies Draughtsman colonies Dome shaped Growth in liquid medium Uniform turbidity Granular turbidity Bile solubility Positive Negative Optochin sensitivity Positive Negative Inulin fermentation Positive Negative Quellung reaction Positive Negative Intraperitoneal mice inoculation Fatal Nonpathogenic Vaccines Pneumococcal vaccines are not recommended for general use, but for persons who are at higher risk of getting pneumococcal infections. Followings are the vaccines used recently: 1. 23-valent pneumococcal polysaccharide vaccine: A 23-valent pneumococcal polysaccharide vaccine for use against pneumococcal infections is now available in many countries. The vaccine contains capsular antigens from each of 23 serotypes of S. pneumoniae most commonly involved in human infections. ■ ■ The vaccine is effective and safe in children older than 5 years. It is not recommended for use in children younger than 2 years, as polysaccharide antigen, being T-cell independent, does not induce adequate immune response in children younger than 2 years. There is no long-lasting immunity, and the antibody level attained is not adequate. 2. 7-valent pneumococcal conjugate vaccine: This is a vaccine made available recently for the immunization of infants and toddlers against invasive pneumococcal disease caused by capsular serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. The polysaccharide antigen is conjugated with a T-cell-dependent protein and so it can be used in children younger than 2 years. Vaccines are recommended for the following groups of persons: 1. Elderly persons aged 65 years or more. 2. Persons aged 2–64 years who are suffering from chronic illnesses, such as chronic cardiovascular disease, chronic pulmonary disease, chronic renal failure, nephrotic syndrome, diabetes mellitus, cirrhosis, and alcoholism. 3. Persons who are splenectomized, particularly those suffering from sickle cell disease. 4. Immunocompromised persons, such as with human immunodeficiency virus (HIV) infection, leukemia, lymphoma, Hodgkin disease, multiple myeloma, malignancy, organ or bone marrow transplantation and persons receiving immunosuppressive chemotherapy. Chapter 25 Inulin fermentation test: Pneumococci ferment inulin; hence inulin fermentation test is a useful test to differentiate pneumococci from streptococci as the latter do not ferment it. Features for distinguishing pneumococci and viridans streptococci Section III Bile solubility test: This is a very useful test to identify S. pneumoniae. It detects an autolytic enzyme, amidase, present in pneumococci, which breaks the bond between alanine and muramic acid of the peptidoglycan of the pneumococcal cell wall. The enzyme amidase is activated by bile salts present in bile, resulting in lysis of pneumococci. The test is carried out by applying a loopful of 10% sodium deoxycholate solution on the young colonies in the blood agar. Most colonies of pneumococci are dissolved within a few minutes. TABLE 25-2 199 200 BACTERIOLOGY Chapter 25 Section III Penicillin-resistant strains: Most pneumococci are susceptible to penicillin. Strains that are susceptible to penicillin are also susceptible to nearly all other antibiotics. But since 1977, penicillin-resistant pneumococci are being increasingly documented. Penicillin-resistant strains may be moderately resistant (minimum inhibitory concentration, or MIC, 0.11 g/ mL) or highly resistant (MIC 2 g/mL). The resistance to penicillin and other beta-lactam antibiotics is not mediated by production of beta-lactamase enzymes, but is due to the modifications of proteins, such as penicillin-binding proteins (PBPs) found on the cell wall. These penicillin-resistant strains are also resistant to multiple drugs, such as cefotaxime, ceftriaxone, erythromycin, tetracycline, macrolides, and trimethoprim–sulfamethoxazole (TMP–SMX). Resistance is seen most often in S. pneumoniae serotypes 6, 9, 14, 19, and 23. The success of antibiotic therapy depends on drug concentrations attained in the affected part of the body, which should be several times higher than the MIC of the organism. Beta-lactam antibiotics are not used alone for the treatment of meningitis caused by penicillin-resistant pneumococci. This is because, adequate bactericidal levels against penicillin-resistant organisms in the central nervous system (CNS) are difficult to achieve with beta-lactam antibiotics. Ceftriaxone can be used for meningitis caused by ceftriaxone-susceptible pneumococci (MIC 0.5 g/mL). Amoxicillin is the drug of choice for treatment of otitis media, sinusitis, and pneumonia caused by penicillin-resistant pneumococci with intermediate resistance. Ceftriaxone is the drug of choice for non-CNS invasive pneumococcal diseases caused by penicillin- and ceftriaxoneresistant pneumococci. Vancomycin is used if the pneumococcus is resistant to ceftriaxone (MIC 0.5 g/mL). Prevention and Control Pneumococcal vaccines play an important role in prevention of pneumococcal diseases. CASE STUDY A 6-year-old child was admitted to hospital with high-grade fever (103°C), headache, stiff neck, vomiting, lethargy, and altered sensorium for 3 days. Gram staining of the CSF smear showed Gram-positive cocci. The same bacteria were isolated from CSF by culture. The patient died despite prompt treatment with ceftriaxone. ■ ■ ■ ■ Which are the most likely bacteria to cause this fulminant condition? What other diseases are caused by this bacterium? What are the vaccines available against the infection caused by the bacteria in children? Do the bacteria show any resistance to antibiotics? 43 26 Neisseria Mycobacterium Leprae Introduction The genus Neisseria consists of Gram-negative, aerobic, nonsporing, nonmotile cocci, typically arranged in pairs (diplococci) with adjacent sides flattened together. The bacteria belonging to this genus are oxidase positive and mostly catalase positive. They ferment sugars with production of acid but no gas. The genus Neisseria consists of 10 species. Neisseria gonorrhoeae and Neisseria meningitidis are the two important species that cause human infections. These two species are strictly pathogens for humans, whereas the other Neisseria species are commensals of the mouth and upper respiratory tract, and hence cause opportunistic infections. Human infections caused by Neisseria are listed in Table 26-1. Neisseria gonorrhoeae N. gonorrhoeae is a strict human pathogen. It is the causative agent of gonorrhea, one of the most common sexually transmitted disease worldwide. Gonococci when transmitted nonsexually from the mother’s genital tract to the newborn during birth cause ophthalmia neonatorum. Properties of the Bacteria ◗ Morphology N. gonorrhoeae shows following features: ■ N. gonorrhoeae are Gram-negative and aerobic diplococci. They are mostly intracellular—found within the polymorphonuclear (PMN) leukocytes—and some cells contain as TABLE 26-1 Human infections caused by Neisseria species Bacteria Diseases Neisseria gonorrhoeae Gonorrhea, disseminated gonococcal infections, ophthalmia neonatorum; and other gonococcal diseases: anorectal gonorrhea, gonococcal pharyngitis, and acute perihepatitis Neisseria meningitidis Meningitis and meningococcemia; other meningococcal diseases: meningococcal pneumonia, septic arthritis, purulent pericarditis, and endophthalmitis Other Neisseria species Opportunistic infections ■ ■ ◗ many as hundred cocci. Smears from the pus sample show the intracellular kidney-shaped cocci, typically arranged in pairs with concave sides facing each other. Freshly isolated bacteria may be capsulated. They do not form endospores. They are nonmotile. Culture N. gonorrhoeae is a fastidious coccus. It requires complex media for growth. The cocci grow on enriched media, such as blood or chocolate agar. These cannot grow on ordinary media, such as nutrient agar or Mueller–Hinton agar. They are aerobes but can also grow anaerobically. They grow optimally at a temperature range of 35–36°C. They fail to grow at temperature less than 25°C or greater than 37°C. The growth of bacteria is enhanced by incubation in humid atmosphere supplemented with 5–10% CO2. 1. Blood agar: On blood agar at 24 hours, N. gonorrhoeae produces convex small colonies measuring 0.6 ⫻ 1.4 ␮m in diameter. These colonies are translucent with entire edges and finely granular surface. They are soft and easily emulsifiable. Gonococci are inhibited by fatty acids and trace metals present in the digested products of peptone found in the blood agar. Addition of soluble starch to the media neutralizes the toxic effects of the fatty acids. 2. Selective media: Thayer Martin medium (chocolate agar medium containing antibiotics, such as colistin, nystatin, and vancomycin) and modified New York City medium (a translucent medium containing vancomycin, colistin, trimethoprim, and either nystatin or amphotericin B) are selective media used for isolation of gonococci from the clinical specimens containing mixed microbial flora. In these media, the growth of contaminating bacteria is suppressed including that of commensal Neisseria. On these media, N. gonorrhoeae produces small, translucent, and convex colonies, which are soft and easily friable. Four types of colonies of gonococci have been recognized. ■ These are T1, T2, T3, and T4. ■ Types 1 and 2 are small and are brown pigmented colonies. The strains producing these colonies possess pili, are virulent and cause acute cases of gonorrhea. ■ Types 3 and 4 are large and are nonpigmented colonies. The cocci producing these colonies do not possess pili and are avirulent. 