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Infections, infertility and assisted reproduction


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  • 1. This page intentionally left blank
  • 2. Infections, Infertility, and AssistedReproductionAssisted reproductive technology (ART) treatmentis vulnerable to the hazard of potential infectionfrom many different sources: patients, samples,staff, and the environment. Culture of gametes andembryos in vitro provides multiple targets fortransmission of potential infection, including thedeveloping embryo, neighbouring gametes andembryos, the couple undergoing treatment, andother couples being treated during the same period.This unique situation, with multifaceted opportuni-ties for microbial growth and transmission, makesinfection and contamination control absolutelycrucial in the practice of assisted reproduction, andin the laboratory in particular. This unique and practical book provides a basicoverview of microbiology in the context of ART,providing an up-to-date guide to infections inreproductive medicine. The relevant facets of thecomplex and vast field of microbiology arecondensed and focused, highlighting informationthat is crucial for safe practice in both clinicaland laboratory aspects of ART. This is an essentialpublication for all ART clinics and laboratories.Kay Elder is Director of Continuing Education atBourn Hall Clinic, Bourn, Cambridge, UK.Doris J. Baker is Chair and Professor, Departmentof Clinical Sciences at the University of Kentucky.Julie A. Ribes is Associate Professor of Pathologyand Laboratory Medicine at the University ofKentucky.
  • 3. Infections,Infertility, andAssistedReproductionKay Elder, M.B., Ch.B., Ph.D.Director of Continuing Education, Bourn Hall Clinic,Cambridge, UKDoris J. Baker, Ph.D.Professor and Chair, Department of Clinical Sciences andDirector of Graduate Programs in Reproductive LaboratoryScience, University of Kentucky, Lexington, KY, USAJulie A. Ribes, M.D., Ph.D.Associate Professor of Pathology and Laboratory Medicine, andDirector of Clinical Microbiology, University of Kentucky,Lexington, KY, USA
  • 4. CAMBRIDGE UNIVERSITY PRESSCambridge, New York, Melbourne, Madrid, Cape Town, Singapore, São PauloCambridge University PressThe Edinburgh Building, Cambridge CB2 8RU, UKPublished in the United States of America by Cambridge University Press, New Yorkwww.cambridge.orgInformation on this title:© K. Elder, D. J. Baker and J. A. Ribes 2005This publication is in copyright. Subject to statutory exception and to the provision ofrelevant collective licensing agreements, no reproduction of any part may take placewithout the written permission of Cambridge University Press.First published in print format 2004ISBN-13 978-0-511-26408-5 eBook (EBL)ISBN-10 0-511-26408-9 eBook (EBL)ISBN-13 978-0-521-81910-7 hardbackISBN-10 0-521-81910-5 hardbackCambridge University Press has no responsibility for the persistence or accuracy of urlsfor external or third-party internet websites referred to in this publication, and does notguarantee that any content on such websites is, or will remain, accurate or appropriate.
  • 5. To: our families,Robbie and BethanyJohn and JustinPaul and Maxwell JamesWith love and thanks for their patience,tolerance, and support.
  • 6. ContentsForeword page xiPreface xiiiAcknowledgements xviiPart I Overview of microbiology1 Introduction 3 History of microbiology 3 History of assisted reproduction 7 Artificial insemination 7 In vitro fertilization 8 Assisted reproductive technology (ART) and microbiology 10 Overview of microbiology 11 References 14 Further reading 15 Appendix: glossary of terms 162 Bacteriology 21 Structure and function of bacteria 21 Bacterial structure 21 Bacterial growth 26 Bacterial metabolism 26 Bacterial classification and identification 27 Nomenclature 27 Identification of bacteria 27 Major groups of organisms 38 Gram-negative bacilli and coccobacilli 38 Gram-negative cocci 42 Gram-positive cocci that are catalase positive 42 vii
  • 7. viii Contents Gram-positive cocci that are 4 Virology 105 catalase-negative 43 Introduction 105 Gram-positive bacilli that are Virus structure 105 non-branching and catalase Host range and specificity 106 positive 44 Viral replication 106 Gram-positive bacilli that are Growth characteristics 106 non-branching and catalase Lytic growth 107 negative 46 Lysogenic growth 107 Gram-positive bacilli that are Latent infections 107 branching or partially Virus classification 107 acid-fast 47 Double-stranded DNA 107 Anaerobic bacteria 47 Single-stranded DNA 109 Mycobacteria and bacteria with Double-stranded RNA 109 unusual growth requirements 50 Single-stranded RNA 109 Normal flora in humans 54 Single-stranded (+) sense RNA Further reading 61 with DNA intermediate 109 Appendix 62 Double-stranded DNA with RNA 2.1 Media used for isolation of intermediate 109 bacteria 62 Laboratory diagnosis of viral 2.2 Biochemical tests for disease 110 identification of bacteria 67 Direct examination 110 2.3 Antibacterial agents 85 Culture 110 Further reading for Appendix 2.3 89 Antigen detection systems 110 Serologic diagnosis 111 3 Mycology: moulds and yeasts 90 Molecular diagnostics 111 Introduction 90 Viruses directly relevant to ART 111 Classes of fungi 91 Double-stranded DNA Zygomycetes 92 viruses 112 Ascomycetes 92 Hepatitis viruses 115 Basidiomycetes 92 Retroviruses 116 Deuteromycetes 92 Human oncornaviruses 117 Laboratory classification of Further reading 118 fungi 92 Appendix: antiviral agents 119 Taxonomic classification 92 5 Prions 122 Clinical classification of fungi 94 Prion protein 122 Infections 94 Prion diseases 122 Contaminants 98 Animal 122 Laboratory identification of fungi 99 Human 122 Direct examination 99 Prion structure 122 Culture 99 Replication 124 Microscopic examination for Transmission 124 fungal structures 100 Clinical presentation 126 Mycology in ART 100 Sporadic Creutzfeldt–Jakob Further reading 100 disease (nvCJD) 126 Appendix: antifungal agents 102
  • 8. Contents ix New-variant Creutzfeldt–Jakob 8 Vaginitis syndromes 199 disease (CJD) 126 Trichomonas vaginalis 200 Pathology 126 Yeast vaginitis 203 Diagnosis 127 Candida spp. 203 References 128 Bacterial vaginosis 207 Further reading 129 Gardnerella vaginalis 0006 Parasitology 131 Vaginal colonization with Group B Introduction 131 Streptococcus (GBS) 209 Terminology 131 Streptococcus agalactiae 209 Classification 133 Further reading 212 Unicellular: protozoa 133 Lobosea (amoeba) 134 9 Genital human papillomavirus Sarcomastigophora (flagellates) 136 (HPV) infections 215 Ciliophora (ciliates) 139 Genital human papillomavirus Apicomplexa (sporozoa) 140 infections (HPV) 215 Coccidia 141 Genital warts and cervical cancer 215 Microsporidia 145 Further reading 219 Multicellular parasites: helminths and arthropods 145 10 Urethritis and cervicitis Nemathelminthes 145 syndromes 220 Platyhelminthes 154 Male urethritis 220 Arthropods 163 Female urethritis/cervicitis 220 Insecta 163 Gonorrheal disease 221 Arachnida 164 Neisseria gonorrhea 221 Crustacea 164 Chlamydial disease 228 Further reading 165 Chlamydia trachomatis 229 Appendix: antiparastic agents 166 Genital mollicutes 234 Mycoplasma and UreaplasmaPart II Infections in reproductive spp. 234medicine References 238 Further reading 2387 Genital ulcer diseases 177 Herpes simplex virus infections 177 11 Pathology of the upper Syphilis 185 genitourinary tract 243 Treponema pallidum 185 Male upper GU infections 243 Chancroid 190 Epididymitis 243 Haemophilus ducreyi 190 Orchitis 244 Lymphogranuloma venereum Prostatitis 244 (LGV) 192 Female upper GU infections 245 Chlamydia trachomatis 192 Salpingitis 245 Granuloma inguinale (Donovanosis) 193 Oophoritis 246 Calymmatobacterium Endometritis 246 granulomatis 193 Pelvic inflammatory disease (PID) 247 References 195 Pelvic anaerobic actinomycetes 247 Further reading 195 Genital tuberculosis 250
  • 9. x Contents Further reading 259 Treatment of HBV seropositive couples 336 References 259 Treatment of HCV seropositive couples 336 Treatment of HIV seropositive 12 Cytomegalovirus and couples 337 blood-borne viruses 262 Semen washing procedures for Cytomegalovirus (CMV) 262 HBV/HCV/HIV serodiscordant Hepatitis B virus (HBV) 270 couples 339 Hepatitis C virus (HCV) 275 Virus decontamination 340 Hepatitis D virus (HDV) 278 Accidental exposure 340 HIV and AIDS 281 HBV prophylaxis 341 Human T-lymphotrophic viruses HCV prophylaxis 341 (HTLV) 290 HIV prophylaxis 341 HTLV-I 291 Air transport of biohazardous HTLV-II 291 materials 342 References 293 Useful addresses for air transport of Further reading 293 hazardous materials 349 Appendix to Part II: specimen culture by Appendix: general laboratory safety body site 299 issues 350 References 350 Part III Infection and the assisted Further reading 351 reproductive laboratory 15 Prevention: patient screening 13 Infection and contamination and the use of donor gametes 353 control in the ART laboratory 305 Routine screening 353 Sources of infection 305 Prevalence of BBV: geographic Sterilization methods 316 distribution 353 Physical methods of sterilization 316 The use of donor gametes 353 Chemical methods of Recruitment of donors 355 sterilization 318 Screening 356 Disinfection and decontamination 320 Procedures and technical Air quality, classification of cleanrooms aspects 357 and biological safety cabinets 321 Use of gametes for donation 357 Biological safety cabinets (BSCs) 322 Treatment evaluation 358 Microbiological testing and Summary of donor testing contamination 325 practices and proposals in the Fungal contamination in the laboratory 325 USA 358 Laboratory cleaning schedules 327 Cryopreservation and transmission of References 330 infection 358 Further reading 331 Tissue banking: ovarian and testicular tissue 361 14 Handling infectious agents in References 362 the ART laboratory 332 Further reading 363 Blood-borne viruses 332 Biosafety levels 333 Biosafety for ART 334 Index 365
  • 10. ForewordRoger G. GosdenThe Jones Institute for Reproductive MedicineNorfolk, VA.Lucinda L. VeeckWeill Medical College of Cornell UniversityNew York, NYWolbachia are gram-negative, intracellular bacte-ria that shelter in the gonads of invertebrates, andhave profound effects on the fertility of their hosts.In some species, infected hosts can only reproduceparthenogenetically, in others cytoplasmic incom-patibility prevents infected males from breedingwith uninfected females, and in some cases geneti-cally determined male embryos are transformed intofemales. Wolbachia engineers effects, as do all par-asites, for selfish ends. Although this bizarre pathol-ogy is unknown in medical science, the relationshipsbetween microbes and human fertility are nonethe-less complex, fascinating and important for the prac-tice of reproductive medicine. Unfortunately, and usually without advance warn-ing, microbes occasionally enter the clinical lab-oratory through infected semen or vaginal tissue.When this occurs, a patient’s treatment outcome maybe seriously compromised because microbes canquickly deplete nutrients in culture media and alterthe pH, and it would be irresponsible to knowinglytransfer an infected embryo or semen to a patient.Bacterial and fungal growth are often obvious andeasily tested, but how often do infectious agentsgo unrecognized and contribute to the problems ofinfertility, treatment failure and even possibly affectthe child-to-be? This is the first book on medical microbiologythat has been written by experts in reproduction forclinical scientists and physicians in their own field.They are to be congratulated on filling a gap in the xi
  • 11. xii Foreword literature between microbiology and assisted repro- In the final section, the practical implications of this duction, which they achieve in three sections. The knowledge are addressed in the context of infertility, first serves as a primer of medical microbiology for and especially the setting of the clinical embryology readers who are unfamiliar or rusty on the subject. laboratory. Every embryologist is trained in sterile The second focuses on microbes that have implica- techniques, filtration of media and prudent use of tions for human reproduction, whether by causing antibiotics to keep out the bugs, but a deeper knowl- infertility (a familiar example being Chlamydia) or edge of the foundations of safe and effective practice by jeopardizing reproductive safety (such as HIV). is an undervalued safeguard for patient care.
  • 12. PrefaceThe world of microbes is intrinsically fascinating.Microbes are abundant in every place on earth wherelarger living creatures exist, and they can also thrivein habitat extremes where no other kind of organ-ism can survive for long: from deep under the sea tothe stratosphere – up to 32 km in the atmosphere,in oil formations and in hot telluric water. It is esti-mated that the total biomass of microbes probablyexceeds that of all the plants and animals in the bio-sphere. This biomass is predominantly composedof bacteria, and these microorganisms play a cru-cial role in recycling much of the organic materialin the biosphere. Despite their minute size, microor-ganisms carry out all the fundamental processes ofbiochemistry and molecular biology that are essen-tial to the survival of all living species. Although theirsize may give them the illusion of being ‘primitive’,their range of biochemical and biophysical capabil-ities is far wider than that of higher organisms. Oneof their most important properties is adaptabilityand versatility, a key feature in their long history ofevolution. Fossil records suggest that at least somemembers of the microbial world, oxygen-producingcyanobacter-like organisms, had evolved 3.46 billionyears ago (Schopf, 1993); a viable fungus, Absidiacorymbifera, was recovered from the right boot thataccompanied the frozen, well-preserved prehistoriccorpse, ‘Ice Man’, aged approximately 5300 years(Haselwandter & Ebner, 1994). Records of microbial disease that probably influ-enced the course of history can be found in archae-ological sites of early civilizations, as well as in later xiii
  • 13. xiv Preface periods of history. A hieroglyph from the capital of with the help of microscopy. Culture of microor- ancient Egypt dated approximately 3700 BC illus- ganisms and of preimplantation embryos in vitro trates a priest (Ruma) with typical clinical signs of requires special media and growth conditions to a viral infection, paralytic poliomyelitis. The mum- promote cell division, and both are visualized and mified body of the Pharaoh Siptah, who died in 1193 assessed at various stages following cell division. A BC, also shows signs of classic paralytic poliomyelitis, knowledge of microbiology is fundamental to the and the preserved mummy of Rameses V has facial safety and success of assisted reproductive tech- pustular lesions suggesting that his death in 1143 BC niques – but the field of microbiology is vast, and con- was probably due to smallpox. This virulent disease tinues to increase in complexity with the discovery was endemic in China by 1000 BC, and had reached of new organisms and implementation of new med- Europe by 710 AD. Hernando Cortez transferred the ical treatments. The field of assisted reproductive disease to the Americas in 1520, and it appears that technology also continues to expand and develop, around 3 500 000 Aztecs died of smallpox within the particularly in areas of science and biotechnology. next two years – arguably precipitating the end of the Members of an assisted reproduction team are not Aztec empire. usually also experts in infectious diseases, and may In the early 1330s an outbreak of deadly Bubonic find it difficult to identify and follow significant plague occurred in China, one of the busiest of the areas of microbiology that can impact upon their world’s trading nations, and rapidly spread to West- practice. ern Asia and Europe. Between 1347 and 1352 this The purpose of this book is to select areas and top- plague, ‘The Black Death’, killed 25 million people – ics in microbiology that are specifically relevant to one-third of the population of Europe – with assisted reproductive technology (ART), in order to far-reaching social, cultural and economic repercus- provide a very basic background of facts and funda- sions. mental principles. A background of understanding can help prevent contamination and transmission The world of assisted reproduction is equally fas- of disease in ART, and also limit the opportunities cinating, and is one that also has a long history of for microbial survival in embryo culture and cryop- evolution. The concept of assisted procreation by reservation systems. The book is divided into three human artificial insemination was a topic of dis- Parts: cussion between Jewish philosophers as early as the third century AD, and tales exist of fourteenth- Part I provides an outline of microorganism century Arab horse breeders obtaining sperm from classification and identification, as a foundation mated mares belonging to rival groups, using the for understanding the relationships and the sperm to inseminate their own mares. Assisted differences between the types of organisms that reproduction explores the fundamental principles may be encountered in routine ART practice. behind the creation of a new life, the intricate bio- The microorganisms that are human pathogens logical mechanisms that are involved when mature or resident flora, and those that are routinely gametes come into contact, combine genetically found in the environment are introduced. Each and set in motion a cascade of events leading to chapter includes an Appendix of antimicrobial the correct expression of genes that form a new drugs and their modes of action. individual. Part II details organisms that cause disease of Microbiology and assisted reproduction both deal the reproductive tract and those that are with a miniature world, magnified for observation blood-borne pathogens, describing their
  • 14. Preface xvetiology, pathogenesis, diagnosis, pathology and REFERENCEStreatment. Haselwandter, K. & Ebner, M. R. (1994). Microorganisms sur-Part III describes the practical application of viving for 5300 years. FEMS Microbiology Letters, 116(2),microbiology principles within an assisted 189–93.reproduction laboratory. Schopf, J. W. (1993). Microfossils of the early Archean apex chart: new evidence of the antiquity of life. Science, 260, 640–6.
  • 15. AcknowledgementsDigital images for illustrations were produced withthe expert help of Stephen Welch and Robbie Hughes.We would like to thank all of our colleagues andfriends for their valuable encouragement, input andcomments throughout the preparation of this book,with particular acknowledgement of the contribu-tions made by Marc van den Berg, Charles Cornwell,Rajvi Mehta, Rita Basuray, George Kalantzopoulos,Dimitra Kaftani and Kim Campbell. Special thanksto Professor Bob Edwards for his personal reflectionson the ‘History of IVF’ and to Alan Smith for his per-spectives on the development of biotechnology. Barbara and Janet – thank you for your endlesspatience and moral support. We are also grateful for the support of Bourn HallClinic, Cambridge, and the Departments of ClinicalSciences and Pathology and Laboratory Medicine,University of Kentucky, and the University ofKentucky Clinical Microbiology Laboratory. xvii
  • 16. Part IOverview of microbiology
  • 17. 1 IntroductionHistory of microbiology pike does through the water. The second sort . . . oft-times spun round like a top . . . and these were far more inThe history of microbiology, the scientific study of number . . . there were an unbelievably great company ofmicroorganisms, had its origins in the second half of living animalcules, a-swimming more nimbly than any I had ever seen up to this time. The biggest sort . . . bent their bodythe seventeenth century, when Anton van Leeuwen- into curves in going forwards . . . Moreover, the otherhoek (1632–1723), a tradesman in Delft, Holland, animalcules were in such enormous numbers, that all thelearned to grind lenses in order to make microscopes water . . . seemed to be alive.that would allow him to magnify and observe a widerange of materials and objects. Although he had no His Letters to the Royal Society also included descrip-formal education and no knowledge of the scientific tions of free-living and parasitic protozoa, spermdogma of the day, his skill, diligence and open mind cells, blood cells, microscopic nematodes, and aled him to make some of the most important discov- great deal more. In a Letter of June 12, 1716, he wrote:eries in the history of biology. He made simple pow- . . . my work, which I’ve done for a long time, was noterful magnifying glasses, and with careful attention pursued in order to gain the praise I now enjoy, but chieflyto lighting and detail, built microscopes that mag- from a craving after knowledge, which I notice resides in menified over 200 times. These instruments allowed more than in most other men. And therewithal, whenever Ihim to view clear and bright images that he studied found out anything remarkable, I have thought it my duty toand described in meticulous detail. In 1673 he began put down my discovery on paper, so that all ingenious people might be informed thereof.writing letters to the newly formed Royal Society ofLondon, describing what he had seen with his micro- At this time, around the turn of the eighteenth cen-scopes. He corresponded with the Royal Society for tury, diseases were thought to arise by ‘spontaneousthe next 50 years; his letters, written in Dutch, were generation’, although it was recognised that cer-translated into English or Latin and printed in the tain clinically definable illnesses apparently did notPhilosophical Transactions of the Royal Society. These have second or further recurrences. The ancient Chi-letters include the first descriptions of living bacte- nese practice of preventing severe natural small-ria ever recorded, taken from the plaque between his pox by ‘variolation’, inoculating pus from smallpoxteeth, and from two ladies and two old men who had patients into a scratch on the forearm, was intro-never cleaned their teeth: duced into Europe in the early 1800s. The English farmer, Benjamin Justy, observed that milkmaids I then most always saw, with great wonder, that in the said who were exposed to cowpox did not develop small- matter there were many very little living animalcules, very pox, and he inoculated his family with cowpox pus to prettily a-moving. The biggest sort . . . had a very strong and prevent smallpox. Long before viruses had been rec- swift motion, and shot through the water (or spittle) like a ognized as an entity, and with no knowledge of their 3
  • 18. 4 Introduction properties, the physician Edward Jenner (1749–1823) bacteria on solid media such as sterile slices of potato was intrigued by this observation, and started the and on agar kept in the recently invented Petri dish. first scientific investigations of smallpox prevention In 1892 he described the conditions, known as Koch’s by human experimentation in 1796. Jenner used Postulates, which must be satisfied in order to define fluid from cowpox pustules on the hand of a milk- particular bacteria as a cause of a specific disease: maid to inoculate the 8-year-old son of his gar- (i) The agent must be present in every case of the dener, and later challenged the boy by deliberately disease. inoculating him with material from a real case of (ii) The agent must be isolated from the host and smallpox. The boy did not become infected, having grown in vitro. apparently developed an immunity to smallpox from (iii) The disease must be reproduced when a pure the cowpox vaccination. Jenner’s early experiments culture of the agent is inoculated into a healthy led to the development of vaccination as protection susceptible host. against infectious disease and laid the foundations (iv) The same agent must be recovered once again for the science of immunology, which was further from the experimentally infected host. developed during the nineteenth century by Pasteur, Koch’s further significant contributions to microbi- Koch, von Behring and Erlich. ology included work on the tubercle bacillus and The nineteenth century was a ‘Golden Age’ in the identifying Vibrio as a cause of cholera, as well as history of microbiology. The Hungarian doctor, Ignaz work in India and Africa on malaria, plague, typhus, Semmelweiss (1818–1865), observed that puerperal trypanosomiasis and tickborne spirochaete infec- fever often occurred when doctors went directly from tions. He was awarded the Nobel Prize for Physiology the post- mortem room to the delivery room, and sel- or Medicine in 1905, and continued his work on bac- dom occurred when midwives carried out deliveries. teriology and serology until his death in 1910. He thus introduced the notion that infectious agents During the 1800s, agents that caused diseases might be transmitted, and suggested hand washing were being classified as filterable – small enough with chlorinated lime water. Significant discoveries to pass through a ceramic filter (named ‘virus’ about bacteria and the nature of disease were then by Pasteur, from the Latin for ‘poison’) – or non- made by Louis Pasteur, Joseph Lister, Paul Ehrlich, filterable, retained on the surface of the filter (bac- Christian Gram, R. J. Petri, Robert Koch (1843–1910) teria). Towards the end of the century, a Russian and others. Louis Pasteur and Robert Koch together botanist, Dmitri Iwanowski, recognized an agent developed the ‘germ theory of disease’, disproving (tobacco mosaic virus) that could transmit disease the ‘spontaneous generation’ theory held at the time. to other plants after passage through ceramic filters Louis Pasteur (1822–1895) developed the scientific fine enough to retain the smallest known bacteria. basis for Jenner’s experimental approach to vaccina- In 1898 Martinus Beijerinick confirmed and devel- tion, and produced useful animal and human vac- oped Iwanowski’s observations, and was the first to cines against rabies, anthrax and cholera. In 1876 describe a virus as contagium vivum fluidum (solu- Robert Koch provided the first proof for the ‘germ ble living germ). In 1908 Karl Landsteiner and Erwin theory’ with his discovery of Bacillus anthracis as Popper proved that poliomyelitis was caused by a the cause of anthrax. Using blood from infected ani- virus, and shortly thereafter (1915–1917), Frederick mals, he obtained pure cultures of the bacilli by grow- Twort and Felix d’Herrelle independently described ing them on the aqueous humour of an ox’s eye. viruses that infect bacteria, naming them ‘bacterio- He observed that under unfavourable conditions phages’. These early discoveries laid the foundation the bacilli could form rounded spores that resisted for further studies about the properties of bacteria adverse conditions, and these began to grow again and viruses, and, more significantly, about cell genet- as bacilli when suitable conditions were restored. ics and the transfer of genetic information between Koch went on to invent new methods of cultivating cells. In the 1930s, poliovirus was grown in cultured
  • 19. History of microbiology 5cells, opening up the field of diagnostic virology. By drug sales in excess of 10 billion US dollars. The firstthe 1950s, plasmids were recognized as extranuclear genetically engineered human protein, insulin, wasgenetic elements that replicate autonomously, and available by 1982, and the first complete genomeJoshua Lederberg and Norton Zinder reported on sequence of a bacterium, Haemophilus influenzae,transfer of genetic information by viruses (Zinder & was published in 1995. Hormones and other proteinsLederberg, 1952). manufactured by recombinant DNA technology are Following the announcement of the DNA double now used routinely to treat a variety of diseases, andhelix structure by Watson and Crick in 1953, the prop- recombinant follicle stimulating hormone (FSH),erties of bacteria, bacteriophages and animal viruses luteinizing hormone (LH) and human chorionicwere fully exploited and formed the basis of a new gonadotrophin (hCG) are available for routine usescientific discipline: molecular biology, the study of in assisted reproductive technology.cell metabolic regulation and its genetic machinery. A parallel line of investigation that was also a keyOver the next 20 years, Escherichia coli and other feature in molecular biology and medicine duringbacterial cell-free systems were used to elucidate the the latter part of the twentieth century came frommolecular steps and mechanisms involved in DNA the study of retroviruses, novel viruses that requirereplication, transcription and translation, and pro- reverse transcription of RNA into DNA for their repli-tein synthesis, assembly and transport. The devel- cation. During the 1960s, Howard Temin and Davidopment of vaccines and antimicrobial drugs began Baltimore independently discovered viral reverseduring the 1950s, and antibiotic resistance that could transcription, and in 1969 Huebner and Todaro pro-be transferred between strains of bacteria was iden- posed the viral oncogene hypothesis, subsequentlytified by 1959 (Ochiai et al., 1959). In 1967, Thomas expanded and confirmed by Bishop and Varmus inBrock identified a thermophilic bacterium Thermus 1976. They identified oncogenes from Rous sarcomaacquaticus; 20 years later, a heat-stable DNA poly- virus that are also present in cells of normal animals,merase was isolated from this bacterium and used including humans. Proto-oncogenes are apparentlyin the polymerase chain reaction (PCR) as a means essential for normal development, but can becomeof amplifying nucleic acids (Brock, 1967; Saiki et al., cancer-causing oncogenes when cellular regulators1988). Another significant advance in molecular bio- are damaged or modified. Bishop and Varmus werelogy came with the recognition that bacteria produce awarded the Nobel Prize for Medicine or Physiol-restriction endonuclease enzymes that cut DNA at ogy in 1989. In 1983, Luc Montagnier discovered aspecific sites, and in 1972 Paul Berg constructed a retrovirus believed to cause the acquired immunerecombinant DNA molecule from viral and bacterial deficiency syndrome (AIDS) – the human immun-DNA using such enzymes (Jackson et al., 1972). The odeficiency virus (HIV). By the end of the twentiethconcept of gene splicing was reported by 1977, and in century, the total number of people affected by thisthat same year Frederick Sanger and his colleagues novel virus exceeded 36 million.elucidated the complete nucleotide sequence of the Around this same time, another novel pathogen ofbacteriophage X174, the first microorganism to a type not previously described also came to light:have its genome sequenced (Sanger et al., 1977). in 1982 Stanley Prusiner discovered that scrapie, aBerg, Gilbert and Sanger were awarded the Nobel transmissible spongiform encephalopathy (TSE) inPrize for Chemistry in 1980. sheep, could be transmitted by a particle that was These discoveries involving microorganisms apparently composed of protein alone, with no asso-established the foundation for genetic engineer- ciated nucleic acid – the prion protein (Prusiner,ing. Gene cloning and modification, recombinant 1982). This was the first time that an agent with nei-DNA technology and DNA sequencing established ther DNA or RNA had been recognized as pathogenic,biotechnology as a new commercial enterprise: by challenging previous dogmas about disease patho-2002 the biotechnology industry had worldwide genesis and transmission.
  • 20. 6 Introduction The field of microbiology continues to grow and vectors, animal reservoirs, or environmental elicit public concern, both in terms of disease pathol- sources of novel pathogens, e.g. prions. ogy and in harnessing the properties of microbes for (iv) Modern air transportation allows large numbers the study of science, especially molecular genetics. of people, and hence infectious disease, to travel During the past 25–30 years, approximately 30 new worldwide with hitherto unprecedented speed. pathogens have been identified, including HIV, hem- Other areas that can contribute to pathogen emer- orrhagic viruses such as Ebola, transfusion-related gence include events in society such as war, civil hepatitis C-like viruses, and, most recently, the coro- conflict, population growth and migration, as well navirus causing sudden acute respiratory syndrome as globalization of food supplies, with changes (SARS). The first SARS outbreak occurred in the in food processing and packaging. Environmental Guangdong province of China in November 2002 changes with deforestation/reforestation, changes and had spread as a major life-threatening penumo- in water ecosystems, flood, drought, famine, and nia in several countries by March 2003. The infec- global warming can significantly alter habitats and tious agent was identified during that month, and exert evolutionary pressures for microbial adapta- a massive international collaborative effort resulted tion and change. Human behaviour, including sex- in elucidating its complete genome sequence only ual behaviour, drug use, travel, diet, and even use 3 weeks later, in mid-April 2003. The genome of child-care facilities have contributed to the trans- sequence reveals that the SARS virus is a novel class mission of infectious diseases. The use of new med- of coronavirus, rather than a recent mutant of the ical devices and invasive procedures, organ or tis- known varieties that cause mild upper respiratory sue transplantation, widespread use of antibiotics illness in humans and a variety of diseases in other and drugs causing immunosuppression have also animals. Information deduced from the genome been instrumental in the emergence of illness due sequence can form the basis for developing targeted to opportunistic pathogens: normal microbial flora antiviral drugs and vaccines, and can help develop such as Staphylococcus epidermidis cause infec- diagnostic tests to speed efforts in preventing the tions on artificial heart valves, and saprophytic fungi global epidemic of SARS. At the beginning of June cause serious infection in immunocompromised 2003, 6 months after the first recorded case, the World patients. Health Organization reported 8464 cases from more Microorganisms can restructure their genomes in than two dozen countries, resulting in 799 deaths. response to environmental pressures, and during These new diseases are now being defined within a replication there is an opportunity for recombina- context of ‘emergent viruses’, and it is clear that new tion or re-assortment of genes, as well as recombi- infectious diseases may arise from a combination of nation with host cell genetic elements. Some viruses different factors that prevail in modern society: (e.g. HIV) evolve continuously, with a high frequency (i) New infectious diseases can emerge from ge- of mutation during replication. Retroviruses are netic changes in existing organisms (e.g. SARS changing extraordinarily rapidly, evolving sporadi- ‘jumped’ from animal hosts to humans, with a cally with unpredictable patterns, at different rates change in its genetic make-up), and this ‘jump’ in different situations. Their genetic and metabolic is facilitated by intensive farming and close and entanglement with cells gives retroviruses a unique crowded living conditions. opportunity to mediate subtle, cumulative evolu- (ii) Known diseases may spread to new geographic tionary changes in host cells. Viruses that are trans- areas and populations (e.g. malaria in Texas, mitted over a long time period (HIV) have a selective USA). advantage even when their effective transmission (iii) Previously unknown infections may appear in rates are relatively low. humans living or working in changing ecologic Assisted reproduction techniques are now being conditions that increase their exposure to insect used to help people who carry infectious diseases
  • 21. Artificial insemination 7(including those that are potentially fatal and may By the turn of the nineteenth century, the use of arti-have deleterious effects on offspring) to have chil- ficial insemination in rabbits, dogs and horses haddren. This potential breach of evolutionary barri- been reported in several countries. In 1866 an Italianers raises new ethical, policy and even legal issues physician, Paolo Mantegazza, suggested that spermthat must be dealt with cautiously and judiciously could be frozen for posthumous use in humans and(Minkoff & Santoro, 2000). for breeding of domestic animals, and in 1899 the Russian biologist Ivanoff reported artificial insem- ination (AI) in domestic farm animals, dogs, foxes,History of assisted reproduction rabbits and poultry. He developed semen extenders, began to freeze sperm and to select superior stal-Assisted reproduction may also be said to have its lions for breeding. His work laid the foundation fororigin in the seventeenth century, when Anton van the establishment of artificial insemination as a vet-Leeuwenhoek first observed sperm under the micro- erinary breeding technique.scope and described them as ‘animalcules’. In 1779, Around this same time in Cambridge UK, theLazzaro Spallanzani (1729–1799), an Italian priest reproductive biologist, Walter Heape, studied theand scientist was the first to propose that contact relationship between seasonality and reproduc-between an egg and sperm was necessary for an tion. In 1891 he reported the recovery of a pre-embryo to develop and grow. He carried out artifi- implantation embryo after flushing a rabbit oviductcial insemination experiments in dogs, succeeding and transferring this to a foster mother with contin-with live births, and went on to inseminate frogs ued normal development (Heape, 1891). His workand fish. Spallanzani is also credited with some of encouraged others to experiment with embryo cul-the early experiments in cryobiology, keeping frog, ture; in 1912 Alain Brachet, founder of the Belgianstallion and human sperm viable after cooling in School of Embryology, succeeded in keeping a rabbitsnow and re-warming. The Scottish surgeon, John blastocyst alive in blood plasma for 48 hours. Preg-Hunter (1728–1793), was the first to report artificial nancies were then successfully obtained after flush-insemination in humans, when he collected sperm ing embryos from a number of species, from micefrom a patient who sufferered from hypospadias and and rabbits to sheep and cows. Embryo flushing andinjected it into his wife’s vagina with a warm syringe. transfer to recipients became a routine in domesticThis procedure resulted in the birth of a child in 1785. animal breeding during the 1970s.The next documented case of artificial inseminationin humans took place in 1884 at Jefferson MedicalCollege in Philadelphia, USA: Artificial insemination A wealthy merchant complained to a noted physician of his By 1949, Chris Polge in Cambridge had developed inability to procreate and the doctor took this as a golden the use of glycerol as a semen cryoprotectant, and opportunity to try out a new procedure. Some time later, his the process of semen cryopreservation was refined patient’s wife was anaesthetised. Before an audience of for use in cattle breeding and veterinary practice. medical students, the doctor inseminated the woman, using The advantages of artificial insemination were rec- semen obtained from ‘the best-looking member of the class’. ognized: genetic improvement of livestock, decrease Nine months later, a child was born. The mother is reputed in the expense of breeding, the potential to increase to have gone to her grave none the wiser as to the manner of her son’s provenance. The husband was informed and was fertility, and a possible disease control mechanism. delighted. The son discovered his unusual history at the age Almquist and his colleagues proposed that bacte- of 25, when enlightened by a former medical student who rial contaminants in semen could be controlled by had been present at the conception. (Hard, AD, Artificial adding antibiotics to bovine semen (Almquist et al., Impregnation, Medical World, 27, p. 163, 1909) 1949). The practice of artificial insemination was
  • 22. 8 Introduction soon established as a reproductive treatment in Alan Parkes and Bunny Austin, continuing to explore humans. Methods for cryopreserving human semen his interest in genetics, mammalian oocytes and the and performing artificial insemination were refined process of fertilization. During this period he started in the early 1950s (Sherman & Bunge, 1953), and expanding his interests into human oocyte matura- a comprehensive account of Donor Insemination tion and fertilization, and with the help of the gynae- was published in 1954 (Bunge et al., 1954). By the cologist Molly Rose began to observe human oocytes mid-1980s, however, it became apparent that donor retrieved from surgical biopsy specimens. In 1962 he insemination had disadvantages as well as advan- observed spontaneous resumption of meiosis in a tages, including the potential to transmit infectious human oocyte in vitro for the first time. After a brief diseases. Before rigorous screening was introduced, period in Glasgow with John Paul, he was appointed HIV, Chlamydia, Hepatitis B and genital herpes as a Ford Foundation Fellow in the Physiological Lab- were spread via donor semen (Nagel et al., 1986; oratory in Cambridge in 1963. By this time Chang Berry et al., 1987; Moore et al., 1989; McLaughlin, (1959) had successfully carried out in vitro fertiliza- 2002). tion with rabbit oocytes and sperm, and Yanagamichi (1964) subsequently reported successful IVF in the golden hamster. Whittingham was working towards In vitro fertilization fertilization of mouse eggs in vitro, and reported suc- cess in 1968. In Cambridge, Bob Edwards began on Advances in reproductive endocrinology, including the slow and arduous road that eventually led to identification of steroid hormones and their role in successful human in vitro fertilization. He contin- reproduction, contributed significantly to research ued his studies using the limited and scarce material in reproductive biology during the first half of the available from human ovarian biopsy and pathology twentieth century. During the 1930s–40s, the pitu- specimens, and published his observations about itary hormones responsible for follicle growth and maturation in vitro of mouse, sheep, cow, pig, rhe- luteinization were identified, and a combination of sus monkey and human ovarian oocytes (Edwards, FSH and LH treatments were shown to promote 1965). Working with several Ph.D. students, Edwards maturation of ovarian follicles and to trigger ovula- fertilized mouse and cow eggs in vitro, and obtained tion. Urine from postmenopausal women was found mouse offspring. With his students, he studied the to contain high concentrations of gonadotrophins, growth of chimaeric embryos constructed by inject- and these urinary preparations were used to induce ing a single or several inner cell mass cells from ovulation in anovulatory patients during the early donor blastocysts into recipient genetically marked 1950s. blastocysts. The birth of live chimaeras confirmed Parallel relevant studies in gamete physiology the capacity of single stem cells to colonize virtually and mammalian embryology were underway by all organs in the recipient, including germline, but this time, with important observations reported by not trophectoderm. They also removed small pieces Austin, Chang and Yanagimachi. In 1951, Robert of trophectoderm from live rabbit blastocysts and Edwards began working towards his Ph.D. project determined their sex by identifying whether they in Edinburgh University’s Department of Animal expressed the sex chromatin body. Embryos with Genetics headed by Professor Conrad Waddington this body were classified as females and the others and under the directon of Alan Beatty. Here he began as males. The sex of fetuses and offspring at birth to pursue his interest in reproductive biology, study- had been correctly diagnosed, signifying the onset ing sperm and eggs, and the process of ovulation of preimplantation genetic diagnosis for inherited in the mouse. After 1 year spent in Pasadena at the characteristics. At this time, anomalies observed in California Institute of Technology working on prob- some rabbit offspring caused them some concern, lems in immunology and embryology, in 1958 he but a large study by Chang on in vitro fertiliza- joined the MRC in Mill Hill, where he worked with tion in mice proved that the anomalies were due
  • 23. In vitro fertilization 9to the segregation of a recessive gene, and not due co-culture allowed the development of stage-specificto IVF. media optimized for embryo culture to the blastocyst In 1968 Edwards began his historic collabora- stage. Advances in technique and micromanipula-tion with Patrick Steptoe, the gynaecologist who tion technology led to the establishment of assistedpioneered and introduced the technique of pelvic fertilization (intracytoplasmic sperm injection, ICSI)laparoscopy in the UK. Using this new technique in by a Belgian team led by Andr´ Van Steirteghem and ehis clinical practice in Oldham General Hospital near including Gianpiero Palermo (Palermo et al., 1992)Manchester, Steptoe was able to rescue fresh pre- by the mid 1990s. Other microsurgical interventionsovulatory oocytes from the pelvis of patients who were then introduced, such as assisted hatching andsuffered from infertility due to tubal damage. Bob embryo biopsy for genetic diagnosis. Gonadal tis-Edwards and his colleague, Jean Purdy, traveled from sue cryopreservation, in vitro oocyte maturation andCambridge to Oldham in order to culture, observe embryonic stem cell culture are now under develop-and fertilize these oocytes in vitro. The team began to ment as therapeutic instruments and remedies forexperiment with culture conditions to optimize the the vitro fertilization system, and tried ovarian stim- The first live calves resulting from bovine IVFulation with drugs in order to increase the number were born in the USA in 1981. This further mile-of oocytes available for fertilization. After observing stone in reproductive biotechnology inspired theapparently normal human embryo development to development of IVF as the next potential commer-the blastocyst stage in 1970, they began to consider cial application of assisted reproduction in domes-re-implanting embryos created in vitro into the uteri tic species, following on from AI and conventionalof patients in order to achieve pregnancies: the first transfer of embryos produced in vivo from superovu-human embryo transfers were carried out in 1972. lated donors. The assisted reproductive techniquesDespite the fact that their trials and experiments continued to be refined so that by the 1990s IVF waswere conducted in the face of fierce opposition and integrated into routine domestic species breedingcriticism from their peers at the time, they contin- programmes. Equine IVF has also been introducedued to persevere in their efforts, with repeated failure into the world of horse breeding (although repro-and disappointment for the next 6 years. Finally, their ductive technology procedures cannot be used for10 years of collaboration, persistence and persever- thoroughbreds). China used artificial inseminationance were rewarded with the successful birth of the to produce the first giant panda cub in captivity infirst IVF baby in 1978. 1963, and assisted reproduction is now used in the The modern field of assisted reproductive technol- rescue and propagation of endangered species, fromogy (ART) arrived with the birth of Louise Brown on pandas and large cats to dolphins. Artificial insem-July 25, 1978. After a 2-year lag, when no funds or ination, and, in some cases, in vitro fertilizationfacilities were available to continue their pioneering are used routinely in specialist zoos throughout thework, Steptoe and Edwards opened a private clinic world.near Cambridge: Bourn Hall Clinic, dedicated solely In the field of scientific research, applicationto treating infertility patients using in vitro fertiliza- of assisted reproductive techniques in animal sys-tion and embryo transfer. The first babies were con- tems has helped to unravel the fundamental stepsceived within days, and many more within 3 months. involved in fertilization, gene programming andTheir rapid clinical success in achieving pregnancies expression, regulation of the cell cycle and patt-and live births led to the introduction of IVF treat- erns of differentiation. Somatic cell nuclear transferment worldwide throughout the 1980s and 1990s. into enucleated oocytes has created ‘cloned’ animalsThe first babies born after transfer of embryos that in several species, the most famous being Dolly thehad been frozen and thawed were born in 1984–85, sheep, who was born in Edinburgh in 1997; Dolly wasand cryopreservation of embryos as well as semen euthanized at an early age in February 2003, afterbecame routine. Experiments with cell cultures and developing arthritis and progressive lung disease.
