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- Sprache: Englisch
A compact and accessible guidebook exploring current understanding of common bacterial and fungal pathogens of animals. Fundamentals of Veterinary Microbiology brings together knowledge and understanding of the bacterial pathogens of animals from disease signs and diagnostic methods to the molecular basis of the host pathogen interaction. A small but focused book, it enables the reader to access important information during diagnosis and treatment in a clinically relevant way. Suitable as a companion for study on the subject and for professional use, the author focuses on the more clinically common diseases at the general and first-referral level. Divided into three sections, the first covers the basics of bacterial microbiology such as structure, growth and genetics. The second examines the commonly encountered bacterial pathogens, with emphasis on current understanding of the cellular and molecular basis of infection and immunity. The third section explores the current state of knowledge of those fungi involved in infection of animals. Specific topics covered in Fundamentals of Veterinary Microbiology include: * Nutrition, sterilization, and disinfection of bacteria, bacterial genes and gene transfer, pathogenicity and host response, and vaccination * Antimicrobials (action, dynamics, and resistance), typing and identification of pathogens, fungi as agents of disease, Salmonella and E. coli in animal disease and public health * Pathogens transmitted by vectors such as Borrelia, those of endogenous origin such as Clostridium and Pasteurella and those able to spread widely such as Leptospira, Mycobacterium and Chlamydia * Fungal pathogens causing superficial, subcutaneous and systemic mycoses Fundamentals of Veterinary Microbiology is an essential resource for veterinary students, veterinary nurses, and veterinary practitioners worldwide, allowing them to quickly establish a foundation of knowledge of bacterial pathogens based on clear understanding gained from studies over recent decades.
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Table of Contents
Cover
Table of Contents
Title Page
Copyright Page
Dedication Page
Preface
About the Companion Website
1 The Bacterial Cell
Bacterial Structure
Shape
Anatomy of the Bacterial Cell
Cytoplasmic Structures: Ribosomes, Nuclear Body
The Bacterial Cell Wall
Capsule
Flagellae
Fimbriae
Spores
Other Forms of Bacteria
References
2 Metabolism, Growth and Culture of Bacteria
Atmospheric Requirements of Bacteria
Nutritional Requirements
pH Requirements
Temperature Requirements
Culture Media
Identifying Bacteria in Culture
References
3 Sterilisation and Disinfection
Wet Heat
Dry Heat
Radiation
Filtration
Chemicals
Sterilising Agents
Disinfectants (for Decontamination)
References
4 Bacterial Genes and Gene Transfer
The Bacterial Genome
Mutation and Phenotypic Adaptation
Gene Transfer in Bacteria
Restriction and Modification
Transposable Elements and Insertion Sequences
References
5 Bacterial Pathogenicity
Intracellular and Extracellular Pathogens
Pathogenicity
Exotoxins
Endotoxin
Immunopathological Effects
References
6 Bacterial Veterinary Vaccines
Inactivated Vaccines
Live‐attenuated Vaccines
Toxoid Vaccines
Subunit Vaccines
Autogenous or ‘On‐Farm’ Vaccines
References
7 Antimicrobials: Action, Dynamics and Resistance
Targets of Antimicrobial Drugs
Antimicrobial Sensitivity
Bacterial Resistance to Antimicrobials
Multiple Antimicrobial Resistance
Methods of Overcoming Antimicrobial Resistance
References
8 Bacterial Typing
Serotyping
Phage Typing
Multi‐locus Enzyme Electrophoresis
Multi‐locus Sequence Typing
Serotyping from Antigen Gene Sequence and PCR
Whole‐genome Sequence Analysis
References
9 Salmonella
Enterobacteriaceae
Salmonella
and Public Health
Classification of Salmonellae
Disease Caused by
Salmonella
Other Enterobacteria
References
10
Escherichia coli
– An Intestinal Pathogen
E. coli
as an Enteric Pathogen
Enterotoxigenic
E. coli
(ETEC)
Enteropathogenic
E. coli
(EPEC)
Enterohaemorrhagic
E. coli
(EHEC)
Enteroaggregative
E. coli
(EAEC)
Enteroinvasive
E. coli
(EIEC)
Diffusely Adherent
E. coli
(DAEC)
References
11
Escherichia coli
as an Extraintestinal Pathogen
Pathogenicity
Pathogenesis
Urinary Tract Infection
Watery Mouth of Lambs
Oedema Disease in Pigs
Wound and Soft Tissue Infections
Bovine Mastitis
Antimicrobial Resistance
References
12
Campylobacter
– Hyperendemic on the Farm
Campylobacter fetus
subsp.