202 BACTERIOLOGY 3. Transport medium: Stuart’s transport medium is used for the collection and transport of clinical specimens to the laboratory for isolation and demonstration of N. gonorrhoeae. ◗ ■ ■ ■ Chapter 26 Section III ◗ Biochemical reactions ■ Gonococci ferment glucose with the production of acid but no gas. They do not ferment maltose, lactose, sucrose, or fructose. This is an important feature to differentiate N. gonorrhoeae from N. meningitidis. N. gonorrhoeae utilizes glucose only, whereas N. meningitidis utilizes both glucose and maltose. They do not reduce nitrates, and they do not produce hydrogen sulfide. They are oxidase and catalase positive. Other properties Susceptibility to physical and chemical agents: The gonococci are highly delicate bacteria. They die rapidly on drying. They are also killed by soap, and many other disinfectants, such as phenol, chlorhexidine, and hexachlorophene and antiseptics. They are killed at a temperature as low as 25°C. Freeze drying or storing in liquid nitrogen are the most effective methods for storage of gonococci for a longer period. Cell Wall Components and Antigenic Properties The cell wall of N. gonorrhoeae like any other Gram-negative bacteria consists of three layers: outer cell surface, middle peptidoglycan layer, and inner cytoplasmic membrane. These contain following proteins (Fig. 26-1). ◗ ■ ■ N. gonorrhoeae shows following features: ■ growth and metabolism of the cocci. They promote intake of iron by binding hemoglobin, transferrin, and lactoferrin. These proteins are of three types: Outer membrane proteins The outer membrane proteins (OMP) are present in the outer membrane. They mediate the uptake of iron essential for Lipopolysaccharide (Endotoxin) The Por proteins The Opa proteins The Rmp proteins The Por proteins: The Por proteins, earlier known as protein I, are porin proteins that form pores or channels in the outer membranes. Por proteins are of two types: Por-A and Por-B, each with a variety of antigenic variations. Strains producing Por-A proteins are commonly associated with disseminated disease because these proteins prevent killing of gonococci in the serum by the serum complement components. The antigenic variations observed in Por proteins form the basis for the serotype classification of N. gonorrhoeae. The Opa proteins: These proteins, also known as opacity protein, were formerly known as protein II. These proteins are found in the membrane and mediate adherence of the bacteria to each other, and also to the eukaryotic cells. Strains producing Opa proteins produce opaque colonies in culture. The Rmp proteins: These proteins, also known as reduction modifiable proteins, were formerly known as protein III. These are proteins found in the outer membrane of gonococci and lead to the production of antibodies that block serum bactericidal activity against gonococci. ◗ Other important gonococcal proteins Lipo-oligosaccharide (LOS) is another major antigen present in the cell wall of the bacteria. This antigen consists of lipid A and oligosaccharide similar to that of lipopolysaccharide (LPS) of Gram-negative bacteria. However, LOS does not show antigenic variation as found in LPS. LOS possesses endotoxic activity. IgA1 protease and beta-lactamase are the other important proteins. IgA1 protease degrades secretory IgA1, whereas beta-lactamase degrades beta-lactam rings in the penicillin. Pathogenesis and Immunity N. gonorrhoeae causes disease both by multiplying in tissues and by causing inflammation. The bacteria do not produce any toxins. Peptidoglycan Protein I Pilus IgA protease Cytoplasmic membrane Cytoplasm FIG. 26-1. Schematic diagram of Neisseria gonorrhoeae. ◗ Virulence factors N. gonorrhoeae produces several virulence factors as mentioned below (Table 26-2): Capsule: N. gonorrhoeae does not form a true carbohydrate capsule unlike N. meningitidis. Instead, it forms a polyphosphate capsule, which is loosely associated with its cell surface. Capsule is most evident in freshly isolated gonococci and is antiphagocytic. It prevents phagocytosis of the gonococci. Pili: Pili are hair-like structures that extend from the cytoplasmic membrane through the outer membrane. The pili are composed of the proteins known as pilins, which are repeating NEISSERIA TABLE 26-2 Virulence factors of Neisseria gonorrhoeae Biological functions Capsule Prevents phagocytosis Pili Mediate attachment of gonococci to nonciliated epithelial cell; prevent ingestion and killing of gonococci by neutrophils Por proteins Confer resistance to serum killing of gonococci by preventing fusion of phagolysome in neutrophils Opa proteins Mediate bacterial adherence to each other, and to the eukaryotic cells Rmp proteins Produce antibodies that block serum bactericidal activity against gonococci Lipo-oligosaccharide (LOS) Possesses endotoxic activity of the bacteria IgA protease Destroys IgA immunoglobulin Beta-lactamase Degrades beta-lactam rings in the penicillin Plasmids Plasmid-borne virulence determinants are associated with antimicrobial resistance ■ ■ They play an important role in the virulence of the bacteria. They mediate attachment of gonococci to nonciliated epithelial cells. They also contribute to virulence by preventing ingestion and killing of gonococci by neutrophils. Other virulence factors: These include: ■ ■ ■ Por protein of outer membrane protein (OMP) confers resistance to serum killing of gonococci by preventing fusion of phagolysosome in neutrophils. Opa proteins mediate bacterial adherence of bacteria to each other and to the eukaryotic cells. Rmp proteins produce antibodies that block serum bactericidal activity against gonococci. Lipo-oligosaccharide of the bacteria possesses endotoxic activity. ◗ Pathogenesis of gonorrhea N. gonorrhoeae causes disease first by attaching themselves to mucosal cells. Subsequently, they enter the cells and multiply inside the cells and pass through the cells into the subepithelial space, thereby establishing the infection. Pili help in attachment of gonococci to mucosal surfaces and also contribute to the resistance by preventing ingestion and killing by PMN leukocytes. The outer membrane proteins, such as Opa proteins, facilitate adherence between gonococci and also increase ◗ Host immunity The main host defense mechanisms against gonococci are antibodies (IgA and IgG), complement, and neutrophils. Antibody response to gonococci is characterized by the production of serum IgG antibodies. IgG3 is the predominant immunoglobulin. Antibody response is strong against Opa proteins and LOS, whereas it is minimal against Por proteins. Antibodies to LOS cause activation of complement, thus producing a chemotactic effect on neutrophils. Gonococcal infection does not confer protection against reinfection. Repeated gonococcal infections occur due to the antigenic changes of the pili and outer membrane proteins. Persons with a deficiency of the late-acting complement components (C6–C9) are at a risk of disseminated infections. Clinical Syndromes N. gonorrhoeae cause following clinical syndromes (Fig. 26-2): (a) gonorrhea, (b) disseminated gonococcal infections (DGI), (c) ophthalmia neonatorum, and (d) other gonococcal diseases. ◗ Gonorrhea Gonorrhea is a sexually transmitted disease. It is primarily a genital infection restricted to the urethra in men and cervix in women. The incubation period varies from 2 to 8 days. Gonorrhea in men: A symptomatic acute infection is seen in approximately 95% of all infected men. Urethritis is the major clinical manifestation, with burning micturition and serous urethral discharge as the initial manifestation. Subsequently, the discharge becomes more profuse, purulent, and even bloodtinged. Acute epididymitis, prostatitis, and periurethral abscess are rare, but are noted gonococcal complications in men. Gonorrhea in women: In women, endocervix is the primary site (80–90%) of infection because gonococci invade only the endocervical columnar epithelial cells. The bacteria cannot infect the squamous epithelial cells in the vagina of postpubescent women. Urethra (80%), rectum (40%), and pharynx (10–20%) are the other sites of infection in women. The infection is mostly asymptomatic in women. The presence of vaginal discharge, dysuria, dyspareunia, and mild lower abdominal pain are the common symptoms in symptomatic women. In 10–20% of infected women, the primary infection may spread Chapter 26 protein subunits. The expression of protein pilin is controlled by P gene complex. The pilins of all the strains of gonococci are antigenically different. There is a marked antigenic variation in gonococcal pili as a result of chromosomal rearrangement. More than 100 serotypes are known. The pili are important virulence factors: adherence to phagocytes. The Opa proteins also facilitate subsequent migration of gonococci into the epithelial cells. The Por proteins inhibit phagolysosome fusion in the phagocytes, thereby protecting the phagocytosed bacteria from intracellular killing. Production of beta-lactamase (penicillinase) by the bacteria also contributes to the invasion. The host response is characterized by infiltration with leukocytes, followed by epithelial sloughing, formation of microabscesses in the submucosa, and production of purulent pus. The LOS of gonococcal cell wall stimulates the production of tumor necrosis factor alpha (TNF-␣) and other inflammatory responses which contribute to most of the symptoms associated with gonococcal infection. Section III Virulence factors 203 204 BACTERIOLOGY Key Points The strains that cause DGI are characterized by their: Pharyngitis ■ ■ Disseminated infection Skin Anorectal infection Genital infection Chapter 26 Section III Arthritis ■ ◗ Resistance to bactericidal action of serum, Marked sensitivity to penicillin, and Auxotropism for arginine, hypoxanthine, and uracil (for growth they require these substances in the medium). Ophthalmia neonatorum Ophthalmia neonatorum is a nonsexually transmitted infection caused by N. gonorrhoeae. This is a condition of bilateral conjunctivitis of a neonate born by vaginal delivery to an infected mother. However, transmission to the newborn can also occur in utero or in the postpartum period. Pain in the eyes, redness, and purulent discharge are the common symptoms. Blindness is an important complication of this condition. Gonococci can cause permanent injury to the eye in a very short time; hence prompt recognition and treatment of the condition are very essential to avoid blindness. ◗ Other gonococcal infections These include the following: FIG. 26-2. Sites of infection caused by Neisseria gonorrhoeae. from urethra and cervix to cause ascending genital infections including salpingitis, tubo-ovarian abscess, and pelvic inflammatory disease (PID). ■ ■ Pelvic inflammatory disease (PID) is the most important complication in females following gonococcal infection. Increased vaginal discharge or purulent urethral discharge, dysuria, lower abdominal pain, and intermenstrual bleeding are the common symptoms of the PID. Tubal scarring, ectopic pregnancy, and infertility are the major complications in women following PID. Gonococcal vulvovaginitis occurs in prepubertal girls through sexual contact. ■ ■ ■ Anorectal gonorrhea and gonococcal pharyngitis occur in homosexual men following rectal intercourse or by orogenital contact, respectively. Pharyngitis is most commonly acquired during orogenital contact. Pharyngitis often is asymptomatic, however, it may present as exudative pharyngitis with cervical lymphadenopathy. Purulent gonococcal conjunctivitis occurs in adults following autoinoculation of gonococci into the conjunctival sac from a primary site of infection, such as the genitals. The conjunctivitis may rapidly progress to panophthalmitis and loss of the eye unless promptly treated. Acute perihepatitis (Fitz–Hugh and Curtis syndrome) occurs due to the direct extension of N. gonorrhoeae or Chlamydia trachomatis from the fallopian tube to the liver capsule and overlying peritoneum. Epidemiology ◗ Disseminated gonococcal infections Disseminated gonococcal infection (DGI) occurs because of hematogenous dissemination of gonococci from the primary site of infection. The symptoms vary greatly from patient to patient. Arthritis-dermatitis syndrome is the classic presentation of DGI. Joint or tendon pain is most common in the early stage of infection. Migratory polyarthralgia, especially of the knees, elbows, and more distal joints, and also tenosynovitis are the common symptoms. The skin lesions include maculopapular to pustular lesions often with a hemorrhagic component. Septic arthritis, especially of the knee, is the next stage of DGI. During