  • 24. 10 Introduction Advances in molecular biology and biotechnol- cover a wide range of expertise, including micro- ogy continue to be applied in ART. Preimplantation biology. Culture media and systems used to pro- genetic diagnosis (PGD), introduced in 1988, is used cess and culture gametes and embryos are designed to screen embryos for sex-linked diseases or auto- to encourage cell growth, but this system can also somal mutations in order to exclude chromosomally encourage the growth of a wide variety of microbes. abnormal embryos from transfer. Molecular biology Whereas cell division in preimplantation embryos is techniques can identify chromosomes with the use relatively slow, with each cell cycle lasting approxi- of fluorescently labelled probes for hybridization, or mately 24 hours, microbes can multiply very rapidly to amplify DNA from a single blastomere using PCR. under the right conditions. Under constant condi- This technique is also used for gender selection, now tions the generation time for a specific bacterium used routinely in animal breeding programmes. is reproducible, but varies greatly among species. The generation time for some bacteria is only 15–20 minutes; others have generation times of hours or Assisted reproductive technology (ART) even days. Spore-forming organisms have the ability and microbiology to go into ‘suspended animation’, allowing them to withstand extreme conditions (freezing, high tem- Assisted reproduction is a multidisciplinary field that peratures, lack of nutrients). This important prop- relies on close teamwork and collaboration between erty allows the organisms to survive in central heat- different medical and scientific disciplines that must ing and air conditioning systems or cooling towers Fig. 1.1. Schematic diagram of a typical eukaryotic cell, illustrating characteristic intracellular organelles.
  • 25. Overview of microbiology 11for indefinite periods: the ‘Ice Man’ fungus survived Overview of microbiologyat least 5300 years, and live bacterial spores werefound in ancient pressed plants at Kew Gardens Naturam primum cognoscere rerumdating back to the seventeenth century. Viruses First . . . to learn the nature of things (Aristotle, 384–322 BC )do not form spores, but some can survive dry on Living things have been traditionally classifiedhandkerchiefs, or cleaning or drying cloths if protein into five biological Kingdoms: Animals (Animalia),is present, e.g. in droplets from a sneeze or cough. Plants (Plantae), Fungi (Fungi), Protozoa (Protista)These properties presents special problems in the and Bacteria (Monera). Animals, plants, fungi andART laboratory since many agents used to inhibit or protozoa are eukaryotic, with nuclei, cytoskele-destroy microorganisms are toxic to sperm, oocytes tons, and internal membranes (see Fig. 1.1). Theirand embryos. An ART laboratory must incorporate chromosomes undergo typical reorganization dur-strict guidelines for maintaining necessary sterile ing cell division. Bacteria are prokaryotes, i.e. theyconditions without compromising the gametes and have no well-defined nucleus or nuclear mem-embryos. brane and divide by amitotic division (binary fis- Patients presenting for infertility treatment often sion) (Fig. 1.2). The world of microbes covers a widehave a background of infectious disease as a factor variety of different organisms within the Kingdomsin their infertility, and it is now acknowledged that of Fungi, Protista and Monera, with a diverse rangesome chronic infectious diseases may be ‘silent’ orinapparent, but transmissible in some patients (e.g.Herpes, Ureaplasma, Chlamydia). Some patientsmay be taking antimicrobial drugs that will have Inner cytoplasmica negative effect on gamete function: for example, membranebacterial protein synthesis inhibitors affect spermmitochondrial function, adversely affecting sperm Cell wallmotility. The trend for worldwide travel also intro-duces new contacts and potentially infectious agents Piliacross previous geographic barriers. Nucleoid There are many factors to be taken into accountwhen assessing the risk from microorganisms, andsome that are unique to ART are complex and mul-tifaceted. Formal assessment for quantifying riskrequires experimental study data, epidemiologicalinformation, population biology and mathematicalmodelling – this is not possible in assisted reproduc-tive practice. Instead, ART laboratory practitionersneed to collate, review and evaluate relevant infor-mation, bearing in mind that ART practises breach Flagellabiological barriers, increasing the risk: Wisdom lies in knowing what one is doing and why one is doing it – to take liberties in ignorance is to court disaster – each fragment of knowledge teaches us how much more we have yet to learn. (John Postgate Microbes and Man, 2000) Fig. 1.2. Schematic diagram of a typical prokaryotic (bacterial) Chance favours the prepared mind (Louis Pasteur, cell, illustrating the nucleoid, inner cytoplasmic membrane, 1822–1895) cell wall, pili and flagella.
  • 26. 12 Introduction Table 1.1. Relative sizes of microbes and cells Microbe Size Cell Size Microbe Cells Prion protein 27–55 kD molecular weight, Red blood cell (yardstick) 7 m 243 amino acids, <15 nm White blood cell ranges 6–25 m Small virus 20 nm Epithelial cells (squamous) 40–60 m (Papillomavirus, Poliovirus) HIV virus 110 nm Human gametes/embryos Large virus (Poxvirus, 250–400 nm Mature spermatozoon 4.0–5.0 m in length; Herpesvirus) head size 2.5–3.5 m in width Bacteria size range 0.25 m to 1 m in width and Mature spermatozoon tail 45 m 1 to 3 m in length length Mycoplasma species 0.3 × 0.8 m Round spermatid 7–8 m Staphylococcus spp. 1–3 m Primary spermatocyte 14–16 m Fungi up to 25 m Human oocyte 100 m–115 m (including ZP) Single-celled yeast 8 m Zygote pronuclei ∼30 m Protozoa 10–60 m Cleaving embryo approx. 175 m, including ZP Amoeboid cyst 10–20 m diameter Trichomonas vaginalis 13 m long Parasite eggs 50–150 m diameter Parasitic adult worms 2 cm–1 m long 1 m = 1 × 10−6 metre = 1 × 10−3 mm; 1 m = 1 × 10−9 metre = 1 × 10−6 mm; ZP: zona pellucida. Fig. 1.3. Size of microorganisms and microscope resolution. Bacteria, protozoa and fungi all can be viewed with light microscopy. The average diameter for protozoa is 12–60 m; the largest parasitic protozoan, Balantidium coli is 60 m × 40 m. Bacteria range in size from 0.25 to 1 m in width and 1 to 3 m in length. Viruses are larger than macromolecules, but smaller than the smallest bacteria. Viruses can be visualized by electron microscopy.
  • 27. Overview of microbiology 13Fig. 1.4. Overview of microorganisms, in order of size and complexity.
  • 28. 14 Introduction of properties. This microcosm is further classified an organism to sustain itself. The authors speculate into basic ‘families’ of phylogenetic classification that these nanoorganisms may represent an earlier based upon evolutionary sequence and character- intermediate form of life (Huber et al., 2002). Their istic properties. finding suggests that there may be other similarly (i) Prokaryotes: Bacteria, Cyanobacteria, Ricket- unusual groups of microbes yet to be discovered. tsiae Pathogenic microbiology primarily deals with gen- (ii) Fungi: (eukaryotes) moulds, mushrooms, yeasts era and species within families. Microorganisms that (iii) Viruses are important in clinical ART practice can be found (iv) Eukaryotic protists (protozoa): unicellular or in all the categories, and they cover a wide range of multicellular sizes. Table 1.1 describes the size range of microbes A virus is a submicroscopic infectious particle com- relative to blood cells, gametes and embryos; posed of a protein coat and a nucleic acid core. Viru- Fig. 1.3 illustrates the relative sizes of protozoa, bac- ses, like cells, carry genetic information encoded in teria, viruses and macromolecules. An overview of their nucleic acid, and can undergo mutations and the microorganisms discussed in Part I, in order of reproduce; however, they cannot carry out meta- size and complexity, is presented in Fig. 1.4. bolism, and thus are not considered ‘alive’ by the classical definition. Viruses are classified by the type of nucleic acid they contain, and the shape of their REFERENCES protein capsule. Prions are a recent addition to the list of ‘micro- Almquist, J. O., Glantz, P. J. & Shaffer, H. E. (1949). The effect organisms’. They do not fit into any of the ‘classical’ of a combination of penicillin and streptomycin upon the families, and present a further challenge to accepted viability and bacterial content of bovine semen. Journal of definitions of ‘life’: although they apparently show Dairy Science, 32: 183–90. multiplication, heredity and variation, they do not Berry, W. R., Gottesfeld, R. L., Alter, H. J. & Vierling, J. M. have the ability to react and adjust to environments. (1987). Transmission of hepatitis B virus by artificial insem- In the late 1970s, a new group of organisms was ination. Journal of the American Medical Association, 257: 1079–81. added to this classification: the Archaea, a group of Brock, T. D. (1967). Micro-organisms adapted to high tempera- bacteria that live at high temperatures or produce tures. Nature (London), 214: 882–5. methane. Because these organisms are biochemi- Bunge, R. G., Keeteel W. C. & Sherman J. K. (1954). Clinical use cally and genetically different from usual bacteria, it of frozen semen. Fertility and Sterility, 5: 520–9. was proposed that life be divided into three domains: Chang, M. C. (1959). Fertilization of rabbit ova in vitro. Nature Eukaryota, Eubacteria and Archaea – rather than (London), 184: 406. the classical five kingdoms. Archaeans have been Edwards, R. G. (1965). Maturation in vitro of mouse, sheep, cow, found in the most extreme environments on the pig, rhesus monkey and human ovarian oocytes. Nature planet, and also in plankton of the open sea and as (London), 208: 349–51. methane-producing organisms inside the digestive (1989). Life Before Birth: Reflections on the Embryo Debate. tracts of cows, termites, and marine life. They live London: Hutchinson. Heape, W. (1891). Preliminary note on the transplantation and in the anoxic muds of marshes and at the bottom growth of mammalian ova within a uterine foster-mother. of the ocean, and even thrive in petroleum deposits Proceedings of the Royal Society, 48: 457. deep underground. In May 2002, the discovery of Huber, H., Hohn, M. J., Rachel, R., Fuchs, T., Wimmer, V. C. & another new member of the Archaea was reported, Stetter, K. O. (2002). A new phylum of Archaea represented the Nanoarchaeota. This microbe has one of the by a nanosized hyperthermophilic symbiont. Nature, 417: smallest genomes known, with 500 000 nucleotide 63–7. bases. The sequence of these nucleotide bases may Jackson, D. A., Symons, R. H. & Berg, P (1972). Biochemical . point to the minimum number of genes needed for method for inserting new genetic information into DNA of
  • 29. Further reading 15 simian virus 40: circular SV 40 DNA molecules containing Advisory Committee on Dangerous Pathogens (2002). Microbi- lamda phage genes and the galactose operon of Escherichia ological risk assessment – an interim report, HMSO Publi- coli. Proceedings of the National Academy of Science, USA, cations. 69: 2904–9. Austin, C. R. (1951). Observations on the penetration of theMcLaughlin, E. A. (2002). Cryopreservation, screening and stor- sperm into the mammalian egg. Australian Journal of Sci- age of sperm – the challenges for the twenty-first century. entific Research, 4: 581–96. Human Fertility, 5 (Suppl.): S61–5. Balen, A. H. & Jacobs, H. S. (1997). Infertility in Practice. Edin-Minkoff, H. & Santoro, N. (2000). Ethical considerations in the burgh: Churchill Livingstone. treatment of infertility in women with human immunode- Bloom, B. R. (2003). Lessons from SARS. Science, 300: 701. ficiency virus infection. New England Journal of Medicine, Brackett, R. G., Bousquet, D., Boice, M. L. et al. (1982). Normal 343: 1748–50. development following in vitro fertilization in the cow. Biol-Moore, D. E., Ashley, R. L., Zarutskie, P. W. et al. (1989). Trans- ogy of Reproduction, 27: 147–58. mission of genital herpes by donor insemination. Journal Cole, H. & Cupps, P. (1977). Reproduction in Domestic Animals. of the American Medical Association, 261: 3441–3. New York: Academic Press.Nagel, T. C., Tagatz, G. E. & Campbell, B. F. (1986). Transmission of Dobell, C. (ed.) (1960). Antony van Leeuwenhoek and his ‘Little Chlamydia trachomatis by artificial insemination. Fertility Animals’. New York: Dover Publications. and Sterility, 46: 959–62. Edwards, R. G. (1972). Control of human development. In Arti-Ochiai, K., Yamanda, K., Kimura, K. & Sawada, O. (1959). Stud- ficial Control of Reproduction; Reproduction in Mammals, ies on the inheritance of drug resistance between Shigella Book 5, ed. Austin C. R. & Short R. V., pp. 87–113. Cambridge, strains and Escherichia coli strains. Nippon Iji Shimpo, UK: Cambridge University Press. 1861: 34–46. Edwards, R. G., Bavister, B. D. & Steptoe, P. C. (1969). Early stagesPalermo, G., Joris, H., Devroey, P & Van Steirteghem, A. C. (1992). . of fertilization in vitro of human oocytes matured in vitro. Pregnancies after intracytoplasmic injection of single sper- Nature (London), 221: 632–5. matozoon into an oocyte. Lancet, 340: 17–18. Epidemiologic Notes and Reports: HIV-1 infection and artificialPrusiner, S. B. (1982). Novel proteinaceous particles cause insemination with processed semen (1990). Morbidity and scrapie. Science, 216: 136–44. Mortality Weekly Report, 39(15): 249,255–6.Saiki, R. K., Gelfand, D. H., Stoffel, S. et al. (1988). Primer-directed Ford, B. J. (1991). The Leeuwenhoek Legacy. Bristol: Biopress and enzymatic amplification of DNA with a thermostable DNA London: Farrand Press. polymerase. Science, 239: 487–91. Gosden, R. G. (1999). Cheating Time: Sex, Science and Aging. NewSanger, F., Air, G. M., Barrell, B. G. et al. (1977). Nucleotide York: W. H. Freeman & Co. sequence of bacteriophage phi X174 DNA. Nature, 165: 687– Haselwandter, K. & Ebner, M. R. (1994). Microorganisms surviv- 95. ing for 5300 years. FEMS Microbiology (Lett). 116: 189–94.Sherman, J. K. & Bunge, R. G. (1953). Observations on preserva- Medvei, V. V. (1993). The History of Clinical Endocrinology. tion of human spermatozoa at low temperatures. Proceed- Carnforth, Lancs. Parthenon. ings of the Society for Experimental Biology and Medicine, Morice, P., Josset, P., Charon, C. & Dubuisson, J. B. (1995). History 82(4), 686–8. of infertility. Human Reproduction Update, 1(5): 497–504.Yanagamachi, R. & Chang, M. C. (1964). IVF of golden hamster Parkes, A. S. (1966). The rise of reproductive endocrinology, ova. Journal of Experimental Zoology, 156: 361–76. 1926–40. Journal of Endocrinology, 34: 19–32.Zinder, N. & Lederberg, J. (1952). Genetic exchange in Polge, C. (1972). Increasing reproductive potential in farm ani- Salmonella. Journal of Bacteriology, 64: 679–99. mals. In Artificial Control of Reproduction, Reproduction in Mammals, Book 5, ed. Austin C. R. & Short R. V., pp. 1–32. Cambridge, UK: Cambridge University Press. Postgate, J. (2000) Microbes and Man, 4th edition. Cambridge,FURTHER READING UK: Cambridge University Press. Robert G. Edwards at 75 (2002). Reproductive BioMedicine Online 4, suppl. 1. Schopf, J. W. (1993). Microfossils of the early Archean apex∼dmsander/WWW/224/ chart: new evidence of the antiquity of life. Science, 260: Classification224.html 640–6.
  • 30. 16 Introduction Steptoe, P. C. & Edwards, R. G. (1970). Laparoscopic recovery of BBV Blood-borne viruses. pre-ovulatory human oocytes after priming of ovaries with biotype biologic or biochemical type of an organ- gonadotrophins. Lancet, i: 683–9. ism. Organisms of a given biotype have identical (1976). Reimplantation of a human embryo with subsequent tubal pregnancy. Lancet, i: 880–2. biologic or biochemical characteristics. Key mark- (1978). Birth after the re-implantation of a human embryo ers are used to recognise biotypes in tracing spread (letter). Lancet, ii: 366. of organism during epidemics. Yanagamachi, R. & Chang, M. C. (1964). IVF of golden hamster candle jar a jar with a lid providing a gas-tight seal ova. Journal of Experimental Zoology, 156: 361–76. in which a small white candle is placed and lit after Whittingham, D. G. (1968). Fertilization of mouse eggs in vitro. culture plates are added. The candle will only burn Nature, 200: 281–2. until the oxygen is reduced to a point that burning is no longer supported; at that point the atmosphere in the jar will have a lower oxygen content than room Appendix: air and ∼3% carbon dioxide. glossary of terms capnophile an organism that requires increased CO2 (5–10%) and approximately 15% O2 . Neisse- abscess local collection of pus. ria gonorrhoeae and Haemophilus influenzae are aerobe a bacterium that grows in ambient air, which capnophilic bacteria. These organisms will grow in a contains 21% oxygen (O2 ) and a small amount candle jar with 3% CO2 or a CO2 incubator. (0.03%) of carbon dioxide (CO2 ). carrier a healthy person who is carrying, and usu- aerosol suspension of small solid or liquid particles ally excreting an infectious agent, but who generally in air; fine spray of droplets. has no symptoms of the infection. anaerobe a bacterium that usually cannot grow catalase bacterial enzyme that breaks down hydro- in the presence of oxygen, but which grows in gen peroxide with release of oxygen. atmosphere composed of 5–10% hydrogen (H2 ), CD4 an antigen found on the surface of T-helper 5–10% CO2 , 80–90% nitrogen (N2 ) and 0% oxygen cells and certain other types of cell such as the mono- (O2 ). cyte, that acts as a receptor for attachment of HIV. antibiotic substance produced by a microorganism CFU Colony forming units; numbers of bacterial that suppresses the growth of, or kills, other microor- colonies on agar cultures, each of which started as ganisms. a single bacterium in the original specimen. antigenaemia the presence of an antigen in the chlamydyospore thick-walled fungal spore formed blood. from vegetative cell. antimicrobial chemical drug produced by a chronic infection persistence of a replicating infec- microorganism that is growth suppressive or tious agent in the host for longer than 6 months. microbiocidal in activity. commensalism a symbiotic relationship in which antiseptic compound that inhibits bacterial growth one organism benefits without harming the host without necessarily killing the bacteria. organism. bacteriostatic agent that inhibits the replication of community-acquired pertaining to outside the target bacteria, but does not kill the organism. hospital (community-acquired infection). bacteriocidal agent that kills the target organism. conidia asexual fungal spores. bacteriophage virus that infects a bacterium. conjugation passing genetic information between bacteriuria bacteria in the urine. bacteria via pili.
  • 31. Appendix: glossary of terms 17contaminant an agent that causes contamination EMB eosin-methylene blue.or pollution. In laboratory cultures, contaminants enterotoxin toxin that affects the intestinalgenerally arise from skin flora or from environmental mucosa.sources. endotoxin substance containing lipopolysaccha-CSF cerebrospinal fluid. ride found in the gram-negative cell bacterial wall;culdocentesis aspiration of cul-de-sac fluid requir- plays an important role in complications of sep-ing puncture of the vaginal wall to enter into the sis (shock, disseminated intravascular coagulation,retroperitoneal space. thrombocytopenia).cystitis inflammation of the urinary bladder. epidemiology the study of occurrence and distribu-cytopathic effect (CPE) changes in cell morphol- tion of disease in populations and factors that contrology resulting from viral infection of a cell culture the presence or absence of disease.monolayer. etiology causative agent or cause of disease.dark-field microscopy technique used to visual- fastidious an organism with very stringent growthize very small or thin microorganisms such as requirements. Certain anaerobes will not grow in thespirochaetes; light is reflected or refracted from the presence of even traces of oxygen.surface of viewed objects. eugonic growing luxuriantly (refers to bacterial cul-decontamination process of rendering an object or tures).area safe by removing microbes or rendering them fermentation anaerobic decomposition of carbo-harmless using biologic or chemical agents. hydrate.definitive host the host in which sexual reproduc-tion of a parasite takes place. fimbrae fingerlike proteinaceous projections that act as a bacterial adherence mechanism.dematiaceous pigmented (dark coloured) moulds,as in those that produce melanin. When examining flagella structures composed mostly of proteinthese moulds, the reverse side of the culture plate responsible for microbe motility.appears dark, indicating a pigmented mycelium. FTA fluorescent treponemal antibody A.dermatophyte parasitic fungus on skin, hair or fulminant a condition or symptom that is of verynails. sudden onset, severe, and of short duration, oftenDFA direct fluorescent antibody test. leading to death.dimorphic fungi fungi with both mould and yeast hyaline non-pigmented, used to refer to fungalphases. organisms that do not produce melanin. Whendisinfectant agent that destroys or inhibits micro- examining the reverse side of the culture plate, theseorganisms. organisms appear tan–white.dysgonic bacteria that grows poorly in culture. iatrogenic caused by medical or surgical interven- tion, induced by the treatment itself.dysuria painful urination. IFA indirect fluorescent antibody.edema excessive accumulation of tissue fluid. immunocompromised a state of reduced resis-Eh oxidation–reduction potential. tance to infection and other foreign substancesendemic occuring in a particular region or popula- that results from drugs, radiation illness, congenitaltion. defect or certain infections (e.g. HIV).elementary body infectious stage of Chlamydia. immunoglobulin antibody; there are five classes:ELISA enzyme-linked immunosorbent assay. IgG, IgM, IgA, IgE and IgD.
  • 32. 18 Introduction inclusion bodies microscopic bodies within body myositis inflammation of muscle. cells thought to be viral particles in morphogenesis. nares nostrils; external openings to nose. incubation period the interval between exposure neonatal first 4 weeks after birth. to an infection and the appearance of the first symp- toms. NGU non-gonococcal urethritis. induration abnormal hardness of tissue resulting non-productive infection failure of a virus-infected from inflammation and hyperaemia. mammalian cell to release progeny virions, usually infection invasion and subsequent multiplication due to the suppression of late gene expression. of microorganisms, causing disease. normal flora the body’s resident microbes, will not indolent a disease process that is failing to heal or cause disease in the ‘normal’ site, but may if dis- has persisted. placed. Some of the organisms that may be encoun- tered in ART may be normal flora (e.g. E. coli and E. latent infection persistence of a non-replicating faecalis found in semen that are normal stool flora) infectious agent in the host. can cause a real problem if they contaminate culture leukocytosis elevated white blood cell count. medium. leukopenia low white blood cell count. nosocomial (Noso, disease, komeo, hospital) media disease acquired by patients during hospitalization– differential permits recognition of organism or clinical manifestations present at least 72 hours group based on recognition of natural organism after admission. Organisms are carried on the hands product identified by incorporating the appropri- of healthcare providers from colonized patients to ate substrate and indicator in media. newly admitted patients, who then become colo- nized themselves. enrichment medium that favours growth of one or more organisms and suppresses growth of com- OPF operation protection factor. peting flora. O&P ova and parasites. selective medium that contains inhibitory sub- stances or unusual growth factors that allow one O–F oxidation–fermentation media. particular organism or group to grow while sup- oncogene regulatory gene of a mammalian cell that pressing most others. has been integrated into the genome of a retrovirus. meningitis inflammation of the meninges of the oncogenic having the potential to cause normal brain. cells to become malignant. metastatic spreading from primary site to distance opportunistic pathogen does not normally pro- focus via the bloodstream or lymphatic system. duce disease. When the host is immunocompro- microconidia small, single-celled fungal spores. mised due to antibiotic therapy, cancer therapy, mixed culture growth of more than one organism steroids or debilitating conditions, opportunists from a single culture. proliferate and cause infection. microaerophile grows under reduced O2 (5–10%). pandemic epidemic that covers a wide geographic Examples are Helicobacter pylori and Campylobacter region or is worldwide. jejuni. It is unlikely that these organisms would be parasite organism that lives on, or within, another encountered in an ART laboratory. organism, at that organism’s expense. mycelium mass of hyphae. parenteral route of drug administration other than mycoses fungal diseases. by mouth (intravenous (IV), intramuscular (IM)).
  • 33. Appendix: glossary of terms 19parotitis inflammation of the parotid gland; mu- prognosis forecast as to outcome of disease.mps is the most common cause. prostatitis inflammation of the prostate gland, usu-paroxysm rapid onset of symptoms (or return of ally caused by infection.symptoms); usually refers to cyclic recurrence of protists eukaryotic microorganisms.malaria symptoms. pure culture growth of a single organism in culture.pathogen an organism that can cause disease. pus inflammatory material consisting of manypathogenesis process by which an organism causes white blood cells, other cellular debris and often bac-disease. teria.pathogenic capable of causing disease. pyuria presence of eight or more white blood cellspathologic caused by, or involving, a morbid con- (WBCs) per cubic mm in uncentrifuged urine.dition such as a pathologic state. purulent containing pus.PCR polymerase chain reaction. reagin an antibody that reacts in various serologicpeptidoglycan murein layer of bacterial cell wall tests for syphilis.responsible for shape and strength to withstand reservoir source from which infectious agent maychanges in osmotic pressure. disseminate.perineum portion of the body bound by the pubic reticulate body metabolically active form in Chla-bone anteriorly and the coccyx posteriorly. mydia life cycle.percutaneous through the skin (e.g. bladder aspi- RPR rapid plasma reagin, non-treponemal tests forration). antibodies to syphilis.petechiae tiny hemorrhagic spots on the skin or routes of infection may bemucous membranes. direct transmissionphage grouping or phagotype bacterial grouping congenitalbased on the ability of certain strains to be lysed sexual contactby specific bacteriophages (useful in epidemiolog- hand-to-hand transmission oral–fecalical tracing of hospital outbreaks). respiratory droplets/secretions indirect transmissionPID pelvic inflammatory disease. fomitespili similar to fimbrae; for transfer of genetic mate- water and foodrial during bacterial conjugation. animals, insect vectors, arthropod vectorsplasmid autonomously replicating DNA molecule airbornefound in bacteria; they may be transferred from cell nosocomial infectionsto cell. community-acquired infectionspleomorphic having more than one form. endogenous infection exogenous infection – obtained or produced out-prion proteinaceous infectious agent associated side the body; not part of the body’s resi-with Creutzfeldt–Jakob disease and other chronic dent flora. Clostridium tetani and Clostridiumdebilitating CNS diseases. botulinum are acquired from the environment.proctitis inflammation of the rectum. E. coli, on the other hand is resident bowel floraprodrome early manifestations of disease before and can cause infection when it reaches anotherspecific symptoms appear. body site (e.g. from ruptured appendix).
  • 34. 20 Introduction saprophytic living off or deriving nutrition from syndrome set of symptoms occurring together. decaying plant or animal matter. teichoic acids part of gram-positive bacteria cell septate having cross walls, as in septate hyphae. wall consisting of glycerol or ribitol phosphate poly- mers combined with various amino acids and sugars. seroconversion the development of antibodies not previously present resulting from a primary infec- teleomorph sexual stage of fungi. tion. thrush Candida infection producing white lesions, serogroup, serotype or serovar grouping of bacte- usually in the oral cavity. ria based upon the antigenic diversity of their surface tolerance a form of induced resistance to antimi- or subsurface antigens. crobials. septicemia or sepsis systemic disease pathogens or transduction moving of genetic material from one their toxins in the blood. prokaryote to another via a bacteriophage. spore reproductive cell of bacteria, fungi or proto- transformation organism takes up free DNA in zoa; may be inactive in bacteria. environment when another organism dies. sensitivity ability of a test to detect all true cases for transposon genetic material that can move from condition being tested; absence of false-negatives. one genetic element to another (plasmid to plas- mid or plasmid to chromosome); called ‘jumping specificity ability of a test to correctly yield a nega- genes’. tive result when condition being detected is absent; Tzanck test stained smear of cells from the base of absence of false-positive results. vesicles, examined for inclusion bodies produced by STD sexually transmitted disease. herpes simplex or varicella-zoster viruses. substrate substance on which enzyme acts. true pathogen causes disease when it enters target suppuration pus formation. body site in sufficient numbers, e.g. hemorrhagic synergism combined effect of two or more agents viruses or syphilis. that is greater than the sum of each separately. viremia the presence of virus in the blood.
  • 35. 2 BacteriologyBacteria are unicellular organisms that belong to the Structure and function of bacteriakingdom of Prokaryotae. Whereas eukaryotes havemembrane-bound cytoplasmic organelles designed Bacterial structureto carry out specific functions, bacteria have no well- Cell walldefined nucleus, nuclear membrane or membrane-bound organelles. Their bacterial genome consists The bacterial cell wall lies close to the cytoplas-of a single, double-stranded, closed circular DNA mic membrane, and gives the organism shape andmolecule that lies within the cytoplasm, along with rigidity. It also acts as a barrier to low molecularmesosomes, ribosomes, and various cytoplasmic weight substances (<10 000 kD), allowing the organ-granules, all of which are enclosed within a thin elas- ism to withstand changes in osmotic pressure thattic trilaminar membrane. The majority of bacteria would cause cell lysis, and serves as an attach-also lack the cytoskeleton that gives support and ment site for bacteriophages and appendages suchstructure to eukaryotes. With the exception of the as pili, fimbriae, and flagella. The cell wall is mademycoplasmas, bacteria contain a cell wall of unique up of a three-dimensional peptidoglycan lattice, achemical composition. They are microscopic in size, biopolymer with alternating units of N-acetyl-D-with species ranging from 0.25 m to 1 m in width muramic acid and N-acetyl-D-glucosamine linkedand 1 m to 10 m in length. Some bacterial species to short peptides, the murein layer. Differences inhave capsules, flagella, or pili, and some species pro- cell wall structure between bacteria are reflectedduce endospores. in their staining properties, and the gram stain is Bacteria are divided into Orders, Families, Gen- used to separate bacteria into groups for identifi-era, and species, and named according to genus cation. Gram-positive and gram-negative bacteriaand species: e.g. Escherichia (genus) coli (species). differ in the amino acid composition of the pep-Bacteria within the same genus and species also tides, and also have different lipoproteins, phospho-may be designated as subspecies, based upon differ- lipids, and lipopolysaccharides (Fig. 2.1). Bacteriaences in geographic distribution, transmission, clini- without cell walls (mycoplasmas), or those lackingcal manifestation, and pathogenicity, e.g. Treponema the peptidoglycan layer (chlamydiae), are referred topallidum subsp. pallidum, and T. pallidum subsp. as gram-null. Since this cell wall structure is uniquepertenue. They may also be classified in serogroups, to bacteria, antimicrobial agents that are targetedserotypes, or serovars based upon surface or subsur- towards its synthesis (e.g. penicillin), or towards trig-face antigens or according to the type of capsule sur- gering of cell wall autolysis will have selective activ-rounding the bacterium. For example, streptococci ity against bacteria, with little or no host toxicity.are grouped on the basis of specific cell wall polysac- Some bacteria such as Mycobacterium tuberculosischaride antigens (Lancefield groups). and members of the Nocardiae have mycolic acid 21
  • 36. 22 Bacteriology Fig. 2.1. Gram-positive and gram-negative bacterial cell envelopes. The outer membrane and periplasmic space are present only in the gram-negative envelope. The murein layer is thicker in gram-positive envelopes. in the cell wall, which allows them to retain the solutes (including antibiotics) that enter the space crystal violet primary stain after acid decolorizing. from the environment. These organisms are ‘acid-fast’ or partially acid-fast, depending on the amount of mycolic acid in the cell Inner cytoplasmic membrane wall. Both gram-negative and gram-positive bacteria have an inner cytoplasmic membrane. This typical cell Outer membrane membrane is a lipid bilayer of 7 nm thickness stud- ded with proteins, many of which are enzymes that Gram-negative, but not gram-positive, bacteria catalyse metabolism within the cell. The membrane have an outer membrane adjacent to the cell serves as an osmotic barrier, allows transport of wall that serves as a selective permeability barrier solutes in and out of the cell, and is the site of to hydrophilic and hydrophobic compounds. This many of the functions that are performed within the bilayered membrane is composed of lipopolysac- organelles of eukaryotic cells, including generation charide (LPS), and is important in the pathogenicity of adenosine triphosphate (ATP), cell motility, sens- of gram-negative organisms. The lipopolysaccharide ing of environmental changes, housing enzymes for layer contains porins, which allow passage of nutri- synthesis of cellular building materials, and media- ents and solutes, as well as antibiotics. Porins aid in tion of chromosome segregation during replication. the attachment of the outer membrane to the cell wall, and they vary among bacterial species. Gram- negative bacteria have a periplasmic space between Appendages the inner surface of the outer membrane and the Capsules outer surface of the cell membrane. This space is filled with the murein layer, a gel-like substance that Capsules are well-defined mucoid structures that helps secure nutrients from the outside, containing surround the murein layer of gram-positive bacteria enzymes that degrade large molecules and detoxify and the outer membrane of gram-negative bacteria.
