Ananthanarayan and Paniker's Textbook of Microbiology [10th, 10 ed.] 9386235250, 9789386235251

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Contents Preface to the Tenth Edition

viii

Preface to the First Edition Acknowledgements

ix xi

Special Acknowledgements

xii

Part I

General Microbiology ~ I n troduction and Bacterial Taxonomy 2.Morphology and Physiology of Bacteria

3 9

..a--s'terilisation and Disinfection

28

4Culture Media

39

5culture Methods

44

Vldentification of Bacteria

48

7 bacterial Genetics

54

Part II

Immunology 8

Infection

~ Immunity ,..-10(Antigens

73

80

89

11

Antibodies-lmmuno globulins

95

12

Antigen-Antibody Reactions

105

13

Complement System

122

1 4 s t r uand c tFunctions u r e of the Immune System

130

15

Immune Response

147

16

Hypersensitivity

163

17

Immunodeficiency Diseases

173

18

Autoimmunity

180

19

Immunology of Transplantation and Malignancy

185

20

lmmunohematology

193

Contents

Part 111

Bacteriology 21 staphylococcus 22 streptococc us

is,,-- Corynebacterium Bacillus

_.:g,-

Anaerobic Bacteria I: Clostridium Bacteria II: Non-sporing Anaerobes / An Anaerobic 28 29 Enterobacteriaceae I: Coliforms-Proteus ../

/ ,...JO' Enterobacteriaceae II: Shigella Enterobacteriaceae Ill: Salmonella

M

v' 32 vibrio 33 Pseudomonas 34 Yersinia, Pasteurella, Francisella

35

Haemophilus

36

Bordetella

37

Brucella

v31f Mycobacterium I: M.tuberculosis

J.9""' Mycobacterium II: Non-Tuberculous Mycobacteria (NTM) Ill: M.leprae 4 ¥ • Spirochetes

Part IV

210

223

23 pneumococcus {1:/Neisseria

26

201

230 239 248 256 273 279 291 296 309 320 325 333 339 345 351 366 371

377

42 43

Actinomycetes

44

Miscellaneous Bacteria

393 398 402

45

Rickettsiaceae

412

46

Chlamydiae

422

Mycoplasma

Virology 47 48 49 50 51 52 53 54

General Properties of Viruses Virus-Host Interactions: Viral Infections Bacteriophages Poxviruses Herpesviruses Adenoviruses Picornavi ruses Orthomyxoviruses

433 450 462 467 472 486 490 502

Contents

vii

55

Paramyxoviruses

56

Arthropod- and Rodent-borne Viral Infections

57

Rhabdoviruses

512 522 534

58

Hepatitis Viruses

544

59

Miscellaneous Viruses

557

60 Oncogenic Viruses 61 Human Immunodeficiency Virus: AIDS

568 574

Medical Mycology 62 General Aspects 63 Superficial and Subcutaneous Mycoses 64 Systemic and Opportunistic Mycoses

Part VI

Applied Microbiology 65 66 67

Part VII

593 599 609

Normal Microbial Flora of the Human Body Bacteriology of Water, Air, Milk and Food Laboratory Control of Antimicrobial Therapy

68 69 70

Biomedical Waste Management

71

Emerging and Re-emerging Infections

72

Recent Advances in Diagnostic Microbiology

lmmunoprophylaxis Healthcare-associated Infections

625 629 639 643 648 657 660 663

Clinical Microbiology 73

Bloodstream Infections

669

74

Respiratory Tract lnfections

672

75 Meningitis

vs/'

76 urinary Tract Infections

675

78

Diarrhea and Food Poisoning ••

79

Skin and Soft Tissue Infections

678 681 684 687

80

Pyrexia of Unknown Origin

689

81

Zoonoses

692

82

Principles of Laboratory Diagnosis of Infectious Diseases

694

77 Jsexually Transmitted Infections

f

Further Reading

698

Index

700

Preface to the Tenth Edition authors The tenth edition of Ananthanarayan and Paniker's Textbook of Microbiology upholds the vision of the pioneering cases clinical including by contents the in changes in brought editions Previous Dr R Ananthanarayan and Dr CK Jayaram Paniker. information, new of deluge the with However, . diseases infectious to pertinent microbiology and relevant to individual organisms, constant compounded with the challenges in contending with infectious diseases and the rapid evolution of microorganisms, there is a diseases, infectious of domain the to subject sed need to revise existing knowledge. As microbiology moves from a laboratory-ba relevant students need to reorient themselves from the concept of microbiology as a non-clinical speciality to that of a clinically quired community-ac and ociated healthcare-ass to diseases infectious in concepts basic from subject, with applications ranging challenges. health public other and management, epidemic and outbreaks prevention, and detection disease infections, applied The Medical Council of India has emphasised the need for integrated teaching, underscoring the requirement for concepts. and themes basic their retaining while reviewed, microbiology. Keeping this in mind, the chapters have been thoroughly Some of the significant updates are listed below. ew concepts in sterilisation and disinfection, including plasma sterilisation and practices in healthcare settings • ew and automated methods for identification of bacteria • • Updated molecular techniques as applied to microbiology and their • Simple diagrammatic presentations of current immunological techniques for antigen and antibody detection, applications • Clinical implications of bacterial organisms, current methods of detection, and suggested antibiotic treatment • Salient features of the Revised National Tuberculosis Program (RNTCP) • Strategies for diagnosis of MDR and XDR tuberculosis, and the STOP TB strategy of WHO the Ebola • New and emerging viral infections such as SARS, MERS-CoV, influenza epidemics, the Zika virus outbreak, and outbreak • NACO guidelines for HIV testing strategies for different categories of the population, and HIV exposure and source codes and • Latest vaccines for immunisation against childhood infections in India, including Rotavirus, Haemophilus influenzae pneumococci pictorial • Healthcare-associated infections leading to CAUTI, VAP, HCA-BSI and SSI, and strategies for prevention with representations • Biomedical waste management rules (2016) • Recent advances in diagnostic microbiology and the work flow in a clinical microbiology laboratory • Quality control and accreditation of diagnostic tests performed by laboratories • Easy-to-unde rstand line diagrams • Flowcharts to make the conceptualisation of processes easy to comprehend by the undergraduat e student • Relevant points boxed as highlights • Recaps updated and retained for quick review by the students right Universities Press and its editorial team deserve special appreciation for the meticulous and methodical editing process book. the of edges rough the fine-tune to helped from the time I took up the assignment. Constant communication and interaction valuable My heartfelt thanks to Dr Sudha Ganesan for her timely feedback, and to Mr Madhu Reddy and Ms Aathira Varma for their pursuing those to and microbiology, of students the to helpful be will edition revised this in made changes inputs. It is hoped that the infectious diseases. We welcome suggestions for further improvement of the book in subsequent editions. Reba Kanungo