this stage, skin lesions usually disappear and blood cultures for gonococci are always negative. The DGI is mostly seen in untreated asymptomatic women and in persons with complement deficiency. ◗ Geographical distribution Gonococcal infection is reported throughout the world. However, the incidence is much lower in the European countries, and this condition has virtually been eliminated in Sweden. The highest incidence of gonorrhea and its complications occurs in developing countries. The median prevalence of gonorrhea in pregnant women has been estimated to be 4% in Asia, 5% in Latin America, and 10% in Africa. ◗ Habitat N. gonorrhoeae is exclusively a human pathogen. The gonococci are only found in infected conditions. In infected women, the gonococci are most commonly found in the endocervix, and in NEISSERIA present at the meatus, urethral specimens are collected by inserting and rotating a small swab 2–3 cm into the urethra. A calcium alginate or Rayon swab on a metal shaft is usually used for this purpose. infected men found in the urethra. In both men and women gonococci can also be found in the pharynx, rectum, and eyes. The gonococci are not found as normal human flora in the mucosa of the urethra, cervix, or vagina. ◗ Reservoir, source, and transmission of infection Only humans, especially asymptomatic infected men and women, are reservoirs of infections. Asymptomatic carriage is more common in women than in men. Purulent urethra or cervical discharge is the most common source of infection. The infection is transmitted: ■ ■ Fomites do not play any role in transmission of the disease, because gonococci die rapidly outside the human body. Strain typing: Strains of N. gonorrhoeae can be typed by (a) auxotyping and (b) serotyping. ■ Laboratory Diagnosis Laboratory diagnosis of gonococcal infection depends on demonstration of N. gonorrhoeae at the site of infection. ◗ After collection, the specimens are transported and processed immediately in the laboratory. If delay is unavoidable, specimens are collected and transported to the laboratory in a transport medium, such as Stuart’s transport medium. ◗ Microscopy Gram stain of urethral exudates: The presence of four or more polymorphonuclear (PMN) leukocytes per oil-immersion field in Gram-stained urethral exudate smear is diagnostic of urethritis: ■ ■ ■ Demonstration of typical Gram-negative intracellular diplococci is characteristic of N. gonorrhoeae (Fig. 26-3, Color Photo 22). Gram stain helps in the presumptive diagnosis of the gonococcal infection. It is more than 90% sensitive and 98% specific for the diagnosis of gonococcal infection in symptomatic males. However, in asymptomatic males, the sensitivity of the Gram stain is only 60% or less. In women, presence of more than 10 PMN per high-power field on an endocervical smear is suggestive of cervicitis. Gram stain of endocervical smears is less sensitive (50–60%) and 82–90% specific in both symptomatic and asymptomatic women. Gram stain is not a sensitive method for detection of gonococci in patients with anorectal gonorrhea, pharyngitis, and Specimens The genital (urethral discharge, cervical discharge, etc.), rectal, and pharyngeal specimens are collected for the isolation and identification of gonococci. ■ ■ In chronic infection, since urethral discharge is less, the exudate after prostatic massage or morning drop of secretion and urine are also examined for the cocci. Rectal specimens are frequently useful for demonstration of gonococci in asymptomatic women and in homosexual and bisexual men. Samples are collected from all possible mucosal sites, such as pharynx, urethra, cervix, and rectum, and from blood and synovial fluid in patients with possible DGI. In acute gonococcal infection, urethral discharge in males and cervical discharge in females are the specimens of choice. High vaginal swab in females is not satisfactory. ■ When collecting specimens, such as endocervical discharge in women, the cervix is first cleaned of the exudate; a swab is then placed into the external os and rotated for several seconds. ■ In males, discharge present at the meatus is collected for examination. The meatus is first cleaned with gauze soaked in saline. The urethral discharge is then collected with the help of a platinum loop. If no discharge is Intracellular gonococci FIG. 26-3. Gram-negative intracellular Neisseria gonorrhoeae in Gram-stained smear of pus exudate (⫻1000). Chapter 26 ■ Auxotyping is based on addition of specific nutrients and cofactors in the medium for the growth of gonococci. There are over 30 auxotypes. The most common auxotypes are prototrophic or wild type (Proto), praline-requiring type (Pro), and the strains requiring arginine, hypoxanthine, and uracil (AHU). Serotyping is based on the OMP “Porin”, which is further divided into serovars (e.g., 1A-4, 1B-12) based on agglutination with a panel of monoclonal antibodies. ■ Section III Primarily by sexual contact. N. gonorrhoeae infection occurs following mucosal inoculation during vaginal, anal, or oral sexual contact. Increased sexual contact with infected partners increases the risk of acquiring the infection. Less frequently, by nonsexual contact. Ophthalmia neonatorum is acquired nonsexually. This infection occurs following a conjunctival inoculation during vaginal delivery. Less frequently, the disease is transmitted through rectum, oropharynx, or through the birth canal. 205 206 BACTERIOLOGY skin lesions. Specificity is also less because commensal Neisseria species in the oropharynx and gastrointestinal tract can be confused with those of N. gonorrhoeae. Wet mount examination of centrifuged deposit of urine sample: In men, the urine sample, preferably 10–15 mL of early morning (the first) voided urine, is collected and centrifuged and examined under high power. The demonstration of 10 or more PMN in the centrifuged urine under high power is suggestive of urethritis. Section III ◗ Chapter 26 Detection of gonococcal antigen The gonococcal antigens can be detected by both direct fluorescent antibody (DFA) test and direct enzyme-immunoassays (EIA) in urethral discharge and endocervical discharge as well as in other clinical specimens. The DFA using fluorescein-conjugated monoclonal antibodies is a rapid and useful method for demonstration of gonococcal antigens in clinical specimens. The EIA using polyclonal antigonococcal antibodies are also used for the detection of gonococcal antigens in clinical specimens. Culture Isolation of N. gonorrhoeae from clinical specimens by culture confirms the diagnosis of gonorrhea. Genital, rectal, and pharyngeal specimens are inoculated on a nonselective medium (e.g., blood agar or chocolate agar) and on a selective medium (e.g., Modified Thayer Martin medium). The colonies of gonococci on chocolate agar after 48 hours of incubation at 35–36°C in the presence of 5–10% CO2 are small, round, translucent, and convex with finely granular surface. On Thayer Martin medium, the colonies show similar morphology as that on chocolate agar. The mixed microbial flora present in the clinical specimens is suppressed by the selective media. However, the vancomycin present in the selective media inhibits some strains of gonococci. ◗ ◗ ◗ Serodiagnosis The serological tests are done to detect gonococcal antigens or specific anti-gonococcal antibodies in the serum for diagnosis of gonorrhea. ELISA and RIA (radioimmunoassays) using whole cell lysates, pilus proteins, and LPS antigens of the gonococci demonstrate antibodies in the serum. These serological tests are not recommended for routine use. These are used only in specific situations, such as chronic gonorrhea, gonococcal arthritis, etc. Molecular Diagnosis DNA probes (Gen probe) are commercially available for the direct detection of bacteria in the genital and other clinical specimens. Identification of bacteria N. gonorrhoeae are identified by the characteristics listed in Box 26-1. They are differentiated from N. meningitidis and other Neisseria species by a variety of tests (Table 26-3). Box 26-1 Identifying features of Neisseria gonorrhoeae ■ These probes are specific for nucleic acid of N. gonorrhoeae and are sensitive and rapid. The results become available within 2–4 hours, but these tests are highly expensive. Treatment 1. Gram-negative diplococci. 2. On blood agar, produces translucent colonies with entire edge and granular surface. 3. Oxidase test positive. 4. Catalase test positive. 5. Ferments glucose with production of acid. 6. Does not ferment maltose or sucrose. TABLE 26-3 ■ Sulfonamides were used as early as in 1935 for treatment of gonorrhea. In the beginning, all the strains of gonococci were sensitive to sulfonamides but subsequently, they developed resistance to these antibiotics. Penicillin is the drug of choice for penicillin-sensitive strains of N. gonorrhoeae. Differential characteristics of Neisseria species Neisseria species Growth on Production of acid from BA at 22°C CHA NA at 35°C Thayer Martin medium Glucose Maltose Sucrose Lactose Neisseria gonorrhoeae ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫺ Neisseria meningitidis ⫺ ⫺ V ⫹ ⫹ ⫹ ⫺ ⫺ Neisseria lactamica V V ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ Neisseria sicca ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ V ⫽ variable. NEISSERIA Penicillin-resistant strains of N. gonorrhoeae: Initially, gonococci were highly sensitive to penicillin (minimal inhibitory concentration, or MIC, 0.005 U/mL). However, since 1957, strains of gonococci with decreased sensitivity (MIC ⬎0.1 U/mL) to penicillin have been documented. The concentration of penicillin required to inhibit the growth of gonococci has increased by many folds and is now considerably higher (2.4–4.8 MU). ■ ■ Most of them are beta-lactamase (penicillinase) producing by the virtue of plasmid transmission. These strains show high level of resistance to penicillin. Some strains of N. gonorrhoeae not producing beta-lactamase but yet showing resistance to penicillin have also been reported. This resistance is mediated chromosomally and is of low level. Resistance to other antibiotics: Chromosomal-mediated resistance to other antibiotics, such as tetracycline, erythromycin, and aminoglycosides, has also been reported. ■ Key Points N. meningitidis causes a spectrum of diseases ranging from meningococcemia (which is rapidly fatal) to a transient bacteremia (which is relatively benign). It is also the second most common cause of community-acquired meningitis in adults. Properties of the Bacteria ◗ ■ ■ ■ ■ ◗ N. meningitidis are Gram-negative, spherical, or oval cocci arranged in pairs with the adjacent sides flattened. The cocci are generally intracellular in PMN in smears from pus cells and other specimens. They measure 0.6–0.8 ␮m in diameter. Freshly isolated bacteria are usually capsulated. They are nonmotile and nonsporing. Culture Meningococci are strict aerobes. They grow optimally at a temperature between 36°C and 39°C and optimum pH of 7.4–7.6. Their growth is enhanced by incubation in a moist atmosphere in the presence of 5% CO2. Meningococci are fastidious bacteria with complex nutritional requirements. They do not grow on ordinary media, but grow well on the medium enriched with blood or serum, such as blood agar, chocolate agar, and Mueller–Hinton agar. The blood or serum promotes growth of bacteria by neutralizing inhibitory substances found in the media rather than by providing additional requirements. 