  • 37. Structure and function of bacteria 23They are composed of high molecular weight environment. There are two types of pili, knownpolysaccharides, and their secretion may depend on as sex (F) pili, and common pili (fimbriae), andenvironmental conditions, especially the nutritional when they are present, cells usually have hundredsenvironment. Unlike the cell wall, the capsule does of pili. Sex pili may participate in deoxyribonucleicnot provide physical strength, and does not func- acid (DNA) transfer, mediating the conjugation oftion as a permeability barrier. The mucoid capsule, donor and recipient cells. Common pili serve as vir-sometimes referred to as the ‘slime layer’, provides ulence factors by mediating adherence to host cellprotection from drying, and helps the coated bac- surfaces, often a first step in establishing infection.teria to evade the host immune system by prevent- Once piliated bacteria are established within theing their engulfment by phagocytic cells. The capsule host, the pili can then undergo antigenic variationmay also inhibit the immune system, and can facil- that allows them to evade the host cell immuneitate bacterial colonization by attaching to surfaces system.on teeth and inanimate devices such as prostheticheart valves. The capsule contributes to the virulence Interior of the cellof certain bacteria: the virulence of both Strepto-coccus pneumoniae and Haemophilus influenzae are The inner cytoplasmic membrane surrounds thedependent on encapsulation. Capsules also give bac- cytosol, which contains polysomes, inclusions,teria a wide antigenic diversity, which contributes to nucleoid, plasmids, and endospores. The cytosol isthe invasiveness of the organism, and makes effec- abundant in enzymes, and nearly all cell functionstive vaccine preparation more difficult. are carried out here, including protein synthesis. The granular appearance of the cytosol is due to the manyFlagella polysomes (messenger ribonucleic acid (mRNA) molecules linked with several ribosomes) that areSome bacteria have flagella embedded in the cell present during translation and protein synthesis,envelope that can enhance their invasive properties and inclusions or storage granules, composed pri-by making them highly motile. The surface of the marily of nutrient and energy reserves such as glyco-flagellum is made up of protein antigens with diverse gen and polyphosphates. The nucleoid containsepitopes that are useful in the identification and clas- highly coiled DNA, intermixed with RNA, polyami-sification of some organisms such as Salmonella. nes and structural proteins. There is usually only oneSpirochetes have periplasmic flagella in the space chromosome per bacterial cell, but during certainbetween the cytoplasmic and outer membranes, and stages of cell division, separate extrachromosomalthis unusual location is responsible for their helical genetic elements known as plasmids may be presentmovement. Bacterial flagella can be arranged in sev- independently, in various numbers or not at all.eral different ways, including: (i) polar: one flagellum located at one end of the cell Endospores (ii) monotrichous: several flagella located at one Certain gram-positive bacteria can sporulate to pro- end of the cell duce endospores when under adverse physical and(iii) lophotrichous: flagella located at both ends of chemical conditions (including ultraviolet radia- the cell tion). Endospores are produced by vegetative cells(iv) peritrichous: the entire cell covered with flagella. as a survival strategy in situations of nutritional depletion. The metabolically active and growing cellPili or fimbrae transforms to a dormant state, with a decrease inPili or fimbrae are hairlike rigid structures up to 2 m cytosol and an increase in the thickness of the cellin length, composed of protein. They originate in the envelope. Endospores are highly resistant to heatcytoplasmic membrane, and extend to the outside and chemical agents, and can survive for years in
  • 38. 24 Bacteriology the environment. Under favourable conditions, the (iii) The bacterial chromosome is preserved during spore will germinate to produce a single vegeta- reproduction, but plasmids may be lost. tive cell that can then multiply. Both environmen- (iv) Bacterial chromosomal genes code for proteins tal contaminants (species of Bacillus) and human essential for viability, while plasmids code for pathogens (species of Clostridium and some species products that mediate transfer between bacte- of Bacillus, e.g. B. anthracis) produce endospores. rial cells, or for survival determinants such as These highly resistant structures present a problem resistance to antibiotics. in sterilization procedures. Unlike plasmids, transposable elements do not exist separately in the cell, but are attached either to chromosomes or to plasmids. These entities move Bacterial chromosomes from one location to another: from plasmid to Bacteria have a single circular chromosome that chromosome and from chromosome to plasmid. contains all the genes necessary for the viabil- There are two types of transposable elements, inser- ity of the organism. Unlike chromosomes in the tion sequences and transposons. Insertion sequence membrane-bound nucleus of eukaryotes, the bac- genes code for information required for movement terial chromosome is ‘naked’ in the cytoplasm. along plasmids and chromosomes, and transposons The single chromosome contains double-stranded, contain genes that code for movement and for supercoiled DNA with a composition and structure drug resistance markers. Genetic exchange among identical to that of eukaryotic DNA. The DNA is com- bacteria is due to these extrachromosomal pieces posed of nucleotides, i.e. purine (adenine and gua- of DNA. nine) and pyrimidine (thymine and cytosine) bases linked to deoxyribose sugar and a phosphate group. Bacterial reproduction Via molecular hydrogen bonds, adenine pairs with thymine, cytosine pairs with guanine, and groups of The process of reproduction in bacteria is similar paired bases code for specific genes. The sequence to that of eukaryotic cells: proteins and enzymes of DNA bases represents the genetic code. All genes are produced in preparation for DNA replication, collectively comprise the organism’s genome, which DNA is replicated and cytoplasmic division fol- is usually expressed as the number of base pairs. lows. The DNA double helix uncoils, each parent Genes are widely distributed among bacteria, and strand then serves as a template for synthesis of similarities or differences in DNA sequence are used a complementary daughter strand, and two new to develop molecular tests for detection and identi- duplicate chromosomes are produced prior to cell fication of pathogenic microorganisms. division. Bacteria also contain extrachromosomal genetic Bacterial growth occurs through an asexual veg- material, located on plasmids and transposable ele- etative process known as binary fission, with each ments, both of which can reproduce. Like the bacte- parent cell producing two daughter cells. The cell rial chromosome, plasmids are double-stranded and enlarges, elongating at designated growing zones, circular, but there are several differences between and cellular components are synthesized and assem- them. bled into a mature cell structure that resembles (i) A plasmid ranges in size from 1–2 kilobases to the parent. Once it reaches a critical size, the cell 1 megabase or more, whereas the bacterial chro- begins to divide, and produces two daughter cells; mosome is about 1300 m in length (a single these may or may not separate physically, depend- base is 3.4 × 10−4 m long or 0.34 nanometres ing on the species. About half of each new cell in length). consists of newly synthesized material, and the (ii) There is only one chromosome per bacterium, other half is made up of pre-existing material from whereas the numbers of plasmids vary. the parent cell. Each daughter cell has a newly
  • 39. Structure and function of bacteria 25replicated copy of the bacteria’s genome. Some the recipient bacterium, and this bridge acts as aspecies grow in filamentous and cordlike structures conduit for DNA to be transferred from the donor(e.g. Mycobacterium), or as filamentous or branch- bacterium to the recipient. Formation of the bridgeing mycelia that eventually lead to spore formation triggers DNA replication; the donor cell producesand a budding or branching process of cell growth one new DNA strand and passes this to the recip-(e.g. Nocardia). In eukaryotic cells, new combina- ient, where a complementary strand is made. Thetions of genes occur via segregation of gametes dur- new DNA is now available to recombine with theing sexual reproduction and by recombination of recipient cell’s genetic material. Bacterial chromo-genes on homologous chromosomes during the somes, plasmids and transposons may participate inprocess of meiosis. Gene exchange in bacteria can conjugation.occur via recombination, and also by three addi-tional mechanisms: transformation, transduction Transcription and translationand conjugation. When cells are not reproducing or preparing to reproduce, they are carrying out routine cellularTransformation activities that are dependent on synthesis of pro-During transformation bacteria that are competent teins and enzymes. Protein synthesis occurs in twocan take in DNA that is free in the environment from stages: transcription and translation. Transcriptionthe lysis of dead bacteria, creating new genetic com- is the process by which DNA, carrying the geneticbinations. Transformation is not limited to trans- code, is copied onto a complementary strand offer of genes among bacteria of the same species, mRNA. Usually only one of the two DNA strandsand thus genetic traits can be disseminated to a serves as a template for RNA transcription. Whenvariety of medically important microbes. Among mRNA is transcribed from DNA, cytosine bases pairthe human pathogens, Haemophilus, Neisseria and with guanine, but uracil is substituted for thymineStreptococcus have competence characteristics. in pairing with adenine. A sequence of three bases is a triplet, and each triplet encodes for a specific amino acid. This sequence is referred to as a codon.Transduction Since there are four bases and the codon consistsTransduction is mediated by bacteriophages (viruses of three bases, there are 64 (43 ) possible codons,that infect bacteria). Viruses integrate their DNA into with some amino acids coded for by more than onethe bacterial chromosome and direct replication of triplet. In bacteria, the mRNA molecule will code forviral DNA, which is then packaged and released from several genes whose protein products are involvedthe bacterial cell during lysis. The virus packages in similar functions. Transfer RNA (tRNA) and ribo-some bacterial DNA along with its own viral DNA, somal RNA (rRNA) are also produced during tran-and the bacterial DNA from the previous host is scription. All three RNA molecules are required forreleased when the virus infects another bacterium, translation when mRNA is decoded by the ribo-introducing foreign bacterial DNA into the newly somes for polypeptide synthesis. Each mRNA codoninfected cell. matches an anticodon located on a specific transfer RNA (tRNA) molecule. The mRNA associates with subunits on the ribosomes, and translation is ini-Conjugation tiated. As the ribosome moves along the mRNA, itConjugation is an exchange of genetic material deciphers the mRNA code and matches it with thebetween two strains of living bacteria, with one strain corresponding anticodon on a tRNA molecule. Thedonating DNA to a recipient strain. During this pro- tRNA molecule picks up the specific amino acid cor-cess the pilus of the donor forms a bridge with responding to the anticodon and attaches it to a
  • 40. 26 Bacteriology undergo a process of differentiation to form resting stages, spores or cysts that can survive for months or Stationary phase Death phase years with no apparent metabolic activity. In non- spore-forming species, if necessary substrates areNumber of cells Exponential (log) phase absent cellular energy supplies are depleted, and the cells enter a death phase. The number of viable cells decreases until no living cells are left, and these dead cells undergo autolysis. Lag phase Bacterial metabolism Time in hours Bacterial metabolism involves four major processes: Fig. 2.2. Bacterial growth curve. When a culture of acquisition of nutrients, metabolism of acquired microorganisms is transferred into a new container, there is an nutrients, energy production to support all cellular initial ‘lag phase’, while the bacteria adjust to their new environment. Having adapted to the new source of nutrients activity, and synthesis of new materials. and new temperature, they then enter an ‘exponential’, or log phase of rapid multiplication. A ‘stationary phase’ begins when Acquisition of nutrients the bacteria have exhausted the nutrient supply, or have produced toxins that limit their growth; metabolism is Nutrients are transported across the bacterial cell adjusted to maintain cells only, without growth. In non-spore membrane. Oxygen, water and carbon dioxide dif- forming species, cells enter a ‘death phase’, where the number fuse into the cell, but energy-requiring active trans- of viable cells decreases and the dead cells undergo autolysis. port is required for the uptake of sugars, amino acids, and organic and inorganic acids. The majority of nutrients are carried across the membrane by car- growing chain of amino acids to form a polypeptide. rier molecules. Translation is terminated when the ‘stop’ codon (UAA) is reached. Metabolism Once inside the cell, nutrients serve as raw materials Bacterial growth for precursor metabolites required for synthesis. The Bacterial growth generally occurs very rapidly, with metabolites are produced by three central pathways: three predictable and recognizable phases over time the Embden–Meyerhof–Parnas (EMP) pathway, the (Fig. 2.2). tricarboxylic acid (TCA) cycle, and the pentose phos- Lag phase: when a bacterium is introduced into a phate shunt. Medically important bacteria are often favourable new growth environment, the cell adapts identified by measuring products and byproducts of to its new surroundings by synthesizing any new cel- these metabolic pathways. lular machinery required. Once it has adjusted opti- mally, there is then a phase of rapid cell growth and Energy production division: the exponential or log phase. Bacterial cell numbers increase exponentially, until an essential Energy production is coupled to the breakdown of nutrient is depleted, or toxic metabolites accumu- nutrients, and involves oxidation–reduction reac- late. Cell growth then slows down and usually stops tions. Energy, primarily in the form of ATP is pro- within a single generation time: the bacteria then duced in the bacterial cell by the breakdown of enter a stationary phase. The cells are still viable, but nutrients via two general mechanisms: oxidative metabolism is readjusted in order to maintain the phosphorylation and fermentative metabolism, cells only, without growth. Certain bacteria can then which does not require oxygen. The pathways used
  • 41. Bacterial classification/identification 27during fermentative metabolism and the end prod- biotype, and serotype or phagotype may be used toucts produced vary among bacteria, and bacteria describe groups below the subspecies level. Speciescan be identified by detecting these end products. with several features in common are grouped into aFor example, the methyl-red test detects use of the genus. Similar genera are grouped into families. Bac-mixed acids pathway, and the Voges–Proskauer test teria are generally referred to by genus and species:detects production of 2,3 butylene glycol. During aer- Escherichia is the genus and coli is the species for theobic respiration, oxidative phosphorylation involves common bacterium E. coli.the electron transport chain and requires oxygen as a Family: Enterobacteriaceaeterminal electron acceptor. During anaerobic respi- Genus: Escherichiaration, the terminal electron acceptor is not oxygen. species: coliThe mechanism used to produce ATP is important Serotype: O157:H7when cultivating organisms in the clinical microbi- Genus: Vibrioology laboratory. species: cholera Biotype: Classical or El TorSynthesis Both genus and species are used routinely in pathogenic microbiology, with names typed in ital-When precursor molecules and energy are avail- ics. The genus is always capitalized, and the speciesable, bacteria assemble the materials into larger is written in lower case, as in Escherichia coli. Themolecules or polymers for cellular structures. first letter of the genus name may be used as abbre-Bacteria vary widely in their biosynthetic capabil- viation: E. coli. When family names are used, theyities – this also is important for cultivation in the are capitalized, typed in italics or underlined. E. coliclinical microbiology laboratory. Some organisms is in the family Enterobacteriaceae. Groups of organ-can synthesize the majority of their requirements, isms are often referred to generically. For example,while others may be unable to synthesize a par- ‘streptococci’ refer to all members of the genus Strep-ticular material, such as an amino acid, and this tococcus. Generic groupings are never capitalized,must be supplied in the laboratory culture medium italicized or underlined. Organism names changein order to ensure growth. Synthesis and assem- when new information becomes available for bet-bly of macromolecules within the bacterial cell is ter classification of species, and these changes areenzyme driven, and the ability of a bacterium to pro- documented in the International Journal for System-duce certain enzymes is another means of laboratory atic Bacteriology.identification. For example, members of the genusStaphylococci produce the enzyme catalase, and thisdifferentiates the genus from the morphologically Identification of bacteriasimilar Streptococcus species, which cannot produce Bacteria are identified by genotypic and pheno-catalase. Detection of catalase is an early test for typic features, and identification of bacteria by aidentification of bacteria shown to be gram-positive clinical microbiologist is still based primarily oncocci on the Gram stain. phenotypic features and physiologic requirements, although molecular technology is now being used more frequently. Bacteria can be grown on a varietyBacterial classification and identification of artificial media under various environmental con- ditions in the microbiology laboratory, and this pro-Nomenclature vides information about the organism’s nutritionalBacteria are classified on the basis of shared proper- requirements and ability to grow at various tem-ties, including genetics, morphology, and physiology. peratures, pH levels, and atmospheric conditions.The basic taxonomic group is the species. Occasion- The macroscopic appearance observed on artificialally a subspecies is recognized within a species and media and the microscopic appearance, observed on
  • 42. 28 Bacteriology stained and/or unstained specimens, are critical for with identification. Anaerobic infections are usu- the identification of an organism. Antigenic proper- ally characterized by foul odor and the presence ties are useful for serologic identification of bacte- of pus. ria, and antibiotic susceptibility and resistance pro- files also aid in identification. More advanced assays Microscopic examination may be necessary for the definitive identification of some bacteria, such as the use of gas liquid chro- The direct microscopic evaluation is usually a Gram matography to identify metabolic end products for stain, but may include a wet mount preparation. certain anaerobes, or molecular analysis of cell wall Since the specimen may contain multiple organ- components to place an organism in a particular isms and other materials, the following information genus. is noted: The process of identifying a bacterium involves the r presence of inflammatory cells, blood or debris routine assessment of specific features by the micro- r Gram reaction biologist: r morphology and arrangement of cells (i) initial macroscopic examination; r relative number of bacterial cells (ii) direct microscopic examination, using the r additional information for specific identification, Gram stain and wet preparation; e.g. presence of endospores or granules. (iii) cultivation The Gram stain (Fig. 2.3) is used to divide bac- (a) nutritional requirements teria into two main groups, based upon cell wall (b) environmental requirements structure and content. The majority of clinically sig- (c) colony appearance on artificial media (e.g. nificant organisms react to the Gram stain; excep- size); tions include those that do not contain a cell wall (iv) post-culture microscopic morphology, includ- (Mycoplasma spp. and Ureaplasma urealyticum), ing size, shape and arrangement of cells and those lacking the peptidoglycan layer (chlamy- other physical features noted, such as endos- diae) and those too small to be visualized by light pores; microscopy (spirochetes). For Gram staining, the (v) results of the Gram stain or other stain such as specimen must be fixed to a glass slide by heat or acid-fast; methanol: methanol fixation preserves the morphol- (vi) special stain for endospores, flagella, capsule; ogy of host cells as well as bacteria. Slides are overlaid (vii) results of biochemical tests, which may be per- with 95% methanol for 1 minute and air dried before formed manually or run as a battery using auto- staining. Crystal violet is added as the primary stain, mated instrumentation. followed by a mordant, Gram’s iodine, that chem- ically bonds the alkaline dye to the bacterial cell wall. Treatment with Gram’s decolorizer then dis- Initial macroscopic examination of tinguishes bacteria as either gram-positive or gram- clinical specimens negative. Following decolorization, safranin is added When a specimen arrives in the clinical microbi- as counterstain. All organisms take up crystal violet, ology laboratory, it is examined macroscopically but only gram-negative organisms will decolorize and evaluated initially by direct microscopic exam- and incorporate the pink-red safranin stain. ination. Preliminary data obtained from the initial Differential staining is based upon differences in macroscopic and microscopic examinations pro- the cell wall. Organisms that stain gram-positive vide direction for culture and future testing. The have thick cell walls with thick layers of peptido- macroscopic appearance of the specimen is noted, glycan and many teichoic cross-bridges; organisms along with additional information such as the pres- that stain gram-negative have a very thin layer of ence of blood, pus or a foul odor that may assist peptidoglycan, and teichoic cross-links resist alcohol
  • 43. Bacterial classification/identification 29 Steps for stainingGram-positive Gram-negative bacteria bacteria Fix cells on slide with heat or methanol Crystal violet Stain purple Stain purple primary stain Gram’s iodine Remain purple Remain purple mordant Alcohol Remain purple Become and/or acetone colourless decolorizer Remain purple Safranin Stain pink counterstainFig. 2.3. Gram stain Fig. 2.4. Bacterial morphologies. Common shapes and1. Heat-fix material on a slide and allow to cool; material also arrangements of bacteria.can be fixed using methanol.2. Flood slide with crystal violet and leave for 10–30 seconds(check staining instructions and run control slides to decolorization. Crystal violet stain washes out of hostdetermine optimum time). Rinse with tap water and shake offexcess. cells, including erythrocytes and white blood cells,3. Flood slide with Gram’s iodine to increase affinity of the allowing the cells to absorb the safranin. These cellsprimary stain and leave for twice as long as the crystal violet should stain pink.remained on the slide. Rinse with tap water and shake off Gram staining should be performed on youngexcess. cultures. Gram-positive bacterial cell walls that are4. Flood with alcohol or acetone decolorizer for 10 seconds; compromised due to age, damage or antibiotic treat-rinse with tap water immediately. Repeat this step until blue ment will lose the ability to retain crystal violetdye no longer runs off the slide when the decolorizer is added; and will appear as gram-negative or gram-variable.rinse with tap water and shake off excess. Gram-negative bacteria, on the other hand, rarely5. Flood with safranin counterstain and leave for 30 seconds. retain crystal violet, if the staining procedure isRinse with tap water and gently blot the slide dry with carried out correctly.absorbent paper or air dry. Air drying is recommended fordelicate smears (e.g. certain body fluids). The Gram stain also allows description of mor-6. Examine microscopically under oil immersion at a phology, or shape: bacterial shape can take the formmagnification of 1000×. Note bacteria, white blood cells and of cocci, bacilli or spirochetes, and the various cellu-other cellular material. lar arrangements provide clues to their identification (Fig. 2.4).
  • 44. 30 Bacteriology Fig. 2.5. Dilution streak method for isolation and semiquantitation of bacterial colonies. (a) = 1+ bacterial growth limited to the first quadrant; (b) = 2+ or moderate bacterial growth extends to the second quadrant; (c) = 3+ or 4+ heavy bacterial growth extends to the fourth quadrant. Cocci are arranged in pairs, tetrads, or clusters. Cultivation These arrangements suggest particular genera (e.g. Cultivation refers to growing microorganisms in cul- Staphylococcus aureus will form grape-like clusters ture: samples are taken from the site of infection in and streptococci will form chains, particularly when vivo and grown in the laboratory in vitro on artificial grown in liquid medium). Bacilli vary in length and media. Bacterial cells in a colony all have the same width: some are so short that they are easily con- genotypic and phenotypic characteristics (a clone). fused as cocci, and are thus referred to as coc- Cultures derived from a single bacterium are pure, cobacilli. This cellular morphology is typical for and those derived from more than one bacterium species of Haemophilus. Bacilli may also be fusiform, will be mixed. Biochemical testing and other identi- with pointed ends typical of the anaerobic genus fication procedures can only be carried out on pure Fusobacterium. Vibrios are bacilli in the shape of colonies. Figure 2.5 illustrates a technique for obtain- a comma. Two Campylobacter bacilli often align to ing isolated colonies. resemble the wings of a seagull. The presence of endospores also provides a key to identification: only Nutritional requirements two common genera of bacteria form endospores, the aerobic genus, Bacillus, and the anaerobic genus The nutritional requirements for most human Clostridium. pathogens are fairly basic, and commercial media
  • 45. Bacterial classification/identification 31have been developed for the cultivation of thioglycollate broth supports the growth of a range ofpathogenic bacteria. Organisms that grow well in a organisms, including aerobes, microaerophiles, fac-basal medium are considered to be non-fastidious. ultative organisms and anaerobes.A typical basal medium consists of a beef extract Enrichment media contain nutrient supplementsthat provides carbohydrates, nitrogen, vitamins and required for the growth of a particular bacterialsalts, as well as peptone to control pH. Pathogens that pathogen, and supports the growth of a particularrequire complex growth factors are considered to be pathogen from a mixture of organisms based on thefastidious and require additional substances, usually pathogen’s requirements. Trypticase soy agar (TSA)blood or serum products or vitamins. Neisseria gon- supports the growth of many fastidious and non-orrhoea requires hemin and nicotine adenine dinu- fastidious organisms. Other examples include thecleotide (NAD), and some nutritionally variant strep- following.tococci require vitamin B6 or thiol for growth. Some Sheep blood agar (SBA) contains blood, and isintracellular bacteria can be cultured only in a cell used for cultivation of most gram-positive andline (Chlamydia) and others cannot be cultured in gram-negative organisms. The blood agar platevitro at all (Calymmatobacterium granulomatis). also supports the growth of most yeasts, but will not support growth for Haemophilus spp. and other fastidious organisms that require heme andTypes of media reduced nicotinamide adenine dinucleotide phos-Commercial media are supplied in the form of a phate (NADPH).liquid broth or a solid agar. Bacterial growth in Chocolate agar (CA) contains lysed blood cells;liquid is determined by turbidity and growth on NAD and heme are released. This medium is usedagar is visualized as a colony derived from a single for cultivation of Neisseria and Haemophilus.bacterium. Commercial media not only support bac- IsoVitaleX contains dextrose, cysteine, vitaminterial growth, but are also used to grow and isolate B12, thiamine, ferric nitrate, and is also used forthe disease-causing pathogens while minimizing the cultivation of Haemophilus.contaminants or colonizers. Colonizers or normal Selective media ‘select’ for particular organisms,flora/resident flora are defined as organisms that inhibiting all organisms except those being sought.normally inhabit a given body site. Staphylococcus Inhibitory agents include dyes, alcohols, acids andepidermidis is resident skin flora; Lactobacillus is antibiotics, e.g.present in high numbers in the healthy vagina. Con- Modified Thayer–Martin agar (MTM) is composedtaminants are organisms that ‘contaminate’ a spec- of chocolate agar supplemented with antibiotics. Itimen. They may be from the environment (Bacillus is selective for Neisseria gonorrhoeae and Neisseriaspores from the air) or from a body site. For exam- meningitides because it supports these Neisse-ple, semen is easily contaminated with fecal flora ria pathogens while inhibiting most other organ-(members of the Enterobacteriaceae or Enterococcus isms, including gram-positive organisms, gram-species). Media also have been developed to select negative bacilli, and yeast. The vancomycin in thefor certain pathogenic organisms and to differentiate medium inhibits gram-positive bacteria, the col-between organisms as an aid in early identification. istin inhibits gram-negative organisms, nystatin There are four general categories of media: sup- inhibits yeasts and trimethoprim inhibits swarm-portive, enrichment, selective and differential (see ing of Proteus.Appendix 2.1). Hektoen enteric media (HE), Salmonella–Shigella Supportive media contain nutrients to support agar (SS), and xylose–lysine–deoxycholate agarthe growth of non-fastidious organisms, providing (XLD) inhibit gram-positive bacteria, and haveno advantage to any specific group. For example, differential biochemical reactions that permit
  • 46. 32 Bacteriology the distinction between pathogenic and non- Organisms that grow best in an oxygen concentration pathogenic coliform bacteria. lower than that of ambient air (5–10% oxygen) are Differential media incorporate an element or fac- microaerophilic. Helicobacter pylori and Campy- tor that allows colonies of one species to exhibit a lobacter jejuni require microaerophilic conditions metabolic trait that distinguishes or differentiates for cultivation. the species from other organisms growing on the Anaerobes usually cannot grow in the presence of medium. Examples include the following. oxygen, and grow in an atmosphere composed of MacConkey agar contains a neutral red indica- 5–10% hydrogen (H2 ), 5–10% CO2 , 80–90% nitro- tor: lactose fermenters are purple and non-lactose gen (N2 ) and 0% oxygen (O2 ). fermenters are clear or light pink. All members of Obligate anaerobes or strict anaerobes require a the family Enterobacteriaceae grow on MacConkey strict anaerobic environment where oxygen is agar: some ferment lactose and others do not. totally absent. Most Bacteroides spp., Clostridium, Crystal violet and bile salts in this medium also Eubacterium, Fusobacterium spp. Peptostrepto- inhibit the growth of gram-positive organisms as coccus spp., Porphyromonas spp., and most strains well as some gram-negative bacilli. Whether or not of Veillonella parvula, an organism found in the a gram-negative bacillus grows on MacConkey is female genital tract, are obligate anaerobes. Some a first step in differentiating gram-negative bacilli anaerobes are aerotolerant, i.e. they can survive that are not members of the Enterobacteriaceae. for short periods in the presence of oxygen, but The media selected for inoculation of a specimen will cannot reproduce under these conditions. The depend on: (i) the body site to be cultured; (ii) infor- Actinomyces spp. and Bifidobacterium spp. can mation from the direct prep; and (iii) physician order grow in the presence of reduced or atmospheric based on his/her observations, patient profile and oxygen, but thrive under anaerobic conditions. any current epidemiology information. Appendix 2.1 Facultative anaerobes have enzyme systems that at the end of this chapter includes media typically allow them to grow under either aerobic or anaer- used in clinical microbiology and gives examples obic conditions. They preferentially use oxygen as of enrichment, supportive, selective and differential a terminal electron acceptor, but in the absence media. Appendix 2.2 lists biochemical tests used for of oxygen can ferment sugars anaerobically. The further identification. Enterobacteriaceae, most staphylococci and strep- tococci are facultative organisms. Environmental requirements Temperature Gases: oxygen and carbon dioxide Microorganisms also have temperature require- Genetic make-up determines the oxygen require- ments. Human pathogens are mesophilic, thriving at ments of microbes. Some organisms require oxygen a temperature range between 30 ◦ C and 45 ◦ C. Some for metabolism, whereas it may be toxic to other strict pathogens, such as N. gonorrhoeae require a species; some grow better in an environment with temperature at or near human body temperature for increased carbon dioxide (CO2 ). growth. Other bacteria that may be pathogenic to An obligate aerobe (fungi and mycobacteria) cannot humans (staphylococci and members of the Enter- grow unless the atmosphere contains 15–21% oxy- obacteriaceae) grow well in vitro at room tempera- gen, the concentration found in air (21% oxygen ture as well as at 37 ◦ C. Some organisms, including and 0.03% CO2 ), or in a CO2 incubator with 15% some human pathogens, can grow at temperatures oxygen and 5–10% CO2 . above and below this range. Psychrophilic bacteria Organisms that require increased carbon dioxide are grow between 4 ◦ C and 20 ◦ C. Yersinia enterocolitica, referred to as capnophilic. Neisseria gonorrhoeae an organism known to contaminate blood products and Haemophilus influenzae are capnophiles. used for transfusion, can grow in refrigerated units
  • 47. Bacterial classification/identification 33of red blood cells. In the laboratory, the relatively from urine to be significant. The isolation of a strictgood growth of this organism in the cold is used to pathogen (e.g. Yersinia pestis) is always significant,enhance detection of Yersinia enterocolitica by ‘cold regardless of the site or number of microorganismsenrichment’. Thermophilic organisms, such as some present.Campylobacter spp. and Rhizomucor (fungus) spp.grow best at temperatures >40 ◦ C. The appearance of organisms growing on media can provide keys to identification, and a report ofSpecial requirements colony morphology should include the followingOrganisms have an optimal pH range. Since most information.clinically relevant bacteria grow best at or near a (i) Colony size: Bacillus colonies are large; thoseneutral pH (6.5–7.5), commercial media are buffered of Haemophilus are small.for neutral pH. Bacterial growth requires moisture, (ii) Pigment: Pseudomonas aeruginosa secretes aand incubation systems have been designed to pre- green pigment and several non-fermentingvent loss of water from the medium. Water is nec- gram-negative bacilli are pigmented. Pigmentessary for bacterial metabolism, and loss of water may provide important clues for identificationincreases the relative concentration of solutes lead- of anaerobes: the genus Porphyromonas anding to osmotic shock and cell lysis. Some bacte- species of Prevotella are pigmented; Clostrid-ria have special environmental requirements, e.g. ium difficile fluoresces yellow–green on bloodspecies of Vibrio require varying concentrations of agar.salt for growth. (iii) Description of the colony shape (form, eleva- tion) and surface (mucoid, dry). Colony shape may be unique: Actinomyces israelii coloniesPost-culture evaluation have a ‘molar tooth’ appearance, ClostridiumPost-culture evaluation documents information septicum has irregular margins resembling agained from the type of media and environmental ‘Medusa head’; Actinobacillus actinomycetem-conditions that have supported growth, and this pro- comitans forms a four- to six-pointed star-likevides important clues in identifying the organism. configuration in the centre of a mature colonyCertain species of Vibrio will only grow in 8% sodium when grown on clear medium. Colonies ofchloride, whereas most other organisms will not tol- Eikenella are sticky and may pit the agar, anderate a high level of salt. Many pathogens require colonies of Proteus swarm over the entire plate.special media (Legionella pneumophilia) and there- (iv) Alterations in medium including pitting of thefore will not be detected on basic media such as sheep agar or hemolysis on blood agar. Hemolysisblood agar. Colony appearance on differential media patterns are the key to identifying species ofis especially important, e.g. if a gram-negative bacil- Streptococcus, and -hemolysis around a large,lus grows on MacConkey agar, it can be placed in a feathered colony suggests Bacillus cereus.major group, and if it also ferments lactose in the (v) Odour is a further clue to organism identifica-MacConkey medium, it is likely to be a member of tion. Pseudomonas aeruginosa smells of grapesthe Enterobacteriaceae. or tortillas, S. aureus smells like dirty athletic In general, the clinical significance of bacterial socks, and Clostridium difficile has the odourgrowth will depend upon the body site from which of a horse organism was cultured and the relative numbers Clinical specimens often reveal mixed cultures,of bacterial colonies on the agar. For example, any with organisms that may represent pathogens,organism isolated from a sterile body site (e.g. blood) contaminants or normal flora. Organisms sus-is significant if not due to contamination, whereas pected of being pathogenic, based on the macro-a certain number of organisms must be isolated scopic appearance and conditions that support
  • 48. 34 Bacteriology (a) Pus from a boil is Gram-stained and plated to sheep blood agar (SBA), chocolate agar (CA) and MacConkey agar (MAC). The following results were obtained: Gram stain gram-positive cocci; clusters suggests staphylococci Note: Staphylococci do not always appear in clusters. Culture results: SBA = yellow-white, round colonies exhibiting a narrow zone of β-hemolysis. This appearance on SBA suggests Staphylococcus aureus Note: Not all strains of S. aureus are beta-hemolytic. CA = heavy growth; expected for Staphylococcus aureus MAC = no growth; expected for Staphylococcus aureus Since this is a gram-positive coccus and the appearance on SBA suggests Staphylococcus aureus, the following tests would be run to confirm: Catalase = positive Modified oxidase = negative Mannitol reduction = positive Coagulase = positivea amost important test since S. aureus is the only staphylococcal species pathogenic to humans that produces the enzyme coagulase. Note: Latex agglutination tests also could be used for identification. Fig. 2.6. Identification of Staphylococcus aureus recovered from an infection site. (a) Method and (b) flowchart.
  • 49. Bacterial classification/identification 35(b) Gram-positive (Growth on blood agar and chocolate agar, no growth on MacConkey agar) Budding yeasts Cocci Bacilli Catalase + Catalase − Staphylococci or micrococci Streptococci and enterococci a Oxidase-negative Oxidase-positivea Staphylococcus species Micrococcus species Mannitol salt fermentation and coagulase test Mannitol fermented Mannitol not fermented Coagulase-positive Coagulase-negative S. aureus S. epidermidis or other Staphylococcus spp. a modified oxidase Note: a gram-positive, β-hemolytic, catalase-positive organism recovered from pus is presumptively S. aureus.Fig. 2.6. (cont.) organism growth (e.g. chocolate agar, but not be visualized using special stains. When blood agar), should be tested. Testing must be mycobacteria or organisms that are par- performed on organisms obtained from an iso- tially acid-fast are suspected (e.g. Nocardia lated colony, and the tests should include: and Rhodococcus), an acid-fast stain is per- (i) Repeat Gram stain, denoting all morpho- formed. logic characteristics (e.g. gram-positive cocci (iii) A wet prep to examine motility can pro- in clusters). vide information in many cases: Helicobac- (ii) Additional staining if indicated: although not ter pylori has a corkscrew movement and a routine procedure in the clinical laboratory, Listeria monocytogenes displays a tumbling flagella, capsules, spores and inclusions can motility.
  • 50. 36 Bacteriology (a) A semen specimen with a foul odour was Gram-stained and plated to sheep blood agar (SBA), chocolate agar (CA) and MacConkey agar (MAC). The following results were obtained: Gram stain Gram-negative, straight bacilli Culture media: SBA = grey, mucoid colonies CA = growth MAC = pink, lactose-fermenting colonies A gram-negative, lactose-fermenting bacillus is likely to be a member of the Enterobacteriaceae. All members of the family Enterobacteriaceae are oxidase-negative. A series of tests were run to speciate the organism and the following results were obtained: Oxidase = negative Indole = positive Motility medium = positive Voges–Proskauer = negative Simmon’s citrate = negative Hydrogen sulfide production = negative Urease = negative Note: testing is usually automated or performed using a panel of multiple tests such as the API strip for the Enterobacteriaceae. The organism is identified as Escherichia coli, a common fecal contaminant in semen. Fig. 2.7. Identification of Escherichia coli recovered from a semen specimen. (a) Method and (b) flowchart. (iv) Preliminary further testing can be carried out Definitive organism identification requires based upon culture and microscopy. additional testing. Biochemical tests to determine (a) Gram-positive cocci should be tested for metabolic activity (e.g. glucose utilization and catalase. All members of the Micrococ- enzyme production) may be performed manu- caceae (this includes staphylococci) are ally or with the use of automated systems. Bio- catalase-positive, streptococci and entero- chemical reactions are listed in Table 2.2 of the cocci are catalase-negative. Appendix to this chapter. Additional media-based (b) All gram-negative bacilli should be tested testing may also be indicated. Vibrios are placed for oxidase. Members of the Enterobacte- in increasing solutions of sodium chloride to help riaceae, the most common gram-negative speciate the genus, and pseudomonads may be bacilli encountered in the clinical labo- subcultured to media containing heavy metals for ratory, are oxidase-negative. The gram- speciation. Antibiotic resistance profiles are also negative bacilli Pseudomonas and Burkho- useful for organism identification. Staphylococcus lderia species are oxidase positive. saprophyticus is resistant to novobiocin, whereas
  • 51. Bacterial classification/identification 37(b) Gram-negative Growth on blood agar, chocolate agar, and no Growth on blood agar, chocolate agar, and growth on MacConkey agar) growth on MacConkey agar Cocci or coccobacilli Bacilli Lactose fermentation + Lactose fermentation − Oxidase − Oxidase + Enterobacteriaceaea Pseudomonas Indole = positive Burkholderia Motility medium = positive Other Voges--Proskauer = negative Simmon s citrate = negative H2S = negative Urease = negative E. coliFig. 2.7. (cont.) other species of coagulase-negative staphylococci enzyme immunoassays, as well as other immunoas- are sensitive. Sensitivity to a panel of antibiotics says targeted at antigen detection. Antibodies to (kanomycin, vancomycin and colistin) helps dif- antigens associated with infectious disease can ferentiate anaerobic bacilli. Figures 2.6 and 2.7 be detected by a variety of serodiagnostic meth- illustrate flowcharts and methods for the isolation ods: agglutination assays, flocculation tests (e.g. and identification of organisms in two different rapid plasma reagin (RPR) and Venereal Disease specimens. Research Laboratory Slide test (VDRL) tests forPhysical characteristics observed on stains and syphilis), counterimmunoelectrophoresis, immun-in culture and results of metabolic characteristics odiffusion, hemagglutination inhibition assays, neu-provide phenotypic information that identifies an tralization assays, complement fixation, enzyme-organism. Other tools are also used to identify bac- linked immunosorbent assays (e.g. for humanteria, including molecular techniques to charac- immunodeficiency virus (HIV)), indirect fluorescentterize part of the bacterium’s genome, and other antibody tests (e.g. fluorescent treponemal antibodyanalytical methods such as electrophoretic analysis absorption test for T. pallidum) radioimmunoassays,and gas–liquid and high-performance liquid chro- fluorescent immunoassays, and Western blots (e.g.matography. Immunochemical methods are impor- for T. pallidum, HIV and herpes simplex virus typestant in identifying organisms that cannot be cul- 1 and 2).tured (e.g. Treponema pallidum). Immunochemical The results obtained from the battery of iden-techniques include detection of antigens using poly- tifying tests place the organisms into their majorclonal and monoclonal antibodies, precipitin and groups, as illustrated on the overview flowchartagglutination tests, immunofluorescent assays and (Fig. 1.4).
  • 52. 38 BacteriologyTable 2.1. Bacteria: Gram-negative bacilliGrow on MacConkey Grow on MacConkey No growth on MacConkey No growth on MacConkeyOxidase-positive Oxidase-negative Oxidase-positive Oxidase-negative (variable) Special media for recovery 1 1 1 3Pseudomonas Acinetobacter Moraxella Haemophilus Campylobacter2Burkholderia1 Chryseomonas1 Elongated Neisseria1 H. ducreyi 3* Helicobacter1Achromobacter grp1 Flavomonas1 Eikenella3 Arcobacter3 A. xylosoxidans Stenotrophomonas1 Pasteurella3 Legionella1 A. dentrificans Escherichia3 Actinobacillus3 Brucella1 E. coli ∗∗∗ Kingella1 Bordetella pertussis1Chryseobacterium1 Shigella3 Cardiobacterium3 Bordetella parapertussis1C. meningosepticum1 Salmonella3 Capnocytophaga1 Francisella1Sphingobacterium1 Citrobacter3 Sphingomonas paucimobilis3 Bartonella1Alcaligene1 s Klebsiella3 Weeksella virosa3 Afipia1Bordetella Enterobacter3 Methylobacterium3 Streptobacillus3 (non-pertussis)1 Serratia3 Bergeyella zoohelcum3 Spirillum minus1Comomona1 Hafnia3Vibrio3 Proteus3Aeromonas3 Providencia3Plesiomonas3 Morganella3Chromobacterium3 Yersinia3Ralstonia picketti1 Edwardsiella3Oligella3Ochrobacterium3Shewanella putrefaciens1Aerobic = 1 ; Aerobic, microaerophilic = 2 ; Facultative = 3urogenital pathogen*.prenatal/neonatal pathogen**.urogenital pathogen and prenatal/neonatal pathogen***. Major groups of organisms These organisms are differentiated by specimen Gram-negative bacilli and coccobacilli source and by further testing. Gram-negative bacilli and coccobacilli are outlined in Table 2.1. Pseudomonas, Burkholderia and Ralstonia Grow on MacConkey agar, oxidase-positive Species of Pseudomonas, Burkholderia and Ral- stonia are important contaminants found in the Pseudomonas Burkholderia Ralstonia pickettii Achromobacter group environment and in water. Burkholderia cepacia, Chryseobacterium, A. xylosoxidans Ralstonia pickettii and Pseudomonas aeruginosa are Sphingobacterium A. dentrificans common contaminants of medical devices and solu- Oligella Ochrobacterium tions. Pseudomonas aeruginosa is an opportunistic Non-pertussis Bordetella Alcaligenes pathogen that is a leading cause of hospital-acquired Vibrio Shewanella putrefaciens infections. These organisms are straight, slender, Plesiomonas shigelloides Comomonas aerobic bacilli that use a variety of carbohydrates; Aeromonas although they are mesophilic, they can survive at low Chromobacterium violaceum temperatures (to 4 ◦ C).