Preface to the First Edition Many of the health problems in developing countries like India are different from those of developed countries. Bacterial diseases still play a considerable role in our country. Topics such as cholera and enteric diseases are important to us though only of less or academic interest to the developed countries. The increasing importance of the newer knowledge in immunology to health and disease is not adequately stressed in most of the extant textbooks. Virus diseases which are responsible for nearly 60 per cent of human illness require wider coverage. The general approach to the teaching of microbiology in our country has also been rather static. All these factors called for a textbook of microbiology more suited to countries like India. We therefore undertook this endeavour based on our experience of teachi ng undergraduates and postgraduates for over two decades. We omitted the discipline of parasitology from our book since we already have an excellent textbook on the subject published in India. This book has taken us over three years to write and over a year in publication. Naturally we would be out of date to a certain and inevitable extent. We do not claim any perfection. On the contrary, we have requested medical students and teachers all over the country to write to us about any shortcomings and give us suggestions as to how to improve the book. We shall spare no pains in seeing that their valuable suggestions are given effect to in our second edition.

R Ananthanarayan CK Jayaram Paniker

Acknowledgements For kind permission to use photographs, the publishers are indebted to the following institutions and individuals: Lister Metropolis Laboratory and Research Centre Pvt. Ltd., Chennai, for figures 32.2, 38.3, 43 .1a, 43. 1b, 4 7 .6, 64.8a; Sudha Ganga! and Shubhangi Sontakke, for figures 10. la, 10.l b, 10.2, I 1.1, 11.2b, 11.3, 11.6, 12. lOb, 13 .1, 13.4, 14.6, 14.8, from Textbook of Basic and Clinical Immunology (2013) published by Universities Press India Pvt Ltd. ; Department of Microbiology, Nizam's Institute of Medical Sciences, Hyderabad, for figures 5.3a, 5.3b, 5.3c, 21.4, 23.1, 26.4, 28.1 , 61.4, 64.1O; Dr Ratna Rao, Senior Consultant Microbiologist, Apollo Hospitals, Jubilee Hills, Hyderabad for figures 2.3a, 2.3b, 2.3c, 2.3d, 21.1 , 38.1, 64.9b; Dr Swarajya Lakshmi, Assistant Professor, Department of Microbiology, MNR Medical College, Andhra Pradesh for figure 64.9a; National Institute of Virology, Pune, for figures 56.1, 56.3; Dr Pallab Ray, Additional Professor, Department of Medical Microbiology, PGIMER, Chandigarh, for figures 6.7, 27.3; Dr Asha Mary Abraham, Professor, Department of Clinical Virology, Christian Medical College, Vellore, for figures 4 7. 7, 52.2a, 52.2b; Dr G Sasikala, Professor of Microbiology, STD Regional Laboratory, Osmania General Hospital, Hyderabad, for figures 27.6, 27.2, 32.3, 33.1 , 38.2, 44.1, 58.1; Dr Subha Parameswaran, Professor and Head, Department of Microbiology, K J Somaiya Medical College, Mumbai, for figures 36.1, 41.2, 51 .3, 51.4, 64.8b; Dr Uma Tendolkar, Department of Microbiology, Lokmanya Tilak Municipal Medical College, Mumbai, for figure 51.5; Belnap, D M, McDermott, 8 M, Jr, Filman, DJ, Cheng, N, Trus, 8 L, Zuccola, HJ, Racaniello, V R, Hogle, J M, and Steven, AC (2000), for figure 53 . 1; World Health Organization, Global Programme for Vaccines and Immunization EPI, for figure 53.2; Centers for Disease Control and Prevention Archives for figure 34.2; FA Murphy, University of Texas Medical Branch, Galveston, Texas, for figures 59.2, 59.3 ; Prof. L. Dar, Department of Microbiology, Al/MS, New Delhi for figure 57.3; Prof. MC Sharma, Department of Microbiology, Al/MS, New Delhi for figure 43.1 (a). www.wikimedia.org for figures I.I, 1.2, 1.3, 1.4, 48.1 and 57.3; www.purifiers.co.za for figure 3.4a; www.pharmlabs.unc.edu for figure 3.4b; www.healthessentials4you.com for figure 3.4c; www.tjclarkinc.com for figure 25.1 ; www.webak.upce.cz for figure 31.1; www.home.kku.ac.th for figure 33.2; www.michigan.gov for figure 38.4; www.leprosyhistory.org for figure 40.1; www.bilkent.edu.tr for figure 40.2b; www.granuloma.homestead.com for figure 40.2a; www.farm1.static.flickr.com for figure 52.2; www.umanitoba.ca for figure 47.3 ; www.biochem.wisc.edu for figure 49.2; www.zunsanwong.blogspot.com for figure 59.4; www.med.ncku.edu.tw for figure 63.2 Every effort has been made to contact holders of copyright to obtain permission to reproduce copyright material. However, if any have been inadvertently overlooked, or have credited the wrong source, the publishers, on information, will be pleased to rectify the error at the first opportunity. Images for which such permission was awaited at the time of publication will be replaced in subsequent editions if such permission is not granted.