1. Blood agar: On blood agar, N. meningitidis produces small, round (1–2 mm in diameter), convex, gray, and translucent nonpigmented colonies with entire edges after 24 hours of incubation. At 48 hours, the colonies become larger with an opaque raised center and crenated with transparent margin. It does not produce any hemolysis on blood agar. Strains of meningococci with large polysaccharide capsule appear as mucoid colonies. Meningococci produce large colonies on chocolate agar. 2. Selective media: Thayer Martin medium with antibiotics (vancomycin, colistin, nystatin, and trimethoprim) and New York City medium are the selective media commonly used for the isolation of the bacteria from clinical specimens containing mixed bacterial flora. Immediate saline irrigation and intravenous ceftriaxone are effective for treatment of gonococcal conjunctivitis. Local application of 0.5% of erythromycin ophthalmic ointment or 1% tetracycline or 1% silver nitrate ointment is effective for treatment of gonococcal ophthalmia neonatorum. PID as such is a mixed infection of gonococci, Chlamydia, and other facultative anaerobic pathogens. The treatment, therefore, is by broad-spectrum antibiotics to cover all infecting organisms. Prevention and Control Currently, there is no effective vaccine available against N. gonorrhoeae. Chemoprophylaxis by the prophylactic use of penicillin is also ineffective and may promote the development of resistant strains. Therefore, (a) health education, (b) early detection of cases, (c) tracing of contacts, and (d) follow-up of screening of sexual contacts is important in the prevention of gonorrheal epidemics. Furthermore, the prevention of gonorrhea involves the promotion of safe sex and individual counseling. Gonococcal conjunctivitis in the newborns is prevented by using erythromycin ointment. Morphology N. meningitidis shows following features: ◗ Biochemical reactions N. meningitidis shows following reactions: ■ N. meningitidis is oxidase and catalase positive. These two tests are important biochemical markers for preliminary identification of this organism. Alcaligenes spp., Aeromonas spp., Vibrio spp., Campylobacter spp., and Pseudomonas spp. are the other bacteria that are oxidase positive. Chapter 26 Alternative drugs in cases of penicillin resistance or in penicillin-allergic individuals: Ceftriaxone, cefixime, ciprofloxacin, or ofloxacin are the alternative drugs in cases of penicillin resistance or in penicillin-allergic individuals. A single-dose regimen of any of these antibiotics is given as an initial therapy in uncomplicated urethritis, cervicitis, or rectal or pharyngeal infections in adults. A single dose of ceftriaxone 125 mg intramuscularly or cefixime (400 mg), ciprofloxacin (500 mg), or ofloxacin (400 mg) as a single dose orally is also effective. Neisseria meningitidis Section III ■ Tetracyclines are no longer given for gonococcal infection because of the prevalence of tetracycline resistance. Resistance to ciprofloxacin has also been increasingly documented in Southeast Asia, Africa, and Australia. 207 208 BACTERIOLOGY Box 26-2 1. 2. 3. 4. 5. 6. Identifying features of Neisseria meningitidis Gram-negative diplococci arranged in pairs. On blood agar, produces convex, gray, and translucent colonies. Oxidase test positive. Catalase test positive. Ferments glucose and maltose with production of acid. Does not ferment sucrose or lactose. Oxidase test: The test can be performed in two ways: In the first method, 1% solution of oxidase reagent (tetramethyl paraphenylene-diamine-dihydrocholoride) is poured on the culture media; the Neisseria colonies turn deep purple. In the second method, a few colonies of Neisseria are rubbed with a glass rod on a strip of filter paper moistened with oxidase reagent. A deep purple color develops immediately. N. meningitidis ferments glucose and maltose with acid but no gas. It does not ferment sucrose or lactose. Fermentation tests are required for final identification of Neisseria species (Box 26-2). It does not produce hydrogen sulphide and does not reduce nitrates. ■ Section III ◗ Chapter 26 ■ Cell Wall Components and Antigenic Structure Other properties Susceptibility to physical and chemical agents: N. meningitidis are highly delicate organisms. They are highly sensitive to heat, desiccation, and disinfectants. The cell wall of pathogenic meningococci contains a toxic LPS or endotoxin. The meningococcal endotoxin is chemically identical to the endotoxin of enteric bacilli. ◗ Antigenic structure Depending on group-specific capsular polysaccharide antigens, meningococci are subdivided into 13 serogroups (A, B, C, D, X, Y, Z, W135, 29E, H, I, K, and L). ■ ■ ■ Meningococci belonging to group A, B, and C are responsible for most of the epidemics and outbreaks of meningitis. Group Y and group W135 meningococci cause disease more commonly than groups X and Z. Meningococci that lack group-specific antigens are considered nonpathogenic. Each serogroup includes many serotypes. The classification of the isolates of meningococcal serogroups into their serotypes is based on the differences in the proteins in the outer membrane and in the oligosaccharide part of LOS. For example, Group A meningococci has a single serotype, whereas group B and C meningococci consist of many serotypes. Serotyping of strains is useful for the identification of virulent strains for epidemiological studies. TABLE 26-4 Virulence factors of Neisseria meningitidis Virulence factors Biological functions Capsule Prevents phagocytosis LOS endotoxin Causes damage of the blood vessels associated with meningococcal infections IgA protease Destroys IgA immunoglobulin, thereby helps gonococci to attach to the epithelial cells of the upper respiratory tract Lipooligosaccharides Stimulates release of TNF-␣, which results in host cell damage Pathogenesis and Immunity N. meningitidis colonizes the human nasopharynx, and under specific conditions, invades the blood stream and then reaches the brain, causing meningitis. ◗ Virulence factors N. meningitidis has three important virulence factors, which are responsible for causing disease. These are (a) capsular polysaccharide, (b) LOS endotoxin, and (c) IgA protease (Table 26-4). Capsular polysaccharide: N. meningitidis is surrounded by a prominent polysaccharide capsule that is antiphagocytic. The capsule is an important virulence factor, which contributes to the virulence by inhibiting phagocytosis. The capsule protects meningococci from destruction by the leukocytes. Inside the phagocytic vesicle of the leukocyte, they survive intracellular death, multiply, and then migrate to subepithelial spaces. LOS endotoxin: LOS endotoxin is present in the outer membrane of N. meningitidis. It is responsible for damage of the blood vessels associated with meningococcal infections. The endotoxin comprises two antigenic determinant components: (a) a protein component and (b) a carbohydrate component. The continuous production and release of endotoxin by N. meningitidis cause severe endotoxin reaction, seen in patients with meningococcal disease. IgA protease: IgA protease is the other important virulence factor. The enzyme acts by clearing the secretory IgA, thus helping the bacteria to attach to the epithelial cells of the upper respiratory tract. ◗ Pathogenesis of meningitis Initially, N. meningitidis causes a localized infection by colonizing the nasopharynx. From this site, the meningococci invade the submucosa by circumventing the host defense mechanisms and gain access to the central nervous system (CNS). Meningococci reach CNS by the following ways: 1. Invasion of blood stream: This is the most common mode of spread of meningococci. Once inside the blood stream, the meningococci escape the immune surveillance (e.g., antibodies, complement-mediated bacterial killing, NEISSERIA neutrophil phagocytosis) of the host and subsequently reach distant sites including the CNS. The specific mechanism by which the meningococci reach the subarachnoid space still remains to be clearly understood. 2. Direct contiguous spread: Meningococci can also reach the CNS by direct contiguous spread from nasopharynx. Inside CNS, the bacteria multiply and survive because host defense mechanisms (such as immunoglobulins, neutrophils, and complement) appear to have limited role in controlling multiplication of the bacteria. Uncontrolled multiplication of bacteria continues in the CSF, which subsequently causes a cascade of meningeal inflammation. Meningococcal infection of the nasopharynx is usually subclinical. Asymptomatic nasopharyngeal carriage of meningococci is of short duration and resolves within several weeks. In a few persons, meningococci invade the circulation and cause clinical disease. ◗ Clinical Syndromes N. meningitidis causes the following conditions: (a) meningitis, (b) meningococcemia, and (c) other syndromes. ◗ Meningitis Meningococcal meningitis caused by N. meningitidis is most common in children and young adults. It is a febrile illness of short duration characterized by headache and stiff neck. Lethargy or drowsiness is frequent. Confusion, agitated delirium, and stupor are rarer. Mental obtundation, stupor, and coma due to increased intracranial pressure are some of the noted complications at the end stage of the disease. Prognosis of meningitis is good, and the patients recover completely on immediate treatment with appropriate antimicrobial therapy. However, prognosis is bad in comatose patients and in patients with local neurological findings. Nonsuppurative arthritis, usually of the knee joint, is seen in approximately 10% of the patients with meningococcal disease. This condition is observed within the first 48 hours of treatment and is believed to be immunologically mediated. Recurrent meningococcal meningitis is another condition which is associated with hereditary deficiency of various components of complement system. Other conditions include meningococcal pneumonia (which probably results from the aspiration of the organisms), septic arthritis, purulent pericarditis, and endophthalmitis. Epidemiology ◗ Geographical distribution Meningococcal disease occurs worldwide. N. meningitidis serogroup A usually causes epidemics, serogroup B causes both epidemics and outbreaks, while serogroup C mostly causes localized outbreaks. Endemic meningitis is more common in children below the age of 5 years and in elderly people. Large outbreaks of meningococcal disease have occurred in central African countries with attack rate as high as 400–500 cases per 100,000 population. Epidemics of meningococcal disease have occurred in many parts of the world. Meningococci of group A are associated with diseases in underdeveloped countries; meningococci of group B, C, or Y are responsible