  • 53. Major groups of organisms 39Achromobacter and Ochrobacterium and all have different morphologies and physiologic features. Chromobacterium violaceum, easily identi-Achromobacter and Ochrobacterium are all envi- fied due to its violet pigment, causes a rare, but veryronmental flora, but may occasionally inhabit the dangerous systemic infection.human gastrointestinal tract. Gram-negative bacilli and coccobacilli that growSphingobacterium and Chryseobacterium on MacConkey agar, oxidase-negativeSphingobacterium and Chryseobacterium are envi-ronmental flora that may occasionally be encoun- Acinetobacter Chryseomonastered in human specimens, and may contaminate Flavomonas Stenotrophomonas Enterobacteriaceae :solutions and surfaces in the laboratory setting. Escherichia ShigellaChryseobacterium meningosepticum is associated Salmonella Citrobacterwith nursery meningitis. The majority of organisms Klebsiella Enterobacterin this group oxidizes glucose, and display a yellow Serratia Hafniapigment. Proteus Providencia Morganella YersiniaOligella, Alcaligenes, Shewanella putrefaciens, EdwardsiellaComomonas, non-pertussis Bordetella andAchromobacter Acinetobacter, Chryseomonas, Flavomonas andOligella, Shewanella putrefaciens, Comomonas and Stenotrophomonasnon-pertussis Bordetella do not utilize glucose, and These either oxidize or do not utilize glucose. Thistheir morphologies and physiologic requirements feature distinguishes them from the largest, andvary. These organisms are found in the environ- most frequently encountered, organisms, mem-ment (soil and water), the upper respiratory tract of bers of Enterobacteriaceae, which do ferment glu-some mammals, and in humans. The habitat of some cose. This group of non-fermenters is importantspecies remains unknown. Achromobacter xylosoxi- because they are associated with nosocomial infec-dans, Alcaligenes fecalis and species of Comomonas tions acquired from the colonization of hospital-are found as contaminants in medical devices ized patients and contamination of medical devices,and solutions, including intravenous and irrigation equipment and fluids. They are separated by source,fluids. Achromobacter xylosoxidans has been doc- enzymes produced and by biochemical testing.umented to contaminate soaps and disinfectants.Oligella species infect humans as the result of manip- Enterobacteriaceaeulations (e.g. catheterization) of the urinary tract. Most members of the Enterobacteriaceae, includ- The organisms listed in above are commonly seen ing Escherichia coli, are normal intestinal flora incontaminating fluids, sinks, and incubators in a lab- humans and other animals. Infections caused byoratory setting. these genera are the result of transmission via the fecal–oral route or from contaminated food andVibrios, Plesiomonas Aeromonas and water. The Enterobacteriaceae family includes mem-Chromobacterium violaceum bers that are true pathogens: Yersinia pestis, theVibrios, Plesiomonas and Aeromonas inhabit sea- agent of bubonic plague, species of Salmonellawater and therefore are associated with diarrhoea that cause gastroenteritis, bacteremia and typhoidor with water wounds. Vibrio cholera is a seri- fever, and Shigella, which causes gastroenteritis andous toxin-producing pathogen. These genera, along dysentery. Members are also associated with waterwith Chromobacterium violaceum, ferment glucose, wounds (Edwardsiella tarda) and a wide variety of
  • 54. 40 Bacteriology nosocomial infections. Species of Citrobacter, Enter- Eikenella corrodens obacter, Klebsiella, Morganella, Proteus, Providencia Normal flora in the human mouth; infections with and Serratia are spread from person to person and this organism result from trauma from human bites may infect the respiratory tract, urinary tract, blood and clenched-fist wounds. and other normally sterile sites in hospitalized and debilitated patients. These organisms are of special concern because they are antibiotic resistant. Since Bergeyella zoohelcum members of the Enterobacteriaceae are found in feces Oral flora in non-human animals. and in clinical settings, they are encountered in the reproductive laboratory, both as fecal and as envi- ronmental contaminants. Weeksella virosa and Methylobacterium Gram-negative bacilli and coccobacilli, do not Both Weeksella virosa and Methylobacterium species grow on MacConkey agar, oxidase-positive are found in the environment and are known to contaminate medical devices and other clinical Sphingomonas paucimobilis Moraxella materials. Elongated Neisseria Eikenella corrodens Weeksella virosa Bergeyella zoohelcum Methylobacterium Pasteurella Pasteurella Actinobacillus Kingella Pasteurella species are normal flora in domestic and Cardiobacterium Capnoctyophaga, wild animals, and infections with these organisms and similar organisms are therefore limited to contact with animals. Organisms in this group utilize glucose differently: Sphingomonas paucimobilis utilizes glucose oxida- Actinobacillus, Kingella, Cardiobacterium, and tively; Eikenella corrodens, Weeksella virosa and Capnocytophaga Bergeyella zoohelcum are asaccharolytic; Pasteurella, Normal flora in the mouth of humans and animals. Actinobacillus, Kingella, Cardiobacterium and Cap- Most human infections are due to endogenous flora nocytophaga all ferment glucose. These organisms entering deeper tissues (e.g. dental procedures). are identified by their morphology and physiologic requirements. Gram-negative bacilli and coccobacilli that do Sphingomonas paucimobilis not grow on MacConkey agar, oxidase variable An environmental inhabitant that may contaminate This group include the genus Haemophilus. There medical devices and solutions; infections include are several species of Haemophilus, including catheter-related bacteremia, and wound and urinary H. influenzae, which causes life-threatening pneu- tract infections. monia and meningitis, and H. ducreyi, asso- ciated with chancroid. With the exception of Moraxella H. aphrophilus, species of this genus require either hemin and/or nicotine adenine dinucleotide (NAD) Species of Moraxella inhabit mucous membranes in for growth. Species are distinguished based on re- humans but rarely cause infections. quirements for these factors, hemolysis on rabbit blood agar and fermentation of sugars. With the Neisseria elongata exception of Haemophilus ducreyi, species of Normal upper respiratory tract flora in humans. Haemophilus are normal upper respiratory flora in
  • 55. Major groups of organisms 41humans. All species may cause infection, either from Helicobacter pylori resides in the human gastricendogenous flora or person-to-person transmission. mucosa and causes gastritis, peptic ulcer disease andOnly the encapsulated strain of H. influenzae is asso- gastric cancer.ciated with life-threatening infections. H. ducreyi iscovered in detail in Chapter 7. Legionella pneumophilia Causes Legionnaire’s disease, a febrile pneumonia.Gram-negative bacilli that are optimally Other Legionella species are responsible for pneu-recovered on special media monia, endocarditis and wound abscesses. Bartonella Afipia Brucella Campylobacter Helicobacter Legionella Arcobacter Cattle, sheep, goats, swine and dogs are hosts for Bru- Bordetella pertussis Brucella cella species. Although some species will grow on Francisella Bordetella parapertussis MacConkey agar, many require enriched media and Spirillum minus Streptobacillus moniliformis special incubation conditions. Brucella is respon- sible for brucellosis. Although this is a zoonosis,Since this group of organisms requires special media humans can become infected via animal contactfor isolation, they are not detected on routine cul- (inhalation, direct inoculation or ingestion of unpas-ture in the clinical laboratory and must be specifi- teurized and contaminated milk or cheese). Incally sought. The majority cause unusual or atypical humans the infection may range from asymptomaticinfections. to a serious debilitating disease.Bartonella Bordetella pertussis and Bordetella parapertussisBartonella species are associated with sand flies, Special media is required for isolation and for cul-human lice and domestic cats, and may cause zoo- tivation of Bordetella pertussis and Bordetella para-notic infections in humans. Trench fever and cat- pertussis, causative agents of whooping cough.scratch fever are caused by species of Bartonella. Francisella tularensisAfipia felis Requires cysteine and a source of iron for growth. F. tularensis, an extremely virulent organism,Once believed to be an agent of cat-scratch fever, causes tularaemia in both animals and humans, abut the exact role in causing human disease is not severe systemic infection sometimes referred to asknown. ‘rabbit bite fever’.Campylobacter, Arcobacter and Helicobacter Francisella phiolmiragiaSpecies of Campylobacter, Arcobacter and Heli- Present in animals and in ground water and has beencobacter are small curved, motile bacilli; the major- associated with infections in near-drowning victims.ity require microaerophilic conditions for isolation.These organisms may be found in humans and other Streptobacillus moniliformis and Spirillum minusanimals. Both Campylobacter and Arcobacter causegastroenteritis and other infections. Campylobac- Agents of rat-bite fever, a serious systemic dis-ter jejuni, subspecies jejuni may cause proctitis in ease affecting many body sites and associated withhomosexual men. complications. Streptobacillus moniliformis requires
  • 56. 42 Bacteriology Table 2.2. Bacteria: gram-negative cocci Table 2.3. Bacteria: gram-positive cocci Aerobic Aerobic 1 Neisseria Catalase-positive Catalase-negative N. gonorrhoeae***1 1 Staphylococcus Streptococcus2 spp. Moraxella catarrhalis aureus1 S. agalactiae2 Coagulase-negative (Group B***) Aerobic = 1 ; Aerobic, microaerophilic = 2 ; Facultative = 3 . staphylococci Enterococcus2 spp. urogenital pathogen*. prenatal/neonatal pathogen**. Micrococcus1 spp. urogenital pathogen; and prenatal/neonatal pathogen***. Aerobic = 1 ; Aerobic, microaerophilic = 2 ; Facultative = 3 . media with blood or serum and incubation under urogenital pathogen*. prenatal/neonatal pathogen**. carbon dioxide for isolation; Spirillum minus has urogenital pathogen; and prenatal/neonatal pathogen***. never been grown in culture. r Staphylococcus aureus (coagulase positive) Gram-negative cocci r Coagulase-negative staphylococci r Micrococcus species Aerobic gram-negative cocci are shown in Table 2.2. r Stomatococcus mucilaginosus These species are oxidase positive and do not elongate when exposed to subinhibitory concentra- Genus identification is based on sheep blood tions of penicillin, unlike the elongated Nesisseria. agar colony appearance, reaction to modified oxi- Neisseria gonorrhoeae and Neisseria meningitides are dase, resistance to bacitracin and susceptibility to pathogenic; other species of Neisseria are normal lysostaphin. inhabitants of the upper respiratory tract in humans. Neisseria gonorrhoeae is sexually transmitted, and causes gonorrhoea: this organism is described in Staphylococcus aureus detail in Chapter 10. Staphylococcus aureus is normal flora in the respi- Neisseria meningitides is a leading cause of bacte- ratory tract and on other mucosal surfaces in some rial meningitis. humans (carriers). Many healthcare workers are car- Moraxella catarrhalis infections are localized to riers of S. aureus. These organisms are transmit- the respiratory tract, and the organism rarely dis- ted from person to person via fomites, unwashed seminates: infections include sinus infections, otitis hands or when the colonizing organism is intro- media and pneumonia. duced to a sterile body site. S. aureus is one of the Species of Neisseria and Moraxella catarrhalis are most successful of all bacterial pathogens, because identified on the basis of appearance (many com- it has a wide range of virulence factors, including mensals are pigmented), growth on Thayer–Martin toxins and enzymes. These virulence factors are agar, growth on nutrient agar at room tempera- responsible for the numerous S. aureus infections, ture and body temperature, utilization of sugars and including skin infections (folliculitis, furuncles or nitrate reduction. boils, carbuncles and impetigo), wound infections, bacteremia, endocarditis, joint infections, scalded skin syndrome in neonates, toxic shock syndrome Gram-positive cocci that are catalase-positive and food poisoning. Since S. aureus colonizes up to Gram-positive cocci that are catalase positive are 40% of all healthcare workers, and can be spread from listed in Table 2.3. Those that are catalase-positive unwashed hands, it is a potentially serious prob- include: lem in the ART laboratory when aseptic technique
  • 57. Major groups of organisms 43is not followed. It is resistant to antibiotics, so that Gram-positive cocci that are catalase-negativeinfection or contamination with the organism is Examples of gram-positive cocci are listed inparticularly serious: the penicillin-resistant variant Table 2.3. Those that are catalase-negative include‘MRSA’ (methicillin resistant S. aureus) has been Streptococcus, Enterococcus and related species.responsible for serious problems in hospital out- Identification of these genera is based on cellularbreaks. morphology, hemolysis pattern on sheep blood agar, growth in 6.5% sodium chloride, and hydrolysis ofStaphylococcus epidermidis pyrrolidonyl arylamidase (PYR).Staphylococcus epidermidis and other coagulase-negative staphylococci are normal skin flora. Infec- Streptococcus pyogenestion with these bacteria usually occurs when the Streptococcus pyogenes (Lancefield Group A) pro-patient’s endogenous strain reaches a sterile site. duces a large number of virulence factors and causesStaphylococcus epidermidis often appears in large a wide array of suppurative diseases and toxinoses,numbers on the human body; it produces slime and as well as some autoimmmune or allergic diseases.can attach to medical devices. Infection often occurs At least 55 different strains are known, causingas the result of medical manipulations (e.g. shunt or diseases that include acute pharyngitis, impetigo,prosthetic device). S. epidermidis is often found in erysipelas, necrotizing fasciitis and myositis, bac-specimens and solutions as a result of contamina- teremia, pneumonia, scarlet fever and streptococcaltion from cutaneous sources. toxic shock syndrome. The organism cross-reacts with antigens on the heart, leading to rheumatic fever; the deposition of streptococcal antigen-Staphylococcus saprophyticus antibody complexes on the glomerulus leads to acuteStaphylococcus saprophyticus, normal flora on the poststreptococcal glomerulonephritis. Althoughskin and the genitourinary tract, is a urinary tract Group A streptococci inhabit skin and the upperpathogen, generally seen in sexually active young respiratory tract in human carriers, this group is notwomen. In comparison to the traditional pathogens considered part of the normal flora. The bacteriumsuch as E. coli and others, S. saprophyticus may is spread by person-to-person contact via secretionsbe present in relatively small numbers in pure cul- or mucus, or by sneezing and coughing.ture and still be a cause of significant disease. For aclean catch urine, 10 000 cfu/ml would be reportedas significant, as opposed to the usual >100 000 Streptococcus agalactiaecfu/ml. Streptococcus agalactiae (Lancefield Group B), nor- Staphylococci are speciated based on coagulase mal flora of the female genital tract and lower gas-production, resistance to novobiocin, growth in salt trointestinal tract, has fewer virulence factors thanand mannitol reduction. Streptococcus pyogenes. Most Group B streptococ- cal infections are associated with the neonate, often preceded by premature rupture of maternal mem-Micrococcacae and Stomatococcus mucilaginous branes. Adult infections can occur postpartum, andMicrococcus species and Stomatococcus mucilagi- include endometritis, which can lead to septic shocknous are normal flora of human skin and the orophar- and additional problems. This species of streptococ-ynx, and rarely cause infection. Both may be isolated cus may also cause infections such as endocardi-as contaminants in clinical specimens. Micrococcus tis and arthritis in immunocompromised patients.species display a variety of pigments. During pregnancy, mothers are tested to determine
  • 58. 44 Bacteriology if they harbour Group B strep as genital flora, and a Enterococcus positive finding should indicate delivery within 24 h Species of Enterococcus are found in food, water, by membrane rupture to prevent neonatal disease. soil and as normal flora in humans and other ani- Details of Group B strep infections are covered in mals. The species associated with human infection, Chapter 8. E. faecalis and E. faecium, are normal flora of the gastrointestinal tract and the female genitourinary Other Lancefield groups tract. Infection tends to occur when the endoge- Other Lancefield groups of streptococci (C, F and G) nous strains gain access to sterile sites. Transmission are normal flora of human skin, nasopharnyx, genital may be from person to person or via contaminated tract and gastrointestinal tract and cause infections medical equipment. There are multidrug resistant that are similar to S. pyogenes and S. agalactiae. strains of Enterococcus. Streptococci and enterococci Group C has been associated with pharyngitis and species are differentiated based on cellular arrange- Group G is associated with underlying malignancies. ment, hemolysis patterns on sheep blood agar (example: Group A is -hemolytic and S. pneumo- Streptococcus pneumoniae niae is -hemolysis), typing for the C-carbohydrate in the cell wall, PYR hydrolysis, growth in 6.5% Streptococcus pneumoniae colonizes the naso- sodium chloride, hippurate hydrolysis and the CAMP pharynx in humans. Exposed individuals (e.g. (Christie/Atkins/Munch–Peterson) beta hemolysis contact with respiratory secretions) may develop enhancement test. infection. Streptococcus pneumoniae has several associated virulence factors, but the polysaccharide Gram-positive bacilli that are non-branching and capsule is the primary factor. This streptococcus catalase positive species does not have C-carbohydrate in the cell wall and therefore is not associated with a Lancefield Gram-positive bacilli are listed in Table 2.4. Bacil- grouping. Streptococcus pneumoniae is a leading lus, Corynebacterium and Listeria (and other related cause of pneumonia in the elderly, with or without organisms) are non-branching and catalase positive. bacteremia, and is also a leading cause of meningitis. Aspiration of the organisms into the lungs leads to Bacillus pneumonia. Streptococcus pneumoniae also causes sinus infections and otitis media. Bacillus is an aerobic genus of bacteria that forms environmentally resistant endospores, and this is the primary virulence factor associated with these Viridans streptococci bacteria. Bacillus species are found everywhere in Viridans streptococci are of low virulence, but they nature. They are commonly found in clinical speci- produce an extracellular carbohydrate complex that mens, and are responsible for contamination in ster- allows the organism to attach to host cell surfaces ile areas. To ensure that bacillus spores are destroyed, and tooth enamel. The organism may cause subacute sterilization methods must be adequate for their endocarditis in patients with damaged heart valves, destruction, and endospore destruction must be and one member of this group, S. mutans, plays a key documented as part of laboratory quality control role in dental caries development. Other streptococci (e.g. include spore strip indicator in all autoclaving (e.g. Leuconostoc spp. and Aerococcus spp.) should be procedures). first considered as contaminants when encountered The genus includes the well-known Bacillus in clinical specimens. Aerococcus urinae is associated anthracis, which causes anthrax. Bacillus anthracis with urinary tract infections. spores are found in the soil. Infection may be caused
  • 59. Major groups of organisms 45Table 2.4. Bacteria: gram-positive bacilli AerobicSpore-forming Non-spore-formingNon-branching Non-branching Non-branching Branching orCatalase-positive Catalase-positive Catalase-negative Acid-fastBacillus1 Listeria1 Erysipelothrix1 Nocardia1 L. monocytogenes**1 Lactobacillus1 Streptomyces1 Corynebacterium1 Arcanobacterium1 Rhodococcus1 Gardnerella vaginalis*1Aerobic = 1 ; Aerobic, microaerophilic = 2 ; Facultative = 3 .urogenital pathogen*.prenatal/neonatal pathogen**.urogenital pathogen; and prenatal/neonatal pathogen***.by inhalation of spores (pulmonary anthrax), pene- Corynebacterium spp.tration of spores (cutaneous anthrax) or ingestion of The Corynebacterium genus does not form endospo-spores (gastrointestinal anthrax). This organism is a res, making it easy to distinguish from Bacillus.true human pathogen, and has been used as an agent Several species responsible for human diseaseof bioterrorism. belong to this genus, including the agent that Bacillus cereus produces toxins that cause food causes diphtheria, Corynebacterium diphtheriae.poisoning, the classical ‘Chinese rice’ gastroenteri- Corynebacteria are found in the environment andtis. B. cereus may also cause infection when intro- some species are normal human skin flora.duced into sterile body sites either by trauma or Corynebacterium jeikeium is found on the skin ofby exposure to contaminated medical supplies and hospitalized patients, usually in axillary, inguinal andequipment. These infections often involve immuno- rectal sites. This organism may be introduced viacompromised patients. Other species of Bacillus may catheters and/or intravenous therapy sites. Multiplebe responsible for the same infections caused by B. antibiotic resistance makes infection with this organ-cereus. ism significant, and in compromised patients it Bacillus anthracis can initially be distinguished can cause septicemia, wound infections and rarelyfrom the more common B. cereus species on the endocarditis. Corynebacteria speciation is basedbasis of motility: (B. anthracis is non-motile, and on morphology, colony appearance on selectivethe more ubiquitous species are motile) and - media, physical characteristics (arrangement ofhemolysis on sheep blood agar (B. anthracis is non- bacteria on Gram stain, pigment) and biochemicalhemolytic and B. cereus is -hemolytic). Definitive reactions.identification of B. anthracis should take place in anauthorized public health laboratory equipped to deal Listeria monocytogeneswith this serious pathogen. Bacillus spp., not other-wise specified, are the most common strains seen Listeria monocytogenes is a significant reproduc-in the clinical laboratory. Contaminating strains of tive pathogen, distinguishable from species ofBacillus spp. are often identified only to the genus corynebacteria on the basis of motility (L. mono-level. cytogenes displays a tumbling motility by direct wet
  • 60. 46 Bacteriology mount and an umbrella-shaped pattern in motility Arcanobacterium pyogenes medium), -hemolysis on sheep blood agar and the Arcanobacterium pyogenes is also carried by, and ability to survive at 4 ◦ C. Listeria monocytogenes colo- causes, disease in animals. Infection in humans nizes a wide variety of animals and is found in soil and is usually cutaneous and, like erysipeloid, proba- vegetable matter. Infection results from ingestion of bly follows an abrasion with exposure to animals. contaminated food or dairy products. This organism Arcanobacterium haemolyticum is normal flora of is intracellular and can cross the placenta of colo- human skin and pharynx. This organism causes nized mothers to infect the fetus. Neonatal infection infections similar to those caused by Group A strep- of the CNS at less than one month of age presents tococci, including pharyngitis and cellulitis. as a meningitis syndrome with gram-positive rods (GPR) seen on Gram’s stain. Intrauterine infections may also cause spontaneous abortions or stillbirth: Lactobacillus spp. and Gardnerella vaginalis granulomatosis infantiseptica is an in utero infec- Both Gardnerella vaginalis and Lactobacillus species tion with systemic dissemination that results in are important in discussions involving reproductive stillbirth. microbiology. Lactobacilli are widely distributed in nature, and are found in foods. They are normal Gram-positive bacilli that are non-branching and flora of the human mouth, gastrointestinal tract and catalase-negative female genital tract. These bacteria are almost always encountered as a contaminant, but may cause Erysipelothrix rhusiopathiae bacteremia in immunocompromised individuals. Arcanobacterium Peroxidase-producing lactobacillus colonization is Gardnerella vaginalis associated with a healthy vagina. Absence, or a Lactobacillus spp. decrease in number of these organisms is associ- ated with an unhealthy state, e.g. lactobacilli num- Identification of these organisms must be con- bers in the vagina are decreased in cases of bacterial sidered along with anaerobic gram-positive bacilli, vaginosis. some of which are catalase-negative and will grow Lactobacillus is a highly pleomorphic organism on routine media in 5–10% carbon dioxide. They are that occurs in long chains as rods, coccobacilli therefore often described simply as ‘gram-positive, and spirals. It appears as pinpoint, non-hemolytic catalase-negative, non-spore-forming rods’. Some colonies on sheep blood agar and can be cultured on of these genera can be separated on the basis of agar with a low pH (e.g. tomato juice agar). biochemical reactions and fermentation of sug- Gardnerella vaginalis is normal vaginal flora and ars, but gas liquid chromatography is required for also may colonize the distal urethra of males. the definitive species identification of the major- The organism is one of a group contributing to ity. Many of these organisms are normal flora in bacterial vaginosis. It is also associated with uri- humans and, in general, are rarely encountered in nary tract infections, and rarely with bacteremia. infection. G. vaginalis organisms are small, pleomorphic and gram-variable. Special media (human blood agar or Columbia colistin-nalidixic acid agar) is required for Erysipelothrix rhusiopathiae cultivation of this organism. On human blood agar, Erysipelothrix rhusiopathiae is carried by, and the bacteria appear as small, opaque, grey colonies causes, disease in animals. The organism causes a surrounded by a diffuse zone of beta hemolysis. A localized skin infection known as erysipeloid, follow- more complete discussion of Gardnerella vaginalis ing a skin puncture wound with animal exposure. is found in Chapter 8.
  • 61. Major groups of organisms 47Gram-positive bacilli that are branching or Streptomycespartially acid-fast Streptomyces are found in sand, decaying vegeta-Table 2.5 outlines gram-positive bacilli. Those that tion and soil. Infection is acquired by traumaticare branching or partially acid-fast include: inoculation of organisms, usually in the lower r Nocardia extremities. Streptomyces is an agent of actinomyce- r Rhodococcus toma and rarely may cause pericarditis, bacteremia r Streptomyces and brain abscess in immunocompromised patients.This group of organisms, collectively referred toas the actinomycetes, include aerobic, facultative Anaerobic bacteriaanaerobic and obligate anaerobic bacteria. Nocar-dia, Streptomyces and Rhodococcus are aerobic. Anaerobic bacteria include the following genera.Nocardia and Rhodococcus, but not Streptomyces,contain mycolic acid in the cell wall and are partially Gram-positive bacilli (Table 2.5)acid-fast. Nocardia species form branched hyphal fil- Clostridium Actinomyces Eubacterium Bifidobacteriumaments that fragment to form rods or coccoid ele- Mobiluncus Propionibacteriumments. These genera are separated on the basis of appear- Gram-negative bacilli (Table 2.6)ance on the Gram stain, colony appearance on rou- Bacteroides Fusobacteriumtine agar, acid-fastness, lysozome resistance, and Porphyromonas Prevotellaurea hydrolysis. Although not frequently encoun- Gram-positive coccitered in the clinical laboratory, these organisms can Peptostreptococcus Peptococcuscause serious infections in humans. Gram-negative cocci VeillonellaNocardia Anaerobes are normally found in the urethra, vagina,Nocardia are inhabitants of soil and water. Follow- and colon, as well as the oral cavity and upper respi-ing traumatic inoculation or inhalation, Nocardia ratory tract. Although they may be part of the normalmay cause infection in both immunocompetent and flora, they can be responsible for significant pathol-immunocompromised individuals. Immunocompe- ogy associated with both male and female infertil-tent individuals may suffer skin infections, lym- ity. Anaerobic infections may be exogenous (usu-phocutaneous infections and mycetoma, a painless ally Clostridium spp.) or endogenous (endogenouschronic, localized, subcutaneous infection. Patients flora gain access) and endogenous infections arewho are immunocompromised may develop an usually polymicrobial. Certain factors predispose theinvasive pulmonary infection and various dissemi- human body to anaerobic infections:nated infections. (i) trauma of mucous membranes, especially rectal and vaginal mucosaRhodococcus (ii) vascular stasisRhodococcus is found in soil and water, and as (iii) tissue necrosisfarm animal flora. Infections with Rhodococcus (iv) decrease in the redox potential of a tissue, whenare usually opportunistic, with the majority occur- other organisms (facultative anaerobes) scav-ring in immunocompromised individuals. Clini- enge the available oxygen and produce an anaer-cal manifestations include pneumonia, bacteremia, obic infections, prostatic abscess, peritonitis and These infections are often polymicrobial, includingcatheter-associated sepsis. streptococci or Enterobacteriaceae.
  • 62. 48 Bacteriology Table 2.5. Bacteria: gram-positive or gram-variable anaerobic bacilli Anaerobic Spore-forming Non-spore-forming Clostridium Actinomyces* Bifidobacterium Eubacterium Mobiluncus* Propionibacterium Bacillus; some species Bacillus Curved bacillus Pleomorphic Curved bacillus Diphtheroid rod curved rods Branching Branching Some strains have Gram-variable Pleomorphic unique morphology Some strains Beaded aerotolerant Some strains Variable gram-variable aerotolerance urogenital pathogen*. prenatal/neonatal pathogen**. urogenital pathogen; and prenatal/neonatal pathogen***. Anaerobic infections commonly follow genital Table 2.5 lists examples of gram-positive or gram- tract surgery or traumatic puncture of the genital variable bacilli. tract, as well as gastrointestinal surgery or traumatic puncture of the bowel (e.g. during an oocyte retrieval Gram-positive anaerobic bacilli procedure). The presence of anaerobes in association with Of the gram-positive anaerobic bacilli, Clostrid- an infection is characterized by specific identifying ium species are easily distinguished by their abil- criteria. ity to form endospores. The remaining gram- (i) Infection is in close proximity to a mucosal sur- positive bacilli can be minimally identified based on face (anaerobes are the predominant microflora Gram stain appearance and characteristics on cul- at mucosal surfaces). ture media. Definitive identification requires more (ii) Infection persists despite antimicrobial therapy. advanced tests, such as gas liquid chromatogra- (iii) Presence of a foul odour (Porphyromonas and phy. The genus Clostridium includes the serious Fusobacterium spp. produce foul-smelling me- pathogens Clostridium tetani, C. botulinum, C. per- tabolic end products). fringens and C. difficile. (iv) Presence of a large quantity of gas (Clostridium (i) Clostridium tetani, found in soil and manure, spp. produce lots of gas). releases a potent neurotoxin that is the primary (v) Presence of black colour or brick-red fluores- virulence factor leading to tetanus. cence (pigmented species of Porphyromonas (ii) Clostridium botulinum causes botulism. The and Prevotella produce a pigment that fluo- infection is acquired from eating vegetables resces brick-red under long-wave ultraviolet or meat-based foods containing a preformed light). neurotoxin. (vi) Presence of sulfur granules (actinomycosis). (iii) Clostridium perfringens produces several toxins (vii) Distinct morphologic characteristics in Gram- and may cause gas gangrene and food poison- stained preps (Bacteroides spp. are pleomor- ing. The organism is normal flora in the intes- phic, Fusobacterium nucleatum is fusiform. tine. In the case of gas gangrene, the infec- Clostridium spp. are large, gram-positive rods tion is usually acquired from a puncture wound, that may or may not contain spores). and food poisoning is due to ingestion of
  • 63. Major groups of organisms 49Table 2.6. Bacteria: gram-negative anaerobic bacilli AnaerobicPleomorphic, non-pigmented Pointed ends, non-pigmented Coccoid or thin rods Bacillus, non-pigmentedBile-tolerant Bile-sensitive Pigmented Bile-sensitive Bile-sensitive or tolerantBacterioides fragilis group Fusobacterium Porphyromonas Biophilia Prevotella preformed toxin in food. This organism may be infections are rarely encountered outside the female acquired from the use of non-sterile operating genital tract, but the exact role of the organism in instruments and is associated with infections gynecologic infections remains unclear. resulting from ‘back alley abortions’.(iv) Clostridium difficile produces a toxin that causes Gram-negative anaerobic bacilli a diarrhoea associated with use of antibiotics that may lead to pseudomembranous colitis, a Gram-negative anaerobic bacilli (Table 2.6) occur life-threatening disease of the colon. This con- throughout the body and are the organisms most dition is a nosocomial concern since it is spread frequently encountered in anaerobic infections. from person to person in the hospital setting. Infections are usually mixed infections with otherClostridium is speciated based on Gram stain mor- anaerobes and facultatively anaerobic bacteria, andphology, arrangement of spores, colony appearance may be contained, e.g. abscesses. Sites of infec-on routine, selective and differential media, aero- tion include cranium, periodontium, thorax, peri-tolerance, pigmentation, fluorescence, resistance to toneum, liver, and female genital tract. These organ-selected antibiotics and production of lecithinase. isms may also be involved in aspiration pneumo- nia, decubitus ulcers, sinusitis, septic arthritis and Actinomyces and Bifidobacterium are normal flora other infections. In general, infections with the gram-in the upper respiratory tract and the intestine. Acti- negative anaerobes are associated with the body sitenomyces species are usually involved in mixed infec- where they reside as resident flora.tions of the oral, thoracic, pelvic and abdominal Bacteroides are intestinal flora.regions. Certain species are involved in periodontal Prevotella are flora in the upper respiratory tract, thedisease. Actinomyces also are associated with pelvic intestine, the external genitalia and the vagina.infections from use of intrauterine devices (discus- Fusobacterium is found in the upper respiratory tractsed in Chapter 11). Bifidobacterium may be encoun- and, to a lesser extent, the intestine.tered in mixed infections of the pelvis or abdomen. Infections resulting from organisms in the Bacte- Eubacterium spp. are resident flora in the upper rioides fragilis group tend to be below the dia-respiratory tract, intestine and vagina. Eubacterium associated with mixed infections of the abdomen, Pigmented Prevotellas, Porphyromonas and Fuso-genitourinary tract and pelvis. bacterium nucleatum are involved in infections above the diaphragm. Propionibacterium is found on skin, in the upper Anaerobic gram-negative bacilli are identified byrespiratory tract and in the vagina. Propionibac- aerotolerance, cell shape on the Gram stain, growthterium species are normal skin flora associated with on routine, selective and differential media, pig-acne. mentation, fluorescence, bile sensitivity, resistance Mobiluncus species are found in the vagina to a selected battery of antibiotics, production ofand contribute to bacterial vaginosis. Mobiluncus enzymes, and motility.
  • 64. 50 Bacteriology Table 2.7. Unusual bacteria Intracellular and non-culturable Cell wall-deficient Mycobacteria Spirochetes 1 Chlamydia Mycoplasma M. tuberculosis complex Treponema1 C. trachomatis*** M. hominis***3 NTM (Non-tuberculosis bacteria)1 T. pallidum***1 Rickettsia M. genitalium***3 includes M. leprae1 Borrelia1 Coxiella Ureaplasma3 Leptospira1 Ehrlichia U. urealyticum3 *** Tropheryma Calymmatobacterium C. granulomatis* Aerobic = 1 ; Aerobic, microaerophilic = 2 ; Facultative = 3 . urogenital pathogen*. prenatal/neonatal pathogen**. urogenital pathogen and prenatal/neonatal pathogen***. Gram-positive anaerobic cocci Mycobacteria and bacteria with unusual growth requirements Gram-positive anaerobic cocci are ubiquitous in the human body. Peptostreptococcus is found in Table 2.7 lists examples of bacteria with unusual the upper respiratory tract, intestine and vagina growth requirements, including: and on skin and external genitalia. Peptostrepto- r Mycobacteria coccus is often found as part of a mixed anaero- r Obligate intracellular and non-culturable agents bic/facultative infection in cutaneous, respiratory, r Cell wall-deficient bacteria: Mycoplasma and Ure- oral and pelvic sites. The same criteria used to iden- aplasma tify gram-positive bacilli are used to identify gram- r Spirochetes positive cocci. Gram-negative anaerobic cocci Mycobacteria Veillonella, genus of anaerobic gram-negative cocci, Mycobacteria are thin, non-motile slow-growing is resident flora in the upper respiratory tract, the bacilli that have an unusual cell wall; it is rich in intestine and the vagina. The organism may be lipids and contains N-glycolymuramic acid instead involved in mixed infections, but rarely plays a key of N-acetylmuramic acid. These properties make role. Identification is based on the same criteria it difficult to stain the cells with the aniline dyes used to identify anaerobic gram-negative bacilli. used in the Gram stain and also make them ‘acid- This organism may be confused with Neisseria fast’ (resistant to stringent decolorization with 3% gonorrhea and must be considered when eval- hydrochloric acid after application of basic fuchsin uating specimens for the presence of Neisseria dye or after heating following dye application). gonorrhoeae. The stringent acid-fast characteristic makes it easy N. gonorrhoea should only be presumptively to distinguish mycobacterium from most other reported if the organisms are kidney-shaped, diplo- organisms, although other organisms such as the cocci and intracellular. Veillonella will not be found nocardial species may also be acid-fast if a less inside cells and the coccus is larger than N. stringent decolorization step is used (modified acid- gonorrhoeae. fast).
  • 65. Major groups of organisms 51 There are more than 70 species of mycobac- Chlamydia trachomatis and Calymmatobacteriumterium including the pathogens Mycobacterium are significant reproductive pathogens. Chlamy-tuberculosis and Mycobacterium leprae. These diae have an unusual life cycle characterized by aorganisms are responsible for a spectrum of infec- small (0.25–0.35 m) infective elementary body (EB)tions in humans and animals, ranging from local- and a large (0.5–1.0 m) reticulate body (RB) thatized lesions to disseminated disease. Mycobacteria multiplies within the host cell. The RBs multiply byare divided into two main groups: binary fission in the host cell and condense to form (i) M. tuberculosis complex, which includes the elementary bodies, which are released when the M. tuberculosis, M. bovis and M. africanum. host cell lyses and dies. These EBs can then infect M. tuberculosis causes primary tuberculosis additional nearby cells. lesions in the lung, but can spread to extra- pulmonary sites including the genitourinary Chlamydia tract, lymph nodes, brain, bones, joints, peri- toneum, pericardium and larynx. Tuberculosis The genus Chlamydia contains the species: C. pneu- infection of the genital tract is covered in moniae, C. trachomatis and C. psittaci. Chapter 11. C. pneumoniae is a human pathogen transmitted (ii) Non-tuberculosis mycobacteria (NTMs). from person to person by aerosol droplets from the The non-tuberculosis mycobacteria group is respiratory tract. The organism may cause pneu- subdivided on the basis of growth rate (slow- monia, bronchitis, sinusitis, pharyngitis and flu- growing vs. rapid-growing) with the slow- like illness. growers further divided based on the organism’s C. psittaci, common in birds and domestic ani- ability to produce pigment either in the presence mals, causes infections in humans that are char- of light (photochromogens) or in the absence acterized by pneumonia, severe headache, hep- of light (scotochromogens). Some mycobac- atosplenomegaly and changes in mental state. The teria do not produce pigments under either infection may range from mild to life-threatening. light or dark conditions and are referred to C. trachomatis has subtypes. as non-photochromogens. M. leprae cannot be Subtypes A, B, Ba and C cause endemic trachoma cultivated in vitro. None of the mycobacte- by spread of the organism from hand to eye ria are associated with reproductive problems, from the environment; it may also be spread by but members of the rapid growing mycobac- flies. teria are associated with postoperative infec- L1, L2 and L3 subtypes are responsible for the tions (M. abscessus, M. chelonae and M. muco- sexually transmitted disease lymphogranuloma genicum). M. mucogenicum also is associated venereum, described in Chapter 7. with catheter-related sepsis. Subtypes D–K may be spread sexually, from hand to eye by autoinoculation of genital secretions, or from eye to eye. These subtypes can cause ure- thritis, cervicitis, pelvic inflammatory diseaseNon-culturable obligate intracellular pathogens and epididymitis. Neonatal transmission causes pneumonia and conjunctivitis in infants. Species of Chlamydia may be grown on cell cultures Chlamydia Rickettsia in the laboratory or identified by direct methods, Ehrlichia Coxiella burnetii including cytologic examination, antigen detection Tropheryma whippelii and nucleic acid hydridization. C. trachomatis and Calymmatobacterium infections caused by subtypes D–K are covered in granulomatis detail in Chapter 10.
  • 66. 52 Bacteriology Rickettsia by host cells and multiplies in vacuoles before being picked up by macrophages and carried through Rickettsia are small (0.3 m × 1.0 × 2.0 m) fas- lymph nodes to the bloodstream. The organism is tidious, pleomorphic, obligate intracellular parasites identified by serology. that survive outside the host for only very short periods of time. The rickettsia divide by binary fission in host cell cytoplasm, and mature rick- Tropheryma whippelii ettsiae are released with lysis of the host cell. The Tropheryma whippelii is the agent of Whipple’s organisms infect wild animals and humans; humans disease, found primarily in middle-aged men and are accidental hosts who become infected follow- characterized by the presence of mucopolysaccha- ing the bite of an arthropod vector, e.g. ticks, lice, ride or glycoprotein in virtually every organ sys- mites, chiggers. Rickettsia are associated with states tem. A cellular immune defect may be involved of crowding and unsanitary conditions including in the pathogenesis, characterized by diarrhea famine, war and poverty. They are responsible for and weight loss, lymphadenopathy, hyperpigmen- spotted fevers, including Rocky Mountain spotted tation, arthralgia, joint pain and a distended tender fever, typhus, and scrub typhus. Rickettsia are iden- abdomen. This organism is phylogenetically a gram- tified by serology or immunohistology. positive actinomycete, unrelated to any other genus known to cause infection. It is detected by PCR assay. Ehrlichia Ehrlichia, also spread to humans via arthropod vec- Calymmatobacterium granulomatis tors, infect leukocytes. Once inside the white blood Calymmatobacterium granulomatis causes the cells, the organisms undergo a developmental cycle sexually transmitted Donovanosis or granuloma similar to Chlamydia: an elementary body infects the inguinale. Donovanosis is common in many parts cell, then multiplies and clusters to form an initial of the world, including India, the Caribbean and body, then a morula that ruptures to release the EBs. Australia, but is rare in the United States. Although Species of Ehrlichia infect monocytes and granulo- it is primarily sexually transmitted, C. granulomatis cytes. may be transmitted via non-sexual modes. The infection is characterized by subcutaneous nodules that form red, granulomatous, painless lesions that Coxiella burnetti bleed easily, and patients often present with inguinal Coxiella burnetti is harboured in farm animals and is lymphadenopathy. C. granulomatosis genital lesions the agent of Q fever, an acute systemic infection that have been mistaken for neoplasms. The organism, a primarily affects the lungs. This organism, which is gram-negative, pleomorphic encapsulated bacillus, smaller than the rickettsia, can live outside cells, but is observed in vacuoles in large mononuclear cells. can only be cultivated on lung tissue. C. burnetii has Since cultivation of C. granulomatis is difficult, a spore-like life cycle and can exist in two antigenic identification is based on visualizing the organism states: in scrapings of lesions stained with Wright’s or Phase I, the highly infectious form, is isolated from Giemsa stain. Disease manifestations and diagnosis animals. of this organism are presented in Chapter 7. Phase II has been grown in culture and found to be non-infectious. Mycoplasma (class Mollicutes) The organism is found in animal milk, urine, feces and birth products. Human infection follows aerosol These include the genera Mycoplasma and Ure- inhalation. Once inhaled, C. burnetti is phagocytized aplasma; they are the smallest (0.3 m × 0.7 m)
  • 67. Major groups of organisms 53free-living organisms and are widespread in nature. either by crossing the placenta of a colonizedThey lack a cell wall, but are related to gram-positive mother or from the birth canal during vaginalbacteria: they appear to have evolved from gram- delivery. Disease in the neonate is systemic andpositive clostridial-like cells by a drastic reduction of includes meningitis, abscess and pneumonia.genome size, resulting in the loss of many biosyn- (iii) U. urealyticum also is associated with devel-thetic abilities – they may be considered as the best opment of chronic lung disease. M. hominis isrepresentatives of the concept of a minimal cell. distinguished from U. urealyticum by colonyThere are many species of mycoplasma in nature; morphology, production of urea and glucosespecies of Mycoplasma and Ureaplasma urealyticum utilization. M. hominis is positive for arginineare important reproductive pathogens. They are also (detected by colour change in liquid medium)a known hazard in cell culture systems, causing and has a ‘fried egg’ morphology; U. urealyticum‘silent’ infections that are not visually obvious, but produces the urease enzyme and the coloniescan alter cell metabolism and induce chromosomal appear as dark clumps. Genital mycoplasma andaberrations. ureaplasma are discussed in Chapter 10. In general, they are aerobic and fastidious, requir-ing sterols in the medium for membrane functionand growth. Due to their size and lack of cell wall, Spirochetestypical methods, e.g. Gram staining cannot be used These are long, gram-negative, helically curvedto identify the organisms. Bacterial colonies must be bacilli. The helical curves are responsible for organ-visualized using a stereo microscope. The bacteria ism motility and give the bacteria a corkscrew shape.are cultured using indicators for different metabolic There are numerous spirochetes in nature, someactivities to detect growth. Serodiagnosis is helpful of which are normal flora in humans, and some offor certain species. which are serious pathogens. (i) Mycoplasma pneumoniae, spread via respira- The genera Treponema, Borrelia and Leptospira tory droplets, causes a community-acquired include human pathogens. They are distinguished pneumonia, primarily in young adults. Infec- by their metabolic and biochemical characteristics, tions with M. pneumoniae are associated with and by the number and tightness of coils: closed populations (e.g. families, dormitories, Treponema are slender with tight coils. military barracks). M. pneumoniae also may Borrelia are thicker than Treponema and have fewer cause upper respiratory tract infections in chil- and looser coils. dren. This organism may be cultured in the lab- Leptospira has thick loose coils and hooked ends. oratory, or serology can be used for diagnosis. (ii) Mycoplasma hominis, M. genitalium, and Ure- Borrelia recurrentis is transmitted via the bite of aplasma urealyticum are genital mycoplasmas. a tick or louse and causes relapsing fever. Borre- M. hominis and U. urealyticum colonize the lia burgdorferi, transmitted by the bite of Ixodes newborn, but colonization does not persist ticks, is responsible for Lyme disease. The organ- beyond the age of two years. Individuals acquire isms are identified using serology and PCR to detect the organisms via sexual contact once they organism DNA. Leptospira interrogans causes lep- reach the age of puberty. These mycoplasma tospirosis that is acquired by contact with infected may cause, or contribute to, urogenital infec- animals. The organism invades the blood and other tions, including prostatitis, bacterial vaginosis, sites throughout the body including the kidneys and pelvic inflammatory disease, non-gonococcal the central nervous system. L. interrogans is iden- urethritis and amnionitis, and may cause inva- tified by direct detection in body fluids, as well sive disease in immunocompromised patients. as by fluorescent antibody staining and molecular The organisms can cause disease in neonates techniques.
  • 68. 2second part of chapter 2 53
  • 69. 54 Bacteriology The reproductive pathogens in this order are mem- antibodies, behaving as antigens to elicit an immune bers of the genus Treponema. response. Colonization by well-adapted flora also T. pallidum subspecies pallidum causes syphilis, excludes other microorganisms from colonizing the a sexually transmitted (or congenital) disease that is site. They compete for attachment sites, and produce limited to humans (covered in detail in Chapter 7). substances that inhibit or kill other bacteria, such as T. pallidum subspecies pertenue and T. pallidum fatty acids, peroxides, or specific bacteriocins. The subspecies endemicum also infect only humans, composition of normal flora varies widely in differ- causing non-venereal diseases of the skin. These ent animal species, and is invariably related to age, organisms have not been cultivated in vitro for more sex, diet and environmental temperature. Some are than one passage. Detection methods include biopsy found regularly at particular sites, and others are and visualization using dark-field or phase-contrast present only occasionally. Table 2.8 lists the normal microscopy. Other treponemes inhabit the mouth flora that may be found at different body sites in the or genital tract in humans, and these organisms human. can be cultured in the laboratory under anaerobic Fortunately, animals that host this large and var- conditions. Organisms from this group, along with ied ecosystem have highly developed and elabo- fusiform anaerobes, cause Vincent’s angina, a gum rate defense mechanisms, which prevent the organ- disease referred to as acute necrotizing ulcerative isms from travelling to areas where they can cause gingivitis. disease, and also provide mechanisms for dealing with infection if the normal defence barriers are breached. Normal flora in humans (i) Mechanical barriers include reflexes, such as Internal tissues such as blood, bone marrow, solid coughing, gag reflex, sneezing and swallow- organs, muscle, pleural, peritoneal, synovial and ing. Intact skin is protected by sebaceous gland cerebrospinal fluid are normally free of microorgan- secretions, as well as continuous sloughing isms. Surface tissues, such as respiratory tract, oral of epithelial cells. Sweat removes microor- cavity, eyes, ears, urinary tract, genital tract, gas- ganisms, and contains the enzyme lysozyme, trointestinal tract and skin, on the other hand, are in which is bacteriostatic for some organisms. constant contact with the environment, and there- The conjuctiva is protected by tears, and by fore are readily colonized by microbes. The mixtures lysozyme. The epithelium of mucous mem- of organisms regularly found at any anatomical site branes is protected by mucus production are known as ‘normal flora’. The microbes present (traps organisms), the ciliary transport sys- as flora establish a dynamic interaction with their tem, lysozyme production, and nasal hairs host, which results in a situation of mutualistic (filter). symbiosis. Many are specifically adapted to host (ii) In the gastrointestinal tract, a number of fac- tissues, with biochemical interactions between the tors are important in maintaining a normal bal- surface components of bacteria and host cell adhe- ance of flora. Saliva, lysozyme, stomach acidity, sion molecules. From the host, flora derive a supply bile, normal peristalsis and maintaining the of nutrients, a stable environment, constant tem- integrity of the mucosal layer all serve to pro- perature, protection and transport. The host also tect epithelial cells from the flora inhabiting the derives some nutritional benefit from his or her gut lumen. flora. In humans, the microbial flora of the gut, (iii) The genitourinary tract maintains its balance for example, provide the host with Vitamin K and with the flushing effect of urine and urine acid- some B vitamins. The flora can also stimulate lym- ity. The vaginal epithelium provides an intact phatic tissue development, producing cross-reactive barrier and vaginal secretions that are high
  • 70. Major groups of organisms 55 in lactose. These secretions promote growth (vii) Host defence mechanisms also include the and colonization by the H2 O2 -producing sophisticated machinery of specific immune lactobacilli that maintain an appropriate vagi- response mechanisms: secretory IgA in mucus nal pH and microbiota in the healthy vagina. secretions can bind some pathogens and pre- Prostatic secretions also contain elements that vent attachment, and serum IgG neutral- inhibit some bacteria. izes viruses, acts as an opsonin for bacteria,(iv) Physiological circulating fluids, such as blood fungi, and parasites, initiates the comple- and lymphatic secretions, contain soluble ment cascade when bound to an antigen, and and circulating non-specific factors, includ- neutralizes some bacterial toxins. Serum IgM ing complement (C1–C9) that lyses bacte- similarly has a significant and complex role. rial membranes, and the complement alter- IgM antibodies represent the first wave of nate pathway at C3 stage. Acid and alkaline serum immunoglobulin production during an phosphatase inactivate Herpes viruses, inter- acute infection; IgM is therefore used as an feron proteins prevent virus re-infection, and early marker for infection and its disappear- fibronectin can act as a non-specific opsonin ance reflects conversion to a chronic infection for some microbes. or convalescent state. (v) Host defence mechanisms also include cellular (viii) The cellular immune system (CMI) orchestrates non-specific immune effectors, such as alveolar an elaborate system of host defence mecha- macrophages in the lung, polymorphonuclear nisms, which include T-lymphocytes (T-helper neutrophils, eosinophils, fixed macrophages cells), cytotoxic T-cells that kill infected cells (histiocytes), lactoferrin that sequesters iron directly, natural killer T-cells (NK) that destroy available to invading pathogens, and an elab- certain bacteria and attack virus-infected cells, orate system of cytokines produced by cells of and T-suppressor cells that downregulate anti- the immune system that can affect the immune body production by B-cells. response, such as Interleukins 1, 6, 8, and In contrast to this complex and highly developed sys- tumour necrosis factor (TNF). tem of physiological defence mechanisms, the pro-(vi) Metabolic or natural defences include body cedures of assisted reproduction involve removing temperature: T. pallidum cannot survive in oocytes from their natural, highly protected environ- patients with a high fever. Specific host cell ment, placing them in contact with semen samples, receptors are important (such as the CD4 and culturing embryos in artificial media in a lab- receptor on helper lymphocytes that allows oratory environment. The oocytes frequently have HIV infection to occur), and a compromised their only source of protection, the barrier offered nutritional and metabolic state of the host can by the zona pellucida, breached with microsurgi- prevent, or facilitate microbial invasion, e.g. cal techniques. In this scenario, there is no defence diabetic ketoacidosis results in low pH and high against any flora in the environment, and therefore glucose levels, an environment where yeast and an understanding of the background, the hazards, fungi thrive. Patients presenting with recurrent and the precautions that may be applied to protect yeast/fungal infections should always be tested the gametes and embryos, is a crucial element of any for diabetes. assisted reproductive practice.