Special Acknowledgements For their most helpful suggestions in the formulation of this edition, the publishers are grateful to Dr Anuradha Makkar, Professor and Head, Department of Microbiology, Army College of Medical Sciences, New Delhi; Dr Prerna Bhalla, Professor, Department of Microbiology, Hindu Rao Medical College, New Delhi (former Head, Maulana Azad Medical College, New Delhi) ; Dr A K Praharaj, Professor and Head, Department of Microbiology, AIIMS , Bhubaneswar, Odisha; Dr Rajni Gaind, Professor and Head, Department of Microbiology, VMMC, New Delhi; Dr Sai Leela Kondapanerti, Professor and Head, Department of Microbiology, KIMS , Hyderabad. For valuable feedback and help in developing the MCQs for the tenth edition, we are profoundly thankful to Dr Sarada Devi K.L, Professor and Head, Department of Microbiology, GMC, Thiruvananthapuram, and her team members Dr Deena Philomina, Professor and Head, GMC Kozhikode; Dr Anitha PM, Additional Professor, GMC Kozhikode and Dr Shabina MB, Associate Professor, GMC Kozhikode. Our special thanks are also due to Dr G Jyothi Lakshmi, Professor, Department of Microbiology, Osmania Medical College, Hyderabad; Lieutenant Colonel Dr N andita Hazra, Sr Adv(Pathology and Microbiology) and Head, Department of Laboratory Medicine, Command Hospital (Central Command) , Lucknow; Dr Thyagarajan Ravinder, Professor and Head, Department of Microbiology, Kilpauk Medical College, Chennai; Dr S Manick Dass, Professor and Head, Department of Microbiology, Apollo Medical College, Hyderabad; Dr G Jayalakshmi, Director and Professor, Department of Microbiology, Madras Medical College, Chennai; Dr Mary Mathews, Retired Professor and Head, Department of Microbiology, Christian Medical College, Vellore.

Part I

General Microbiology 1

Introduction and Bacterial Taxonomy

3

2

Morphology and Physiology of Bacteria

9

3

Sterilisation and Disinfection

28

4

Culture Media

39

5

Culture Methods

44

6

Identification of Bacteria

48

7

Bacterial Genetics

54

Introduction and Bacterial Taxonomy HISTORY CLASSIFICATION, NOMENCLATURE AND TAXONOMY Bacterial classification Nomenclature Type cultures

HISTORY Medical microbiology is the study of microbes that infect humans, the diseases they cause, and their diagnosis, prevention and treatment. It also deals with the response of the human host to microbial and other antigens. As microbes are invisible to the unaided eye, definitive knowledge about them had to await the development of the microscope. The credit for having first observed and reported bacteria belongs to Antony van Leeuwenhoek, a draper in Delft, Holland, whose hobby was grinding lenses and observing diverse materials through them (Fig. 1.1 ) . In fact, even before the microbial cause of infections had been established, Ignaz Semmelweis in Vienna (1846) had independently concluded that puerperal sepsis was contagious. Semmelweis also identified its mode of transmission by doctors and medical students

Fig. 1.1

Antony van Leeuwenhoek

attending on women in labour in the hospital and had prevented it by the simple measure of washing his hands in an antiseptic solution, for which service to medicine and humanity, he was persecuted by medical orthodoxy and driven insane. The development of microbiology as a scientific discipline dates from Louis Pasteu.r 1822-95) introduced techniques ot &_terilisatjon and develope the d steam steriliser, hot-air oven and autoclave.3 e also established the d i f f e growth ring needs of different bacteria and contriEuted to the knowledge hy'crrophobia. accidental observation that chicken -cholera

~ ~ lost their to prot

for s

t

of th

of attenuation

and the developmentof live vaccinattenuated es cultures of the anthrax bacillus by incubation at high temperature- (~43°C) and pr~d that inoculation of such cultures_in animals induced specific protection against anthrax. It was Pasteur who coined the term vaccine for such prophylactic preparations to commemorate the first of such preparations, namely cowpox, employed by Edward Jenne or protection against smallpox. The greatest impact on medicine was made by Pasteur's development of a vaccine for hydrophobia.

Fig. 1.2

Louis Pasteur

Part I

GENERAL MICROBIOLOGY

An immediate application of Pasteur's work was the introduction of antiseptic techniques in surgery by Joseph Lister (186 7) , effecting a pronounced drop in mortality and morbidity due to surgical sepsis. Lister's antiseptic surgery involving the use of carbolic acid was a milestone in the evolution of surgical practice, from the era of 'laudable pus' to modern aseptic techniques. While Pasteur in France laid the foundations of microbiology, Robert Koch (184 3-1910) in Germany perfected bacteriological techniques duringffjts stud ies on the culture and _life cycle pf the anthrax baciltechniques and W.. (1876). He introduced.Stain ing methods of obtaining bacteriajn pure culture using solid media (Fig. 1.3). e discovered the bacillus of tuberculosis ( 1882) and the cholera vibrio ( 1883). Roux and yersin (1888) identified a new mechanism of pathogenesis when they discovered the diphtheria toxin. Similar toxins were identified in tetanus and some other bacteria. The toxins were found to be