for most (90%) of the cases of meningococcal diseases in the developed countries (Table 26-5). ◗ Habitat N. meningitidis is primarily a pathogen of humans. Meningococci are found in the nasopharynx and oral cavity. Asymptomatic carriage of N. meningitidis varies from as low as 1% to as high as 40% in the population. The carriage rates are highest in schoolgoing children, in young adults, and in the population with low economic status. Chapter 26 The presence of meningococci in the nasopharynx induces a humoral antibody response, and most people acquire immunity to meningococcal disease by age of 20 years. Maternal antibodies provide protection to infants for the first 3–6 months of life. Later, colonization with nonpathogenic meningococci appears to produce cross-reacting, protective antibodies. Specific IgG antibodies are produced against meningococcal polysaccharides in combination with the complement mediate bactericidal activity against the meningococci. Individuals lacking the bactericidal antibodies and those suffering from complement deficiencies—such as C5, C6, C7, or C8 components of the complement—show increased susceptibility to meningococcal disease. An episode of meningitis confers group-specific immunity, but a second episode may be caused by another meningococcal serogroup. Other syndromes Section III Host immunity Meningococcemia Meningococcemia with or without meningitis is a life-threatening condition. The condition presents as an acute fever with petechial rash. Small petechial rashes are continuously found on the trunk and lower extremities; subsequently the rashes may coalesce to form large hemorrhagic lesions. Waterhouse–Friderichsen syndrome is an overwhelming systemic infection caused by N. meningitidis. This condition is characterized by severe disseminated intravascular coagulation, shock, and multisystem failure including destruction of adrenal glands. The condition, associated with circulatory collapse with intravascular coagulation, is invariably fatal. It is most commonly seen in persons suffering from deficiency of C5–C9 components of the complement. The vascular damage seen in this condition is caused primarily by the action of LOS endotoxin present in the meningococci. ◗ ◗ 209 210 BACTERIOLOGY TABLE 26-5 Serogroups Disease A Meningococcal disease in underdeveloped countries B Meningitis and meningococcemia; most (⬎90%) cases of meningitis in developed countries C Meningitis and meningococcemia; most (⬎90%) cases of meningitis in developed countries Y Meningococcal pneumonia W135 Meningococcal pneumonia Chapter 26 Section III ◗ Reservoir, source, and transmission of infection Human is the only reservoir of meningococcal infection. Nasopharyngeal secretion is the most common source of infection. Meningococci are transmitted by airborne droplets of infected nasopharyngeal secretions (the most common source of infection). Family members living in crowded conditions or the people who live in close populations (such as military barracks and prisons) and older people are more susceptible to infection. Laboratory Diagnosis Laboratory diagnosis depends on demonstration of meningococci in clinical specimens by microscopy and culture. ◗ Specimens Cerebrospinal fluid and blood are the specimens of choice for demonstration of meningococci in the early stage of meningitis. Nasopharyngeal swabs are useful to detect carriers. The CSF is collected by lumbar puncture and blood by venipuncture in strict aseptic conditions. CSF is never refrigerated as Haemophilus influenzae, another agent of meningitis, may die at the cold temperature. CSF specimens are transported immediately to laboratory for processing. Similarly, blood is collected in blood culture media containing either glucose broth or sodium taurocholate broth. Nasopharyngeal specimens are collected using sterile swabs and are transported in Stuart’s transport medium to the laboratory. CSF Meningococcal meningitis produces various inflammatory changes in the CSF: ■ ■ ■ ■ 2. The second part of CSF is used for direct culture. 3. The third part is incubated overnight with an equal volume of glucose broth and then subcultured onto the blood agar and chocolate agar. Epidemiology of Neisseria meningitidis serogroups The CSF in bacterial meningitis is more turbid. It contains more than 1000 WBC/␮L, and the cells are predominantly PMN cells. The total protein content is increased. The total glucose level, which is normally 60% of simultaneous blood glucose level, is lowered (hypoglycorrhachia). The intracranial pressure may be elevated. CSF received in the laboratory is processed in three parts: 1. First part is centrifuged and smear is prepared from the deposit for Gram staining. The supernatant is tested for meningococcal antigens. ◗ Microscopy Gram staining of the CSF is a very useful method for detection of meningococci. Meningococci are seen as Gram-negative diplococci present mainly inside the leukocytes and some may even be present extracellularly. These cocci can be demonstrated in the CSF in approximately 50% of the patients with meningococcal meningitis. In fulminant meningococcemia, Gram staining of the peripheral blood buffy coat may reveal Gram-negative diplococci. ◗ Culture Isolation of N. meningitidis from the CSF, blood, and other clinical specimens by culture confirms the diagnosis of meningococcal infection. The CSF is inoculated immediately on a nonselective medium, such as blood agar or chocolate agar, and incubated at 35–36°C under 5% CO2 for 18–24 hours. The colonies of meningococci are small, round, translucent, and convex with a smooth glistening surface. Blood is inoculated immediately into blood culture bottles containing either glucose broth or sodium taurocholate broth and incubated at 35–36°C. Subcultures are made on blood agar or chocolate agar from these broths and are reincubated overnight at 35–36°C in the presence of 5% CO2. The cultures should be incubated for 4–7 days with daily subculture. Blood culture is often positive during early stage of meningitis and in meningococcemia. ◗ Other specimens Other specimens, such as nasopharyngeal swabs and petechial exudates are processed in a similar way as described earlier for CSF. ◗ Identification of bacteria N. meningitidis are identified by the characteristics listed in Box 26-2. Serogrouping of the bacterial isolates grown on culture is carried out by slide agglutination with specific hyperimmune serum. ◗ Antigen detection Detection of soluble polysaccharide antigen in the CSF is a useful method for diagnosis of meningococcal meningitis. Counter-current immunoelectrophoresis, latex agglutination test, and bacterial coagglutination test using specific antibodies are the rapid tests frequently used to detect the soluble antigen in the CSF. Antigen detection is useful when bacteria are scanty in the CSF. However, antigen detection is not useful in the meningitis caused by Group B meningococci because N. meningitidis serogroup B is relatively nonimmunogenic and does not react with specific antibodies. NEISSERIA ◗ Serodiagnosis Vaccines Indirect hemagglutination test and ELISA are useful for the demonstration of antibodies against specific polysaccharide antigen in the serum. Serodiagnosis is useful in the cases of chronic meningococcal infection where cultures have proved negative for meningococci. PCR has been used for detection of N. meningitidis DNA in clinical specimens. The test is useful to detect small amounts of meningococcal DNA in CSF. It is a more sensitive test for diagnosis of meningococcal meningitis than the culture. The high cost of the test and the expertise necessary to operate a PCR assay are the disadvantages of the test. The test, therefore, is only used in a large-scale outbreak when a number of specimens are to be analyzed, and in a tertiary healthcare center. Treatment This includes chemoprophylaxis and vaccines. ◗ Chemoprophylaxis Antimicrobial chemoprophylaxis of close contacts is the key factor for preventing secondary cases of sporadic meningococcal disease. Person-to-person transmission can be interrupted by administration of antibiotics, which eradicate the asymptomatic nasopharyngeal carrier state. Sulfonamides, rifampin, minocycline, ciprofloxacin, and ceftriaxone are the drugs frequently used to eradicate meningococci from the nasopharynx. However, ciprofloxacin is not recommended for children, because it has been found to cause cartilage damage in immature experimental animals. ◗ Immunoprophylaxis Immunoprophylaxis by vaccination with group-specific meningococcal capsular polysaccharides of groups A, C, Y, and W135 meningococci is very much useful for prevention of meningococcal disease. Other species of the genus Neisseria rarely cause human disease. They are found as part of normal bacterial flora mostly of the respiratory tract. These commensal Neisseria are Neisseria flavescens, Neisseria sicca, Neisseria lactamica, and Neisseria subflava. The commensal Neisseria differ from pathogenic Neisseria species by following properties: ■ ■ ■ They can grow on ordinary agar not enriched with blood and serum and they can also grow at 22°C. They do not require high percentage of CO2 for their growth. They produce greenish yellow or yellow colonies on the media. N. flavescens and N. sicca have been associated with isolated cases of meningitis, osteomyelitis, acute otitis media, and acute sinusitis. But true incidence of respiratory tract infection caused by these Neisseria species is not known. Most of these strains are susceptible to penicillins. N. lactamica is frequently isolated from the nasopharynx and is a nonvirulent Neisseria; however, it is closely related to pathogenic Neisseria. Neisseria catarrhalis—which was later designated as Branhamella catarrhalis and is now renamed as Moraxella catarrhalis—is a commensal of the upper respiratory tract and, occasionally, is found in female genital tract. It is a recognized respiratory opportunistic pathogen in immunocompromised host and hospitalized people. M. catarrhalis is multidrug resistant and grows on ordinary media, such as nutrient agar and MacConkey agar. It causes infections (e.g., otitis media, maxillary sinusitis, meningitis, septic arthritis, endocarditis, sepsis, etc.) in immunocompromised patients and in children. Some strains are susceptible to cephalosporins, chloramphenicol, and tetracycline. Chapter 26 Prevention and Control Other Neisseria Species Section III Quadrivalent meningococcal polysaccharide vaccine (MPSV4): It has been shown to be highly effective in preventing disease caused by A, C, Y, and W135 serogroups of meningococci. The vaccine is given intramuscularly. Use of this vaccine is indicated for population at risk during outbreak of infection caused by one of these serogroups of meningococci. These vaccines developed against group A, C, Y, and W135 are poorly immunogenic under 2 years of age. These vaccines, however, produce good antibody response in children above 2 years of age. Tetravalent meningococcal polysaccharide-protein conjugate vaccine (MCV4): Recently, in 2005, MCV4 is being used for the persons aged 11–55 years for vaccination against meningococci in the United States. This is recommended for groups of population at risk, which include (a) military recruits, (b) travelers to areas hyperendemic or epidemic for meningococcal disease, (c) patients with anatomic or functional asplenia, (d) patients with terminal complement deficiency, and (e) microbiologists who are routinely exposed to meningococci. No vaccine against group B meningococci is available because group B meningococcal capsular antigen is not immunogenic. However, of late some progress has been made in preparation of vaccine for group B meningococci. The vaccine, which is at the experimental stage, consists of outer membrane proteins that are capable of inducing group-specific bactericidal antibodies. Molecular Diagnosis Prompt and specific antimicrobial therapy of meningococcemia or meningococcal meningitis is most crucial. Intravenous penicillin G is the recommended drug for the treatment of meningococcal disease. The MIC of penicillin usually ranges from 0.01 to 0.05 ␮g/mL against meningococcal isolates. Chloramphenicol, rifampicin, erythromycin, tetracycline, and cephalosporins (ceftriaxone, cefotaxime, and cefuroxime) are useful in treatment of bacterial meningitis. Ceftriaxone has an additional advantage of eradicating the nasopharyngeal carriage of meningococci. Chloramphenicol is useful for patients who are allergic to penicillin. Meningococci are not susceptible to vancomycin and polymyxin. Meningococci resistant to sulfadiazine (MIC ⱖ0.128 ␮g/mL) have been documented recently. 