  • 71. Table 2.8. Normal human flora Organisms which are present orSite/Specimen Colonization if applicable may be present CommentsBlood Sterile Although organisms may appear in the blood transiently (e.g. post-dental manipulation), any organism that multiplies and causes symptoms would be considered pathogenic in this site. Parasites may be found in the blood in transit to another site, but this is not a state of good health.Cerebral Spinal Fluid Sterile(CSF) and CNSPleural fluid SterilePeritoneal fluid SterilePericardial fluid SterileSynovial fluid SterileBone SterileBone marrow SterileSinuses SterileEyes sparse flora Staphylococcus epidermidis Other organisms may colonize: Lactobacillus spp. Propionibacterium acnes Staphylococcus aureus (<30% population) Haemophilus influenzae (up to 25% of population) Moraxella catarrhalis, some Enterobacteriaceae, various streptococci (S. pyogenes, S. pneumoniae, other -hemolytic and -hemolytic strep) are found in a very small percentage of peopleEars sparse flora Staphylococcus epidermidis May also see organisms similar to those found in the Lactobacillus spp. conjunctival sac listed in the column above, but the following are seen more often: Streptococcus pneumoniae Propionibacterium acne Staphylococcus aureus Enterobacteriaceae Pseudomonas aeruginosa is found on occasion Candida spp. (non-Candida albicans) are also common
  • 72. Skin Diphtheroids Staphylococcus epidermidis Other coagulase-negative staphylococci Propionibacterium acneUpper respiratory tract Actinobacter spp. These organisms are found in the nasopharynx and Viridans strep. oropharynx of healthy people, but are possible β-hemolytic strep. pathogens Streptococcus pneumoniae Staphylococcus aureus Nesisseria spp. Mycoplasma spp. Haemophilus influenzae Haemophilus parainfluenzae Moraxella catarrhalis Candida albicans Herpes simplex virus Enterobacteriaceae Mycobacterium spp. Pseudomonas spp. Burkholderia cepacia Klebsiella ozaenae Eikenella corrodens Bacteroides spp. Peptostreptococcus spp. Actinomyces spp. Capnocytophaga spp. Actinobacillus spp. Filamentous fungi (cont.)
  • 73. Table 2.8. (cont.) Organisms which are present orSite/Specimen Colonization if applicable may be present CommentsUpper respiratory tract Non-hemolytic strep. These organisms are found in the nasopharynx and Staphylococci oropharynx of healthy people, and may be possible Micrococci pathogens, but these are rare. Corynebacterium spp. Coagulase-negative staph Neisseria species other than N. gonorrheae Lactobacillus spp. Veillonella spp. Spirochetes Rothia dentocariosa Leptotrichia buccalis Selenomonas Wolinella Stomatococcus mucilaginosus Campylobacter spp.Lower respiratory tract To cause disease in the lower respiratory tract, organisms (either those potential pathogens in the URT or true pathogens) must possess traits or produce products that promote colonization, multiplication and subsequent infection in the host.
  • 74. Upper urinary tract – (ureters sterileand kidneys)Lower urinary tract: Note: All areas above the urethra are sterilebladder Bladder sterileurethra Urethra Coagulase-negative The short female urethra lies in close proximity to the staphylococci, perirectal region, which has many microbes present. excluding S. saprophyticus Potential pathogens may be present as transient Viridans and non-hemolytic colonizers. These include gram-negative aerobic bacilli strep (primarily Enterobacteriaceae), and occasional yeasts. Lactobacilli Diphtheroids (Corynebacterium spp.) Non-pathogenic Neisseria saprophytic spp. Anaerobic cocci Propionibacterium spp. Anaerobic gram-negative bacilli Commensal Mycobacterium spp. Commensal Mycoplasma spp.Genitourinary tract Commensals Note: Vulva and penis of the uncircumcised male mayUrethra (covered above) Coag-neg staph harbour Mycobacterium smegmatis and otherLining of the genital tract Corynebacteria gram-positive bacteria AnaerobesPre-pubescent and Same flora as seen on Staphylococci The flora of the female genital tract is dependent on pHpost-menopausal female surface epithelia Corynebacteria and estrogen concentration, which are dependent on the age of the female. Lactobacillus spp. are the primary organisms in normal, healthy vaginal secretions with hydrogen peroxide-producing lactobacilli associated with a healthy state. (cont.)
  • 75. Table 2.8. (cont.) Organisms which are present orSite/Specimen Colonization if applicable may be present CommentsReproductive age females Large numbers of facultative The number of anaerobes remain constant throughout bacteria the monthly cycle. Enterobacteriaceae Note: Many women carry Group B -hemolytic strep Streptococci (S. agalactiae) which may be transmitted to the Staphylococci neonate. Anaerobes Lactobacilli Non-spore forming Note: Although yeasts (from the GI tract) may be found bacilli and cocci transiently in the female vagina, they are not normalGI tract Clostridia flora.Upper small intestine Babies are colonized by Sparse flora (101 to In the large intestine, anaerobes outnumber aerobes human epithelial flora 103 /mL)consisting of 1000:1 (staphylococci, streptococci, lactobacilli, yeasts Corynebacterium spp., 106 to 107 /mLLower small intestine other gram-positive Predominantly:(distal ileum) organisms Enterobacteriaceae (bifidobacteria, BacterioidesLarge bowel clostridia, lactobacilli, Predominantly anaerobic streptococci) a few hours species: after birth; Bacterioides Over time the content of Clostridium the intestine changes. Peptostreptococcus The normal flora of the Bidifobacterium adult large bowel is Eubacterium established relatively Facultatives include: early in life. Escherichia coli Other Enterobacteriaceae Enterococci Streptococci
  • 76. Further reading 61FURTHER READING Forbes, B. A., Sahm, D. F. & Weissfeld, A. S. (2002). Diagnostic Microbiology, 11th edn. St. Louis: Mosby Publishers.Alberts, B., Johnson, A., Lewis, J. et al. (2002). Molecular Biology Leland, D. S. (1996). Clinical Virology. W. B. Saunders Co. of the Cell. 4th edn. New York: Garland Science. Morello, J. A., Mizer, H. E., Wilson, M. E. & Granato, P. A. (1998).de la Maza, L M., Pezzlo, M. T. & Baron, E. J. (1997). Color Microbiology in Patient Care, 6th edn. Boston, MA: McGraw- Atlas of Diagnostic Microbiology. St. Louis: Mosby Publi- Hill. shers. Sarosi, G. A. & Davies, S. F. (1994). Therapy for fungal infections.Delost, M. D. (1997). Introduction to Diagnostic Microbiology. Mayo Clinic Proceedings, 69: 1111–17. St. Louis: Mosby Publishers. Schaechter, M., Medoff, G. & Schlessinger, D. (1989). Mech-Forbes, B. A., Sahm, D. F. & Weissfeld, A. S. (1998). Diagnostic anisms of Microbial Disease. Baltimore, MD: Williams & Microbiology. 10th edn. St. Louis: Mosby Publishers. Wilkins.
  • 77. Appendix 2.1. Media used for isolation of bacteria Differential appearance ofMedium Components Isolation/primary purpose colonies on agarBacteroides Bile esculin agar (BBE) Tryptic soy agar (TSA) with ferric Selective and differential for Bacteroides B. fragilis = dark colonies ammonium citrate, hemin, bile fragilis group; presumptive salts and gentamicin identification based on growth on BBE.Bile esculin agar (BEA) Nutrient agar with ferric citrate, Differential isolation and presumptive Medium turns black if esculin is esculin, bile and sodium identification of Group D streptococci hydrolysed; if esculin is not hydrolysed deoxycholate. Sodium deoxycholate and enterococci there will not be a blackening of the inhibits many organisms medium. Examples: Group D streptococci and enterococci will grow in the presence of bile salts and hydrolyse esculin, turning the medium black; other groups of streptococci will be inhibited by the bile in the medium.Bismuth sulfite agar (BS) Peptone agar with dextrose, ferrous Selective for isolation of Salmonella sulfate and brilliant green, spp. from stool Gram-positive organisms and members of the family Enterobacteriaceae other than Salmonella are inhibited by bismuth sulfite and brilliant green.Blood agar (sheep blood)(BA) TSA with blood (may also have Cultivation of fastidious organisms and Non-hemolytic = no zone of hemolysis (SBA) Brucella agar or beef heart infusion demonstration of hemolysis around colony with 5% sheep blood) Alpha-hemolysis = greenish-brown area surrounding colony Beta-hemolysis = clear area around colony Examples: Non-hemolytic = Group D streptococci Alpha-hemolytic = Streptococcus pneumoniae Beta-hemolytic = Streptococcus pyogenes (Note: Blood agar is required for growth of Streptococcus pyogenes)
  • 78. Bordet-Gengou Potato-glycerol-based medium with Isolation of Bordetella pertussis defibrinated blood and methicillinBuffered charcoal yeast extract Yeast extract, agar, charcoal, salts, Enrichment medium for Legionella spp. L-cysteine HCl, ferric pyrophosphate, ACES-buffer, and alpha-ketoglutarateCampy-blood agar Brucella agar base with vancomycin, Selective for Campylobacter spp. trimethoprim, polymixin B, amphotericin B, and cephalothinCenters for Disease Control and SBA with hemin, L-cysteine and Isolation of anaerobes; enhanced growth Prevention anaerobic blood vitamin K of peptostreptococci agarCefsulodin–igrasan–novobiocin Peptone base with yeast extract, Selective for Yersinia spp.; isolation of agar (CIN) mannitol, bile salts, cefsulodin, Aeromonas spp. igrasan, novobiocin; neutral red and crystal violet indicatorsChocolate agar (CA) Peptone base with 2% hemoglobin or Cultivation of Haemophilus spp. and IsoVitaleX (BBL) pathogenic Neisseria spp.Columbia–Colistin–nalidixic acid Columbia agar base with colistin, Selective isolation of gram-positive cocci (CNA) nalidixic acid and 5% sheep bloodCycloserine–cefoxitin–fructose Egg yolk base with fructose, Selective for Clostridium difficile C. difficile produces yellowish rhizoid agar (CCFA) cycloserine, and cefoxitin; neutral colonies red indicatorCystine–tellurite blood agar Infusion agar base in 5% SBA with Isolation of C. diphtheriae C. diphtheriae reduces potassium potassium tellurite tellurite, producing black colonies.Eosin methylene blue (EMB) Peptone base with lactose and Isolation and differentiation of Fermenters = purple sucrose; eosin and methylene blue lactose-fermenting and (Note: E. coli has green, metallic sheen) indicators non-lactose-fermenting enteric bacilli non-lactose fermenter (NLF) = clear, pink (cont.)
  • 79. Appendix 2.1. (cont.) Differential appearance ofMedium Components Isolation/primary purpose colonies on agarGram-negative broth (GN) Peptone base with glucose, mannitol, Selective (enrichment) liquid medium citrate and sodium deoxycholate for enteric pathogens inhibits many organismsHektoen agar (HE) Peptone base with bile salts, lactose, Differential and selective medium for Salmonella and Shigella = blue-green, and sucrose; bromothymol blue isolation and differentiation of clear and acid fuchsin indicators Salmonella spp. and Shigella spp. from Fermenters = yellow-orange other gram-negative enteric bacilli NLF = clear, greenKanamycin-vancomycin- Brucella agar base with kanamycin, Selective isolation of Bacteroides spp. Prevotella melaninogenica produces a laked blood agar (KVLB) vancomycin, vitamin K, and 5% and Prevotella spp. pigment on this medium laked bloodLowenstein–Jensen agar (LJ) Egg-based medium; malachite green Isolation of mycobacteria inhibitorMacConkey agar Peptone base with lactose, crystal Isolation and differentation of Lactose fermenters = red violet and bile salts; neutral red lactose-fermenting and Non-lactose fermenters = clear, pink indicator non-lactose-fermenting enteric bacilli (take on colour of the medium) Bile salts and crystal violet inhibit Examples: Klebsiella spp. are gram-positive organisms lactose-fermenters. Proteus spp. are non-lactose-fermenters.Mannitol salt agar (MSA) Peptone base, mannitol, and 7.5% Selective isolation of staphylococci Medium turns yellow when mannitol is salt; phenol red indicator hydrolysed. Examples: Staphyloccus aureus hydrolyses mannitol, giving a positive reaction. Staphylococcus epidermidis does not hydrolyse mannitol, giving a negative reaction.Middlebrook 7H10 agar Complex base with albumin, salts, Isolation of mycobacteria; antimicrobial and digest of casein; malachite susceptibility testing can be performed green inhibitor on colonies growing on this medium
  • 80. Modified Thayer–Martin agar BA with hemoglobin, supplement B; Selective for Neisseria gonorrhoeae and (MTM) colistin, nystatin, vancomycin and Neisseria meningitidis trimethoprim Colistin inhibits growth of gram-negative organisms other than Neisseria gonorrhoeae and Neisseria meningitidis, nystatin inhibits yeast, vancomycin inhibits gram-positive organisms and trimethoprim inhibits swarming Proteus spp.New York City agar (NYC) Peptone agar with cornstarch, yeast Selective for Neisseria gonorrhoeae; dialysate, 3% hemoglobin, horse genital mycoplasmas also grow plasma, vancomycin, colistin, amphotericin B, and trimethoprimSalmonella–Shigella agar (SS) Peptone base with lactose bile salts, Selective for Salmonella spp. and Shigella Salmonella = clear with black centres brillant green and sodium citrate; spp. Shigella = clear neutral red indicator; brilliant green Lactose-fermenters = pink and bile salts inhibit coliforms NLF = clearSelenite broth Peptone base broth with sodium Enrichment for isolation of Salmonella selenite. Sodium selenite is toxic for spp. most EnterobacteriaceaeSchaedler agar Peptone and soya base with yeast Non-selective for recovery of anaerobes extract, dextrose, buffers, hemin, and aerobes L-cysteine and 5% bloodSkirrow agar Peptone and soya protein base with Selective for Campylobacter spp. lysed horse blood; vancomycin, polymyxin B, and trimethoprimStreptococcal selective agar (SSA) SBA with colistin, trimethoxazole Selective for Streptococcus pyogenes and (SXT) and crystal violet Streptococcus agalactiaeTetrathionate broth Peptone base with bile salts and Selective for Salmonella spp. and Shigella sodium thiosulfate spp. Bile salts and sodium thiosulfate inhibit gram-positive organisms and Enterobacteriaceae spp. other than Salmonella and Shigella. (cont.)
  • 81. Appendix 2.1. (cont.) Differential appearance ofMedium Components Isolation/primary purpose colonies on agarThioglycollate broth Pancreatic digest of casein, soya Supports growth of anaerobes, aerobes, broth, and glucose microaerophilic organisms and Thioglycollate and agar reduce redox fastidious organisms potential (Eh)Thiosulfate citrate–bile salts Peptone base with yeast extract, bile Selective and differential for vibrios Sucrose-fermenters = yellow (TCBS) salts, citrate, sucrose, ferric citrate, Non-sucrose-fermenters = green and sodium thiosulfate; bromthymol blue indicatorTrypticase soya agar (TSA) Nutrient agar For isolation of non-fastidious organisms Examples: Bacillus species and coliforms will grow on this mediumVaginalis (V) agar Columbia agar with 5% human blood Selective and differential for Gardnerella G. vaginalis produces small, grey, opaque vaginalis colonies surrounded by a diffuse -hemolytic zoneXylose lysine deoxycholate agar Yeast extract agar with lysine, xylose, Isolation and differentiation of Salmonella spp. = pink-red with black (XLD) lactose, sucrose, ferric ammonium Salmonella and Shigella spp. from centre citrate; phenol red; sodium other gram-negative enteric bacilli Shigella spp. = clear deoxycholate inhibits Lactose-fermenters = yellow gram-positive organisms NLF = red, yellow or clear with or without black centres
  • 82. Appendix 2.2. Biochemical tests for identification of bacteriaTest Principle/major use/examples of key positive and negative organisms when applicableAcetamide utilization Determines if an organism can use acetamide as the sole source of carbon. Organisms that grow on this medium are able to deaminate acetamide and release ammonia, causing a change in pH. The change in pH results in a colour change in the medium from green to blue. Interpretation: Positive: Deamination of acetamide results in a blue colour. Negative: No change in colour. Examples: Positive: Pseudomonas aeruginosa Negative: Stenotrophomonas maltophiliaAcetate utilization Determines if an organism can use acetate as the sole source of carbon. The organism that is able to utilize acetate as a sole carbon source will break down the sodium acetate in the medium causing an increase in pH that turns the indicator from green to blue. Interpretation: Positive: Growth of the organism results in an alkaline (blue) medium. Negative: No growth or growth without change in indicator (no colour change). Examples: Positive: Escherichia coli Negative: Shigella flexneriBacitracin susceptibility Determines an organism’s ability to tolerate bacitracin (0.04 units). A standard quantity of a bacterial suspension is streaked on agar (either tryptic soya agar or sheep blood agar, depending on the organism’s growth requirements) and streaked in three directions to produce a lawn of growth. A bacitracin disc is placed in the centre of the streaked plate. Following incubation and bacterial growth, zones of inhibition are measured around the bacitracin disc to determine susceptibility. (cont.)
  • 83. Appendix 2.2. (cont.)Test Principle/major use/examples of key positive and negative organisms when applicable Interpretation: Positive: Zone of inhibition around the disc (must measure size) indicates susceptibility. Negative: No zone of inhibition around the disc indicates resistance. Examples: Positive (any zone of inhibition): Group A streptococci; some strains of Lancefield Groups B, C, and F may be susceptible Positive (with zone>10 mm): Micrococcus species Negative (no zone of inhibition): Staphylococcus speciesBile esculin Determines both an organism’s ability to grow in the presence of bile (4% oxgall) and its ability to hydrolyse the glycoside esculin to esculetin and dextrose. Interpretation: Positive for growth in bile: growth, but no change in colour of medium. Positive for growth in bile and esculin hydrolysis: growth plus medium turns black. Negative: No growth in the medium Examples: Positive: Group D streptococci and Enterococcus species will grow in the bile and will hydrolyse esculin. Negative: Lancefield Groups other than Group D streptococciBile solubility test Determines if an organism is lysed by bile or a bile salt solution such as sodium desoxycholate. Lysis is dependent on the presence of intracellular autolytic enzymes. Bile salts lower the surface tension between the cell membrane of the bacteria and the medium, activating the organism’s natural autolytic process. The amidase enzyme splits the muramic acid–alanine bond in peptidoglycan in the cell wall resulting in cell lysis. This test is used to differentiate Streptococcus pneumoniae from other species of -hemolytic streptococci. Interpretation: Positive: Colonies disintegrate with an imprint of the lysed colony remaining within the zone. Negative: Colonies remain intact. Examples: Positive: Streptococcus pneumoniae. Negative: -hemolytic streptococci other than S. pneumoniae.
  • 84. CAMP test Determines if an organism produces a diffusible extracellular (CAMP factor) protein that acts synergistically with the beta-lysin strain of Staphylococcus aureus to cause enhanced lysis of red blood cells in the sheep blood agar medium when the organisms are streaked perpendicular to each other. Interpretation: Positive: Enhanced hemolysis will appear as an arrowhead-shaped zone of -(clear) hemolysis at the juncture of the two organisms. Negative: No increased hemolysis. Examples: Positive: Group B streptococcus. Negative: Streptococci other than Group B.Catalase Catalase is used to distinguish the families Streptoccoaceae from Micrococcaceae. The test determines the ability of an organism to produce the enzyme catalase that can be detected by adding a 3% solution of hydrogen peroxide to a suspension of the organism to be tested. A positive is indicated by the formation of bubbles. Catalase-producing bacteria +3% H2 O2 − − − − →H2 O and O2 (bubbles form) −−−− Catalase production may function to inactivate toxic hydrogen peroxide and free radicals formed by the myeloperoxidase system with phagocytic cells after ingestion of the microorganisms. Interpretation: Positive: Bubbles are produced when H2 O2 is added to a bacterial colony. Negative: No bubbling when H2 O2 is added to the bacterial colony. Examples: Positive: Staphylococci and Micrococci Negative: StreptococciCetrimide Determines an organism’s ability to grow in the presence of cetrimide, a toxic substance that inhibits the growth of most bacteria. Interpretation: Positive: Organism growth on the agar. Negative: No growth on the agar. Examples: Positive: Pseudomonas aeruginosa Negative: Escherichia coli (cont.)
  • 85. Appendix 2.2. (cont.)Test Principle/major use/examples of key positive and negative organisms when applicableCitrate utilization Determines an organism’s ability to use sodium citrate as the sole source of carbon for metabolism.(Simmon’s citrate) Bromothymol blue is included in the medium as an indicator to detect the breakdown of citric acid. Citric acid (citrate) → breakdown products (green = acid) (blue = alkaline) Interpretation: Positive: Intense blue colour. Negative: Growth, but no colour change. Examples: Positive: Klebsiella pneumoniae Negative: Escherichia coliCoagulase test Coagulase is an enzyme that acts on fibrinogen to form a clot. This test is used to differentiate Staphylococcus aureus, which is positive, from staphylococci that are negative for the enzyme. S. aureus produces two forms of coagulase; bound (clumping factor) and free. Bound coagulase is bound to the bacterial cell wall and reacts with fibrinogen, causing it to precipitate the bacterial cell wall. This reaction can be visualized as clumping when bacterial cells are mixed with plasma. Bound coagulase + fibrinogen → fibrin clot Interpretation: Positive: Macroscopic clumping in ≤ 10 seconds in coagulated plasma drop Negative: No clumping (Note: must confirm negative test for bound coagulase with a tube test for free coagulase) Free coagulase is an extracellular enzyme that causes clot formation when S. aureus colonies are incubated with plasma. Free coagulase first binds with a fibrinogen precursor, prothombin, to activate a plasma coagulase-reacting factor (CRF), which is a modified thrombin molecule, to form a CRF–coagulase complex. This complex in turn reacts with fibrinogen to produce a fibrin clot. Prothrombin + free coagulase → CRF CRF + fibrinogen → clot
  • 86. Free and bound coagulase may act to coat the bacterial cells with fibrin, rendering them resistant to opsonization and phagocytosis. Positive: Staphylococcus aureus. Negative: Other species of Staphylococcus (e.g. S. epidermidis)CTA sugars Cysteine trypticase agar (CTA) sugars are used for differentiation of Nesisseria species by observing the production of acid from metabolism of carbohydrates in the CTA. CTA supports the growth of the fastidious Neisseria species. The sugars (glucose, maltose, fructose and lactose) are added to yield a 1% concentration in the CTA medium. Following inoculation of organism to the CTA medium and subsequent incubation in a non-CO2 incubator, the CTA is examined for the production of acid in the top portion of the tube. Interpretation: Positive: Yellow in the top of the tube, indicating that acid has been produced Negative: No colour change in the medium. Examples: Positive for glucose only = Neisseria gonorrhoeae Positive for glucose and maltose = Neisseria meningitidis Postive for glucose, maltose and lactose = Neisseria lactamica Positve (+/−) for glucose, maltose, lactose and sucrose = Neisseria sicca Negative for all four = Moraxella catarrhalis, NeisseriaflavescensDecarboxylase tests The test measures the enzymatic ability of the organism to decarboxylate or hydrolyse an amino acid to form an amine. Decarboxylase media contains glucose, bromcresol purple, a nitrogen source, cresol red indicator, the enzyme activator pyridoxal and the amino acid to be tested. Decarboxylation results in an alkaline pH change, causing a shift in the pH indicators (bromocresol purple and cresol red) to dark purple (colour of the control is pale purple). The amino acids tested are lysine, ornithine and arginine. Lysine is decarboxylated to cadaverine; ornithine is decarboxylated to putrescine and arginine undergoes a dihydrolase reaction to form citrulline, which is decarboxylated to ornithine. For each amino acid to be tested, it is necessary to inoculate both a tube with basal medium (control to ensure that the organism does not form alkaline end products in the absence of the amine end product) and one with basal medium plus the amino acid to be tested. An uninoculated control is compared to any positive tubes. Interpretation: Positive: Blue-purple colour compared to the uninoculated control (pale purple). Negative: No colour change or yellow in both the test and inoculated control tubes. Glucose is fermented during the early part of the decarboxylation process, leading to a yellow colour. As the pH rises, an optimal environment for decarboxylation occurs and decarboxylation of the amino acid leads to an increase in pH and a purple colour. If a test result is yellow, it should be reincubated for up to 4 days. At the end of the incubation, the acid from glucose fermentation would not mask the alkaline colour change resulting from a positive decarboxylation reaction. (cont.)
  • 87. Appendix 2.2. (cont.)Test Principle/major use/examples of key positive and negative organisms when applicable Uninoculated control: pale purple Inoculated control: yellow if the organism is a glucose fermenter. Examples: Arginine Positive: Enterobacter cloacae Negative: Klebsiella pneumoniae Lysine: Positive: Klebsiella pneumoniae Negative: Enterobacter cloacae Ornithine: Positive: Enterobacter cloacae Negative: Klebsiella pneumoniaeDNA hydrolysis The test determines if an organism has the DNAse enzyme and is therefore able to hydrolyse DNA. Bacteria are streaked onto a medium containing a DNA–methyl green complex. When DNA is hydrolysed, methyl green is released and combines with the highly polymerized DNA at pH of 7.5, turning the medium colourless around the test organism. If DNA is not degraded, the medium remains green. Interpretation: Positive: Medium is colourless around the organism. Negative: Medium remains green. Note: If toluidene blue is used instead of methyl green, it complexes with polymerized DNA (control) and a royal blue colour results. Where DNA is hydrolysed by the toluidene blue, the dye complexes with the oligo- and mononucleotides resulting in a change in dye structure and absorption spectrum, yielding a bright pink colour. Interpretation: Positive: Medium turns pink. Negative: Medium remains blue. Examples: Positive: Staphylococcus aureus; Serratia marcescens Negative: Staphylococcus epidermidis
  • 88. Flagella stain (RYU) The stain is used to determine the arrangement of flagella for motile bacteria. A wet-mount of organism is stained with RYU flagella stain and the motile bacteria are evaluated for: (1) presence or absence of flagella; (2) number of flagella per cell; (3) location of flagella as either peritrichous, lophotrichous or polar; (4) amplitude of wavelength (short or long) and (5) whether or not tufted. Interpretation: Positive: Flagella are present and can be described. Negative: Organism is non-motile; no flagella present Examples: Positive: Peritrichous: Escherichia coli Polar: Pseudomonas aeruginosa Negative: Klebisella pneumoniaeGelatin hydrolysis The test determines the ability of an organism to produce proteolytic enzymes (gelatinases) that liquefy gelatin. An inoculated tube along with an uninoculated control is incubated for growth, then removed, placed at 4 ◦ C and subsequently observed for liquefaction. Interpretation: Positive: Partial or total liquefaction of the inoculated tube; note: control tube must be completely solidified. Negative: Complete solidification of the tube at 4 ◦ C. Examples: Positive: Proteus vulgaris Negative: Enterobacter aerogenesGrowth at 42 ◦ C Determines an organism’s ability to grow at 42 ◦ C. Following inoculation and incubation of two trypticase soy agar tubes, one tube is incubated at 35 ◦ C and the other is incubated at 42 ◦ C. The presence of growth is recorded for each tube after 18–24 hours. Positive: Good growth at both at 35 ◦ C and 42 ◦ C. Negative: Good growth at 35 ◦ C but not at 42 ◦ C. Examples: Positive: Pseudomonas aeruginosa Negative: Pseudomonas fluorescens (cont.)
  • 89. Appendix 2.2. (cont.)Test Principle/major use/examples of key positive and negative organisms when applicableHemolysis patterns Determines if bacteria produce extracellular enzymes that lyse red blood cells in agar (hemolysis). This can result in complete clearing of the erythrocytes (beta hemolysis) or only a partial clearing ( -hemolysis) around the bacterial colony. Interpretation: Positive for -hemolysis: Complete lysis of red blood cells seen as clearing around the bacterial colony. Positive for -hemolysis: Partial lysis of red blood cells seen as green discoloration around the bacterial colony. Negative for hemolysis: No effect on red blood cells and no halo around the colony. This is referred to as non-hemolysis or -hemolysis. Examples: Positive for -hemolysis: Group A streptococcus Positive for -hemolysis: Streptococcus pneumoniae and other alpha streptococci Gamma or non-hemolytic: Group D streptococci and Enterococcus speciesHippurate hydrolysis Tests for the presence of the constitutive enzyme, hippuricase, which hydrolyses the substrate hippurate to produce the amino acid, glycine. Glycine is detected by oxidation with ninhydrin reagent, resulting in a deep purple colour. Ninhydrin is added to hippurate that has been inoculated with colonies of the organism and observed for a colour change. Sodium hippurate → sodium benzoate + glycine Glycine + ninhydrin reagent → purple colour Interpretation: Positive: Deep purple colour. Negative: Light purple or no colour change. Examples: Positive: Streptococcus agalactiae, Campylobacter jejuni, Listeria monocytogenes Negative: Streptococcus pyogenes; other streptococci
  • 90. Hydrogen sulfide This test detects hydrogen sulfide that is produced from the degradation of sulfur-containing amino acids (in peptone). When H2 Sproduction combines with a heavy metal such as iron or lead in the medium, ferrous sulfide, a black precipitate is produced. Interpretation: Cysteine or methionine → pyruvic acid + H2 S +ammonia Positive: Black H2 S + Fe(NH4 )2 (SO4 )2 − − − − − − − → FeS(black precipitate) −−−−−−− Desulfurase enzyme Negative: No colour change in medium. Examples: Positive: Salmonella typhi, Edwardsiella spp. Negative: Shigella spp.Indole production Determines if an organism produces the enzyme tryptophanase and is therefore able to split the amino acid tryptophan to form the compound indole. The test is used to presumptively identify Escherichia coli, the gram-negative bacillus most commonly encountered in diagnostic microbiology. A suspension of the organism is added to a trytophan broth and incubated. Following incubation either Kovak’s reagent (for Enterobacteriaceae) or Ehrlich’s reagent after xylene extraction (for other gram-negative bacilli) is added and the mixture is observed for the presence of a red ring in the upper layer of the aqueous mixture. Tryptone- → tryptophan → indole + pyruvic acid + NH3 Interpretation: Positive: Red ring at the interface. Negative: No colour reaction. Variable: Orange colour (indicates production of skatole, a methylated intermediate that may be a precursor to indole production). Examples: Positive: Escherichia coli Negative: Klebsiella pneumoniaeLitmus milk Determines an organism’s ability to metabolize litmus milk. Lactose fermentation is noted by the litmus turning pink from acid production. If sufficient acid is produced, casein in the milk will coagulate and solidify the milk. Some organisms will shrink the curd and form whey at the surface; other organisms hydrolyse the casein, producing a straw-coloured turbid product. Some organisms reduce litmus, making the medium colourless at the bottom of the tube. Following incubation of the organism in litmus milk, the product is observed for 7 days and all changes recorded. Multiple changes may occur during the observation period. (cont.)
  • 91. Appendix 2.2. (cont.)Test Principle/major use/examples of key positive and negative organisms when applicable Interpretation: Positive reactions for litmus indicator: Alkaline reaction: Litmus turns blue. Acid reaction: Litmus turns pink. Negative reaction for litmus indicator: Purple: Identical to uninoculated control Positive reactions for milk appearance: Coagulation (occurs in acid or alkaline conditions): clot Dissolution of clot: clear, greyish, watery fluid with a shrunken, insoluble pink clot (in acid environment; record as digestion). Dissolution of clot: clear, greyish, watery fluid with a shrunken, insoluble blue clot (in alkaline environment; record as peptonization). Examples: Alkaline: Alcaligenes faecalis Acid: Enterococcus faecium Peptonization: Burkholderia cepaciaLysostaphin Determines if an organism is susceptible to lysostaphin. Lysostaphin is an endopeptidase that cleaves the glycine-rich pentapeptide cross-bridges in the staphylococcal cell wall peptidoglycan, rendering the cells susceptible to osmotic lysis. Following the addition of a commercial lysostaphin to a suspension of organisms and subsequent incubation, the tube is observed for clearing of the suspension. Interpretation: Positive: Clearing of the suspension; susceptibility to lysostaphin. Negative: No clearing of the suspension; resistance to lysostaphin. Examples: Positive (Susceptible): Micrococcus spp. Negative (Resistant): Staphylococcus spp.Methyl red/Voges– Determines ability of an organism to produce and maintain stable acid end products from glucose fermentation and the ability of someProskauer (MRVP ) tests organisms to produce neutral end products (acetylmethylcarbinol or acetoin) from glucose fermentation. Following inoculation of MR–VP medium and subsequent incubation, the sample is split and one tube is used to perform the MR test and the other to perform the VP test. Methyl red is added to the MR tube and observed for a colour change to red; Barritt’s reagents A (alpha-naphthol) and B (KOH) are added to the VP tube, shaken and observed for a colour change to red. Mixed acids + methyl red → red colour
  • 92. MR Intepretation: Positive: Bright red colour (indicative of mixed acid fermentation). Weakly positive: Red-orange colour. Positive: Escherichia coli Negative: Enterobacter cloacae Voges–Proskauer-glucose→ 2, 3 butylene glycol + acetoin → red colour VP interpretation: Positive: Red colour indicative of acetoin production. Negative: Yellow colour. Delayed reaction: Orange colour at surface; reincubate. Positive: Enterobacter cloacae Negative: Escherichia coliMotility Determines motility of bacteria. A bacterial colony is inoculated (using a needle to stab a straight line to the bottom to the agar) to a medium containing a small amount of agar and 1% triphenyltetrazolium chloride, a colourless dye that bacteria incorporate and reduce to a red pigment. Motile bacteria move away from the line of the inoculum, and non-motile bacteria grow only along the line of the inoculation. Interpretation: Positive: Motile; diffuse growth extending laterally from the line of inoculation. Negative: Non-motile; growth only along the line of inoculation. Examples: Positive: Escherichia coli Negative: Klebsiella pneumoniaeNitrate reduction The test is used to determine an organism’s ability to reduce nitrate. Reduction of nitrate to nitrite is determined by adding sulfanilic acid and alpha-naphthylamine. Sulfanic acid and nitrite react to form a diazonium salt that couples with the alpha-naphthylamine to produce a red, azo dye. Nitrate broth with a Durham tube (inverted small tube at bottom of large tube to detect gas) is inoculated with organism and incubated. Following incubation, the sample is observed for the presence of bubbles in the Durham tube, indicating gas production, and Reagent solution A (sulfanilic acid) and Reagent Solution B (alpha-naphthylamine) are added and observed for the development of a red colour, indicating the presence of nitrite. If no colour develops, zinc powder is then mixed with the broth to which Solutions A and B have (cont.)
  • 93. Appendix 2.2. (cont.)Test Principle/major use/examples of key positive and negative organisms when applicable already been added. If a red colour then develops, this is due to zinc catalysing the conversion of nitrate to nitrite, confirming that nitrate was still present and had not been reduced to nitrite. NO3 + 2e + 2H → NO2 + H2 O → N2 nitrate nitrite nitrogen gas Interpretation: Positive: Nitrate to nitrite: Red colour when Reagents A and B are added; no gas. Positive: Nitrate to nitrite and nitrite to gas: Red colour when Reagents A and B are added plus gas. Positive: Nitrate to nitrite and nitrite to nitrogen gas: No red colour when Reagents A and B are added; gas present (nitrate has been reduced to nitrite and nitrite to nitrogen gas). Negative: Nitrate not reduced: No red colour when Reagents A and B are added; no gas. Negative confirmation: Nitrate not reduced: No red colour when Reagents A and B are added, but red colour when zinc is added to catalyse the conversion of nitrate to nitrite. Examples: Positive: NO3 + (gas): Pseudomonas aeruginosa Positive: NO3 + (no gas): Escherichia coli Negative: NO3 − (no gas): Acinetobacter spp.Novobiocin susceptibility Determines an organism’s susceptibility to novobiocin. This test is used to differentiate Staphylococcus saprophyticus from other coagulase-negative species of staphylococci. A standard quantity of a bacterial suspension is streaked on sheep blood agar in three directions to produce a lawn of growth. A 5 g novobiocin disc is placed in the centre of the streaked plate. Following incubation and bacterial growth, zones of inhibition are measured around the novobiocin disc to determine susceptibility. Interpretation: Positive: Zone of inhibition around the disc that is > 16 mm. Negative: No zone of inhibition or zone of inhibition ≤ 16 mm around the disc. Examples: Positive: Staphylococcus saprophyticus Negative: Other coagulase-negative staphylococci
  • 94. ONPG The ONPG (o-nitrophenyl- -D-galactopyranoside) test is used to determine the ability of an organism to produce the enzyme, -galactosidase, which hydrolyses the substrate ONPG to form orthonitrophenol, which is a visible, yellow product. ONPG is a rapid test for the detection of -galactosidase. The test is used primarily to determine if an organism is a slow lactose-fermenter or a non-lactose fermenter. All lactose fermenters (rapid and slow) produce -galactosidase, but rapid fermenters also produce the enzyme permease that transports the lactose across the bacterial cell membrane. If permease is not produced, lactose must diffuse into the cell, making fermentation of lactose a much slower process. Beta-galactosidase, if present, acts on the substrate ONPG in the same way the enzyme hydrolyses lactose to form galactose and glucose; the end product of ONPG hydrolysis is a visible (yellow) compound, orthonitrophenol. Interpretation: Positive: Yellow. Negative: Colourless. Examples: Positive (rapid lactose-fermenter (with permease): Escherichia coli Negative (non-lactose fermenter ( -galactosidase not produced): Salmonella typhimuriumOptochin This test is used to determine the effect of optochin (ethylhydrocurpreine hydrochloride) on an organism. Optochin lyses pneumococci, but other alpha-streptococci are resistant. Lysis is indicated by a zone of inhibition around an optochin (P) disc. Colonies suspected of being Streptococcus pneumococci are streaked on sheep blood agar in three directions to produce a lawn of growth. An optochin (P) disc is placed in the centre of the streaked plate. Following incubation in CO2 and bacterial growth, zones of inhibition are measured around the optochin disc to determine susceptibility. Interpretation: Positive: Inhibition indicated by a zone of hemolysis ≥14 mm in diameter for 10 g P disc and ≥ 10 mm in diameter for 6 g P disc. Negative: No inhibition zones or zones < those listed as positive. Examples: Positive: Streptococcus pneumoniae. Negative: Other alpha-hemolytic streptococci.Oxidase (Kovac’s method) The test is initially used for differentiating between groups of gram-negative bacteria. The test detects the presence of the enzyme cytochrome oxidase. Cytochrome oxidase reacts with oxygen (terminal electron acceptor) in the process of oxidative phosphorylation in aerobic bacteria. Cytochrome oxidase is detected by using reagents that are normally colourless but become coloured when oxidized. It also is used as a key reaction for the identification of Neisseria species. The test detects the presence of the bacterial enzyme cytochrome oxidase using the substrate is 1% N,N,N ,N -tetra-methyl-p-phenylenediamine dihydrochloride to indophenol, a dark, purple-coloured end product. A drop of oxidase (1% tetraethyl-p-phenylenediamine dihydrochloride in dimethyl sulfoxide) is flooded onto bacterial colonies and observed for a colour change. Alternatively, colonies can be placed onto a filter paper impregnated with the reagent and observed for a colour change. (cont.)
  • 95. Appendix 2.2. (cont.)Test Principle/major use/examples of key positive and negative organisms when applicable Interpretation: Positive: Dark purple colour indicating the presence of oxidase. Negative: No colour development, indicating the absence of the enzyme. Examples: Positive: Pseudomonas spp, Aeromonas spp., Campylobacter spp., Neisseria spp. Negative: Enterobacteriaceae, Stenotrophomonas maltophilia, Acinetobacter spp.Modified oxidase A drop of modified oxidase (6% tetraethyl-p-phenylenediamine dihydrochloride in dimethyl sulfoxide) is added to a colony of organism that has been placed on filter paper and observed for a colour change. Interpretation: Positive: If oxidase is present the colourless reagent will be oxidized and will turn purple or blue black. Negative: No colour change. Examples: Positive: Micrococcus spp. Negative: Staphylococcus spp.Oxidation-fermentation Oxidation–fermentation determines the oxidative or fermentative metabolic capabilities of an organism. The test determines whether an(OF) organism uses carbohydrate substrates to produce acid byproducts. Typically, non-fermentative bacteria are tested for their ability to produce acid from glucose, lactose, maltose, sucrose, xylose and mannitol. A tube of each type of sugar is inoculated with organism along with a control tube containing OF base, but no carbohydrates. The OF medium contains an indicator, 0.2% peptone and 1% of the carbohydrate to facilitate the oxidative use of carbohydrates by non-fermenting, gram-negative bacilli. The carbohydrate is in the deep portion of the tube and the amines are near the top. OF glucose is used to determine if an organism ferments or oxidizes glucose or if it is a non-glucose utilizer. Two tubes of OF glucose and one OF basal medium are inoculated. One tube is overlaid with mineral oil to create anaerobic conditions. Following incubation all tubes are observed for changes in colour. Interpretation: Positive for fermentation: Yellow throughout the medium in both tubes due to acid production from the fermentation of glucose under anaerobic conditions. Positive for oxidation: The tube exposed to oxygen is yellow at the top, indicating oxidative glucose utilization; the tube under anaerobic conditions will remain green in colour.