specifically neutralised by their antitoxins. Paul Ehrlich who studied toxins and antitoxins in quantitative terms laid the foundations of biological standardisation (Fig. 1.4) . Ernst Ru ska (1934) developed the electron microscope, enabling visualisation of the microbes we now call viruses. The development of tissue culture techniques has permitted the cultivation of viruses. The causative agents of various infectious diseases were being reported by different investigators in such profusion that it became necessary to introduce criteria for proving the claims that a microorganism isolated from a disease was indeed causally related to it. These criteria were enunciated by Koch and are known as Koch's postulates. According to these, a microorganism can be accepted as the causative agent of an infectious disease only if the following conditions are satisfied The bacterium should be constantly associated with the lesions of the disease It should be possible to isolate the bacterium in pyre culture from the lesions . of such pure culture into suitable inoculation laboratory animals should reproduce lesions of the disease. --->1t should be possible to re-isolate the bacterium in pure culture from the lesions produced in experimental animals, An additional criterion introduced subsequently requires that specific antibodies to the bacterium should be demonstrable in the serum of patients suffering from the disease. Though it may not always be possible to satisfy all the postulates in every case, they have proved extremely useful in sifting doubtful claims made regarding the causative agents of infectious diseases. The study of immunity had to await advances in protein chemistry. The pioneering work of Karl Landsteiner laid the foundations of immunochemistry. In 1955, Niels Jerne proposed the natural selection theory of antibody synthesis which attempted to explain the chemical specificity and biological basis of antibody synthesis, signifying a return to the original views of antibody formation proposed by Ehrlich (1898). Frank Burnet (195 7) modified this into the clonal selection theory, a concept which, with minor alterations, holds sway even now. The last few decades have witnessed an explosion of conceptual and technical advances in immunology. Immunological processes in health and disease are now better understood following

---

Flg. 1.3 Robert koch

Fig. 1.4

Paul Ehrlich

Introduction and Bacterial Taxonomy

the identification of the two components of immunity-humoral or antibody-mediated processes and cellular or cell-mediated processes-which develop and manifest in separate pathways, Alexander Fleming (1929) made the accidental discovery that the fungus Penicillium produces a substance that destroys staphylococci. Work on this at Oxford by Florey, Chain and their team during the Second World War led to the isolation of the active substance penicillin and its subsequent mass production, This was the beginning of the antibiotic era, Other similar antibiotics were discovered in rapid succession, With the sudden availability of a wide range of antibiotics with potent antibacterial activity, it was hoped that bacterial infections would be controlled within a short period, But soon the development of drug resistance in bacteria presented serious difficulties, With the development of a wide variety of antibiotics active against the whole spectrum of pathogenic bacteria, and of effective vaccines against most viral diseases, expectations were raised about the eventual elimination of all infectious diseases. The global eradication of smallpox inspired visions of similar campaigns against other major pestilences. However, when new infectious diseases began to appear, caused by hitherto unknown microorganisms, or by known microbes producing novel manifestations, it was realised that controlling microbes was a far more difficult task than was imagined , The climax came in 1981 when AIDS was identified in the USA and began its pandemic spread, Unceasing vigil is essential to protect humans from microbes, Apart from obvious benefits such as specific methods of diagnosis, prevention and control of infectious diseases, medical microbiology has contributed to scientific knowledge and human welfare in many other ways, Microorganisms constitute the smallest forms of living beings and, therefore, have been employed as models of studies on genetics and biochemistry, As nature's laws are universal in application, information derived from the investigation of microbes holds true, in the main, for humans as well, Studies on microorganisms have contributed, more than anything else, to the unravelling of the genetic code and other mysteries of biology at the molecular level. Bacteria and their plasmids, yeasts and viruses are routinely employed as vectors in recombinant DNA technology. They have made available precious information and powerful techniques for genetic

'

,5 ,, ,

.

manipulation and molecular engineering. They need to be used wisely and well for the benefit of all living beings. The number of Nobel laureates in Medicine and Physiology awarded the prize for their work in microbiology, listed in Table I. l , is evidence of the positive contribution made to human health by the science of microbiology.

CLASSIFICATION, NOMENCLATURE AND TAXONOMY All organisms are classified primarily to enable easy identification, all classification systems aim to group organisms with similar properties and to separate those that are different. The basic taxonomic unit in bacteria is the species; two species differ from one another in several features determined by genes. The method most widely adopted is presented in successive editions of Bergey's Manual of Determinative Bacteriology. Bacterial taxonomy or systematics comprises three components: • Classification, or the orderly arrangement of units. A group of units is called a taxon (pl taxa) , irrespective of its hierarchic level. • Identification of an unknown with a defined and named unit. • Nomenclature, or the naming of units.

Bacterial classification A kingdom is divided successively into division, class, order, family, tribe, genus and species. An important difference between the classification of bacteria and that of other organisms is that in the former, the properties of a population are studied, and not of an individual. • A population derived by binary fission from a single cell is called a clone. • A single bacterial colony represents a clone. Though all the cells in a clone are expected to be identical in all respects, a few of them may show differences due to mutation. • A population of bacteria derived from a particular source, such as a patient, is called a strain. The general absence of sexual reproduction in bacteria serves to keep their character constant. But bacteria possess several features that contribute to some degree of heterogeneity in their populations. Their short generation time and high rate of mutation