211 212 BACTERIOLOGY CASE STUDY A 22-year-old female complained of lower abdominal pain on and off for the last 3 months. She complained of a feeling of heaviness in the pelvis and pain during sexual intercourse. On examination, a tender mass was found to the right side during examination. Gram staining of cervical swab showed plenty of pus cells and a few Gram-negative cocci. She gave a history of allergy to penicillins. ■ ■ ■ ■ Chapter 26 Section III ■ Which is the most likely genital infection the patient is suffering from? Which is the most likely bacterium to cause this genital condition? What other diseases are caused by this bacterium? How you will confirm diagnosis of this condition in the laboratory? What antibiotics you can use in this patient for treatment of the condition? 27 Corynebacterium Introduction Corynebacterium diphtheriae The genus Corynebacterium consists of a diverse group of bacteria including animal and plant pathogens, as well as saprophytes. Some corynebacteria are found as part of the normal flora of humans in the skin, upper respiratory tract, and urogenital tract. Corynebacteria (from the Greek words koryne, meaning club, and bacterion, meaning little rod) are Gram-positive, aerobic or facultative anaerobic, nonmotile, and catalase-positive rod-shaped bacteria. They have a cell wall with arabinose, galactose, meso-diaminopimelic acid, and short-chain mycolic acids. They do not form spores or branch as do the actinomycetes, but they have the characteristic of forming irregular-shaped, club-shaped, or V-shaped arrangements in normal growth. Gram staining shows bacteria in short chains or clumps resembling characteristic Chinese letters. The genus Corynebacterium consists of 46 species, of which at least 30 species are known to be associated with human diseases. Corynebacterium species that can cause infections in humans are summarized in Table 27-1. Corynebacterium diphtheriae, the causal agent of the disease diphtheria is the most widely studied species. Nondiphtherial corynebacteria— collectively referred to as diphtheroids—originally were believed to be mainly contaminants. These diphtheroids have recently been recognized as pathogenic, especially in immunocompromised hosts. TABLE 27-1 C. diphtheriae is the most important species causing diphtheria. Diphtheria is an acute upper respiratory tract illness characterized by sore throat, low-grade fever, and an adherent membrane on the tonsil(s), pharynx, and/or nose. Properties of the Bacteria ◗ Morphology C. diphtheriae shows following features: ■ ■ ■ ■ C. diphtheriae is a Gram-positive bacillus showing maximum pleomorphism on Gram staining. The bacteria characteristically appear in palisades or as individual cells lying as sharp angles to each other in V and L formation. This Chinese letter pattern formation or cuneiform arrangement is caused by the incomplete separation of the daughter cells during division when the organism is grown on nutritionally inadequate media, such as coagulated egg medium or Loeffler’s coagulated serum. The bacterium measures 3–6 ⫻ 0.6–0.8 ␮m and is slender and sometimes has swollen ends. Most of the bacteria have 2–3 granules at the swollen ends, which give reddish purple color when stained with Loeffler alkaline methylene blue. Rest of the bacterium is unevenly stained with the dye. Human infections caused by Corynebacterium species Bacteria Diseases Corynebacterium diphtheriae Diphtheria (respiratory and cutaneous); diphtheria of other sites (external ear, the eye) and the genital mucosa; pharyngitis, and endocarditis Corynebacterium ulcerans Cutaneous infections; diphtheria-like lesions, such as pharyngitis and respiratory disease Corynebacterium jeikeium (group JK) Wound infections, septicemia, foreign body (catheter, prosthesis) infections, and endocarditis Corynebacterium urealyticum (group D2) Urinary tract infection Corynebacterium pseudotuberculosis Native and prosthetic valve endocarditis, pneumonia, lung abscesses, tracheobronchitis, and suppurative lymphadenitis Corynebacterium haemolyticum Pharyngitis Corynebacterium striatum Respiratory tract infections and foreign body infections Corynebacterium pseudodiphtheriticum Pneumonia, lung abscesses, tracheobronchitis, endocarditis, and lymphadenitis 214 ■ ■ ◗ BACTERIOLOGY The granules are also known as metachromatic granules, BabeErnest’s granules, or volutin granules. These granules, which are the accumulation of polymerized phosphates, are responsible for the beaded appearance of the bacteria. The granules can be demonstrated by special staining techniques, such as Albert stain, Neisser stain, and Ponder stain. They are nonmotile and nonsporing. Culture Chapter 27 Section III C. diphtheriae is an aerobic and facultative anaerobic organism but grows best under aerobic conditions. It grows at 37°C and at a pH of 7.2–7.4 on media enriched with blood, serum, or egg. 1. Loeffler’s serum slope: Loeffler’s serum slope is an enriched medium frequently used for the growth of C. diphtheriae. The characteristic morphology of the bacteria is best seen on Loeffler’s serum slope. The bacteria, on this medium, produce a luxuriant growth in 6–8 hours at a temperature of 37°C. Initially, the colonies are small, circular, opaque, and white, but on prolonged incubation the colonies become larger in size and show a distinct yellow tint. Loeffler’s serum slope does not support the growth of streptococci and pneumococci. 2. MacLeod’s or Hoyle’s tellurite blood agar media are the examples of selective media used for the culture of C. diphtheriae. Tellurite (0.04%) present in the media inhibits growth of other contaminant bacteria. Most strains of the bacteria require nicotinic and pantothenic acids for their growth; some also require thiamine, biotin, or pimelic acid. For the optimal production of diphtheria toxin, it is essential to supplement the medium with amino acids and a source of iron. On the tellurite agar, C. diphtheriae produces characteristic gray or black colored colonies after 48 hours of incubation (Fig. 27-1, Color Photo 23). C. diphtheriae reduces tellurite to metallic tellurium, which is incorporated in the colonies, thereby giving them a characteristic gray or black color. TABLE 27-2 Salient features of various biotypes of Corynebacterium diphtheriae Features Gravis Intermedius Mitis Size Short rods Long rods Long curved rods Pleomorphism ⫹ ⫹⫹⫹ ⫹⫹⫹⫹ Granules Few or no Few Prominent Staining reaction Uniform Irregular Irregular Colony Daisy head Frog’s egg Poached egg Surface Malt Shining Glossy Consistency Brittle Weak buttery Buttery Hemolysis Variable Nonhemolytic Hemolytic Starch fermentation ⫹ ⫺ ⫹ Phage types 14 3 4 Toxigenicity of strains 95% 99% 85% Antigenic types 13 4 40 In broth medium Pellicle formation and granular deposits No pellicle, Diffuse turbidity On tellurite agar Granular deposit C. diphtheriae is classified into three distinct biotypes (mitis, intermedius, and gravis) based on the colony morphologies on cysteine-tellurite agar (Table 27-2): ■ ■ ■ Mitis colonies are small, round, convex, and black. Intermedius colonies are small, flat, and gray. Gravis colonies are large, irregular, and gray. These morphological types of the strains were initially correlated with severity of the disease. Mitis strains were thought to produce mildest variety, gravis to produce the most serious disease, while the intermedius to produce the disease of intermediate severity, but now these distinctions are not considered valid. These morphological types, however, are useful for the epidemiological classification of C. diphtheriae isolates. ◗ Biochemical reactions C. diphtheriae shows the following reactions: ■ ■ ◗ FIG. 27-1. Potassium tellurite agar showing black colonies of Corynebacterium diphtheriae. C. diphtheriae ferments many sugars (glucose, galactose, maltose, and dextrin) producing acid but no gas. Hiss’s serum water is always used for testing fermentation of sugars. The bacteria do not ferment lactose, mannitol, and sucrose. Some strains of virulent C. diphtheriae ferment sucrose. They do not hydrolyze urea or form phosphatase and they lack proteolytic activity. Other properties Susceptibility to physical and chemical agents: Diphtheria bacilli are readily killed by heating at 58°C for 10 minutes and at 100°C for 1 minute. They are destroyed by the usual strengths of antiseptics. They are resistant to the action of 215 CORYNEBACTERIUM The cell wall contains neuraminidase, arabinose, galactose, mannose, corynemycolic acid, and corynemycolenic acid. The cell walls of the diphtheria bacilli are antigenically heterologous. Pathogenesis and Immunity Diphtheria is a classic example of toxin-mediated bacterial disease. ◗ Virulence factors ■ ■ ■ ■ ■ ■ Diphtheria toxin is a powerful exotoxin, as little as 1–10 millionths (i.e., 0.0000001) of a gram can cause death in a guinea pig weighing 250 g in 96 hours. It is a protein with a molecular weight of 58,000 Da. It is synthesized in precursor form on membrane-bound polysomes in C. diphtheriae and is cotranslationally secreted as a single polypeptide chain made up of 535 amino acids. When released, the native toxin molecule is nontoxic until exposure of the active enzymatic site to mild trypsin treatment. The biologically active molecule consists of two functionally distinct polypeptide chain fragments A (24,000-Da protein) and B (38,000-Da protein), linked by a disulfide bridge. Neither of the fragments are toxigenic on their own, but act together to cause toxigenicity. Intradermal inoculation of toxigenic culture or toxin