  • 96. Negative for glucose utilization. Neither tube shows a colour change in the presence of bacterial growth, indicating that the organism is not breaking down glucose, but instead is utilizing the amines in the top of the tube. Examples: Fermentative: Enterobacteriaceae Oxidative: Pseudomonas aeruginosa Non-glucose utilizers: Alcaligenes faecalisPhenylalanine deaminase Tests for the organism’s ability to oxidatively deaminate phenylalanine to phenylpyruvic acid. Phenylalanine → phenylpyruvic acid + FeCl3 → green end product Ferric chloride is added to a phenylalanine slant that has been incubated with organism and observed for a green colour. Interpretation: Positive: Green colour develops after ferric chloride is added. Negative: Slant remains original colour after addition of ferric chloride. Examples: Positive: Proteus vulgaris Negative: Escherichia coliPYR hydrolysis Detection of the enzyme pyrrolidonyl arylamidase is useful in differentiating streptococci and enterococci. The enzyme L-pyrroglutamyl-aminopeptidase hydrolyses the substrate L-pyrrolidonyl- -naphthylamide (PYR) to produce -naphthylamine. Broth containing substrate L-pyrrolidonyl- -naphthylamide (PYR) is inoculated with the organism and incubated. During the incubation PYR is hydrolysed to produce free -naphthylamine which is then detected by addition of a diazo dye coupler, N,N-dimethylaminocinnamaldehyde, producing a colour change to red. Interpretation: Positive: Red colour. Negative: Slight orange colour or no colour. Examples: Positive: Streptococcus pyogenes, Enterococcus spp. Negative: other streptococciPyruvate broth The test determines if an organism is able to utilize pyruvate. The test aids in the differentiation of Enterococcus faecalis and Enterococcus faecium. Pyruvate broth with indicator is inoculated with organism and incubated to allow for organism growth. Following incubation, the tube is observed for a change in colour. (cont.)
  • 97. Appendix 2.2. (cont.)Test Principle/major use/examples of key positive and negative organisms when applicable Interpretation: Positive: Yellow. Negative: Medium remains green in colour or turns yellow-green (a weak reaction that is interpreted as negative). Examples: Positive: Enterococcus faecalis Negative: Enterococcus faeciumSalt tolerance (growth in Determines the ability of an organism to grow in high concentrations of salt. The test is used to differentiate enterococci from6.5% NaCl) non-enterococci. Organisms are incubated in a tube containing 6.5% NaCl and in a control tube containing broth without salt. Following incubation tubes are examined for growth. Interpretation: Positive: Growth is equivalent in both the control tube and the tube containing 6.5% NaCl. Negative: Growth in the control, but scant or no growth in the tube containing 6.5% NaCl. Examples: Positive: Enterococci Negative: Non-enterococciSXT (sulphamethoxazole- Determines an organism’s ability to tolerate SXT (1.25 g). A standard quantity of a bacterial suspension is placed on sheep blood agar, andtrimethoprim) resistance streaked in three directions to produce a lawn of growth. A SXT disc is placed in the centre of the streaked plate. Following incubation and bacterial growth, zones of inhibition are measured around the SXT disc to determine susceptibility. Interpretation: Positive: Zone of inhibition of any size indicates susceptibility. Negative: No zone of inhibition around the disc. Examples: Positive with zone of inhibition of any size: Typical for beta-hemolytic non-Group A, non-Group B streptococci. (Groups C, G, and F) Negative: Beta-hemolytic streptococci Group A and Group B
  • 98. Triple sugar iron (TSI ) TSI is used to differentiate gram-negative bacilli. The test detects the ability of these organisms to utilize glucose, lactose and sucrose fermentatively and also detects the organism’s ability to form hydrogen sulfide. TSI contains 1% lactose and sucrose, 0.1% glucose, peptone, phenol red as an indicator of acidification and ferrous sulfate as an indicator of hydrogen sulfide production. The test detects: (1) the inability of the organism to ferment any of the sugars, using peptone for energy instead; (2) fermentation of glucose only; (3) fermentation of lactose and/or sucrose in addition to glucose; (4) production of gas; and (5) production of hydrogen sulfide. The TSI butt and slant are inoculated with organism and incubated. When glucose is fermented, the entire medium becomes yellow (acidic) within 8–12 hours of incubation. The butt of the tube will remain yellow after 24 hours incubation due to the organic acids building up for the anaerobic fermentation of glucose, but the slant will become red or alkaline from oxidation of the fermentation products under aerobic conditions and the oxidation of peptones in the medium. If lactose and/or sucrose are fermented, the large amount of fermentation products (10× amount of lactose/sucrose than glucose in the medium) formed on the slant will more than neutralize the alkaline amines and the slant will become acid or yellow at 18–24 hours. The formation of CO2 and H2 (hydrogen gas) is indicated by bubbles or cracks in the agar. The production of H2 S requires an acidic environment and is demonstrated by blackening of the butt of the medium. Interpretation: Amines are utilized; the organism is a non-utilizer of carbohydrates. The butt and slant are red at 18–24 hours. Glucose is fermented: Yellow slant and yellow butt at 8–12 hours; red slant and yellow butt at 18–24 hours. Glucose, lactose and/or sucrose fermentation: The butt and the slant are yellow at 24 hours. Black precipitate in butt: Ferrous sulfide and hydrogen sulfide gas are produced. Bubbles or cracks: CO2 or H2 is produced. Examples: Acid/Acid with gas = Escherichia coli Alkaline/Acid with hydrogen sulfide production = Salmonella typhi Alkaline/no change in butt = Pseudomonas aeruginosaUrea hydrolysis The test determines the organism’s ability to produce the enzyme urease, which hydrolyses urea to produce CO2 and ammonia. NH3 + H2 O NH3 + CO2 + H2 O (NH4 )2 CO3 Organisms are inoculated into urea broth containing a phenol red indicator and incubated. Following incubation the medium is observed for a change in colour from yellow to bright pink (magenta) when the alkaline ammonia is produced from the breakdown of urea. (cont.)
  • 99. Appendix 2.2. (cont.)Test Principle/major use/examples of key positive and negative organisms when applicable Interpretation: Positive: Change in colour from light orange to magenta. Negative: No change in colour. Examples: Positive: Proteus vulgaris Negative: Escherichia coliX and V factors X and V factors are used to speciate Haemophilus. X factor (hemin) is derived from the digestion or degradation of blood. V factor (NAD) is obtained from yeast or potato extract and is produced by some bacteria (Staphylococcus aureus). These factors are required either singly or in combination by various species of Haemophilus. X and V factors are impregnated on filter strips that can be placed on a nutrient-poor medium along with the organism to be speciated. Following incubation, the plates are observed for growth of organism around the factors. Interpretation: X factor is required: Growth around the X factor disc and around the XV factor disc; no growth around V factor disc. V factor is required: Growth around V factor disc and around XV factor disc; no growth around X factor disc. X and V are required: Growth around the XV factor disc, but not around the X factor disc nor the V factor disc. Examples: Requires X factor only: Haemophilus ducreyi Requires V factor only: Haemophilus parainfluenzae Requires both X and V factors: Haemophilus influenzae
  • 100. Appendix 2.3. antibacterial agentsClass Agents Mode of action Spectrum of activity Limitations/Comments Cell wall synthesis inhibitorsBeta-lactams Penicillins Peptidoglycan cannot be produced, r Gram-positive bacteria Advantages: Penicillin inhibiting cell wall synthesis r Gram-negative bacteria r Broad spectrum antibiotics; Ampicillin most bacteria have a cell wall and are Piperacillin therefore susceptible Mezlocillin r Penicillins are inexpensive Cephalosporins Disadvantages: Cefazollin r Many organisms produce penicillinase Cefuroxime r Many mutant organisms are resistant to Cefotexan penicillins Cefotaxime Ceftriaxone Ceftrazidime Cefepime Carbapenems Imipenem Meropenem Monolactams AztreonamGlycopeptides Vancomycin Prevents incorporation of r Gram-positive bacteria only precursors into growing cellBacitracin Inhibits recycling of metabolites r External use only, due to toxicity needed for synthesis of cell wall peptidoglycan (cont.)
  • 101. Appendix 2.3. (cont.)Class Agents Mode of action Spectrum of activity Limitations/Comments Cell membrane function inhibitorsPolymyxins Polymyxin B Cell membrane disruption r Gram-negative bacteria r Ineffective against most gram-positive Colistin bacteria r Toxicity is a concern since human cell membranes may be affected Protein synthesis inhibitorsAminoglycosides Gentamicin Bind to ribosomal 30S subunit and r Gram-positive bacteria r Not effective against anaerobes Kanamycin inhibit protein synthesis r Gram-negative bacteria r Used in conjunction with beta-lactams for Streptomycin (bacteriocidal) maximum killing Tobramycin Amikacin NetilmicinTetracyclines Paromomycin* Bind (doxycycline) to ribosomal r Gram-positive bacteria 30S subunit and inhibits protein r Gram-negative bacteria synthesis (bacteriostatic) r Intracellular pathogens (e.g. chlamydia, rickettsia and rickettsia-like organisms)Chloramphenicol Binds to ribosomal 50S subunit and r Gram-positive bacteria r Toxicity is a concern inhibits protein synthesis r Gram-negative bacteria (bacteriocidal)Macrolides- Macrolides Bind to ribosomal 50S subunit and r Most gram-positive bacteriaLincosamides Erythromycin inhibit protein synthesis r Some gram-negative bacteria Azithromycin Clarithromycin Lincosamide Clindamycin
  • 102. Nitrofuran Furazolidone Binds to 30S subunit and block r GI pathogens translation Salmonella Shigella Proteus Aerobacter aerogenes Vibrio cholerae Giardia lambliaStreptogramins Quinupristin/ Binds to ribosomal 50S subunit at r Gram-positive bacteria dalfopristin two different sites, inhibiting protein synthesisOxazolidinones Linezolid Binds to ribosomal 50S subunit and r Gram-positive bacteria r Effective against organisms resistant to interferes with initiation of protein other agents synthesis Inhibitors of DNA and RNA synthesisRifampicin Binds to DNA- dependent RNA r Gram-positive bacteria r Used only in combination with other polymerase and inhibits synthesis r Gram-negative bacteria drugs because resistance can develop of RNA (certain organisms) rapidly r Used in treatment of tuberculosis and meningitis due to Neisseria meningitidisFluoroquinolones Ciprofloxacin Bind to DNA gyrases to inhibit DNA r Gram-positive bacteria r Broad-spectrum but spectrum may vary Norfloxacin synthesis r Gram-negative bacteria with individual antibiotics OfloxacinMetronidazole Disrupts DNA; exact mechanism r Gram-negative bacteria r Most effective against anaerobes; not known r Gram-positive bacteria activation requires low redox potential (certain genera only) r Anaerobes (mainly gram-negative) (cont.)
  • 103. Appendix 2.3. (cont.)Class Agents Mode of action Spectrum of activity Limitations/Comments Inhibitors of metabolic processesNitrofurantoin May directly damage DNA; exact r Gram-positive bacteria r Used only to treat urinary tract infections mechanism not known r Gram-negative bacteriaSulfonamides Interfere with folic acid production r Gram-positive bacteria r Not effective against Pseudomonas r Many gram-negative bacteria aeruginosaTrimethoprim Interferes with folic acid r Gram-positive bacteria r Used in combination with sulfonamide production r Many gram-negative bacteria (e.g. sulfamethoxazole) to target two different sites∗ Paromomycin is an antiparasitic, not an antibacterial, target two sites
  • 104. Further reading 89FURTHER READING FOR ANTIBACTERIAL Kasten, M. J. (1999). Clindamycin, metronidazole, and chlo-AGENTS APPENDIX 2.3 ramphenicol. Mayo Clinic Proceedings, 74: 825– 33. of contents2.htm Marshall, W. F. & Blair, J. E. (1999). The cephalosporins. Mayo Clinical Proceedings, 74: 187–95. 202668.html Osmon, D. R. (2000). Antimicrobial prophylaxis in adults. Mayo Clinic Proceedings, 75: 98–109. Atovaquonecd.shtml Physician Desk Reference, 54th edn. (2000). Des Moines, Iowa: Medical Economics Co. parahd.htm Smilack, J. D. (1999). The tetracyclines. Mayo Clinic Proceedings,Alvarew-Elcoro, S. & Enzler, M. J. (1999). The macrolides: 74: 727–9. erythromycin, clarithromycin, and azithromycin. Mayo Virk, A. & Steckelberg, J. M. (2000). Clinical aspects of Clinic Proceedings, 74: 613–34. antimicrobial resistance. Mayo Clinic Proceedings, 75:Hellinger, W. C. & Brewer, N. S. (1999). Carbapenems and 200–14. monobactams: imipenem, meropenem, and aztreonam. Walker, R. C. (1999). The fluoroquinolones. Mayo Clinic Proceed- Mayo Clinic Proceedings, 74: 420–34. ings, 74: 1030–7.Henry, N. K., Hoecker, J. L. & Hable, R. K. (2000). Antimicro- Wilhelm, M. P., Estes, L. & Pharm, D. (1999). Vancomycin. Mayo bial therapy for infants and children: guidelines for the Clinic Proceedings, 74: 928–35. inpatient and outpatient practice of pediatric infectious dis- Wright, A. J. (1999). The penicillins. Mayo Clinic Proceedings, 74: eases. Mayo Clinic Proceedings, 75: 86–97. 290–370.
  • 105. 3 Mycology: moulds and yeasts Introduction harsh conditions. Because fungal cell walls and membranes differ from those of bacteria, they Medical mycology studies fungi that may produce are insensitive to antibiotics. Fungal infections disease in humans and other animals. Fungi are require treatment with antifungal agents, many not related to bacteria: bacteria are prokaryotes, of which are targeted toward sterol production. without a membrane-bound nucleus or intracel- Changes in the ergosterol composition of the mem- lular organelles; fungi are eukaryotes that have brane can lead to drug resistance. Since fungi are both sexual and asexual reproductive phases, and eukaryotic, side effects are common with many have membrane-bound organelles including nuclei, treatments that are targeted towards eukaryotic mitochondria, golgi apparatus, endoplasmic retic- cells. ulum, lysosomes, etc. Initially, fungi were thought Fungi can be unicellular or multicellular /filamen- to be part of the Kingdom of Plantae, albeit lower tous, bear conidia (spores) and usually reproduce by members of this Kingdom. When the Five Kingdom both asexual and sexual processes. Asexual repro- division of life forms came into general use, how- duction involves simple nuclear and cytoplasmic ever, the fungi were separated into their own King- division, and sexual reproduction involves the fusion dom, separate from the plants, due to their lack of nuclei from two cells to form a zygote. The pro- of chloroplasts or chlorophyll, the composition of cesses are not exclusive, and a fungus may reproduce their cell wall and their asexual reproduction by in either, or both, ways. means of spores. Whereas eukaryotes such as plants Fungi appear in two basic forms: yeasts and and algae contain chlorophyll that allows them to moulds. generate energy by photosynthesis (autotrophic), 1. Yeasts are unicellular (single vegetative cells) that fungi lack chlorophyll: they are heterotrophic, and generally form smooth colonies like bacteria. Like must absorb nutrients from their environment or bacteria, identification is based on macroscopic host. They can be saprophytes, living on dead and microscopic morphology and biochemical organic matter (e.g. mushrooms, toadstools, bread testing. Yeasts reproduce by simple budding to mould) or parasites utilizing living tissues (e.g. form blastoconidia. A few species, such as some yeast infections). Fungi are aerobic and non-motile; Saccharomyces used in wine-making and baking, they have rigid cell walls composed of complex reproduce by fission. polysaccharides such as chitins or glucans and 2. Moulds are multicellular, composed of a vege- their plasma membrane contains sterols, principally tative growth of filaments. Fungal filaments are ergosterol. They grow best at a neutral pH in a known as hyphae, and a mass of hyphae make up moist environment, but can tolerate a range of pH. the mycelium. ‘Hyphae’ and ‘mycelium’ are terms Fungal conidia (spores) can survive in dry and that are used interchangeably.90
  • 106. Classes of fungi 91Table 3.1. Mycology overview: moulds and yeastsZygomycota Ascomycota Basidiomycota DeuteromycotaMoulds Moulds Yeasts Moulds Moulds YeastsAbsidia Ajellomyces Saccharomyces Amanita Acremonium CandidaBasidiobolus (Blastomyces and Histoplasma (Cryptococcus Alternaria CryptococcusConidiobolus teleomorph stages) teleomorph stage) Aspergillus HansenulaCunninghamella Arthroderma Bipolaris MalasseziaMucor (Trichophyton and Microsporium Chrysosporium RhodotorulaRhizopus teleomorph stages) Filobasidiella Cladosporium TorulopsisSaksenaea *Coccidioides Trichosporon Pseudallescheria Curvularia Emericella Epidermophyton (Aspergillus teleomorph stage) Exophilia Eurotium Fonsecaea (Aspergillus teleomorph stage) Fusarium Neosartorya Paecilomyces (Aspergillus teleomorph stage) *Paracoccidioides Philophora Scedosporium Scopulariopsis *Sporothrix Wangiella* Thermally dimorphic. Dimorphic pathogenic fungi express one distinct the ART laboratory and may cause disease in indi-form in tissue (the parasitic yeast form) and another viduals with a compromised immune system.form (saprobic or mould) when grown in the envi-ronment or in the laboratory on artificial mediumunder appropriate conditions. Classes of fungi Fungal infections may be pathogenic for immuno-competent individuals, with immunocompromised The Kingdom of fungi is divided into five phyla, basedindividuals, post-surgery patients, patients under- on the method of spore production of the perfectgoing radiation therapy and chemotherapy and or sexual state (known as the teleomorphic state).those receiving corticosteroids at much greater risk Of these five phyla, only four are known to containfor infection. In medical mycology both laboratory human pathogens outlined in Table 3.1: Zygomycota,and clinical classification must be considered. Lab- Ascomycota, Basidiomycota and ‘Fungi Imperfecti’, ororatory classification is based on taxonomy and Deuteromycota. The fifth phylum is the Mycophyco-organism characteristics. Four clinical classifica- phyta, or Lichens, which represent a symbiosis of twotions are based on type of infection and body sites organisms – a fungus and an algae. Mycologists nowinvolved, and both yeasts and moulds are included suggest eliminating the Lichens as a Phylum, andin these categories. Medically important organisms instead reclassifying each individual lichen accord-may include those that are usually encountered as ing to its fungal component.contaminants, as well as those that are associated The majority of clinically significant fungi arewith disease. They are a source of contamination in members of Deuteromycota, but members of
  • 107. 92 Mycology: moulds and yeasts Zygomycota, Ascomycota and Basidiomycota also Deuteromycotia may cause infection. Deuteromycotia or Fungi Imperfecti (also known Zygomycetes as Hyphomycetes or conidial moulds) contain the largest number of organisms (17 000 species) that Bread or pin moulds belong to the Zygomycetes. are responsible for cutaneous, subcutaneous and They are rapidly growing, normally found in soil and systemic mycoses. They have septate hyphae and decaying vegetable matter and may be pathogens reproduce asexually via conidia on hyphae (conidio- in immunocompromised patients. Their hyphae are phores) or from spore-bearing (conidiogenous) cells. aseptate or sparsely septate, producing profuse grey Sexual spore production has not yet been observed to white aerial mycelia. They reproduce asexually, for this group. but compatible mating strains can reproduce sex- ually to produce zygospores. Clinically important zygomycetes include those that can produce sub- Laboratory classification of fungi cutaneous and systemic infections, such as Mucor, Rhizopus, Absidia and rarely other species. Taxonomic classification Taxonomically the four divisions: Zygomycetes, Ascomycetes Ascomycetes, Basidiomycetes and Fungi Imperfecti are distinguished on the basis of: (a) type of colony Ascomycetes are mostly terrestial saprophytes or produced; (b) type of mycelia present; (c) reproduc- parasites of plants: there are 28 650 known species. tion and characteristic spores (conidia) and (d) rate They have septate hyphae, can reproduce asexually of growth. via conidiospores, fission, or fragmentation or sex- ually via ascospores produced in sac-like structures Type of colonies known as asci. Asci are frequently located in a fruiting body, or ascocarp. Examples include the dermato- Type of colonies produced can be described in terms phyte Trichophyton spp., Pseudallescheria boydii, of texture, topography, and colour: and the yeast that is such a well-known favourite to Texture describes the height of aerial hyphae, which cell biologists, molecular biologists and biochemists: can be cottony/woolly, velvety, granular/powdery, Saccharomyces cerevisiae. or glabrous/waxy. Topography describes hills and valleys seen on fungal cultures; these are often masked by aerial hyphae, Basidiomycetes but can be seen on the reverse side of the colony. Mushrooms and toadstools belong to the Basidi- They can be flat, rugose (deep radiating furrows), omycetes. There are 16 000 species, saprophytic or umbonate (button-like) or verrucose (wrinkled). parasitic, especially of plants. Their hyphae are sep- Colonies have a wide variety of colours, and their tate and they reproduce sexually via basidiospores. front and reverse sides may be of different colours. Occasionally they reproduce asexually via budding, Dematiaceous fungi produce melanin pigment in the conidia or mycelial fragmentation. They are rarely vegetative mycelium, with dark hyphal elements isolated in clinical labs, but the pathogenic species and a dark colour on the reverse side of the cul- Filobasidiella neoformans is the sexual form of ture plate. Hyaline moulds are non-pigmented, the basidiomycete yeast Cryptococcus neoformans, producing white or colourless vegetative mycelia a major threat as an opportunistic infection in seen on the reverse side of clear agar culture immunocompromised patients, especially patients medium. These may, however, have highly pig- with HIV or AIDS. mented surface fruiting structures (spores).
  • 108. Laboratory classification of fungi 93Type of mycelia (i) holoblastic: all layers of the parent cell are involved in developing daughter conidia (e.g.Hyphae may intertwine to form a mycelium. In penicillium),culture, aerial hyphae extend above the medium (ii) enteroblastic: only the inner cell wall layers areand account for the macroscopic appearance of included (e.g. philoconidia).the colony. There are two kinds of hyphae: sep- During thalic division, the septum forms first, and atate hyphae have frequent cross-walls, and non- growing point ahead of it becomes a daughter cellseptate (coenocytic) or sparsely septate hyphae have (e.g. budding yeasts). This may be:a few irregularly spaced cross-walls. The septae (i) holothalic, with the formation of microconidiadivide hyphae into compartments, but not into and macroconidia, orcells. Hyphae may be light or dark. Hyaline hyphae (ii) arthric, where daughter cells fragment withinare clear or non-pigmented whereas dematiaceous the hyphal stand before dispersing into arthro-ones have melanin in their cell walls, and produce conidia. Arthric may be holoblastic (e.g. Geo-highly pigmented dark brown, green-black, or black trichum) or enteroblastic (e.g. Coccidioidescolonies. This pigmentation refers to the vegetative immitis).mycelium, and not the asexual fruiting structures. Specialized fruiting bodies (e.g. sporangia) may alsoBoth hyaline and dematiaceous moulds may pro- be involved.duce pigmented spores, but dematiaceous moulds The different types of conidia formed arealone produce melanin pigment in the hyphae. Veg- described by their shape and the manner in whichetative hyphae extend downwards into the medium they are formed.and absorb nutrients, and can form specialized (i) Blastoconidia are produced from buddingstructures of different shapes, important for organ- as in the case of yeasts or moulds such asism identification: antler, racquet, spiral, nodular, or Cladosporium.rhizoid (root-like). (ii) Poroconidia are formed when the daughter Aerial hyphae can be seen macroscopically cells push through a minute pore in the par-extending above the surface of the colony, and these ent cell (e.g. Dreschlera). The parent may be amay support structures involved in both asexual specialized conidiogenous cell or a long stalk,and sexual reproduction. Asexual reproduction is a conidiophore (e.g. Alternaria).nuclear, and sexual reproduction and cytoplasmic (iii) Philoconidia are elicited from a tube-likedivision occurs when two nuclei of closely related structure called a phialide (e.g. Penicillium).compatible strains fuse to form a zygote. (iv) Annelloconidia are grown inside a vase- shaped conidiogenous annelide (e.g. Scopular- iopsis).Reproduction and characteristic spores (conidia) (v) Macroconidia arise from conversion of an entire hyphal element into a multi-celled coni-Asexual reproduction dium (e.g. Microsporum canis). MacroconidiaDuring asexual reproduction conidiogenous parent may be thin-walled or thick-walled, spiny orcells give rise to conidia. Conidia may originate blas- smooth, club-shaped or oval, sessile or sup-tically or thalically and all layers of the parent cell ported by conidiophores, and may appear asmay be involved (holo) or only the inner cell layers individuals or in clusters.(entero). In blastic division, the parent cell enlarges (vi) Microconidia develop from the conversionand a septum separates the enlarged portion into a of an entire hyphal element, but the newdaughter cell. This division may occur in differing conidium remains aseptate (e.g. Trichophytonarrangements: rubrum). Microconidia are one-celled and may
  • 109. 94 Mycology: moulds and yeasts be round or oval, sessile or support, and appear (iii) Two compatible hyphae can each form an ex- individually or in clusters. tending arm, the zygophore. When the zygo- (vii) Chlamydoconidia (e.g. Gliocladium) are phores meet, they fuse to form a thick-walled thick-walled survival conidia formed during protective zygosporangium, within which zygo- unfavourable conditions that germinate and spores develop. This process can happen within produce conidia when conditions are more the same, or between two different compatible favourable. colonies. (viii) Arthroconidia are produced by fragmenting e.g. the zygomycetes, including Mucor, Rhizo- from hyphae through the septation points (e.g. pus, and Absidia. Coccidioides immitis). They may be rectangu- lar or barrel-shaped and maybe separated by Growth rate disjunctor cells. (ix) Sporangiospores are formed by internal cleav- Fungi may grow rapidly or slowly. Moulds may grow age of a sac, called a sporangium (e.g. Zygo- rapidly (3–4 days) or may require 3–4 weeks. Rate myces). of growth is affected by type of media, incubation temperature and inhibitors in the patient’s speci- men. The majority of yeasts and many of the moulds considered to be clinical contaminants require only Sexual reproduction 2–3 days for growth. Forms of sexual reproduction are important for divi- sions in taxonomy. Three different types of sexual Clinical classification of fungi reproduction (teleomorphic states) can be recog- nized: Infections (i) A nucleus from a male cell (antheridium) passes through a bridge into a female cell (ascogo- The majority of fungi are mesophilic, and cannot nium); male and female cells may be from the grow at 37 ◦ C; many are saprophytic, and grow more same colony, or from two compatible colonies. efficiently on non-living substrates than on living Following fusion to form the zygote, the female tissue. The human body also has a highly efficient cell becomes an ascus. The diploid zygote defence mechanism to combat fungal proliferation. nucleus divides by meiosis to form 4 haploid Therefore, in general the development of fungal dis- nuclei, which then divide by mitosis to form 8 ease is related to the immunological status of the nuclei. Each new nucleus is then walled inside host and environmental exposure, rather than to the the ascus to form an ascospore, e.g. the yeast infecting organism. The relatively small number of Saccharomyces cerevisiae and the mould Pseu- fungi that can cause disease have unique enzyme dallescheria boydii. pathways, exhibit thermal dimorphism, and are able (ii) Two compatible hyphae or yeast cells can fuse to block cell-mediated immune defences in the host. to form a binucleate mycelium; the terminal end ‘Opportunistic’ fungi cause infections in debilitated of the mycelium enlarges into a club-shaped patients with compromised immune systems. Alto- structure, a basidium. The two nuclei within gether, around 200 human pathogens have been rec- the basidium fuse to form a zygote that then ognized from among an estimated 1.5 million species undergoes meiosis to produce 4 haploid nuclei, of fungi. Human fungal diseases include dermato- or basidiospores that extend out of the basid- mycoses (skin, hair and nail diseases), yeast infec- ium. tions, pulmonary mycoses, subcutaneous mycoses e.g. Filobasidiela neoformans, the sexual stage (infection is traumatically introduced into tissues) of Cryptococcus neoformans. and opportunistic fungal disease.
  • 110. Clinical classification of fungi 95Superficial tation of a foreign object into deeper tissue. Because the etiologic agents are found in decaying vegetationSuperficial infections are confined to the outermost and soil, the extremities are usually involved. Infec-layer of skin or hair (e.g. epidermis and hair shaft). tions are characterized by nodular lesions that canLiving tissue is not invaded and there is no cellular suppurate and ulcerate and by the presence of drain-response from the host. Symptoms are primarily cos- ing sinus tracts. Subcutaneous infections include:metic: discoloration or depigmentation and scaling Sporotrichosis or ‘rose gardener’s disease’ caused byof skin. These fungi are usually identified in the prac- Sporothrix schenckii.titioner’s office on the basis of clinical appearance. Chromoblastomycosis, caused by dematiaceousExamples of superficial fungi are: mould that produces sclerotic bodies such as Cla-Malassezia furfur, which causes tinea versicolor. dosporium carionii or Philaphora verrucosa.Piedraia hortae, which causes black piedra on scalp Mycetomas may be caused by bacteria (Actino- hair. mycetes) or fungi (eumycotic mycetoma). MaduraTrichosporon beigelii, a yeast that causes white foot is an example of eumycotic mycetoma. piedra of facial and genital hair. Subcutaneous phaeohyphomycosis, caused byExophilia werneckii, which causes tinea nigra of the species of Exophilia and by Wangiella dermatidi- soles and palms. tis, is a more serious infection than superficial phaeohyphomycosis (tinea nigra and blackCutaneous piedra), and this disease is becoming a major concern for immunocompromised patients.Cutaneous infections are caused by dermatophytesand affect the keratinized layer of skin, hair, andnails. No living tissue is invaded, but the presence of Systemicthe fungus and its metabolic products causes itch- Systemic infections affect internal organs and/oring, scaling, broken hair, and thick and discoloured deep tissue. They may be caused by almost any fun-nails. Three genera are etiologic agents of dermato- gus if the patient is immunocompomised, but tra-phytoses: ditionally the systemic infections refer to infectionsTrichophyton species affect both endothrix and that initially occur in the lungs and disseminate ectothrix hair, skin and nails. They typically cause hematogenously with symptoms of fatigue, fever, ‘jock itch’ and ringworm, but may be responsi- chronic cough and chest pain. They are caused by ble for more serious infections, including pustules the dimorphic fungi, Histoplasma capsulatum, Coc- covering the entire body as well as permanent cidioides immitis, Blastomyces dermatidis, and Para- alopecia. coccidioides brasiliensis, all of which have endemicEpidermophyton primarily infects adults, causing areas. ‘jock itch’ and ‘athlete’s foot’.Microsporium species primarily infect children, and may be spread from person to person or via cats Atypical fungus and dogs. Pneumocystis carinii, an opportunistic fungus that infects immunocompromised individuals, remains unclassified. The organism has several characteris-Subcutaneous tics in common with parasites: it has cyst and tropho-Subcutaneous infections are chronic localized infec- zoite stages, and responds to antiparasitic agents andtions affecting deeper skin layers such as muscle and not to antifungal agents. It was originally thoughtconnective tissue, and usually do not disseminate. to be a trypanosome, but DNA sequencing studiesInfection is frequently the result of traumatic implan- reveal that the organism has greater DNA homology
  • 111. 96 Mycology: moulds and yeasts with fungi than with parasites, and is an atypical fun- immunosuppressive therapies. C. albicans is the gus with cholesterol instead of ergosterol in the cell fourth most common cause of blood-borne infec- membrane. tion in the USA, and infections with other Candida It is now classified somewhere between the species is also increasing, e.g. C. tropicalis, C. parap- ascomycetes and the basidiomycetes. P carinii is . silosis: these are very aggressive and difficult to treat. found worldwide, and it replicates in the alveoli of The clinically significant yeasts include: the lung to produce pneumonia when it is inhaled by immunocompromised hosts (particularly HIV/AIDS Candida albicans C. tropicalis patients). P carinii cysts and trophozoites can be . C. parapsilosis C. krusei detected on Giemsa-stained BAL or biopsy speci- C. guilliermondi C. lusitaniae mens. Antigen detection systems can also be used Cryptococcus neoformans Rhodotorula spp. for diagnosis, and nucleic acid amplification systems Candida (Torulopsis) glabrata Sacccharomyces cerevisiae are under development. Geotricum sp. Trichosporon beigelii Malassezia furfur Yeasts and the dematiaceous yeasts, Wangiella dermatiditis Yeasts are classified as ‘yeasts’, meaning that they and Exophilia. reproduce sexually by forming either ascospores or Candida albicans occurs naturally as a commen- basidiospores or as ‘yeast-like fungi’, which either sal of mucous membranes and in the digestive tract cannot reproduce sexually or the sexual stage has yet of humans and animals, but it accounts for up to be demonstrated. In the laboratory all are referred to 70% of Candida species isolated from sites of to simply as yeasts. Yeasts grow on bacteriological infection. Environmental isolates are usually from media as well as on mycology media, and growth can sources contaminated by human or animal excreta, be seen in 2–3 days at 25−30 ◦ C. They generally form such as polluted water, soil, air and plants. It causes smooth colonies that resemble bacteria, and species all types of candidiasis (also known as moniliasis), have characteristic colours: white to cream or tan, with diseases ranging from superficial skin infec- a few pink to salmon or red, and some are dema- tions, oral infections (thrush) and gastritis, to dis- tiaceous (pigmented). Isolates vary in texture, from seminated disease. Oral thrush may be an indication butter-like to velvety or wrinkled, and some (Crypto- of immunosuppression, and immunocompromised coccus) are mucoid due to capsule production. Strain patients can develop disseminated candidiasis. The variation may be seen; phenotypic switching occurs majority of HIV patients develop thrush, which leads so that two colony types will be noted on subculture. to retrosternal odynophagia (pain on swallowing) Evaluation of yeasts in the clinical laboratory gener- in 80% of cases. Patients undergoing chemother- ally includes a germ-tube or other assay to rapidly apy may also acquire thrush that can disseminate. identify C. albicans, microscopic exam to determine Increased blood glucose levels in diabetic patients yeast size, a nitrate and urease test (if Cryptococcus predispose to thrush infections, as do riboflavin neoformans is suspected), a sugar assimilation assay deficiency, oral contraceptives, and antibiotic ther- and a Tween Cornmeal (TOC) agar culture to deter- apy. C. albicans, and other species of Candida mine tertiary structure formation such as pseudohy- are discussed in detail in Chapter 8 as agents of phae and chlamydospores. vaginitis. Microscopic morphology of C. albicans shows spherical to subspherical budding yeast-like cells Candida spp. or blastoconidia, 2.0–7.0 × 3.0–8.5 m in size, with The incidence of clinically significant yeast infec- pseudohyphae and true hyphae; it grows at 37 ◦ C, tions has increased dramatically, as a result of 42 ◦ C, and 45 ◦ C. The majority of cases can be
  • 112. Clinical classification of fungi 97diagnosed by a Germ tube or rapid biochemical Can- requires intravenous (TOC) culture and sugar assim-dida albicans screen. ilation. Candida tropicalis causes aggressive infections, Cryptococcus neoformans is another importantincluding oral thrush, vaginitis, endophthalmitis, pathogenic yeast that causes pulmonary infectionendocarditis, arthritis, peritonitis and mycotic ker- and disseminated infections in immunocompro-atitis. It is a major cause of septicemia and dissem- mised patients. This yeast is a soil contaminant frominated candidiasis, especially in patients with lym- pigeon droppings, and represents a major cause ofphoma, leukemia and diabetes. It is found as part opportunistic infections in AIDS patients, includ-of the normal human mucocutaneous flora, but is ing meningitis. It is characterized by the presencethe second most frequently encountered medical of a slime capsule with anti-phagocytic propertiespathogen, second to C. albicans. that serves as a virulence factor. Non-pathogenic In culture, it resembles C. albicans, but has only strains of the other Cryptococcus spp. do not havepseudohyphae, and no true hyphae. Sugar assim- this capsule. C. neoformans is nitrate and urease pos-ilation is required to correlate with the TOC cul- itive, produces only blastoconidia (budding yeasts)ture for definitive identification of all non-albicans on TOC culture and requires sugar assimilation foryeasts. Infections are resistant to amphotericin B, definitive identification to separate it from the non-and are difficult to treat with traditional antifungal pathogenic cryptococci that are seen as contami-therapy. nants. Candida glabrata is one of the most common yeast Geotrichum candidum is part of the normal flora inspecies found on the body surface and is often iso- the human mouth and skin and can be found in thelated as an incidental finding from skin and urine. stool. It most commonly causes bronchial infections,It accounts for about 25% of all non-albicans fungal but can cause oral, intestinal, vaginal, hand and skininfections seen in the clinical laboratory. Its micro- and (rarely) systemic infections. It is found as a con-scopic morphology shows numerous ovoid, budding taminant in soil, cottage cheese, milk and decayingyeast-like cells or blastoconidia, 2.0–4.0 × 3.0–5.5 m foods such as tomatoes. Laboratory identificationin size. No pseudohyphae or other tertiary structures is by direct mount examination, where fragment-are produced on TOC culture. This organism uti- ing hyphae can be seen – these have non-alternatinglizes only glucose and trehalose in sugar assimilation rectangular arthroconidia with rounded ends. Blas-assays. toconidia are not produced. Candida parapsilosis is a major cause of outbreaks Wangiella dermatitidis and Exophilia jensmaleiiof nosocomial infections in hospitals; it can cause are two dematiaceous yeast-like moulds that areendophthalmitis, endocarditis, vaginitis, external pathogenic to humans, and may cause cutaneousear infections and septicaemia. Nosocomial infec- and subcutaneous mycoses. They grow very slowly,tions are frequently associated with prolonged use producing black yeast-like colonies whose reverseof central venous catheters and can lead to fungemia is also black after 3–4 weeks. They also colonize thein immunocompromised patients. In the laboratory gut and are frequently cultured from stool specimensit grows at 37 ◦ C, but not at 42 ◦ C and 45 ◦ C. Sugar if mycology cultures are ordered. These two organ-assimilation and TOC culture are required for defini- isms can be differentiated from one another on thetive identification. basis of their optimal growth temperature (Exophilia Other Candida spp. are occasionally seen in the grows only up to 38 ◦ C, while Wangiella grows up toclinical microbiology laboratory, either as contami- 42 ◦ C), and on the basis of casein, tyrosine and xan-nants or in immunocompromised patients such as thine hydrolysis patterns. They may be found as lab-those with diabetes, malignancies, patients receiving oratory contaminants.prolonged corticosteroids or antibiotics and intra- Trichosporon beigelii and Malassezia furfur arevenous (IV) drug abusers. Definitive identification agents of superficial mycoses. Malassezia furfur is
  • 113. 98 Mycology: moulds and yeasts Table 3.2. Organisms which are contaminants, but may cause infection Zygomycetes Aseptate hyphae, Sexual reproduction via zygospores Asexual sporangia (or similar specialized structures) bear asexual conidia Mucorales Endomorphorales Other hyaline contaminants Dematiaceous moulds Rhizopus (#1 cause of Basidiobolus Penicillium Alternaria infection) subcutaneous disease Paecilomyces Aureobasidium Mucor Conidiobolus Scopulariopsis Bipolaris Absidia (#2 cause of infection) rhinofacial disease Gliocladium Cladosporium Rhizomucor r Aseptate (less so than the Fusarium Curvularia Apophysomyces Mucorales) Acremonium Dreshlera Saksenaea r Splendore–Hoppeli material Chryseosporium (resembles Epicoccum Mortiterella present Blastomyces dermatitidis) Nigrospora Syncephalastrum r Infections in Sepedonium (resembles Stemphylium Cunninghamella immunocompetent Histoplasma capsulatum) Ulocladium Cokeromyces individuals Aspergillus fumigatus r Cause infections in r Not angioinvasive Aspergillus niger immunocompromised Aspergillus flavus individuals Aspergillus terreus r Splendore–Hoppeli material Aspergillus nidulans not present r Angioinvasive further implicated in causing line sepsis in neonates are frequent contaminants. The penicillin produc- receiving lipid supplemented hyperalimentation. ers are hyaline moulds that frequently have pig- mented microconidia and surface colony coloration on the reverse side of the plate. They have septate Contaminants hyphae with rather uniform diameters. Penicillin producers grow rapidly producing flat colonies that Zygomycetes are frequently powdery, and all have characteristic Contaminants include members of the Zygomycetes, microscopic morphology. Members include Penicil- other hyaline contaminants (including Aspergillus lum spp., Paecilomyces spp., Scopulariopsis spp. and species and penicillin producers), and dematiaceous Gliocladium spp. or dark moulds (Table 3.2). The Zygomycetes are hya- Aspergillus species are hyaline moulds with sep- line moulds with aseptate, ribbon-like hyphae with tate hyphae. Colonies are low growing and vel- great variation in width. They grow rapidly and pro- vety, with highly pigmented surfaces. The Aspergillus duce an abundant mycelium that frequently pushes species reproduce asexually by producing strings of the lid off the petri dish. The Zygomycetes repro- conidia from conidigenous cells arranged in swollen duce sexually via zygospores and asexual sporangia vesicles. Aspergillus fumigatus, A. niger, A. flavus, (or similar specialized structures) and bear asexual A. nidulans, and A. terreus may be seen as con- conidia. Rhizopus, Mucor, Absidia and Rhizomucor taminants in the laboratory, and are discussed in
  • 114. Laboratory identification of fungi 99Chapter 13. Other hyaline moulds seen as contam- (ii) clues as to appropriate media for inoculation;inants include species of Fusarium, Acrimonium, (iii) possible evidence of a positive culture when theChrysoporium, and Sepedonium. Each of these patient is on antifungal medication.moulds produces a septate mycelium and may bedifferentiated on the basis of their microscopic mor- Direct examinationphology and sporulation characteristics. Dematiaceous moulds seen as contaminants in- Direct examination for fungal elements generallyclude species of Alternaria, Bipolaris, Cladospo- uses some form of wet preparation, including salinerium, Curvularia, Aureobasidium, Dreshlera, Epic- wet preparations, potassium hydroxide (KOH) prepsoccum, Nigrospora, Stemphylium and Ulocladium. or KOH with calcofluor and India ink preparations.The dematiaceous fungal contaminants are dark KOH dissolves keratin and other cellular elements inon top and dark on the reverse. Both conidia and the sample and calcofluor binds to polysaccharideshyphae are generally pigmented. Organisms are present in the chitin of the fungus cell wall. Sincedifferentiated on the basis of colony characteris- any element with a polysaccharide skeleton will flu-tics, conidia morphology and distribution of coni- oresce, fungal elements must be visualized. India inkdia and macroconidia. Aureobasidium pullans pro- stain is used to observe yeast cells and the capsuleduces thick-walled dematiaceous fungal hyphae and of Cryptococcus neoformans in cerebral spinal fluid,oval blastoconidia. The organism grows as a yeast- although it should be noted that the capsule may notlike black colony whose reverse is also black. They be present, especially in the strain that tends to infecthave a rapid growth rate, with colonies visible within patients with AIDS.7 days. Aureobasidium pullans is often seen as a Several additional stains are commonly used tobiofilm producer and thus may be cultured from detect fungal elements in tissue sections.water baths, incubator water pans and other moist (i) The Giemsa or Wright’s Giemsa stain detectsenvironments. Histoplasma capsulatum and other yeast forms Although the fungi listed in this section are gen- in blood or bone marrow.erally considered contaminants in clinical spec- (ii) The Masson–Fontana stain stains melanin inimens, most can produce disease in humans the cell wall and is used to detect dematiaceousgiven the correct conditions (Table 3.2). Many are fungi.considered opportunists, producing disease in (iii) Periodic-acid–Schiff (PAS) attaches to polysac-immunocompromised individuals. AIDS-defining charides in the fungal wall, staining fungal ele-opportunistic pathogens are described in Chapter ments a magenta color.12, in Table 12.1. (iv) The Gormori methenamine–silver (GMS) stain, probably the most commonly used fungal stain, detects fungal elements by staining the fungalLaboratory identification of fungi wall septations grey-black against a background of green tissue elements.Specimens must be properly collected for fungalidentification and should be transported to the lab- Cultureoratory and processed as quickly as possible. Directmicroscopic examination of specimens provides a Direct examination for fungi should always be linkedrapid report to the physician and provides important to appropriate culture techniques, since the directinformation leading to identification, including: exam is relatively insensitive and cannot speciate (i) documentation of specific morphological char- any organisms detected. Because cultures must be acteristics that might provide a clue to genus held for a long time, specimens from sites that identification; might be colonised with bacteria must be plated
  • 115. 100 Mycology: moulds and yeasts onto antibiotic-containing medium. Unlike bacte- the type of preparation, the following characteristics ria, fungi do not have a wide range of nutritional and should be observed: environmental requirements. Therefore, only a few (i) septate vs. non-septate hyphae, types of media are needed for primary isolation, such (ii) hyaline or dematiaceous hyphae, as: (iii) types, size, shape and arrangement of conidia. (i) Sabouraud dextrose agar or SDA, a nutrition- Yeasts are cultured on cornmeal agar medium, and ally poor medium with pH 5.6 used for the ini- identification includes their biochemistry as well as tial isolation of pathogens and saprobes. the appearance of specific structures. (ii) Sabouraud brain heart infusion agar (SABHI), Germ tubes, hyphal-like extensions of yeast cells are more enriched than SDA for initial isolation. formed with no constriction at the point of origin (iii) Brain heart infusion agar with Blood (BHIAB), of the cell. They have parallel sides, and are non- which is of limited use since contaminants and septate. These differ from pseudohyphae, formed pathogens will grow (used for the isolation of when blastoconidia elongate. Pseudohyphae con- Histoplasma and Nocardia). strict at the point of origin and true hyphae do not; (iv) BHIAB with antibiotics, used to recover slow- they may elongate, and may be septate. growing dimorphic pathogens while inhibiting Blastoconidia are formed from true budding. Fur- the growth of bacteria in clinical specimens. ther identification relies on capsule and ascopore (v) Inhibitory mould agar contains gentamicin, production, as well as assimilation reactions, ure- and is used for initial isolation of fungi, again ase and nitrate production, temperature sensitiv- inhibiting bacteria present in the sample. ity and cycloheximide tolerance. (vi) Dermatophyte test medium replaces SDA and Special media (Birdseed agar) is used to culture is used to recover dermatophytes. Cryptococcus neoformans. Oxidase production by C. (vii) Cornmeal agar enhances blastoconidia forma- neoformans is observed as a brown colour on the tion of yeasts isolated in culture. agar. (viii) Birdseed agar is used for the isolation and iden- tification of Cryptococcus neoformans from yeast colonies identified on initial culture. Mycology in ART (ix) Chrom-agar is now available for rapid identifi- cation of yeasts. In the context of ART treatment, there are two areas Fungi grow optimally at 25–30 ◦ C, with slower growth of mycology that must be considered: at the lower temperature. If a dimorphic fungus (i) contamination of laboratory equipment or cul- is suspected, cultures should also be incubated at ture systems by moulds or yeasts, from the envi- 37 ◦ C. Cultures are maintained for 4–6 weeks, and for ronment or via clinical/laboratory staff. This 12 weeks if Histoplasma capsulatum is suspected, topic is covered in Chapter 13; with twice weekly examination to record rate of (ii) clinically significant yeasts, especially those growth and gross morphology of the colony (e.g. responsible for vaginitis are discussed in colour, topography). Chapter 8. Culture systems can easily be infected with yeasts transferred during oocyte retrieval procedures (see Chapter 13, Fig. 13.2). Microscopic examination for fungal structures Several types of preparations can be made, including tease preparation, cellophane tape preparation and FURTHER READING slide culture. Slide culture is ideal for observing fun- gal structures, but since it requires culture it is more Anaissie, E. J., McGinnis, M. R. & Pfaller, M. A. (2003). Clinical difficult and delays the clinical report. Regardless of Mycology. New York: Churchill Livingston, Elsevier Science.