Part I Table 1.1

GENERAL MICROBIOLOGY

Nobel laureates in Physiology and Medicine

1901 Emil Avon Behring 1902 Ronald Ross 1905 Robert Koch 1907 CL A Laveran 1908 P Ehrlich and E Metchnikof 1913 Charles Robert Richet 1919 Jules Bordet 1926 Johannes Fibiger 1928 Charles Nicolle 1930 Karl Landsteiner 1939 Gerhard Domagk 1945 A Fleming, E Boris Chain and Howard Walter Florey 1951 Max Theiler 1952 Selman A Waksman 1954 Franklin Enders, T H Weller and F C Robbins 1958 George Beadle & E L Tatum and J Lederberg 1960 FM Burnet and P B Medawar 1965 Francois Jacob, Andre Lwoff and Jacques Monod 1966 Peyton Rous 1969 M Delbruck, AD Hershey and S E Luria 1972 1975

Gerald M Edelman and Rodney R Porter D Baltimore, R Dulbecco and H Martin Temi n

1976

Baruch S Blumberg and D Carleton Gajdusek

1978 1980 1984 1987 1989 1996 1997 2005 2008

Werner Arber, Daniel Nathans and Hamilton O Smith Baruj Benacerraf, Jean Dausset and George D Snell Niels K Jerne, Georges J F Kohler and Cesar Milstein Susumu Tonegawa J Michael Bishop and Harold E Varmus Peter C Doherty and Rolf M Zinkernagel Stanley B Prusiner Barry J Marshall and J Robin Warren Harald Hausen and Francoise Barre-Sand L Montagnier

2011

Bruce A Beutler, Jules A Hoffmann, Ralph M Steinman

2012

Sir John B Gurdon, Shinya Yamanaka

2015

William C Campbell and Satoshi Omura Youyou Tu

lead to the presence, in any population, of cells with altered characters. Methods of genetic exchange such as transformation, transduction and conjugation cause differences in character. Prophage and plasmid DNA can induce new properties. Phylogenetic classification: The hierarchical classification represents a branching tree-like arrangement, one characteristic being employed for division at each branch or level. This system is called phylogenetic because it implies an evolutionary arrangement of

Serum therapy Malaria Tuberculosis Role of Protozoa in causing diseases Immunity Anaphylaxis Immunity Spiroptera carcinoma Typhus Discovery of human blood groups Prontosil Penicillin Yellow fever Streptomycin Poliomyelitis growth in tissue Gene action and genetic recombination Acquired immunological tolerance Genetic control of enzymes Tumour-inducing viruses Replication mechanism and the genetic struct ure of vi ruses Chemical structure of antibodies Interaction between tumour viruses and the genetic material of the cell New mechanisms of infectious disease dissemination Restriction enzymes Immunological regulation by cell surface Control of immune system and monoclonal antibodies Generation of antibody diversity Origin of retroviral oncogenes Specificity in cell mediated immune defence Prions Helicobacter pylori Human papi lloma viruses, human immunodeficiency virus Activation of innate immunity,-and the dendritic cell and its role in adaptive immunity Mature cells can be reprogrammed to become pl uripotent Discoveries concerning a novel therapy against infections caused by roundworm parasites Discoveries concerning a novel therapy against Malaria

species. Here some characteristics are arbitrarily given special weightage. Depending on the characteristic so chosen, the classification would give differ ent patterns . While classification based on a weighted characteristic is a convenient method, it has the serious drawback that the characters used may not be valid. Fermentation of lactose, in the example cited, is not an essential and permanent characteristic. It may be acquired or lost, upsetting the system of arrangement.

Introduction and Bacterial Taxonomy Percentage similarity

40

50

60

70

80

90 100 Strains

A B E

..-+--+- C .----t-----t-

F

-_ -+ - JG .___ _ _ D H X

y

Fig. 1.5 Adansonian classification

Adansonian classification: This avoids the use of weighted characteristics (Fig. 1.5) . It takes into account all the characteristics expressed at the time of study. The availability of computers has extended the scope by permitting comparison of very large numbers of properties of several organisms at the same time. This is known as numerical taxonomy. Molecular or genetic classification: This is based on the degree of genetic relatedness of different organisms. Since all properties are ultimately based on the genes present, this classification is said to be the most natural or fundamental method. DNA relatedness can be tested by studying the nucleotide sequences of DNA and by DNA hybridisation or recombination methods. The nucleotide base composition and base ratio (adenine-thymine: guanine-cytosine ratio) varies widely among different groups of microorganisms, though it is constant for members of the same species. Molecular classification has been employed more with viruses than with bacteria. lntraspecies classification: For diagnostic or epidemiological purposes, it is often necessary to subclassify bacterial species. This may be based on biochemical properties (biotypes) , antigenic features (serotypes) , bacteriophage susceptibility (phage types) or production of bacteriocins (colicin types). A species may be

divided first into groups and then into types, as for example, in streptococci. Much greater discrimination in intraspecies typing has been achieved by the application of newer techniques from immunology, biochemistry and genetics. Investigations of epidemiology and pathogenesis using these techniques have been collectively referred to as molecular epidemiology. The methods used are of two types: phenotypic (study of expressed characteristics) and genotypic (direct analysis of genes, chromosomal and extrachromosomal DNA). Molecular phenotypic methods include electrophoretic typing of bacterial proteins and immunoblotting. Genotypic methods include plasmid profile analysis, restriction endonuclease analysis of chromosomal DNA with Southern blotting, PCR and nucleotide sequence analysis.

Nomenclature The need for applying generally accepted names for bacterial species is self-evident. The scientific name usually consists of two words, the first being the name of the genus and the second the specific epithet (for example, Bacillus subtilis). The generic name is usually a Latin noun. The specific epithet is an adjective or noun and indicates some property of the species (for example, albus, meaning white), the animal in which it is found (for example, suis, means pig) , the disease it causes (tetani , of tetanus) , the person who discovered it (welchii, after Welch) or the place of its isolation (London). The generic name always begins with a capital letter and the specific epithet with a small letter, even if it refers to a person or place (for example, Salmonella London).