causes local erythematous lesion within 48 hours. In animals, toxin causes congestion of adrenal glands with scattered hemorrhages in medulla or cortex or both. Regional lymph nodes as well as internal organs are also congested. ■ ■ Causes local tissue destruction at the site of membrane formation (the upper respiratory tract), facilitating replication and transmission. Is also absorbed into the blood stream and distributed, resulting in systemic complications of diphtheria including demyelinating peripheral neuritis and myocarditis. Cell membrane 2 Diphtheria toxin A B A B 1 4 A Receptor for toxin A B B 5 NAD+ TABLE 27-3 EF-2 Virulence factors of Corynebacterium diphtheriae Virulence factors Biological functions Diphtheria exotoxins Neuro and cardiotoxin; inhibits protein synthesis by inactivating elongation factor Cell 3 FIG. 27-2. Nicotinamide A 6 EF-2 - ADPR Cellular mechanism of Corynebacterium diphtheriae toxin. Chapter 27 Key Points C. diphtheriae usually enters the body through the upper respiratory tract but can also enter through the skin, genital tract, or eye. Infection begins by adherence of the bacteria at the infected site. The initial lesion usually occurs on the tonsils and oropharynx, and from this site it may spread to the nasopharynx, larynx, and trachea. The organisms multiply rapidly in the epithelial cells, forming a local lesion and secrete exotoxins that cause necrosis of the cells in that area. The combination of cell debris and exudative inflammatory response (leading to accumulation of red blood cells, necrosed cells, bacteria, fibrin, and lymphocytes) result in the formation of the characteristic pseudomembrane. The pseudomembrane of diphtheria is thick, leathery, grayish-blue or white. The membrane adheres very tenaciously to the underlying mucosa and if attempts are made to forcibly remove it, raw bleeding surface is exposed. Spreading of the membrane down the bronchial tree can occur, causing respiratory tract obstruction and dyspnea. Growth of C. diphtheriae is restricted to oral cavity, but toxemia and systemic manifestation of diphtheria occurs due to absorption of toxin from the site of membrane. The toxin binds to a specific receptor (now known as the HB-EGF receptor) present on susceptible cells and enters by receptormediated endocytosis (Fig. 27-2). Apparently as a result of activity on the endosomal membrane, the A subunit is cleaved and released from the B subunit. Fragment A then gains entry into the cell and catalyzes ADP ribosylation. This leads to the inhibition of the NAD in protein synthesis. Ultimately, inactivation of all the host cell EF-2 molecules causes cell death, which clinically manifests as the necrotic lesion of diphtheria. Diphtheria toxin: Section III Diphtheria toxin, the exotoxin produced by C. diphtheriae, is the key virulence factor of the bacteria (Table 27-3). Diphtheria toxin: Diphtheria toxin is produced only by strains of C. diphtheriae that are lysogenized with bacteriophages that contain the structural gene (tox gene) for the toxin molecule (tox⫹ strains). When DNA of the phage becomes integrated into the genetic material of C. diphtheriae, the bacteria develop the capability of producing the polypeptide toxin. The gene for toxin production occurs on the chromosome of the prophage, but a bacterial repressor protein controls the expression of this gene. The repressor is activated by iron, and it is in this way that iron influences toxin production. High yields of toxin are synthesized only by lysogenic bacteria under conditions of iron deficiency. Pathogenesis of diphtheria A Cell Wall Components and Antigenic Structure ◗ B light, desiccation, and freezing. They remain fully virulent and viable in floor dusts and in blankets even for up to 5 weeks. 216 BACTERIOLOGY C. diphtheriae does not cause any invasion of the blood to produce systemic manifestation of the diphtheria. ■ ■ ■ Chapter 27 Section III ◗ Host immunity In diphtheria, immunity against clinical diseases depends on the presence of antitoxin in the blood stream, in response to clinical or subclinical disease or active immunization. Infants below 6 months of age carry IgG antibodies derived from the immune mother either transplacentally or through breast-feeding. The antibodies later developed are IgG and IgA type. In areas where diphtheria is endemic and mass immunization is not practiced, most young children are highly susceptible to infection. Individuals who have fully recovered from diphtheria may continue to harbor the organisms in the throat or nose for weeks or even months. In the past, it was mainly through such healthy carriers that the disease was spread, and toxigenic bacteria were maintained in the population. The immune status of the individuals is assessed by the presence of antitoxin levels or by Schick’s test. Schick’s test: Schick’s test was introduced by Schick in 1913 to assess the immunity among children. The test is performed by injecting 0.1 mL of highly purified toxin (1/50 minimum lethal dose) into one forearm and 0.1 mL of heat-inactivated toxin into another forearm as a control. This brings about four types of reactions: (a) positive reaction, (b) negative reaction, (c) pseudoimmunereaction, and (d) combined reaction. 1. Positive reaction: This is characterized by a local inflammatory reaction that reaches maximum intensity in 4–7 days in the test arm and then reduces gradually. This indicates absence of immunity to C. diphtheriae. 2. Negative reaction: Absence of any inflammatory reaction is suggestive of a negative reaction. This indicates the presence of antitoxin in the individual, which neutralizes the toxin injected. Such an individual is immune to C. diphtheriae infection. 3. Pseudoimmunereaction: In endemic areas, allergy to the toxin is seen among children and adults. Even though the individual is immune, yet an allergic reaction is observed in both the test as well as the control arm. The inflammatory reaction reaches its peak in 36 hours and subsides in 72 hours in both the arms. This reaction is called pseudoimmune reaction and it indicates that the individual is immune but hypersensitive. 4. Combined reaction: This is the condition in which an individual injected with the toxin develops inflammation in the test arm, which increases in intensity by 4–7 days. In the control arm, the inflammation is seen for a maximum of 48–72 hours and then subsidies. It indicates the individual is not immune and is hypersensitive. Clinical Syndromes The clinical manifestations of diphtheria depend upon the following: (a) immune status of the patient, ( b) virulence of the bacteria, and (c) the site of the infection. Toxigenic strains (tox⫹) of C. diphtheriae cause: Serious, sometimes fatal, disease in nonimmune patients. Mild respiratory diseases in partially immune patients. Asymptomatic colonization in fully immune individuals. Nontoxigenic strains (tox⫺) cause a mild disease, such as cutaneous diphtheria. ◗ Respiratory diphtheria Incubation period varies from 2 to 5 days. Sore throat, in the absence of systemic complaints, is the usual initial symptom. Fever, if occurs, is usually lower than 102°F, and malaise, dysphagia, and headache are frequently present. Respiratory diphtheria is: ■ ■ Characterized by the formation of a fibrinous pseudomembrane on the palate, pharynx, epiglottis, larynx, or trachea and may extend to the tracheobronchial tree. The pseudomembrane is generally a firmly adherent, thick, fibrinous, gray-brown membrane. This membrane may cause bleeding if disturbed. Respiratory distress may occur if the membrane breaks loose and occludes the airways. Associated with marked edema of the tonsils, uvula, submandibular region, and anterior neck (bull neck). Complications of respiratory diphtheria include the following: ■ ■ ■ ◗ Myocarditis—the main complication, which may occur in as many as two-thirds of the patients. Circulatory collapse, heart failure, atrioventricular blocks, and dysrhythmias may also occur. Involvement of the cranial nerves (leading to paralysis of the soft palate with resultant difficulty in swallowing and nasal regurgitation of the fluids) and polyneuritis of the lower extremities. Recovery is complete in both cases. Cutaneous diphtheria Cutaneous diphtheria is generally caused by nontoxigenic strains (tox⫺ strains) of C. diphtheriae. The condition is an indolent nonprogressive infection characterized by a superficial, nonhealing ulcer with a gray-brown membrane. The condition may occur at one or more sites—usually confined to the areas with previous mild trauma or bruising. Extremities are affected more often than the trunk or head. Pain, tenderness, erythema, and exudate are the typical presentations. Respiratory tract colonization or symptomatic infection and toxic complications occur in a minority of patients with cutaneous diphtheria. Cutaneous diphtheria may persist for weeks to months. Cutaneous diphtheria often causes no toxicity while producing natural immunity; however, it may cause epidemics in poorly immunized populations. ◗ Diphtheria of other sites External ear, eye (usually the palpebral conjunctivae), and genital mucosa are the other sites of diphtheria. Rare sporadic cases of endocarditis usually due to nontoxigenic strains have been reported. Septicemia caused by C. diphtheriae is rare but is invariably fatal. CORYNEBACTERIUM Epidemiology ◗ Geographical distribution During the early 1990s, diphtheria was still endemic in many parts of the world including the Indian subcontinent, Indonesia, Philippines, Brazil, Nigeria, and republics of the former Soviet Union. ■ ■ ◗ Habitat ◗ Reservoir, source, and transmission of infection Humans are the only natural host of C. diphtheriae and thus are the only significant reservoirs of infection. Infective droplets or nasopharyngeal secretions are the common sources of infection. ■ ■ ■ Direct human contact facilitates transmission of the disease. Patients with active infection are more likely to transmit diphtheria. C. diphtheriae is most commonly transmitted by close contacts through droplets of nasopharyngeal secretions or infected skin lesions. It is known that toxigenic strains may directly colonize the nasopharyngeal cavity. In addition, the tox gene can be spread indirectly by the release of toxigenic corynebacteriophage and by lysogenic conversion of nontoxigenic autochthonous C. diphtheriae in situ. Asymptomatic respiratory carrier states are believed to be important in transmitting diphtheria and immunization appears to reduce the likelihood of carrier state. Dust and clothing also may contribute to the transmission. The organism can survive up to 6 months in dust and fomites. When diphtheria was endemic, the disease was most commonly seen in children younger than 15 years, but recently, the epidemiology has shifted to adults because this group of population lacked natural exposure to toxigenic C. diphtheriae in the vaccine era and also received less booster doses. In serosurvey in the ◗ Typing C. diphtheriae strains are classified into various serotypes by agglutination reactions. Mitis strains have been classified into 40 