  • 116. Further reading 101Chandler, F. W. & Watts, J. C. (1987). Pathologic Diagnosis of Fun- Koneman, E. W., Allen, S. D., Janda, W. M., Schreckenberger, gal Infections. Chicago: ASCP Press. P. C. & Winn, W. C. (1997). Mycology. In Color Atlas andEllis, D. H. (2001). An introduction to medical mycology. Mycol- Textbook of Diagnostic Microbiology, 5th edn, pp. 983–1070. ogy Unit, Women’s and Children’s Hospital, Adelaide. From: Philadelphia PA: Lippincott. Mycology Online, Larone, D. H. (1993). Medically Important Fungi: A GuideFisher, F. & Cook, N. (1998). Fundamentals of Mycology. Philadel- to Identification, 2nd edn. Washington, DC: ASM phia, PA: W. B. Saunders Co. Press.Forbes, B. A., Sahm, D. F. & Weissfeld, A. S. (2002). Diagnostic Ribes, J. A., Vanover-Sams, C. V. & Baker, D. J. (2000). Zygomycetes Microbiology, 11th edn. St. Louis: Mosby Publishers. in human disease. Clinical Microbiology Reviews 13, 236–Kern, M. E. & Blevins, K. S. (1997). Medical Mycology: A Self- 301. Instruction Text, 2nd edn. Philadelphia, PA: F. A. Davis Co. St-Germain, G. & Summerbell, R. (1996). Identifying Filamen-Kwon-Chung, K. J. & Bennett, J. E. (1992). Medical Mycology. tous Fungi: A Clinical Laboratory Handbook. Belmont, CA: Philadelphia, PA: Lea & Febiger. Star Publishing Co.
  • 117. Appendix 3.1. antifungal agentsClass Agents Mode of action Indications Limitations/commentsPolyenes Amphotericin Binds to ergosterol in cell wall of susceptible Most species of fungi causing human infection Resistance is a problem with Pseudallescheria B deoxycholate fungi resulting in alteration of membrane are susceptible to amphotericin B boydii, Fusarium species, Candida lusitaniae, permeability; leakage of cellular contents deoxycholate; main indications are: Trichosporon species and some agents of leads to cell death. r Candidiasis chromoblastomycosis and phaeohyphomy- r Cryptococcosis cosis as well as occasional isolates of other r Aspergillosis Candida species, including C. albicans and r Blastomycosis Aspergillus species r Histoplasmosis r Toxicity is a problem, especially r Coccidioidomycosis nephrotoxicity Amphotericin Treatment of invasive fungal infections in Lipid vehicles; less nephrotoxic than B lipid complex patients refractory to, or intolerant of, amphotericin B deoxycholate amphotericin B deoxycholate Amphotericin B Invasive aspergillosis in patients with renal Lipid vehicles; less nephrotoxic than cholesteryl sulfate impairment, those where amphotericin B amphotericin B deoxycholate deoxycholate has failed, or toxicity precludes use Liposomal Aspergillosis, candidiasis or cryptococcosis in Lipid vehicles; less nephrotoxic than amphotericin B patients who cannot tolerate or who have not amphotericin B deoxycholate responded to amphotericin B deoxycholate Mycostatin Candida species Topical useFlucytosine Two primary mechanisms: Cryptococcus neoformans and Candida species Usually administered with amphotericin B for 1. Conversion by cytosine deaminase into In conjunction with amphotericin B systemic mycoses, especially cryptococcal 5-fluorouracil with subsequent conversion deoxycholate for invasive aspergillosis. meningitis through intermediates into Possible role as sole therapy for the treatment of 5-fluorouridine triphosphate; chromoblastomycosis incorporation into fungal RNA leads to miscoding. 2. Conversion by uridine monophosphate phophosphorylase into 5-fluoro- deoxyuridine monophosphate, inhibits thymidylate synthetase and DNA synthesis.Azoles Ketoconazole Interfere with synthesis and permeability of Histoplasmosis – second line agent Due to newer triazoles, ketoconazole is now used fungal cell membranes. Inhibits the Blastomycosis – second line agent infrequently for systemic fungal infections; cytochrome P-450 enzyme responsible for Candidiasis – as alternative to fluconazole considered an alternative agent. conversion of lanosterol to ergosterol, the Coccidioidomycosis – second line agent Not recommended for patients with C. immitis major sterol found in most fungal cell Paracoccidioidomycosis – second line agent meningitis or those who are seriously ill with membranes. Pseudallescheria boydii any type of Coccidioides infection. Not available in parenteral form.
  • 118. Fluconazole Esophageal, oropharyngeal, vaginal, peritoneal, Broad spectrum (triazole derivative) genitourinary and disseminated candidiasis Generally safe and well tolerated Cryptococcal meningitis No advantage over topical agents for Superficial dermatophyte infections (tinea cruris superficial infections and tinea versicolor) Can be administered intravenously Coccidioidomycosis May be alternative to amphotericin B in patients Paracoccidioidomycosis with coccidioidal meningitis, disseminated histoplasmosis in AIDS patients, blastomycosis, sporotrichosis Studies suggest this as the drug of choice for paracoccidioidomycosis Iatraconazole Superficial mycoses, including dermatophytosis, Broad spectrum (triazole derivative) oral and vaginal candidiasis, mucocutaneous Highly lipid soluble candidiasis and tinea versicolor Studies suggest use for chromoblastomycosis Onychomycosis due to Fonsecaea, especially Cladosporium Sporotrichosis Some success in treatment of HIV-positive Histoplasmosis patients with disseminated Penicillium Blastomycosis marneffei infection Aspergillosis Drug of choice for paracoccidiomycosis Cryptococcosis Coccidioidomycosis Candidiasis Paracoccidioidomycosis Chromoblastomycosis Clotrimazole Broad spectrum for topical use Vaginal cream Miconazole Broad spectrum for topical use Vaginal creamOther Terbinafine Hypothesized to inhibit squalene epoxidase, Nail infections – topical form blocking biosynthesis of ergosterol, an essential component of the fungal cell wall.Other Ciclopirox Nail infectionsOther Tolfinafine Treatment of tinea pedisOther Naftidine Dermatophyte infections, especially ringworm Candida infectionsOther Griseofulvin Inhibits synthesis of hyphal cell walls; binds Dermatophyte infections to RNA, inhibiting microtubule function and nucleic acid synthesis.
  • 119. 104 Mycology: moulds and yeasts FURTHER READING Patel, R. (1998). Antifungal agents part I, Amphotericin B preparations and Flucytosine. Mayo Clinic Proceedings, 73, ANTIFUNGAL AGENTS 1205–25. of contents2.htm Physician Desk Reference. (2000). 54th Edn, Des Moines, IA: Medical Economics Co. 202668.html Sarosi, G. A. & Davies, S. F. (1994). Therapy for fungal infections. Mayo Clinic Proceedings, 69, 1111–17. Atovaquonecd.shtml Terrell, C. L. (1999). Antifungal agents II, the azoles. Mayo Clinic Henry, N. K., Hoecker, J. L., Rhodes, K. & Hable, M. D. (2000). Proceedings, 74, 78–100. Antimicrobial therapy for infants and children: guidelines Virk, A. & Steckelberg, J. M. (2000). Clinical aspects of for the inpatient and outpatient practice of pediatric infec- antimicrobial resistance. Mayo Clinic Proceedings, 75, tious diseases, Mayo Clinic Proceedings, 75, 86–97. 200–14.
  • 120. 4 VirologyIntroduction double-stranded, single-stranded, segmented or non-segmented, with positive or negative strand ori-Viruses are obligate intracellular parasites, i.e. they entation. Viruses have no ribosomes, enzymes orrequire an animal, plant or bacterial cell for growth, ATP synthesizing machinery and use those of theirsurvival and replication. The virus genome can con- host cell to carry out the functions necessary for theirsist of either DNA or RNA, and they use the host cell replication. The viral genome is enclosed by a pro-replication and protein synthesis machinery for their tein coat, the capsid, which is made up of morpho-own growth and replication. Viruses do not have fea- logic protein subunits, the capsomeres. The com-tures that previously were thought to be necessary bination of the viral genome inside the capsid isfor any living organism: they do not respire, move or known as the nucleocapsid. Some viruses have angrow, produce waste products, utilize energy or dis- additional outer membrane, the viral envelope, andplay irritability; they do, however, have the ability to viruses that have a viral envelope require this mem-replicate inside eukaryotic or prokaryotic host cells. brane for infectivity. The combination of nucleocap-Viruses that infect bacteria are known as bacterio- sid and viral envelope is referred to as the completephages, and there are large numbers of viruses that virion. For non-enveloped viruses, the nucleocapsidinfect plants, with significant impact on horticulture alone represents the infective virion. The viral enve-and crops. lope is derived from either the nuclear or the cyto- Until the recent discovery of prions, viruses plasmic membrane of the host cell, and it has embed-were described as the smallest known entities ded virus-specific proteins. It is gathered at the endthat can cause disease. Their size ranges from 20 of the replication process when the virus exits thenm (poliovirus) to around 400 nm (poxviruses). cell. Some envelopes contain ‘spikes’ (peplomers),The smallest known bacterium is around 200 nm which are used to attach to the host cell. Peplomers(mycoplasma), and the larger ones such as staphy- determine host specificity, and also induce neutral-lococci are around 500 nm in size. Viruses there- izing antibody. Stripping off the envelope results infore pass readily through bacterial filters (normally loss of virus infectivity.220 nm) and they can only be visualized by electron Virus morphology can take polygonal, helical ormicroscopy, using negative staining (see Fig. 1.3). complex forms. The virus capsid is usually symmet- rical, either icosahedral (shell-like) or helical (tube- like). The capsid may determine host specificity, andVirus structure it increases efficiency of infection, protects nucleic acids from degradation, and can also induce theThe viral nuclei acid genome may consist of formation of virus-neutralizing antibodies in theDNA or RNA (but never both), and this can be host. 105
  • 121. 106 Virology Host range and specificity being released from the cell. Viral proteins are always responsible for this final stage, although Viruses infect and multiply in specific host cells only. host proteins and other factors may be associ- This specificity is determined by specific interaction ated. Not all particles released are infectious and in virus attachment to host cells, or by the availabil- particle/infectivity ratios are highly variable, ity of cellular factors required for viral replication. with defective particles produced for numerous Therefore, viruses that infect bacteria cannot infect reasons. Many infections are abortive. animal or plant cells, and animal viruses generally Information about the reproductive cycle of viruses infect a specific range of cells only, i.e. viruses that has been gained from the study of synchronously infect nerve cells may not necessarily infect the cells infected cells, and specific phases have been identi- of the GI tract. This feature is known as cell tropism. fied. (i) Shortly after infection, for a period of minutes to hours (depending on the virus being stud- Viral replication ied), only low amounts of parental infectious material can be identified: this is the eclipse Viral replication consists of specific stages. phase. Genome replication has been initiated (i) Attachment to host cell and entry. (Since plant but progeny virus has not yet formed. cells are surrounded by a thick wall of cellulose, (ii) This stage is followed by a maturation phase plant viruses rely on mechanical breach of the when viral material accumulates exponentially cell wall, either by an insect vector or by mechan- within the cell or surrounding medium. ical damage). (iii) After a few hours, cells infected with a lytic virus (ii) Replication of viral nucleic acids. become metabolically disordered and then die. (iii) Synthesis of viral proteins with three sets of No further virus is produced, and titres slowly functions: drop. (a) ensure replication of the genome, (iv) Cells infected with non-lytic viruses can con- (b) package the genome into virus particles, tinue to produce viral particles indefinitely. (c) alter the metabolism of the infected cell so This reproductive cycle is variable, lasting less than that virus particles are produced. an hour with many bacteriophages, 6–8 hours in (iv) Assembly of viral components. Picornaviridae and more than 40 hours in Her- (v) Escape from host cells. pesviridae. Cells infected with poliovirus can yield This process takes place in three different more than 100 000 copies of virus per infected cell. phases: (i) The initiation phase introduces the genetic material of the virus into the cell, with attach- Growth characteristics ment, penetration and uncoating of the viral genome. Different viruses have different modes of reproduc- (ii) The replication phase involves synthesis of tion within a host cell – this is known as virus DNA, RNA and proteins. In a few cases cellular replication and the replication strategy of each virus enzymes replicate the viral genome assisted by depends upon the nature of its genome. Viral growth viral proteins (e.g. parvovirus), but in the major- may be described by the overall effect the infection ity, viral proteins are responsible for genome has on the host cell’s survival. Not all viruses manifest replication with the help of cellular proteins. these growth characteristics and some, such as the (iii) Virus particles are assembled during the release Herpesviridae, may demonstrate each growth phase phase and undergo a maturation process before during host infection.
  • 122. Virus classification 107Lytic growth infection of neighbouring cells. The IFN peptide is now produced by recombinant technology for use inThe virus replicates inside the host cell and then the treatment of a variety of viral diseases.releases virus particles by causing cell lysis. Thismay be seen microscopically as a ‘cytopathic effect’(CPE). Virus classificationLysogenic growth Virus classification is helpful in order to make predic- tions about details of replication, pathogenesis andThe lytic portion of the cycle is suppressed. Repli- transmission: this is particularly important whencation produces new virions without killing the host a new virus is identified. As with other microor-cell. Viral DNA may be incorporated into the host cell ganisms, viruses are also classified into order, fam-DNA and transmitted to the daughter cells. Lysogenic ily, subfamily, genus, species and strain/subtype.viruses may also have lytic phases. Families have the suffix -viridae (Herpesviridae, Poxviridae, Retroviridae, Picornaviridae), and genera have the suffix virus (Herpes virus, Hepatitis virus,Latent infections Lentivirus).Latent infections are persistent asymptomatic infec- Taxonomic classification is based upon a numbertion of the host cell by a virus, typical of the Her- of different properties:pesviridae. Appropriate stimulation induces reacti- (i) morphology: size, shape, enveloped/non-vation to lytic growth. Viral DNA may be incorpo- enveloped, tails, spikes,rated into the host cell nucleus and passed on to (ii) physicochemical properties: molecular mass,daughter cells. buoyant density, pH, thermal sensitivity, ionic stability, Viruses have specific tissue tropism: this property (iii) biological properties: host range, transmission,dictates the type of specimen required for diagnosis tropism etc.,of a specific virus. Conversely, the system involved in (iv) genome: RNA, DNA, segmented sequence,the disease process will direct the differential diag- restriction map, modification, etc.,nosis for possible viruses involved. Viruses are com- (v) macromolecules: protein composition andmonly implicated in hepatitis, respiratory illnesses, function,diarrhoea, skin vesicular lesions, aseptic meningitis, (vi) antigenic properties.pharyngitis and conjunctivitis. An in vivo cytopathic The Baltimore Classification divides viruses intoeffect (CPE) may be demonstrated by direct exam- seven (arbitrary) groups (see Table 4.1). By conven-ination of biopsy or cytology preparations; these tion, the top strand of coding DNA (5 –3 ) is (+) sense,may show nuclear or cytoplasmic inclusion bodies, and mRNA is (+) sense.giant cell formation or cell lysis. In vitro cell cultureshows characteristic cellular changes: plaque for- Double-stranded DNAmation, swelling, granulation/inclusion formation,giant cells, cell lysis, and hemadsorption or hemag- Double-stranded DNA viruses, e.g. Adenoviruses,glutination. Interferon (IFN) is a protein (peptide) Herpesviruses, Poxviruses, Papovaviruses (Poly-produced by cells after they have been penetrated omavirus). Adeno- and Herpes viruses use hostby viruses. It cannot stop virus replication in that cellular proteins to replicate in the nucleus of theircell, but it is released to adjacent cells and stimu- host cell. Poxviruses replicate in the cytoplasm andlates the production of antiviral factors that prevent make their own enzymes for nucleic acid replication.
  • 123. 108 Virology Table 4.1. Representative viruses grouped by genome structure (ICTV, 1995) DNA RNA DS-DNA (−) sense RNA (+) sense RNA Family Viral members Family Viral members Family Viral members Poxvirus Variola Orthomyxovirus Influenza A, B and C Togavirus Western and Eastern Vaccinia equine encephalitis *Molluscum **Rubella contagiosum Herpesvirus ***Herpes simplex type 1 Paramyxovirus Parainfluenza Flavivirus St. Louis encephalitis ***Herpes simplex type 2 Mumps Yellow fever Varicella Zoster Measles Dengue ***Cytomegalovirus Repiratory Syncytial ***Hepatitis C (CMV) Virus (RSV) Epstein-Barr (EBV) Human Herpesviruses 6, 7, 8 Adenovirus Human adenoviruses Bunyavirus California and La Crosse Calcivirus Gastroenteritis- serotypes 1–48 encephalitis causing Rift Valley fever calciviruses Other fever agents Sin nombre and related hantaviruses Papovavirus *Human papilloma virus Rhabdovirus Rabies Astrovirus (HPV) Polyomaviruses JC and BK Arenavirus Lymphocytic Coronavirus SARS polyomaviruses choriomeningitis Lassa fever agents Other hemorrhagic agents Hepadnavirus ***Hepatitis B Filovirus Ebola hemorrhagic Picornavirus Enterovirus fever Polio Marburg hemorrhagic Coxsackie B fever Echovirus Enterovirus 68–71, 72 (hepatitis A) Rhinovirus SS DNA DS RNA SS (+) with DNA Intermediate (Reverse transcribing) Parvovirus **B-19 Reovirus Rotavirus Retrovirus Human Colorado Tick Fever T-lymphotrophic virus (HTLV-I and II) ***HIV * Urogenital pathogen/STD; **prenatal/neonatal pathogen; ***both urogenital pathogen and prenatal/neonatal pathogen DS, double stranded; SS, single stranded. Viruses, like bacteria, are divided into genera and species, but the majority are referred to by common names that have been used for some time. For example, the genus Simplexvirus contains herpes simplex virus. Common names are also used for the virus families (poxvirus refers to members of the Poxviridae family).
  • 124. Virus classification 109Single-stranded DNA are copied to (−) strands, which are copied to pro- duce more (+) strands.Single-stranded (+) sense DNA, e.g. Parvoviruses.Replication takes place in the nucleus, where a (−)strand of DNA is synthesized to serve as a template Negative (−) sense RNAfor the synthesis of (+) strand RNA and DNA. Negative (−) sense RNA, e.g. Paramyxoviruses (measles, mumps), Orthomyxoviruses (influenza),Double-stranded RNA Rhabdoviruses (vesicular stomatitis, rabies), etc.Double-stranded RNA, e.g. Reoviruses (rotavirus), These viruses have a virion RNA-directed RNA poly-Birnaviruses. The double-stranded RNA cannot merase to copy their (−)RNA into mRNA.function as mRNA, and these viruses carry an RNApolymerase to make their mRNA after infecting a Segmented (Orthomyxovirus)cell. Their genome is segmented, and each segmentis transcribed separately to produce monocistronic The (−) sense RNA genome is first transcribed by themRNAs. virion RNA-dependent RNA polymerase to produce monocistronic mRNA, which also serves as the tem-Single-stranded RNA plate for genome replication.Positive (+) sense RNA Non-segmented (Rhabdovirus)Positive (+) sense RNA, e.g. Picornaviruses (Hepati-tis A, Foot and Mouth Disease), Coronavirus (SARS), Replication also requires a virion RNA-dependentArteriviruses (Equine Viral Arteritis), Togaviruses, RNA polymerase, and monocistronic mRNAs areFlavivirus, (Hepatitis C, West Nile Virus, Dengue, Yel- produced.low Fever). There are two types. Single-stranded (+) sense RNA with DNANon-segmented RNA intermediater Hepatitis A is a Picornavirus with Polycistronic Single-stranded (+) sense RNA with DNA interme- mRNA, where the genome RNA = mRNA. This diate in their life cycle: Retroviruses. Their genome means that the naked RNA is infectious and there is (+) sense, but unique among viruses, the genome is no virion-associated polymerase. Translation is diploid, and serves as a template for reverse tran- results in the formation of a polyprotein product, scription instead of as mRNA. which is then cleaved to form the mature proteins.r Coronaviruses (e.g. SARS) have a non-segmented (+) RNA that is translated to produce a viral poly- Double-stranded DNA with RNA intermediate merase, which then produces a full-length (−) Hepadnaviruses sense strand. This is used as a template to produce This group also relies on reverse transcription, but mRNA as an overlapping ‘nested set’ of mono- this takes place inside the virus particle on matura- cistronic transcripts, and individual proteins are tion. When the virus infects a new cell, a gap in the produced from each subgenomic transcript. genome is first repaired, and this is followed by tran- scription to RNA.Togaviruses and FlavivirusesTogaviruses and Flaviviruses have complex tran- ‘Subviral agents’ are classified separately: thisscription; two or more rounds of translation are nec- group includes satellites, deltavirus (Hepatitis D),essary to produce the genomic RNA, i.e. (+) strands viroids and prions.
  • 125. 110 Virology Table 4.2. Virus Characteristic CPE features Optimal cell line Herpesvirus (HSV) Rapid onset, starts at edges, entire culture affected, rounding, swelling and Fibroblasts granularity of infected cells Cytomegalovirus (CMV) Slow onset, small plaques involved, usually near the tube butt. Rounding, Fibroblasts swelling and granularity of infected cells Varicella Zoster Virus (VZV) Slow onset, elongated granular plaques Fibroblasts Laboratory diagnosis of viral disease Classical tube culture Classical tube culture is accomplished by inoculat- Direct examination ing specimens directly over cells that have been Histology grown in monolayers inside culture tubes. The cells are incubated at 37 ◦ C for 1 to 21 days, and Tissue sections may show characteristic cytopathic reviewed microscopically to detect cytopathic cel- changes in infected tissues; these are generally seen lular changes (CPE) in specific cell lines. Table 4.2 as intranuclear or intracytoplasmic inclusions. summarizes the comparative CPEs for several viruses that may be grown in fibroblast cell lines. Since these Cytology changes may subtle and not always definitive, pos- itive CPEs are confirmed using immunostains spe- Characteristic changes may be seen in cellular flu- cific for the virus suspected. ids and smears (bronchoalveolar lavage specimens, cerebrospinal fluid, Papanicolau smears, urine, etc.). Shell vial culture The shell vial culture with immunostaining is a Electron microscopy spin amplification culture system that combines Viral particles may be identified by size, location enhanced infectivity with fluorescence monoclonal within the cell and presence or absence of envelopes. antibody staining for specific identification of viral Although a positive result (visualizing particles or antigens (Fig. 4.1). The specimen is inoculated into a components) confirms the diagnosis, a negative vial with a coverslip at the bottom, on which permis- result does not exclude it. Direct detection may also sive cells have been previously grown to near con- be non-specific, in that more than one virus may fluence. The specimen is slowly centrifuged onto the have a similar morphology, i.e. the different Her- cell monolayer, flattening the cells and opening cel- pesviridae may not be distinguishable on the basis lular membrane pores that allow more efficient virus of electron microscopy. infection. After one or more days of incubation, the cells are fixed and stained with antibodies to detect specific viruses. This culture system may be used to Culture detect a number of viruses, but is most important in detecting CMV rapidly and in distinguishing HSV-1 Specimen handling is very important to ensure that from HSV-2. the virus can be cultured. Many viruses are labile, and the specimen should be inoculated into cul- Antigen detection systems ture medium as soon as possible. Specimens must be transported in the correct support medium, on Antigen detection systems use enzyme immuno the correct swabs, at the correct temperatures. assays (EIAs) designed to detect viral antigens
  • 126. Viruses directly relevant to ART 111 Centrifugation and fluorescent isothiocyanate (FITC) labeled anti- growth, incubation human IgG or IgM antibodiesSpecimen (ii) colorimetric or radiometric immunoassays,inoculation EIAs or RIAs (iii) complement fixation for viral antibody titres (iv) Western blot for HIV diagnosis Monoclonal antibody (v) recombinant immuno blot assay (RIBA) for HCV staining diagnosis. Molecular diagnostics The advent of nucleic acid technology has now revo- HSV-1 HSV-2 lutionized the diagnosis of viral disease, and sophis- ticated specific test kits for many common viruses have been developed. Both qualitative and quanti- tative assays are available, including: (i) PCR, probe amplification, probe capture assays,Fig. 4.1. Shell vial culture for virus identification. Schematic ligase chain reaction, and real-time light cyclerdiagram illustrating shell vial culture, a spin amplification PCR assays. These are used for a number ofculture system that combines enhanced infectivity with viral agents, including human papillomavirusfluorescent monoclonal antibody staining in order to identify (HPV), human immunodeficiency virus (HIV),specific viral antigens. This system is important in detecting HCV and HBV,CMV rapidly and in distinguishing HSV-1 from HSV-2. (ii) DNA probes (not amplified), (iii) Dot blot/Southern blot assay (Human papil-present in direct specimens or in cultured speci- loma virus),mens. These are often useful for point of care testing (iv) line probe assays (to detect HCV genotypes),for Influenza A and B, Rotavirus, Herpes, RespiratorySyncytial Virus (RSV) etc. Viruses directly relevant to ARTSerologic diagnosis Viruses directly relevant to ART practice include:Serologic diagnosis can be qualitative or quantita- (i) double-stranded DNA virusestive, and is used to test acute and convalescent sera: Herpes viruses (i) IgM to detect acute infections Herpes Simplex (ii) IgG to assess acute infection, convalescence and Cytomegalovirus past exposures: a fourfold, or greater, rise in titre Papillomavirus indicates an acute infection Hepatitis B virus (partially double-stranded)(iii) Sequential antibody production to key antigens (ii) single-stranded RNA viruses can help in the diagnosis of specific viral infec- Hepatitis C and D tions such as Hepatitis B and Epstein–Barr Virus. (iii) retrovirusesMethods of detection used in serologic diagnosis human immunodeficiency virus (HIV) -1 and -2include: human T-cell lymphotrophic virus (HTLV) -I (i) immunofluorescent antibody detection (IFA): and -II. patient-derived antibodies reacted with virus- This section covers a brief generic description of the infected cells, and antibodies identified using virus groups in order to clarify relationships and to
  • 127. 112 Virology provide an overall perspective for the purpose of (iv) matrix protein in between the capsid and the comparison. Pathology, consequences and diagno- envelope (tegument = fibrous structure). sis for the viruses that have a direct impact upon ART, particularly the sexually transmitted viruses and the The Herpes family blood-borne pathogens, will be discussed in detail Herpesvirus replication in Chapters 7 and 12. (i) Peplomers attach to specific virus receptors on the cell surface of susceptible cells; at least nine different glycoproteins are present. Double-stranded DNA viruses (ii) The nucleocapsid enters the host cell through fusion of virus envelope and cell membrane. Herpesvirus (iii) An unidentified host enzyme uncoats the viral The Herpesvirus family is one of the most important DNA. virus families in medical practice. They are ubiqui- (iv) Virus core enters the nucleus through a nuclear tous, infecting virtually everyone, and induce a wide pore and viral DNA is circularized. variety of diseases. The viruses enter into a ‘latent’ (v) Viral DNA is transcribed by a host DNA- phase after recovery from primary infections; var- dependent RNA polymerase, producing 50 dif- ious types of stress can then induce the virus to ferent messenger RNAs; the viral genome is tran- start replicating again, causing symptomatic disease. scribed in three blocks sequentially: Eight species of human pathogens in the Herpesvirus (a) immediate mRNA (alpha): codes for regula- family have been identified: tory genes that control transcription, (i) Herpes Simplex Type 1 (HSV-1): cold sores, but (b) delayed early mRNA (beta): codes for can also cause genital ulcers, enzymes, non-structural regulatory pro- (ii) Herpes Simplex Type 2 (HSV-2): genital herpes, teins and minor structural proteins, occasionally oral herpes, (c) late mRNA (gamma): codes for the major (iii) Varicella-zoster (VZV): chicken pox, shingles, structural proteins of the virus. (iv) Epstein–Barr (EBV): glandular fever, tumours, (vi) Once enough virus structural protein has been (v) Cytomegalovirus (CMV), made, host cell protein synthesis shuts down (vi)–(viii) Human Herpes 6, 7, and 8 (HHV 6, 7, 8). and the cell eventually dies. The viral core and The Herpesvirus genome is a single molecule capsid assemble in the nucleus, with the struc- of linear double-stranded DNA of approximately tural proteins condensing around viral DNA to 120–220 kbp, with terminal repeats at each end of form nucleocapsids. The genomic concatomers the duplex that allow the molecule to form a circle. are cleaved and packaged into pre-assembled HSV-1 and −2 are closely related, overlapping in both capsids. structure and in disease pathology; 50% of their DNA The viral envelope is acquired from the inner lamella sequence is shared. There is very little DNA homol- of the nuclear membrane as the nucleocapsid ‘buds ogy between the other six viruses. out’ of the nucleus, and particles then assemble The virion has four structural components: in the space between the inner and outer nuclear (i) core: consists of viral proteins with viral DNA lamellae before being transported to the cell surface. wrapped around them to form an icosahedral The proteins in this membrane are almost entirely capsid, 100 nm in diameter, replaced by viral proteins. Mutations in certain enve- (ii) envelope derived from the host nuclear mem- lope glycoproteins interfere with viral transport. The brane, containing viral proteins, remaining particles are released when the cell lyses, (iii) surface projections: peplomers, around 24 hours after infection. The vesicular fluid
  • 128. Viruses directly relevant to ART 113of active herpetic lesions contains HSV particles in Varicella Zoster virus (VZV)high concentrations.r Only approximately 25% of viral DNA/protein pro- This virus infects virtually every human, in the form of chicken pox. Although it is mostly a child- duced is incorporated into virions – infection is a hood disease (highly contagious!) and is gener- ‘wasteful’ process.r The rest of the DNA/protein accumulates in the ally mild in healthy children, VZV infections can be severe and even fatal in high-risk groups: cell, which eventually dies. This process produces neonates, immunocompromised patients and sus- characteristic nuclear inclusion bodies, which can ceptible adults. Recovery from primary infection be seen by electron microscopy. leads to latency and virus reactivation results in Her- pes Zoster (shingles). Shingles can be a painful andViral latency debilitating disease: the vesiculopapules are local-Certain host cells (nerve cells) can prevent the tran- ized with dermatomal distribution, causing a neu-scription of delayed early and late genes and the viral ronitis that may be associated with protracted pain.genome persists in these as an episome (i.e., not inte-grated into the host cell DNA). Under these condi- Cytomegalovirus (CMV)tions, the virus does not replicate and the cell does CMV, the largest of the Herpesviruses, is a mem-not die: the virus instead remains ‘latent’ within the ber of the beta-Herpesviridae subfamily. The com-cell and can be reactivated into active infection under plete nucleotide sequence of the virus is knownthe right circumstances, such as stress, exposure to and its expression has been studied in detail: it hasUV light, or immunocompromise. the largest genome of all herpesviruses (230 kbp vs. 160 kbp for HSV-1), with an arrangement of unique As mentioned above, Herpes simplex viruses 1 and and inverted repeats, including four genome iso-2 are closely related. HSV-1 is ubiquitous, causing mers. Human CMV infection is widespread through-disease in over 60% of adult humans worldwide, out the world, but is usually asymptomatic or onlywhile HSV-2 is seen primarily as a sexually transmit- mildly symptomatic in immunocompetent individ-ted pathogen infecting about 20% of adults in the uals. Infected persons may excrete virus in urine orUnited States. saliva for months and the virus can also be found in cervical secretions, semen, feces and milk. CMV isHSV-1 capable of reactivating throughout life to produceHSV-1 is acquired primarily during childhood and asymptomatic shedding or symptomatic diseaseadolescence and causes oral and ocular lesions. (particularly in the immunocompromised host). TheIncreasingly, HSV-1 is implicated in causing a signif- virus is an important pathogen that may produceicant proportion of newly diagnosed and recurrent severe disease in the newborn when transmitted dur-genital ulcer disease. ing pregnancy. The clinical picture of CMV infection, diagnosis, and treatment is described in Chapter 12.HSV-2 Epstein–Barr Virus (EBV)HSV-2 causes genital and anal lesions and may betransmitted to the neonate during parturition. The Like the other Herpesviruses, EBV is also ubiqui-incidence of genital herpes has increased signifi- tous and worldwide in distribution, with virtuallycantly during the last few decades. all humans infected by the EBV (∼90% of most Detailed clinical aspects of Herpes genital ulcer populations). The virus causes infectious mononu-disease are discussed in Chapter 7. cleosis (glandular fever) and has been strongly
  • 129. 114 Virology associated with nasopharyngeal carcinoma, Burkitt’s to, but distinct from, HHV-6, with some antigenic lymphoma, salivary gland carcinoma, Hodgkin’s dis- cross-reactivity. There is as yet no clear evidence for ease, chronic fatigue syndrome and hairy leuko- any relationship to human clinical disease, but it may plakia. The complete nucleotide sequence of the act as a co-factor in HHV-6 related syndromes. virus is known, and has been shown to have many internal repeats, with numerous genomic variants. Human Herpesvirus-8 ( HHV-8 Replication/latency has been studied in transformed Kaposi’s sarcoma-related virus) human cell lines. The virus has two target cells: r epithelial cells of the oropharynx – virus replica- HHV-8 is correlated with Kaposi’s sarcoma in AIDS tion occurs here, but the infection is subclinical; patients and can also be isolated from primary bone r human B-lymphocytes, where it results in a non- marrow cells (PBMC). This virus contains ‘pirated’ productive infection. When B-lymphocytes are cellular genes, in an ‘oncogenic cluster’. It contains infected in vitro, the lymphocytes proliferate a vGPCR gene (viral G-protein coupled receptor), rapidly and persistently (immortalization or trans- which acts as a vascular switch, turning on VEGF formation), instead of releasing progeny virus and (vascular endothelial growth factor), which is then lysing. Each transformed lymphocyte has up to responsible for the development of Kaposi’s sar- several hundred EBV genomes and the early gene coma. It resembles the Epstein–Barr virus in hav- products. ing a tropism for epithelial and B-cells and may The virus causes polyclonal B-cell activation and also cause other tumours, e.g. B-cell lymphoma. The benign proliferation; the infected B-lymphocytes virus is normally kept under control by the host’s spread via blood and lymphatics to activate immune system and only becomes a problem dur- T-lymphocytes and cause foci of lymphoprolifera- ing immunosuppression. In non-endemic parts of tion. This process may be subclinical, or may pro- the world, HHV-8 has been identified in semen of duce the symptoms of infectious mononucleosis. In high-risk patients (i.e. AIDS patients with Kaposi’s the setting of HIV, EBV may cause a malignant clone sarcoma), but has not been identified in low-risk of immortalized plasma cells to emerge, leading to populations such as semen donors. However, in high the development of a primary B-cell lymphoma, usu- prevalence regions of the world such as Sicily, HHV-8 ally involving the central nervous system. has been demonstrated in semen even in the absence No specific antiviral therapy is effective, and no of HIV infection and Kaposi’s sarcoma. vaccine is yet available. Papillomavirus Human Herpesvirus 6 (HHV-6) Papillomavirus belongs to a different family of HHV-6 was isolated in 1986, associated with lym- double-stranded DNA viruses, the Papovaviruses, phoreticular disorders. It has a tropism for CD4+ which were the first viruses shown to be onco- lymphocytes and is now recognized as a universal genic in the laboratory: SV40 virus, one of the most human infection: roseola infantum, or ‘fourth dis- well studied of this family of viruses, is known to ease’. Childhood infection is mild, and adult infec- cause tumours in mice. The papillomaviruses are tion rare but more severe, causing mononucleosis/ spherical, icosahedral, non-enveloped viruses, with hepatitis. This virus is a problem in immunocompro- a single molecule of circular double-stranded DNA mised patients. as their genome. There are at least 60 different types of HPV: the target cells are cutaneous and Human Herpesvirus-7 (HHV-7) mucosal squamous epithelium, with many of the HHV-7 was first isolated from CD4+ cells in 1990. viral serovars having marked regional tropism (i.e. Its entire genome has been sequenced: it is similar for hands, feet, laryngeal mucosa, or genital sites).