Type cultures As a point of reference, type cultures of bacteria are maintained in international reference laboratories. The type cultures contain representatives of all established species. The original cultures of any new species described are deposited in type collections. They are made available by the reference laboratories to other workers for study and comparison.

Part I GENERAL MICROBIOLOGY

RECAP • • • •

• • • • • •

•

Over the centuries, the experiments and work of a number of individuals from many different countries have provided a scientific basis to the study of diseases. Louis Pasteur (1822-1895) discovered methods of sterilisation and developed methods for culture of microbes, showed that microorganisms cause disease and established the principles of immunisation. Joseph Lister (1827-1912) introduced 'antisepsis', wherein he sprayed the patient and operating field with carbolic acid . Robert Koch (1843-1910) defined the criteria used to attribute a disease to an organism (Koch's postulates): ❖ The organism must be found in all cases of the disease; the distribution in the body should correspond to that of lesions observed. ❖ The organism should be cultured outside the body in pure culture for several generations. ❖ The organism should reproduce the disease in other susceptible animals. ❖ The organism should be isolated in pure culture from the lesion in animals. (an additional postulate, not fo rmulated by Koch, is added-specific antibody to the organism should develop during the course of the disease). Ruska (1934) developed the electron microscope enabling visualisation of the microbes we now call viruses. The development of tissue culture techniques has permitted the cultivation of viruses. Roux and Yersin (1888) demonstrated that the harmful effects of diphtheria are caused by the exotoxin produced by Corynebacterium diphtheriae during its growth. Paul Ehrlich (1854-1915) was a pioneer in the study of antitoxin and toxin neutralisation. Sir Alexander Fleming (1885-1955) discovered that the fungus Penicillium produces a substance, penicillin, that destroys staphylococci; this discovery led to the formulation of other antimicrobials. Classification is t he arrangement of organisms into related groups or taxa; taxonomy is the science of classification. Since all organisms are classified primarily to enable easy identification, all classification systems aim to group organisms with similar properties and to separate those that are different. However, the bes_t classification schemes are those that are based on evolutionary relatedness. The basic taxonomic unit in bacteria is the species; two species differ from one another in several features determined by genes.

SHORT NOTES

1. 2. 3. 4. 5. 6.

Robert Koch Louis Pasteur Paul Ehrlich Joseph Lister Koch 's postulates Bacterial classification

Morphology and Physiology of Bacteria MORPHOLOGY OF BACTERIA SIZE OF BACTERIA MICROSCOPY STAINING TECHNIQUES Gram stain Acid fast stain

SHAPE OF BACTERIA BACTERIAL ANATOMY Cell wall Cytoplasmic membrane Cytoplasm Ribosomes Mesosomes (chondroids) lntracytoplasmic inclusions Nucleus Slime layer and capsule Flagella Fimbriae Spore Pleomorphism and involution forms L forms

cation under the Plant and Animal kingdoms proved unsatisfactory; they were then classified under a third kingdom, Protista. Based on differences in cellular organisation and biochemistry, this kingdom has been divided into two groups: prokaryotes and eukaryotes (Table 2.1 ). Bacteria and blue-green algae are prokaryotes, while fungi, other algae, slime moulds and protozoa are eukaryotes (Fig. 2.1 ) . Bacteria are prokaryotic microorganisms that do not contain chlorophyll. They are unicellular and do not show true branching, except in the so-called 'highi::r bacteria' (actinomycetales).

MORPHOLOGY OF BACTERIA SIZE OF BACTERIA The unit of measurement used in bacteriology is the .micron (micrometre, µm) . Prokaryote Single, circular chromosome

Cell wall

PHYSIOLOGY OF BACTERIA GROWTH AND MULTIPLICATION OF BACTERIA Cell division Growth Bacterial growth curve Bacterial counts

Plasmid

Eukaryote

*

Mitochondrion (site of cellular respiration)

BACTERIAL NUTRITION Factors that affect growth

BACTERIOCINS

~ - ~-

--=:>t-"- Nuclear membrane

Lysosome Cytoplasm

INTRODUCTION

Rough endoplasmic reticulum (ribosomes)

Smooth endoplasmic reticulum Golgi apparatus

Microorganisms are a heterogeneous grou_p of several distinct classes of living beings. The original classifi-

Fig. 2.1 prokaryote and eukaryote cells

Part I

GENERAL MICROBIOLOGY

Some differences between prokaryotic and eukaryotic cells

Table 2.1

Nucleus Nuclear membrane

nucleolus ~xyribonucleoprot ein Chromosome

Absent Absent Ab_sent One (circular)

Present Present Present More than one (linear) Present

MICROSCOPY The morphological study of bacteria requires the use of microscopes. Microscopy has come a long way since Leeuwenhoek first observed bacteria over three hundred years ago using hand-ground lenses. The following types of microscopes are in use today:

Optical or light microscope: Bacteria may be examined under the compound microscope, either in the Absent Mitotic diyision living state or after fixation and staining. Examination cytoplasm Present of wet films or 'hanging drops' indicates the shape, Absent ytoplasmic streaming Present Absent pinocytosis arrangement, motility and approximate size of the cells. Present Absent mitochondria But due to lack of contrast, details cannot be appreciPresent Absent \.j:ysosomes ated (Fig. 2.2a). Present Absent apparatus golgi Present Absent '--'Endoplasmic reticulum Phase contrast microscopy: This improves the contrast and makes evident the structures within cells Chemical composition Present Absent Sterols that differ in thickness or refractive index. Also, the Present ent abs muramic acid in refractive index between bacterial cells differences and the surrounding medium make them clearly visible. Retardation, by a fraction of a wavelength, of the 1 micron (µ) or micrometre (µm) = one thousandth rays of light that pass through the object, compared of a millimetre to the rays passing through the surrounding medium, 1 millimicron (mµ) or nanometre (nm) = one thouproduces 'phase' differences between the two types of sandth of a micron or one millionth of a millimetre rays. In the phase contrast microscope, 'phase' dif1 Angstrom unit (A) = one tenth of a nanometre ferences are converted into differences in intensity of The limit of resolution with the unaided eye is about light, producing light and dark contrast in the image. 200 microns. Bacteria, being much smaller, can be visFluorescent microscope: This uses light of a high ualised only under magnification. Bacteria of medical source which excites a fluorescent agent intensity importance generally measure 0.2-1.5 µmin diameter . -===which in turn emits a low energy light of a longer wave,and about 3- 5 µmin length.