types, intermedius into 4 types, and gravis into 13 types. Gravis type II strains are found worldwide, while types I and III are commonly found in Great Britain. Type IV is mainly found in Egypt, while type V in the United States. Typing of C. diphtheriae strains can also be done by biotyping, lysotype, and by using molecular biology techniques. The latter includes techniques, such as restriction endonuclease digestion patterns of C. diphtheriae chromosomal DNA and genetic probe for cloned corynebacterial insertion sequences. Laboratory Diagnosis Initial treatment of diphtheria is based on the high clinical suspicion or the clinical diagnosis of the condition. Treatment is started without waiting for the result of the laboratory test because definitive results take as long as a week. Hence, laboratory diagnosis is carried out not for treatment of individual cases, but for the epidemiological purposes and for the initiation of control measures of the disease. ◗ Specimens These include swabs from the nose, throat, pieces of pseudomembrane, if possible even from beneath the membrane, biopsy tissue, etc. At least two swabs from the infected site are obtained. The first swab is used to make a direct smear and the other is used for the culture. The specimens are collected as soon as possible when diphtheria is suspected, even if treatment with antibiotics has been started. These are then transported to the laboratory in a sterile empty container or in silica gel sachets for immediate processing. ◗ Microscopy Gram staining of the smear shows Gram-positive bacilli. Albert, Neisser, or Ponder stain of direct smears shows metachromatic granules (Fig. 27-3). However, diphtheria bacilli may not always be demonstrated in the smears, and also it may be difficult to differentiate the bacilli from those of commensal corynebacteria frequently found in the throat. Hence, staining of the smears alone is not specific to C. diphtheriae. The smear examination, however, is valuable to identify Vincent’s spirochetes and fusiform bacilli, the causative agents of Vincent’s angina. ◗ Culture Culture of specimens for C. diphtheriae is essential to confirm the diagnosis of diphtheria. The specimens are inoculated on nonselective media (e.g., blood agar) as well as on selective Chapter 27 The upper respiratory tract of an infected host is the primary habitat of C. diphtheriae. The bacteria also inhabit the superficial layers of the skin lesions. United States and other developed countries, such as Sweden, Italy, and Denmark, 25% to more than 60% of adults did not show protective antitoxin levels in their serum with particularly low levels found in elderly persons. Section III ■ The largest outbreak of diphtheria in the developed world occurred from 1990 to 1996 throughout the states of the former Soviet Union. Most cases were reported among adolescents and adults, rather than children. More than 110,000 cases of diphtheria and 2900 fatalities from diphtheria were reported during the epidemic. Incidence declined in 1996, possibly due to immunization and early detection activities that were carried out following the outbreak. Outbreaks have also been reported in Central Asia, Algeria, and Ecuador. In the United States, Europe, and Eastern Europe, recent outbreaks of diphtheria have occurred largely among alcohol and/or drug abusers. Since 1994, with the advent of active immunization procedures, case fatality due to diphtheria has reduced significantly. 217 218 BACTERIOLOGY ◗ Toxigenicity testing All strains of C. diphtheriae are tested for production of toxins. Production of toxins can be demonstrated by the following tests: 1. In vivo test (a) Subcutaneous test (b) Intradermal test 2. In vitro test (a) Elek’s gel precipitation test (b) Tissue culture test 3. Molecular diagnosis In vivo test Chapter 27 Section III FIG. 27-3. Albert-stained smear showing granules of Corynebacterium diphtheriae (⫻1000). media (e.g., tellurite agar) or enriched media (e.g., Loeffler, Hoyle, Mueller, or Tinsdale medium). Tellurite medium is very useful for isolation of C. diphtheriae from contacts, carriers, and convalescents, in which clinical specimens contain a number of other bacteria. The bacteria on tellurite medium produce characteristic black to grayish black colored colonies after 48 hours of incubation. On Loeffler’s serum slope, C. diphtheriae grows after 4–6 hours at 37°C producing small, circular, and white opaque colonies. If no colonies are seen, the medium is reincubated further for 24 hours. ◗ Identification of bacteria The identifying features of C. diphtheriae colonies are summarized in Box 27-1. ■ ■ ■ C. diphtheriae may be identified as mitis, intermedius, or gravis biotype on the basis of their (a) growth characteristics on tellurite medium, (b) carbohydrate fermentation patterns, and (c) hemolysis on sheep blood agar plates. Potentially toxigenic species (e.g., C. diphtheriae Corynebacterium ulcerans, Corynebacterium pseudotuberculosis) have cystinase, but no pyrazinamidase activity. More recently, 16S ribosomal ribonucleic acid (rRNA) probes have been designed for the identification of genus and species of corynebacteria. Box 27-1 Identifying features of Corynebacterium diphtheriae 1. Gram-positive, nonmotile, nonsporing bacilli showing a Chinese letter pattern formation or cuneiform arrangement. 2. On blood agar, produce variable hemolysis depending on whether it is mitis, intermedius, and gravis. 3. Ferment serum sugars, such as glucose, galactose, maltose, and dextrin, producing acid but no gas. 4. Demonstrate cystinase, but not pyrazinamidase activity. 5. Toxins are demonstrated by in vivo tests (e.g., subcutaneous and intradermal tests in guinea pigs) and in vitro test (e.g., Elek’s gel precipitation test). Subcutaneous test: In this test, growth from overnight culture of C. diphtheriae on Loeffler’s slope is emulsified in 2–4 mL broth. Two guinea pigs are injected subcutaneously with 0.8 mL of the emulsion. One of these is protected with 500 U of diphtheria antitoxin, which is injected intraperitoneally before 18–24 hours of the test. The other guinea pig is not protected. If the strain is virulent, the unprotected animal will die within 4 days, showing the typical findings. The protected guinea pig shall remain normal without showing any sign of toxemia. Intradermal test: In this test, 0.2 mL of the emulsion obtained from 18 hours’ growth of test bacteria cultured on Loeffler’s serum slope is injected intradermally into shaven sites of two albino guinea pigs (or rabbits), so that each animal receives 0.1 mL into two different sites. The control animal is given 500 U of antitoxin the previous day. The other is given 50 U of antitoxin intraperitoneally 4 hours after the skin test, in order to prevent death. Toxigenicity is indicated by an inflammatory reaction at the site of injection progressing to necrosis in 48–72 hours in the test animal, and there is no change in the control animal. This method is advantageous, since large number of strains can be tested simultaneously and also the animals do not die. In vitro test Elek’s gel precipitation test: The Elek’s test is an immunoprecipitation test for demonstration of biological activity of the toxin, initially described in 1949. It is an in vitro neutralization reaction between toxin and antitoxin. The test is performed on a Petri dish containing horse serum agar. A rectangular strip of filter paper impregnated with diphtheria antitoxin (1000 IU/mL) is placed across the medium before the medium is solidified. The strain of C. diphtheriae to be tested for toxicity is streaked on the medium at right angles to the filter paper strip. A known toxigenic strain of C. diphtheriae (positive controls) and nontoxigenic strain of C. diphtheriae (negative control) are also inoculated along with the test strain at right angles to the strip. The plate is incubated at 37°C for 24–48 hours. After incubation, the toxin produced by the growth of the test strain diffuses into the agar and meets the antitoxin at the optimal concentration, and the line of precipitation can be seen. In strains that are negative, no precipitin lines are seen. CORYNEBACTERIUM Tissue culture test: Many eukaryotic cell lines (e.g., African green monkey kidney, Chinese hamster ovary) are sensitive to diphtheria toxin, enabling in vitro tissue culture tests to be used for detection of toxin production. The toxigenicity of diphtheria bacilli is demonstrated by incorporating strains in agar overlay of cell culture monolayers. The toxin produced by the bacteria diffuses into the cells below and kills the cells in the monolayer. Penicillin and erythromycin are the only antibiotics recommended for treatment. Both antibiotics are equally effective in resolving fever and local symptoms; however, erythromycin has been shown to be marginally superior in eradicating the carrier state. ■ ■ Molecular Diagnosis Polymerase chain reaction (PCR): It is a useful test to detect toxigenic strain of C. diphtheriae directly in a clinical specimen. The assay allows detection of the diphtherial toxin gene (TOX) in the bacteria. Added advantage of the PCR is that it can detect nonviable C. diphtheriae organisms in specimens collected after starting of antibiotic therapy. The test, however, is available only in a few laboratories abroad. Treatment 1. Antitoxin therapy and 2. Antibiotics therapy ◗ ■ ◗ Antitoxin appears to be of no value in treatment of cutaneous diphtheria. The antitoxin is also not recommended for treatment of asymptomatic carriers. Antibiotics therapy Antimicrobial therapy is useful in treatment of diphtheria. Antibiotics: ■ ■ ■ ◗ Limit the production of toxin, Eradicate diphtheria bacteria from infected hosts, and Prevent transmission of the bacteria to patient contacts. Active immunization Active immunization by vaccination with diphtheria toxoid is the key in preventing diphtheria. Vaccines consist of microorganisms or cellular components that act as antigens. Administration of the vaccine stimulates the production of antibodies with specific protective properties. Serum antitoxin concentration of 0.01 IU/mL is usually accepted as the minimum protective level, and 0.1 IU/mL provides a definitely protective level. ■ ■ Vaccination is important, especially for high-risk groups (such as children, elderly individuals, and immigrants from areas of continued endemic infections). Active immunization by vaccination increases resistance to C. diphtheriae infection. Vaccines consist of microorganisms or cellular components that act as antigens. Vaccines DTP vaccines: It is typically combined with tetanus toxoid and acellular pertussis (triple DTaP vaccine) and is the vaccine of choice for children aged 6 weeks to 6 years. Alum-adsorbed combination toxoid vaccine against diphtheria is available. There are three types of preparation: 1. DTP (with tetanus toxoid and pertussis vaccine). 2. DTaP (with tetanus toxoid and acellular pertussis vaccine). 3. DT and Dt (with tetanus toxoid for adult and pediatric use, respectively). Diphtheria toxoid is prepared by treating the exotoxin with formaldehyde. This treatment