  • 130. Viruses directly relevant to ART 115Several HPV serovars produce genital warts, which produce a chronic syndrome associated with cir-are sexually transmitted (see Chapter 9). rhosis and hepatocellular carcinoma. HBV infec- tions are seen worldwide, but prevalence varies geo- graphically. While the USA, Western Europe andHepatitis viruses Australia have very low prevalence rates, other pop-At least six different human pathogenic viruses that ulation in Africa, South America, South East Asia,cause acute and chronic viral hepatitis have been Greenland and the Eskimo populations in Northidentified: America demonstrate prevalence rates of 8% orHAV = picornavirus, RNA genome, greater. An effective vaccine is available for HBV andHBV, HGV = hepadnaviruses, partially double- its use globally may help to diminish the burden of stranded DNA genome, human disease with this virus. Features of HBV infec-HCV, HDV, HEV: RNA genome, tion, transmission and diagnosis are described inThere may be others as yet unidentified. Chapter 12.Hepatitis A and E are mainly spread by the fecal–oralroute and do not induce a chronic carrier state. HBV, Hepatitis C virus (HCV)HCV and HDV produce chronic hepatic infections,and HGV produces no known hepatitis symptoms. HCV is the predominant agent of post-transfusion Hepatitis (formerly known as ‘non-A–non-B hepati- tis’). The genes of HCV were characterized by molec-Hepatitis A virus (HAV) ular cloning in 1989, as a result of which it wasHepatitis A virus (HAV) is a very small picor- included in the family of Flaviviridae. Although it isnavirus (27 nm) with non-segmented single- less efficiently transmitted than HBV, it is seriallystranded positive-sense RNA as its genome (repre- transmissible in chimpanzees, inducing hepatitis.senting mRNA). The virus is icosahedral and not HCV has been identified in semen specimens fromenveloped. Its primary target is the lymphoid tissue infected males and is considered to be a blood-borneof the gastrointestinal tract and it is commonly pathogen. Properties of the virus and its associatedacquired from contaminated food or water. Immu- disease are described in Chapter 12.nization is readily available by a Hepatitis A vaccine,as well as by passive immunization with gamma Hepatitis D (delta) virus (HDV)globulin. HAV vaccination is given routinely duringchildhood in some parts of the world. HDV is a defective single-stranded RNA virus, a 36 nm particle encapsulated by HBsAg: it needs HBV as a helper in order to replicate. HDV uses HBV inHepatitis B virus (HBV) order to synthesize a capsid made up of HBsAg andHBV belongs to the hepadnavirus family. It is a this is used to make up the infective virions for HDV.42 nm spherical particle with a lipoprotein envelope For this reason, HDV infection is seen only in peo-composed of hepatitis B surface antigen (HbsAg); ple also infected with HBV. For example, an individ-the partially double-stranded DNA genome is con- ual with chronic HBV infection may be infected withtained within a 27 nm hexagonal nucleocapsid. The HDV, or an individual may become coinfected withouter shell has virus-specific glycosylated proteins both viruses. HDV viral RNA is complexed with HDV-and lipid, so that the shell is equivalent to a viral specific protein, the HDV antigen, without form-envelope; removal of this shell by detergents results ing the specific morphology of a nucleocapsid: it isin loss of infectivity. The complete infectious virion thought perhaps to be a also known as the ‘Dane Particle’. HBV infec- A viroid is a small molecule of single strandedtion can stimulate an acute hepatitis, as well as RNA, less than 0.5 kb in size, which can replicate in
  • 131. 116 Virology susceptible cells. The RNA has internal regions that not available commercially, but is used for research are complementary, and this allows the molecule purposes. to self-anneal by internal base pairing. This yields a stable rod-like structure, with complementary Retroviruses sequences at both ends of the molecule, allowing it to form a circle. How they multiply remains unresolved: Prior to the discovery of HIV in the etiology of their genome is too small to code for RNA replicase, AIDS, retroviruses were of major interest to exper- and host cells do not have RNA replicase. All viroids imental oncologists, but of minor importance in identified so far are plant pathogens. medical practice. The AIDS epidemic has brought HDV is implicated in causing a fulminant or acute the retroviruses to the forefront of medical virol- hepatitis when coinfecting with HBV, and often pro- ogy, and they have unique features that should be duces a chronic HDV infection in the presence of considered in the perspective of assisted reproduc- a previously acquired chronic HBV infection (see tion and surgical micromanipulation of oocytes/ Chapter 12, Fig. 12.8). Compared to HBV, the dis- embryos. tribution of endemic HDV is much more limited. A Retroviruses use DNA as an intermediate in their more complete discussion of HDV infection may be replication: their genome consists of single-stranded found in Chapter 12. RNA, which is reverse transcribed into DNA that inte- grates into the host cell DNA. This process depends on a virus-associated, virus-specified polymerase, Hepatitis E virus (HEV) the reverse transcriptase enzyme. Integrating a copy Hepatitis E virus (HEV) is enterically transmitted, of their genome into the host cell chromosome and is responsible for a ‘non-A–non-B’ hepatitis. can result in cell transformation (unrestricted cell The virus is spherical, non-enveloped, with a single- division), and they are thus oncogenic in a num- stranded RNA genome. It has been provisionally clas- ber of vertebrate species, causing leukemia, sar- sified as a calcivirus, but its genome is substantially coma and mammary carcinoma. The key reverse different from other calciviruses. HEV has no sero- transcriptase enzyme has three different enzymatic logical cross-reaction with HAV, HBV, HCV, or HDV activities. and its pathogenesis may be similar to that of HAV. (i) It uses RNA as a template to synthesize a com- However, a high rate of fulminant and often fatal plementary single-stranded DNA. infection is seen with HEV infection in the pregnant (ii) It hydrolyses the RNA primer molecule: this is woman. known as RNA-ase H activity. (iii) It synthesizes a complementary DNA sequence to form a double-stranded molecule that can be Hepatitis G virus (HGV) integrated into the host genome. Hepatitis G virus (HGV) is an enveloped virus with- The enzyme has a further unique feature: it requires out surface projections, slightly pleomorphic, with a particular cellular tRNA to use as its primer for DNA occasional filamentous forms. The genome consists synthesis. of circular DNA, which is partially double-stranded and partially single-stranded: neither strand is cova- History of retroviruses lently closed. Its acute disease spectrum is unknown, but it is found with an incidence of 0.3% in cases In 1904, Ellerman and Bang observed that a lym- of acute viral hepatitis, frequently as a co-infection phoproliferative disease in chickens could be trans- with HCV. It is transmitted via blood-borne transmis- mitted by filtrates of cell extracts: this agent was sion. Its role as a sexually transmitted virus is sug- identified as avian myeloblastosis virus (AMV). Paul gested by the relatively high rate of seroprevalence Ehrlich then proposed the theory of immune surveil- seen in prostitutes. Enzyme immunoassay testing is lance in 1905, which subsequently led to the idea
  • 132. Viruses directly relevant to ART 117that these infectious agents escaped immune attack Retrovirus geneticsby becoming associated with their host cell genome. Retroviral genetics is very complex! The viruses havePeyton Rous (1911) found that sarcomas in chickens an extremely high mutation rate: reverse transcrip-could be transmitted by cell filtrates and Rous Sar- tion is apparently error-prone and recombinationcoma virus (RSV) was identified. During the 1960s, also occurs during the process. Interactions occurHoward Temin inhibited retrovirus replication with with the host cell DNA, including insertional muta-experiments using actinomycin D, which inhibits tions and transductions.DNA synthesis, and he then proposed the concept Retrotransposons are endogenous retrovirus-likeof reverse transcription. In 1969, Huebner & Todaro genetic elements, which make up 5–10% of the mam-proposed the viral oncogene hypothesis and David malian genome: these can move around to differentBaltimore and Howard Temin were awarded the positions on the DNA.Nobel Prize in 1975 for their work on retroviruses and Transposons are short segments of DNA that canreverse transcriptase. Human T-cell lymphotrophic move around within a cell to many different posi-virus (HTLV) was identified as the first pathogenic tions on the chromosome. They exist in all organ-human retrovirus in 1981, and HIV was discovered isms, from bacteria to humans, and provide a rapidin 1983. genetic diversity that acts as a selective pressure for survival of the fittest. They are responsible forRetrovirus classification the rapid evolutionary changes seen in bacteria (i.e. antibiotic resistance). IS is a gene that encodes anTwo species of virus have been grouped into three enzyme responsible for the movement of the trans-subfamilies. poson, by inactivating genes into which they land. (i) Oncornaviruses: HTLV-I and HTLV-II, have a Composite transposons have also been identified, centrally located nucleoid e.g. antibiotic resistance flanked by 2 IS genes. (ii) Lentiviruses: HIV-1 and HIV-2, have a cylindri- An ancient retrotransposon insertion has been cal nucleoid reported to be the cause of Fukuyama-type muscular(iii) Spumaviruses – thought to be associated with dystrophy, the most common autosomal recessive chronic fatigue syndrome disorder in Japan (Kobayashi et al., 1998).They have also been grouped according to their mor-phology by electron microscopy. (i) A-type: non-enveloped, intracellular immature Retrovirus pathogenesis particles. Oncogenes hijack normal control of cell division and (ii) B-type: enveloped, extracellular particles, cause cell transformation so that the cells continue with spikes (Mouse mammary tumour virus, to divide indefinitely. Normal cells also contain Onc MMTV). sequences, which are genes that are fundamentally(iii) C-type: similar to B-type particles, but have a important in regulating cell growth. Oncogenes have central core and barely visible spikes. The major- some form of altered regulation that causes abnor- ity of mammalian and avian retroviruses are mal growth. Due to their ability to insert into the host C-type particles: murine leukemia (MuLV), cell genome, genetically modified retroviruses are avian leukemia (ALV), HTLV, HIV. under active development as vectors for gene ther-(iv) D-type: slightly larger, spikes less prominent: apy, with the aim of replacing defective genes with Mason–Pfizer Monkey Virus (MPMV), HIV. ‘good’ copies.However, molecular genetics has now replacedmorphological classification. Comparisons are now Human oncornavirusesmade largely on the basis of sequence conserva-tion in their genomes, particularly involving the viral Two viruses of the oncornavirus group have beengenes that code for gag, pol, and env. identified in humans: Human T-cell lymphotrophic
  • 133. 118 Virology virus, HTLV-I and HTLV-II. HTLV-I is associated Bobroski, L., Bagasra, A. U., Patel, D. et al. (1998). Localization with adult T-cell leukemia (ATL), as well as tropical of human herpesvirus type 8 (HHV-8) in the Kaposi’s sar- spastic paralysis (TSP), a Caribbean chronic degen- coma tissues and the semen specimens of HIV-1 infected erative neurological disease. In Japan, it has also and uninfected individuals by utilizing in situ polymerase chain reaction. Journal of Reproductive Immunology, 41: been found in association with HTLV-I-associated 149–60. myelopathy: this may be the same disease as TSP. Calabro, M. L., Fiore, J. R., Favero, A. et al. (1999). Detection HTLV-II is closely related to HTLV-I, with 65% homol- of human herpesvirus 8 in cervicovaginal secretions and ogy between their genomes. Although very little is seroprevalence in human immunodeficiency virus type known about its pathogenesis and epidemiology, its 1-seropositive and -seronegative women. Journal of Infec- molecular biology has been described: the genome tious Diseases, 179: 1534–7. of both viruses contains four genes: gag, pol, env, and Cann, A. J. (1997). Principles of Molecular Virology, 2nd edn. pX, flanked by terminal repeats. It is a cell-associated Chapter 4. New York: Academic Press. virus, and is transmitted by cell-to-cell contact: it Cannon, M. J., Dollard, S. C., Black, J. B. et al. (2003). Risk factors is infectious for a wide variety of mammalian cells for Kaposi’s sarcoma in men seropositive for both Human by co-cultivation. However, only infected T-cells are Herpesvirus 8 and Human Immunodeficiency Virus. AIDS, 17(2): 215–22. transformed and immortalized. The virus has an Diamond, C., Brodie, S. J., Krieger, J. N. et al. (1998). Human extremely long incubation period, in the order of herpesvirus 8 in the prostate glands of men with Kaposi’s 50 years! Infection is usually acquired at birth and sarcoma. Journal of Virology, 72: 6223–7. T-lymphocytes of infected persons, found in blood, Gnann Jr., J. W., Pellett, P. E. & Jaffe, H. W. (2000). Human Her- semen, milk and cervical secretions are the source of pesvirus 8 and Kaposi’s sarcoma in persons infected with infection. Transmission of HTLV-I by blood transfu- human immunodeficiency virus. Clinical Infectious Dis- sion is well documented. Details of the pathogene- eases, 30, Suppl 1: S72–6. sis and diagnosis of HTLV-I and -II are presented in Huang, Y. Q., Li, J. J., Poiesz, B. J., Kaplan, M. H. & Friedman- Chapter 12. Kien, A. E. (1997). Detection of the herpesvirus-like DNA sequences in matched specimens of semen and blood from patients with AIDS-related Kaposi’s sarcoma by polymerase Lentiviruses: human immunodeficiency virus chain reaction in situ hybridization. American Journal of (HIV) Pathology, 150:147–53. Two species of human lentivirus are well charac- ICTV (1995). Virus Taxonomy. The Sixth Report of the Interna- terized: HIV-1 and HIV-2. Since their discovery, the tional Committee of Taxonomy of Viruses. HIVs have established a worldwide distribution, with Kelsen, J., Tarp, B. & Obel, N. (1999). Absence of human her- pes virus 8 in semen from healthy Danish donors. Human the vast burden of human disease focused in sub- Reproduction, 14: 2274–6. Saharan Africa, southern and Southeast Asia and Kobayashi, K., Nakahori, V., Miyake, M. et al. (1998). An portions of South America. The number of new infec- ancient retrotransposal insertion causes Fukuyama-type tions seen each year with these viruses has increased congenital muscular dystrophy. Nature (London), 394: 388– dramatically since the early 1980s. In 1999, new cases 92. were estimated at nearly 4 000 000 in Sub-Saharan Leland, D. S. (1996). Clinical Virology. Philadelphia, PA: W.B. Africa alone. Properties of these two viruses, together Saunders Co. with details of HIV pathogenesis and diagnosis are Pellett, P E., Spira, T. J., Bagasra, O. et al. (1999). Multicenter com- . described in Chapter 12. parison of PCR assays for detection of human herpesvirus 8 DNA in semen. Journal of Clinical Microbiology, 37: 1298– 301. FURTHER READING Temesgen, Z. & Wright, A. J. (1999). Antiretrovirals. Mayo Clinic Proceedings, 74: 1284–300. Baron, E. J., Chang, R. S., Howard, D. H., Miller, J. N. & Turner, Viviano, E., Vitale, F., Ajello, F. et al. (1997). Human herpesvirus J. A. (eds.)(1994). Medical Microbiology: A Short Course. type 8 DNA sequences in biological samples of HIV-positive New York: Wiley-Liss. and negative individuals in Sicily. AIDS, 11: 607–12.
  • 134. Appendix: Antiviral agents 119Appendix 4.1: antiviral agents(Note: all antivirals inhibit specific steps in the process of viral replication; no agent is active against latent or non-replicating viruses.)Class Agents Mode of Action Indications Comments Amantadine and Inhibit transmembrane protein Influenza A CNS toxicity common with Rimantadine M2-mediated conversion of amantadine and rare with hemagglutinin from native to low pH rimantadine conformation, resulting in reduced uncoating of viral genome in cellular lysosomes Zanamivir Sialic acid analogue; specifically Influenza A and B inhibits influenza virus neuraminidase Oseltamivir Pro-drug of sialic acid analogue, Influenza A, B Effective for prevention and GS4071; selective inhibitor of treatment of influenza neuraminidase in both influenza A, B Acyclovir Guanosine analogue Herpes simplex virus 1, 2 Human herpesvirus 6 is resistant Inhibits viral DNA synthesis by Varicella-zoster virus inhibiting viral DNA polymerase and acting as a DNA chain terminator Valacyclovir Pro-drug of acyclovir; inhibits viral Herpes simplex viruses Shown to prevent CMV disease in DNA synthesis Varicella-zoster virus high-risk renal transplant recipients Ganciclovir Acyclic nucleoside analogue of Herpesviruses Active against herpesvirus 6 guanine; inhibits DNA polymerase Varicella-zoster virus FDA approved for CMV retinitis in (inhibits cellular more than viral) immunocompromised patients and for patients with advanced HIV ?for prevention of CMV in transplant patients Penciclovir Analogue of acyclic guanine; action Herpes simplex virus 1, 2 Similar to acyclovir similar to acyclovir, but does not act as Varicella-zoster virus Some activity against HBV DNA-chain terminator Lesser activity against Limited activity against CMV Epstein–Barr virus Famciclovir Pro-drug of penciclovir Herpes simplex virus 1, 2 Varicella-zoster virus Lesser activity against Epstein–Barr virus Ribavirin Purine nucleoside analogue; Influenza A, B Broad spectrum against DNA and mechanism of action may be Respiratory Syncytial RNA viruses competitive inhibition of host enzymes virus FDA approved only for serious resulting in reduced intracellular Hemorrhagic fever RSV and in combination with concentrations of guanosine Hepatitis A, B, C interferon alpha-2b for treatment triphosphate and decreased DNA Lassa fever of chronic hepatitis C synthesis, inhibition of viral RNA Hepatitis Teratogenic, mutagenic, polymerase complex and inhibition of C virus embryotoxic and gonadotoxic in mRNA. Others small mammals Contraindicated during pregnancy
  • 135. 120 Virology Appendix 4.1: (cont.) Class Agents Mode of Action Indications Comments Foscarnet Inorganic pyrophosphate Herpes simplex virus 1, 2 analogue; non-competitive Varicella-zoster virus inhibitor of viral DNA CMV polymerase and reverse Epstein–Barr virus transcriptase Influenza A, B HBV HIV Lamivudine Deoxynucleoside analogue; Hepatitis B inhibits DNA polymerase; inhibits HIV reverse transcriptase Cidofovir Acyclic nucleoside Herpes simplex virus 1, 2 Potent phosphonate derivative Varicella-zoster virus Carcinogenic and Epstein–Barr virus teratogenic CMV Produces hypospermia in lab animals Interferons: 3 major Interferon-alpha 4 Glycoproteins with antiviral Hepatitis B classes (interferon- , commercially activity; produced in host in Hepatitis C interferon- , and available: response to inducer. Interferon-alfa-2b and interferon- ); only Alfa-2a Promotes cell resistance to alfa-n3 approved for interferon- is Alfa-2b viruses via production of treating condyloma approved for viral Alfacon-1 proteins that inhibit RNA acuminatum due to treatment. Alfa-n3 synthesis and enzymes that human papillomavirus cleave cellular and viral DNA; inhibits mRNA; cell membrane alterations inhibit release of replicated virions. Trifuridine Pyrimidine nucleoside Herpes simplex Topical use only for ocular herpes infection Reverse transcriptase Zidovudine Block reverse transcriptase HIV inhibitors Didanosine activity by competing with Zalcitabine natural substrates, Stavudine incorporated into viral DNA Lamivudine to act as chain terminators Abacavir in proviral DNA synthesis Non-reverse Nevirapine Bind directly transcriptase inhibitors Delavirdine (non-competitively) to Efavirenz reverse transcriptase Protease inhibitors Saquinavir Inhibit HIV-1 protease HIV Ritonavir Indinavir Nelfinavir Amprenavir FDA: Food and Drug Administration.
  • 136. Appendix: Antiviral agents 121FURTHER READING Physician Desk Reference. (2000). 54th Edn Des Moines, IA: Med- ical Economics Co.ANTIVIRAL AGENTS Temesgen, Z. & Wright, A. J. (1999). Antiretrovirals. Mayo Clinic Proceedings, 74, 1284–300. of contents.html Virk, A., Steckelberg, J. M. (2000). Clinical aspects of antimi- crobial resistance. Mayo Clinic Proceedings, 75, 200– 202668.html 14.Keating, M. R. (1999). Antiviral agents for non-human immun- odeficiency Virus Infections. Mayo Clinic Proceedings, 74, 1266–83.
  • 137. 5 Prions Prion protein century. Affected sheep rub or ‘scrape’ their coat against a tree, as if it itches – hence it was given the ‘Prions’ (proteinaceous infectious particles) are par- name ‘scrapie’ in England. In France, it was known ticles made up of an abnormal glycoprotein that is as ‘La tremblante’ because the animals shake due capable of causing a cell to produce more abnormal to ataxia. In 1966, Alper showed that the infectious protein. The Prion particle is a unique agent, infec- agent responsible for scrapie in sheep was very UV- tious by biological and medical criteria, but differ- resistant, compared with known viruses. This finding ent from all known conventional microbes because led to the suggestion that scrapie might be infectious it contains no elements of nucleic acid genetic mate- without the involvement of a nucleic acid. A range of rial. Prusiner identified and classified prion diseases different TSEs has subsequently been identified in in 1982, and his work in describing prions as infec- animals (Table 5.1). tious protein particles that cause neurodegenerative disorders (Prusiner, 1991, 1995) gained the Nobel Human Prize in 1997. All known prion diseases are fatal; they are known as ‘spongiform encephalopathies’ The human form, Creutzfeldt–Jakob Disease (CJD) because they cause the brain to be riddled with holes was described in Germany and Austria in the or ‘spongy’, with accompanying symptoms of pro- 1920s, and is now recognized in different forms gressive neurological degeneration. The spongiform (Table 5.2). change occurs without inflammation, inclusion bod- ies, or apparent immune response. Prion structure Prion diseases Normal cell surface glycoprotein = PrPc Infectious Prion agent = PrPsc Prion diseases, known as transmissible spongi- Prion protein = PrP form encephalopathies, (TSEs) can be infectious/ Prion protein gene = Prnp iatrogenic (5%), inherited (10%), or sporadic (‘Clas- Prusiner showed the Prion agent PrPsc to be a sical’, 85%). protease-resistant glycoprotein in brains of scrapie- infected animals, not found in normal animals. The infectious Prion agent PrPsc has now been iden- Animal tified as a conformationally modified isoform of The first neurological disease of this kind was a normal cell-surface glycophosphatidyl inositol- described in sheep during the early eighteenth anchored glycoprotein, PrPc . This protein occurs122
  • 138. Prion structure 123Table 5.1. Prion diseases in different animal speciesSpecies Prion disease EtiologySheep and goats Scrapie Infection seen in genetically susceptible sheepCows Bovine spongiform encephalopathy (BSE), Acquired from ingestion of prion protein in infected feed ‘mad cow disease’. First observed in the supplements. Incubation period 3 to 6 years UK in 1972, diagnosed in 1986Mink Transmissible mink encephalopathy (TME) Probably acquired from sheep or cattleDeer and elk Chronic wasting disease (CWD) Origin unknownCats Feline spongiform encephalopathy (FSE) Acquired by ingestion of BSE-contaminated food productsUngulates Exotic ungulate encephalopathy (EUE) Probably same source as seen in cattleRodents, pigs Experimental scrapie or BSE Experimentally induced Can be infected experimentally, but have not been diagnosed with a naturally occurring prion disease, to dateTable 5.2. Human prion diseasesSporadic CJD (‘Classical’ CJD) Caused by a somatic mutation or spontaneous conversion of a normal cellular prion protein (PrPc ) to the pathogenic isoform (PrPsc ).Iatrogenic CJD Linked to receipt of prion contaminated human pituitary-derived hormones, dura mater or transfer of infection via contaminated operating room instrumentation during cranial surgeries.Kuru Human infection linked to cannibalism – particularly brain tissuesFamilial CJD Due to germline mutations in a gene on chromosome 20 producing the abnormal PrPsc proteinFatal familial insomnia (FFI) Germline mutation in PrP geneGerstmann–Str¨ ussler– a Germline mutation in PrP gene Scheinker syndrome (GSS)New variant CJD Human equivalent to ‘mad cow disease’, bovine spongiform encephalopathy (BSE) linked to the consumption of infected high risk bovine derived food productsnormally in the brain, cornea, spinal cord, pituitary transported to the brain via the lymphatic system.gland, neural ganglia, spleen, lymphocytes, lung, and The PrP gene (Prnp) has been cloned, and it is nowmuscle of all animals. Its true function is unknown, clear that PrP is host encoded. The Prnp gene is iden-but it may have a role in copper transport, nerve con- tical to Sinc, a gene that controls scrapie replicationduction, cell signalling, regulation of circadian activ- in mice. Prusiner (1995) identified 15 amino acidsity and antioxidant reactions. Pathogenic prions are at the N-terminal end of the PrP protein, and thisbent, curved, or widened beta-rich oligomers that sequence was used to construct molecular probes toconvert the normal prions by changing their tertiary study the sequences of the normal vs. the mutatedstructure and integrating them into the growing PrPsc form of the gene. A point mutation substitutes theaggregate, where it acquires the properties associ- amino acid proline for leucine, and this mutationated with the abnormal protein. The pathogenic encodes additional copies of an octapeptide repeatprions gain entry to the body through tonsils or towards the 5 end (Krakauer et al., 1998). The normalPeyer’s patches of the small intestine, and they are protein consists mainly of alpha helices with a spiral
  • 139. 124 Prions backbone, and the new mutated prion protein is pre- the protease-resistant protein in the sample corre- dominantly beta sheets with a fully extended back- sponded to newly converted protein. The investiga- bone. This suggests that the protein undergoes post- tors describe this procedure as protein-misfolding translational modification. It has a molecular weight cyclic amplification (PMCA), conceptually analo- of 27–30 kD, and may aggregate into ‘prion rods’, or gous to the PCR procedure for nucleic acid ampli- fibrils. Deposition of these fibrils in neuronal tissues fication, and propose that it may used as an aid in provided early evidence that the diseases were due possible TSE diagnosis to detect low quantities of the to prion proteins. abnormal protein before symptoms are apparent. The normal protein; PrPc is protease sensitive, and dissolves in non-denaturing detergents. Pathogenic PrPsc has quite different properties. Transmission (i) It is relatively protease K-resistant – infectivity is not blocked by treating infective materials with Transmission of a TSE was first discovered in humans proteases. with the identification of an elusive and bizarre dis- (ii) It does not dissolve. ease that appeared in New Guinea in the early 1900s. (iii) It is resistant to acid and alkali treatment In 1957, Gadjusek and Zigas discovered an epidemic between pH 2 and 10. form of the disease in a New Guinea tribe, and this (iv) It has survived two-year immersions in formol disease, termed ‘Kuru’, was reported as endemic saline (Mims & White, 1984). in a particular district consisting of approximately 8000 individuals, the South Fore. The majority of affected individuals were women and children. Epi- Replication demiological studies of the tribes indicated that this tribal group practised ritual mortuary cannibalism The replication of the abnormal prion protein in which the women and children consumed the involves recruiting normal PrPc proteins, and ‘flip- brain of diseased males as a sign of respect, and as ping’ them into a rogue prion-like shape that can a way to acquire some of the diseased tribal mem- infect other cells and animals (Mestel, 1996), in the ber’s exceptional qualities. This ingestion of infected absence of a nucleic acid template. The resulting neural tissues was later identified as responsible for PrPsc is a four-helix bundle protein, with four regions transmitting the fatal Kuru epidemic. The disease vir- of secondary structure (H1–H4). This change starts tually disappeared in New Guinea when cannibalism a chain reaction, and newly converted prions con- was terminated, but its study laid the foundation for vert other proteins on contact, on the interior of the understanding the pathology and the nature of prion cell membrane. In cell culture experiments, the con- disease. version occurred inside neuronal cells. PrPsc accu- BSE, the bovine prion, is transmitted via con- mulated in lysosomes, eventually filling them until taminated foodstuffs. Shortly after BSE was recog- they lysed and released prions to attack other cells nized, epidemiological studies suggested that the (Prusiner, 1995). source of infection was meat and bone meal (MBM) Saborio et al. (2001) reproduced prion replica- supplements fed to the cows as dietary protein tion in vitro: PrPsc aggregates from a scrapie brain supplements. It was originally thought that scrapie homogenate were disrupted by sonication, and brain was introduced into the MBM supplements when homogenates from healthy hamsters were used as a changes in the rendering processes were made less source of PrPc . In the presence of a minute amount of stringent, permitting infective prion survival into the disrupted PrPsc aggregate as template, a large excess final extracted product. Scrapie had not naturally of PrPc was rapidly converted into protease-resistant crossed the species barrier into cows prior to this PrPsc . Using cycles of incubation-sonication, 97% of change in practice, perhaps because large amounts
  • 140. Transmission 125of the protein were required for infection to occur in adolescent was diagnosed with a new variant CJDcows. It is now thought that exotic ungulates in the (nvCJD). Examination of this and subsequent casesLondon Zoo (Nyala or Kudu antelopes from Africa) demonstrated that BSE, FSE and nvCJD were allare more likely to be the source of the bovine infec- the same disease process crossing species bound-tion. The protein would have been introduced into aries. As of September 2002, 121 cases of nvCJD hadthe bovine population when the zoo animals died been identified in the UK, 6 in France, 1 in Ireland,from TSE and were then introduced into a render- and 1 in Italy. Prions have been detected in cer-ing process for the production of MBM products tain high-risk tissues in infected animals, and con-used in cow feed. BSE infected bovine tissue then sumption of prion-infected tissues is the probablere-entered the rendering process, with subsequent means of transmitting the disease to humans andamplification of prion in the animal feed MBM sup- other animals at risk. High risk materials includeplements, expanding the risk for bovine infection brain, spinal cord and eyes, but prion protein hasfrom these supplements. Infected meats or bone been found in lesser quantities in lymphatic tissues,meal supplements in animal feeds have allowed BSE dura mater, pineal glands, pituitary glands, CSF, pla-to cross species barriers into other animal popu- centa, adrenal gland, portions of the intestine, bonelations, including large and small cats, mink and marrow, nasal mucosa, liver, lung, pancreas, and thy-additional exotic ungulates at the London Zoo. The mus. It has been suggested that individuals consum-first detected case of BSE in Japanese cattle revealed ing prion-infected beef products were made morethat lipid and protein supplements given to calves in susceptible to infection due to inflammation stimu-their milk feeds are the probable source of the island lated by Group A pharyngeal infections. Inflamma-nation’s outbreak. tion enhances the rate of transmission of the prion Prions are stable in soil and secreted in feces, pla- into the lymphoreticular system, from which it even-centa, or amniotic fluid, and there may be mater- tually reaches neurons and the central nervous sys-nal transmission to the embryo or calf. Following a tem. Although no prion has been detected in blood,dialysis enrichment procedure, they have also been serum or most solid organs, there is a theoretical riskdetected in the urine of scrapie-infected hamsters, for person-to-person transmitted disease via bloodBSE-infected cattle and humans suffering from CJD transfusion or tissue transplantation. Of note for(Shaked et al., 2001). The isoform detected in urine the assisted reproduction laboratory, prion proteinsUPrPsc appears to have lower infectivity than brain have not been detected in the testes, ovary or uteriPrPsc , and may differ in its conformation. Normal of infected animals.PrPc is is expressed on blood cells and infectivity Iatrogenic CJD has been described after neuro-can be transmitted via blood products, although surgery, corneal and dura mater grafts, and followingthe infectious PrPsc agent has not been identified in treatment with human pituitary-derived hormonesblood. There are currently no tests sufficiently sen- such as early preparations of gonadotrophins andsitive to detect infectivity via blood levels of PrPsc . growth hormones. Because of the very long incu-The factors involved in determining whether a prion bation period, there may be a reservoir of asymp-can cross a species barrier are unknown, but a possi- tomatic individuals who are infected but do not yetble mechanism has been suggested in a yeast model. display symptoms of the disease. These may servePrion-like activities have been identified in the yeast as a reservoir of CJD or nvCJD that may be transmit-S. cerevisiae, in the form of two proteins that have ted to patients iatrogenically; therefore, individualsnon-Mendelian inheritance. These prions can adopt who have ever received human-derived growth hor-multiple structures and interact with prions from mones, at any time, are excluded from blood or organother species (Davenport, 2001). donation. The USA also banned blood donations The first evidence that BSE had crossed the species from people who lived in Europe during the heightbarrier into humans was seen in 1995 when an of the BSE crisis, linking the risk of infection to the
  • 141. 126 Prions amount of beef products consumed (proportional to tially younger than the mean age of 60 for spo- the amount of time spent in the region). All donors radic disease. The clinical manifestations are also who spent more than 3 months cumulative time in quite different when the two groups of patients are Britain or Europe have been eliminated from the compared. Patients with nvCJD are more likely to donor population in order to address this theoret- present with psychiatric manifestations of disease ical risk. such as depression, social withdrawal, and other The risk of in vitro transmission after attempted behavioural abnormalities. Sensory abnormalities ‘decontamination’ is difficult to evaluate. Assess- such as paresthesia are also common in this patient ment has been attempted by mixing infectious parti- population. Survival time is more prolonged for cles with cellular products, submitting this to inacti- nvCJD with a mean survival time of 14 months, com- vating treatments and then inoculating animals with pared with 5 months for the sporadic disease. Each fractions of the preparation. These protocols have of the patients diagnosed with nvCJD had a history identified some ineffective treatments, for example, of beef consumption in England during the BSE epi- soaking in aldehyde. Obviously, application of uni- demic. versal precautions of asepsis and hygiene is essential, especially in anatomy/pathology laboratories and for the re-use of medical devices in neurosurgery or Pathology in ophthalmology. The molecular mechanisms underlying the patho- genesis of the human prion diseases are not under- Clinical presentation stood and there are currently no effective strategies for early diagnosis or treatment of TSEs. Although Sporadic Creutzfeldt–Jakob disease (CJD) they are diseases of the central nervous system, the Sporadic CJD is most frequently diagnosed in spleen and lymph nodes are involved in early stages patients between the ages of 50–70 years. Initial of the infection. For the infectious aetiologies such symptoms are vague and non-specific. Approxi- as nvCJD, prions are transported to the lymphoretic- mately one-third of patients present with fatigue, ular system (LRS) following infection, where they anorexia and sleep disturbance. Another third suffer replicate efficiently in the tonsils, spleen, appendix, confusion and memory loss, and other individuals and lymph nodes. The host fails to mount a clas- present with muscle wasting, motor neuron dysfunc- sic immune response. The use of transgenic and tion and aphasia. Disease progress is sustained, with- knockout technology has shown that neither B nor out remission, and is marked by cognitive deficits T cells are competent alone for prion replication. It such as dementia and behavioural abnormalities, has been suggested that uptake of prions may take the development of myoclonus and the presence of place through cellular interaction, perhaps through other neurological abnormalities such as pyramidal an immune complex-type interaction. After replica- and cerebellar dysfunction. In the terminal stage of tion in the LRS, they invade the CNS, probably via disease, patients lose voluntary muscle control and the sympathetic peripheral nervous system. So far, become mute. The mean time from onset of symp- PrPsc has not been found in the autonomic nervous toms until death is 5 months and most patients are system. There is evidence that neurons are involved dead within 1 year. in spreading the infection along specific pathways to the spinal cord and brain. The first change is abnormal accumulation of PrPsc probably on the New-variant Creutzfeldt–Jakob disease (nvCJD) surface of neurons. This is followed by release of In contrast to sporadic CJD, the mean age for PrPsc into intercellular spaces, with formation of fib- onset of symptoms in nvCJD is 29 years, substan- rils (prion rods). The abnormal protein-mediated
  • 142. Diagnosis 127change in conformation in the host’s native PrPc mouse and then monitoring the animal. Becauseleads to production of further abnormal PrPsc such tests can take up to 12 months to yield a result,conformation. Neuronal cells show vacuolization, the development of assays for rapid detection is agliosis, accumulation of the protease-resistant high research priority. Western blotting or enzyme-abnormally folded prion isoform, and cell death. The linked immunosorbent assay (ELISA) techniques aredisease has a very long incubation period of more quicker and more economical, but they are less sen-than 10 years in humans, presenting with symptoms sitive, and it is not clear whether they can be used toof dementia, ataxia and psychiatric symptoms. Once identify animals incubating the disease. These assayssymptoms are recognized, nvCJD and the other prion involve digesting infected tissue with a protease, anddiseases are fatal within a few months to 2 years. then identifying the protease-resistant prion with the Genetic disposition, as well as a helper protein (X) use of antibodies. To date (2003), the European Com-appear to be required for establishment of the infec- mission (EC) has approved three such tests: the Bio-tion. Rieger et al. (1999) suggest that Laminin recep- ` Rad test developed by the Commisariat a l’Energietor protein (LRP), a ribosomal protein and cell sur- Atomique (CEA) in France; Prionics-Check devel-face receptor for infectious agents may act as a helper oped by Prionics AG in Switzerland; and the Enfer testprotein. LRP belongs to a family of cell adhesion system developed by Enfer Technology in Ireland.molecules, and works as a receptor for alphaviruses. New regulations in Europe require that brain tissueIt is also associated with the metastatic potential of from slaughtered cattle be tested using one of thesesolid tumours. Genetic predisposition is also sug- approved tests before the animals can be sold as beef.gested by the different rates at which individuals Testing became mandatory on January 1 2001 for alldevelop nvCJD. Individuals who are homozygous for ‘at risk’ animals and as of July 1 2001, based on factmethionine at codon 129 of the PrPc gene (40% of that the incubation period for BSE ranges from 4–6the population) have been shown to be at greatest years, all cows aged over 30 months have to be tested.risk for the early development of symptomatic dis- However, the tests are based upon immunologicalease. The first case of nvCJD has been detected in a reagents, and it is apparently difficult to developheterozygous individual; although the onset of dis- reagents that reliably differentiate between the twoease may be slower in this genetic population, it is isoforms. Veterinary authorities across the EC havenot eliminated. Individuals with other amino acids at reported hundreds of false positives using these tests.codon 129 (methionine/valine or valine/valine) are A new approach was developed by Paramithiotisalso susceptible to nvCJD, but the incubation period et al. (2003), who found that hydrophobic PrP tyro-is longer. sine residues that are sequestered, and thus ‘hidden’ in native PrPc become exposed to solvent during the conformational conversion to PrpSc . This observa-Diagnosis tion suggested that a conserved tripepetide, Tyr–Tyr– Arg might become accessible to antibodies raisedDue to the potential danger of transmitting pri- against synthetic peptides containing this sequence.ons from infected but asymptomatic individuals, as Polyclonal and monoclonal antibodies raised againstwell as the risk associated with in using any human this repeat motif recognized the pathological iso-or animal-derived products used in transfusion or form but not the normal PrPc , as assessed byblood and urine pharmaceutical products, research immunoprecipitation, plate capture immunoassayinto a preclinical test for the mutated prion has and flow cytometry. Many of the polyclonal antibod-attracted significant commercial interest. The stan- ies were cross-reactive with PrPSc from mice, ham-dard experimental method used to test for TSE infec- sters, sheep, cattle and humans. The antibodies alsotivity involves inoculating a sample of infected mate- recognized misfolded but protease-sensitive PrP a ,rial (neuronal tissue) into the brain of a laboratory molecular species that has recently been identified
  • 143. 128 Prions as characteristic of certain prion strains, early prion been derived both from urine and from culture tech- infection and interspecies prion transmission. The niques that utilize serum supplements, and serum authors suggest that this protease-sensitive prion supplements are also used in embryo culture sys- may represent a transient intermediate between nor- tems. Although the risk of prion transmission in ART mal structure and the abnormal aggregated isoform may be very small, the highest possible levels of that is protease resistant. This new approach, using safety must be maintained by continued care and antibodies against conformation-selective exposure vigilance, both by manufacturers of pharmaceuticals of peptide sequences, may provide tools for diag- and supplies, and by healthcare personnel involved nostics, research and therapies in the future. Non- in their use. immunological tests are also being investigated, including the use of plasminogen, which sticks to rogue but not normal protein (Fischer et al., 2000). REFERENCES Miele et al. (2001) reported that EDRF protein, an erythroid-specific marker, may act as a diagnostic Davenport, R. J. (2001). Getting yeast prions to bridge the species marker for infection in cattle; this possibility is a sub- gap. Science, 291: 1881. ject of further ongoing research. Fischer, M. B., Roecki, C., Parizek, H. P. S. & Aguzzi, A. (2000). For the diagnosis of human disease, CSF may be Binding of disease-associated prion protein to plasmino- tested for the detection of 14-3-3 protein, which gen. Nature (London), 408: 479–83. appears to act as a marker of neuronal cell death. Gadjusek, D. C. & Zigas, V. (1957), Degenerative disease of the This assay detects the beta isoform of the protein central nervous system in New Guinea: the endemic occur- using antibody-based technologies (ELISA or West- rence of ‘kuru’ in the native population, New England Jour- ern blot). Although detection of the 14-3-3 beta iso- nal of Medicine, 257, 974–8. form is very sensitive in the evaluation of demen- Krakauer, D. C., de Zanotto, P. M. & Pagel, M. (1998). Prion’s tia, including Alzheimer’s disease, patients with HSV progress: patterns and rates of molecular evolution in encephalitis may produce some false positive results. relation to spongiform disease. Journal of Molecular Evo- lution, 47: 133–45. Tissue diagnosis of CJD demonstrates characteristic Mestel, R. (1996). Putting prions to the test. Science, 273: spongiform changes in the brain. In sporadic CJD, 184–9. amyloid plaques may be seen in the brain tissue, Miele, G., Manson, J. & Clinton, M.