Ocular lens

Ocular lens

Condenser lens

Fig. 2.2 (a) Principle of bright-field (light) microscopy

Morphology and Physiology of Bacteria

.

length that produces the image. The fluorescent light can be separated from the su,rrounding radiation using filters designedfor that specific wavelength, allowing the viewer to see only that which shows fluorescence. Microorganisms in a specimen can be stained with a fl uorescent dye. On exposure to excitation light, organisms are visually detected by the emission of

fluorescent light by the dye with which they have been stained (Fig. 2.2b). This can be of two ~ : fluorochroming and immunofluorescence. Fluorochroming involves the non-specific staining of any bacterial cell with •a. fluorescent dye. immunofluorescen ce uses antibodies labelled with fluorescent dye (a conjugate) to specifically stain a particular bacterial species (Fig. 2.2c). Dark field( Darkground microscope: Another method of improving the contrast is the dark field (dark ground) microscope in which reflected light is used instead of the transmitted light used in the ordinary microscope. The essential part of the dark field microscope is the dark field condenser with a central circular stop, which illuminates the object with a cone of light, without letting any ray of light fall directly on the objective lens. Light rays falling on the object are reflected or scattered on to the objective lens, with the result that the object appears self-luminous against a dark background. The contrast gives an illusion of increased resolution, so that very slender organisms such as spirochetes, not visible under ordinary illumination, can be clearly seen under the dark field microscope (Fig. 2.2d). The resolving power of the light microscope is limited by the wavelength of light. In order to be seen and delineated (resolved) , an object has to have a size of approximately half the wavelength of the light used . With visible light, using the best optical systems, the limit of resolution is about 300 nm. If light of shorter wavelength is employed, as in the

Barrier filter Fluorescent ---- - light

Light source

Lightwave ,__ _ splitting mirror Exciter filter Excitation light

Specimen (Conta ins microorganisms stained with fluorochrome) -

(b)

~

Fluorochroming

Target bacteria to be stained

Staining results

....

ooo oOO 9

Fluorescent dye

All bacteria stain and fluoresce Antigen

lmmunofluorescence

+ Specific antibody

(

)

(c)

Fig. 2.2 (b) Principle of fluorescent microscopy; (c) Principles of fluorochroming and immunofluoresence

Part I GENERAL MICROBIOLOGY

Light that - + - strikes specimen

-Objective lens

Specimen

ring

Gas molecules scatter electrons, and it is therefore necessary to examine the object in a vacuum. Hence, only dead and dried objects can be examined in the electron microscope. This may lead to considerable distortion in cell morphology. A method introduced to overcome this disadvantage is freeze-etching, involving the deep-freezing of specimens in a liquid gas and the subsequent formation of carbon-platinum replicas of the material. Since such frozen cells may remain viable, it is claimed that freeze-etching enables the study of the cellular ultrastructure as it appears in the living state. The recent development of very high voltage electron microscopes may eventually render the examination of live objects possible. The scanning electron microscope is a useful innovation that permits the study of cell surfaces with greater contrast and higher resolution than with the shadow-casting technique.

Fig. 2.2 (d) Dark field microscopy

STAINING TECHNIQUES ultraviolet microscope, the resolving power can be proportionately extended. Two specialised types of microscopes are the interference microscope which not only reveals cell organelles but also enables quantitative measurement of the chemical constituents of cells such as lipids, proteins and nucleic acids, and the polarisation microscope which enables the study of intracellular structures using differences in birefringence. Electron microscope: Here, a beam of electrons is used instead of the beam of light used in the optical microscope. The electron beam is focused by circular electromagnets, which are analogous to the lenses in the light microscope. The object which is held in the path of the beam scatters the electrons and produces an image which is focused on a fluorescent viewing screen. As the wavelength of electrons used is approximately 0.005 nm, as compared to 500 nm with visible light, the resolving power of the electron microscope should theoretically be 100,000 times that of light microscopes but in practice, the resolving power is about 0.1 nm. The technique of shadow-casting with vaporised heavy metals has made pictures with good contrast and three-dimensional effect possible. Another valuable technique in studying fine structure is negative staining with phosphotungstic acid.

Live bacteria do not show much structural detail under the light microscope due to lack of contrast. Hence it is customary to use staining techniques to produce colou_r contrast Bacteria may be stained in the living state, but this type of staining is employed only for special purposes. Routine methods of staining of bacteria involve drying and fixing smears-procedures that kill them. Bacteria have an affinity to basic dyes due to the acidic nature of their protoplasm. The following are staining techniques commonly used in bacteriology.

Simple stains • Methylene blue or basic fuchsia are used for simple staining They provide colour contrast, but imp1;1rt the same colour to all bacteria.

Negative staining • Indian ink or nigrosin are emulsified with the sample or organism to provide a uniformly coloured background against which the unstained bacteria stand out in contrast. This is particularly useful in the demonstration of bacterial capsules which do not take simple stains. Very slender bacteria that are not demonstrable by such as spirochetes simple staining methods can be viewed by negative staining.

Morphology and Physiology of Bacteria

Impregnation methods

that resist deco!ourisation and retain the primary stain appearing v i o Gram-negative let. bacteria are decolourised by organic solvents and, therefore, take the counterstain, appearing red (Figs 2.3a and 2.3b). C

• Silver impregnation cells and structures too thin to be seen under the ordinary microscope may ·he rendered visible if they are thickened by impregnation of silver on the surface. Such -methods are used for the demonstration of spirochetes and bacterial flagella.

Differential stains • These stains impart different colours to di(ferent bacteria or bacterial struc.tures. The two most widely used differential stains are the Gram stain and the a c ifast d stain. 'C--"'"

Gram stain The Gram stain was originally devised by the histologist christian Gram (1884) as of staining bacteria m tissues.
f/" walls are about 10-25 nm ' - A cytoplasmic or pJasma membrane. (beneath thick and account for about 20-30 per cent of the dry the cell wall) weight of the celL . emically the cell wail is composed • Components of the cell interi.w of mucopeptide( e tido I can or murein) scaffolding The cell envelope en~loses the protoQlasm, comprising the cytoplasm, cytoplasmic inclusions such as _ _ N-acetyl ribosomes and mesos9mes, granules, vacuoles and muramic acid the nuclear body. · · • Additional structures The cell may be enclosed in a viscid layer, which may be a loose slime layer, or organised as a £filllil)e. Some bacteria carry filamentous appendages ..R!:9truding from the cell s~rface-the flagella which are organs ofTQ_comotion and the fimbriae which~ a r to be organs fur _adhesion (Fig. 2.6). \..,-P'

. ,£ell wall

The cell wall accounts for the shape af the h::icterial cell and confers on it,idity and ductility The cell wall cannot be seen by irect light microscopy and does not stain with simple stains.

- - - - - Tetrapeptide chain - - - - - Pentapeptide cross-bridge

Fig. 2.7 Chemical structure of bacterial cell wall

Cytoplasmic membrane Periplasmic space

Flagellum

Mesosome

Fat globule

Chromosome Inclusion body

Fig. 2.6

Diagram of an idealised bacterial cell

Part I

GENERAL MICROBIOLOGY

Thickness (peptidoglycan) Variety of amino acids Aromatic and sulphur containing amino acids LPS and outer membrane Teichoic acid

.:x...x

~ C)

formed by i:':-a~etyl glucosamine and N-acet,y] mqramjc acid molecules alternating in chains, which are crosslin~ed by 9eptide chains (Fig. 2. 7). The inter~of this scaffolding contain ather chemicals, varying in the different species. ..,. In general, the walls of Gram-positive bacteria hfil1! a simP.leLchemical nature than those of Qram-negative bacteria (Table 2.2). The cell wall carries bacterial antigens that are important ·inVirulence and immunity. · < •

-

Cell wall of Gram-positive bacteria: ~ptidoglycan layer: This is ~ r ( 15-80 nm) than that of Gram'-negative ~ a (2-3 nm) • Teichoic acid: Teichoic acid is a ma·or surface antigen of Gram- ositive bacteria containin ribito or glycerol polymers. The cell wall contains a significant amount of teichoic acid. Two types of teichoic acid are present: C_@ wall teichoic acid - linked to peptidoglycan - Membrane teichoic acid (iipoteichoic acid) - linked to membrane ~jycolipid (Fig. 2.8a) . . ;=-Cell wall of Gram-negative bacteria: • Lipopolysaccharides (LPS) present on the cell ~alls of Gram- negative bacteria account for their A1f''L 1ndotoxic activi!)' and 9 antigen specificity. They ~ cJ' were formerly known as the _Boivin antigen. The . 2( · LPS c~nsists of three regions: 'D y-r« ~egionJ is the O polysaccharide portion determining O antigen specificity. ~egion p is the core polysaccharide. Region IU is the glycolipid portioIJ (lipid A) and is responsible for the endotoxic actiyitjes , that is, pyrogenicity,~ar effect, l-ri:ssue necrosis, anticomplementary activity, \.fr cell mitogenicity, immunoadjuvant property and barrtitumour activity. • Oµter membrane: The outermost layer oLJhe Gram-negative bacterial cell wall is called the outer membrane, which contains var~us proteins.Jill.Qw as outer membrane proteins · (OM,P) . ~ong these are porins which f.2!:!ll,Jransmembrane pores ~

Thicker Few Absent Absent Present

Thinner Several Present Present Absent

~ e as diffusion channels for small molecu)es, ~ey also serve as specific receptors for §Orne bacteriophages. ~ tpoprotein: Attaches the_m:otei_n of_p~p~id_oglycan to lipid of outer membrane . ptidoglycan:· Is· thin (2-3 nm) and is boun 0

e

Cl

0

·ro ~

TERMS RELATED TO GENETICS

E

Codon

C '

- -

0

e ...0

*

Cl

C

~

Genetic information is stored in the DNA as code, the unit of the code consisting of a sequence of three bases. ~ach triplet (codon) transcribed on mRNA specifies for a single amino acid. More than one codon may exist for the _same amino acid . Thus, the triplet AQA codes for arginine but the triplets 1Q._9, CGU, CGC, CGA and CGG also code for the same amino ~ The code is non-overlapping, each triplet being a distinct entity, and no base in one codon is employed as part of the message of an adjacent codon. Three codons (UM, UAG and U GA) do not code for any amino acid and are called 'nonsense codons'. They act as Pl!nctuation ~ s (stop codons) ter.m.ina1ing ~ message for the synthesis of a polypeptide.

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