fetus
Campylobacter jejuni
Other Curved Gram‐negative Bacteria
References
13
Leptospira
– Using Urine to Spread
Classification of
Leptospira
The Maintenance Host and the Accidental Host
Transmission
Pathogenesis
Pathogenicity Factors of
Leptospira
Leptospirosis in Dogs
Leptospirosis in Cats
Leptospirosis in Cattle
Leptospirosis in Pigs
Zoonotic Disease
Other Risk Factors
References
14 Lyme Disease – Ticks and Dogs
Borrelia burgdorferi
References
15 Brachyspira
Brachyspira hyodysenteriae
Brachyspira pilosicoli
References
16 Pasteurella
Pasteurella multocida
Fowl Cholera
Haemorrhagic Septicaemia
Atrophic Rhinitis
Rabbit Snuffles (Enzootic Pasteurellosis)
Secondary Infection
References
17
Pseudomonas
and
Burkholderia
Pseudomonas
Burkholderia
References
18 Bordetella
Bordetella bronchiseptica
Other
Bordetella
Species
References
19 Delicate Gram‐negative Bacteria
Brucella
Moraxella bovis
Taylorella equigenitalis
References
20
Mannheimia
,
Actinobacillus
and Other
Pasteurellaceae
Mannheimia
Bibersteinia trehalosi
Histophilus somni
Actinobacillus
Glaesserella parasuis
Gallibacterium anatis
Avibacterium paragallinarum
References
21
Chlamydia
– A Stealthy Pathogen
Animal Disease
Human
Chlamydia
References
22 Bovine Tuberculosis and Johne’s Disease
Acid‐fast Bacteria
Bovine Tuberculosis
Tuberculosis in Other Animals
Johne’s Disease
References
23 Bacillus anthracis
Capsule
S‐Layer
Toxin
Anthrax
Other
Bacillus
Species
References
24 Clostridium
Clostridial Disease
Clostridium perfringens
The Histotoxic Clostridia
The Neurotoxic Clostridia
Diagnosis of Clostridial Disease
References
25
Staphylococcus
– Skin and Soft Tissue Infection
Coagulase‐positive Staphylococci
Major Pathogenicity Factors
Methicillin‐resistant
Staphylococcus aureus
Coagulase‐negative Staphylococci
Staphylococcus schleiferi
Staphylococcus hyicus
References
26 Streptococcus
Classifying Streptococci
Lancefield Groups
Habitat and Pathogenicity
Streptococci Causing Animal Disease
References
27
Nocardia, Actinomyces
and
Dermatophilus
– The Filamentous Pathogens
Nocardia
Actinomyces
Dermatophilus
References
28 Prescottella (Rhodococcus) equi
Pathogenesis
Pathogenicity
Immune Response
Human Disease
References
29
Corynebacterium
– CLA
Corynebacterium pseudotuberculosis
References
30
Listeria
– Growing in the Fridge
Transmission
Pathogenicity
References
31
Erysipelothrix
and
Trueperella
Erysipelothrix rhusiopathiae
Trueperella pyogenes
References
32
Mycoplasma
– Cell‐associated Pathogens
Pathogenicity of Mycoplasmas
Identification of Mycoplasmas
Bovine Mycoplasmas
Avian Mycoplasmas
Porcine Mycoplasmas
Mycoplasma
Infecting Other Species
Haemotropic Mycoplasmas
References
33
Rickettsia
– Arthropod Vector‐borne Pathogens
Ehrlichia ruminantium
Anaplasma phagocytophilum
Anaplasma marginale
Neorickettsia risticii
Coxiella burnetii
References
34 Fungi as Agents of Disease
Fungal Morphology
Fungal Pathogenicity
References
35
Aspergillus
– Strength in Numbers
Avian Aspergillosis
Mammalian Aspergillosis
Canine Nasal Aspergillosis
Equine Guttural Pouch Mycosis
Bovine Mycotic Abortion
References
36 Dermatophytes – Keratinolytic Fungi
Dermatophytosis
References
37 Yeasts: Malassezia, Candida and Cryptococcus
Malassezia pachydermatis
Candida albicans
Cryptococcus neoformans
References
38 Dimorphic Fungal Infections
Blastomycosis
Histoplasmosis
Epizootic Lymphangitis
Coccidioidomycosis
Sporotrichosis
References
Index
End User License Agreement
List of Tables
Chapter 22
Table 22.1 Susceptibility to different mycobacterial pathogens.
Chapter 24
Table 24.1 The toxin types of
Clostridium perfringens
.
Table 24.2
Clostridium novyi
and
C. haemolyticum
(C.h) toxin types.
List of Illustrations
Chapter 1
Figure 1.1 Bacterial cells stained by Gram stain and seen by light microscop...
Figure 1.2 Transmission electron microscope picture of a bacterial cell. The...
Figure 1.3 Bacteria show different shapes: cocci, rods and curved rods.
Figure 1.4 Prototypic bacterial cell to show the common subcellular features...
Figure 1.5 The cytoplasmic membrane: phospholipid bilayer embedded with prot...
Figure 1.6 The cell wall peptidoglycan surrounds the cytoplasmic membrane as...
Figure 1.7 Peptidoglycan: long, linear glycan chains cross‐linked by short p...
Figure 1.8 Simplified Gram‐positive cell envelope structure: thick peptidogl...
Figure 1.9 Simplified Gram‐negative cell envelope structure: thin peptidogly...
Figure 1.10 Lipopolysaccharide structure.
Figure 1.11 Negatively stained image of capsules of
E. coli
. Particles of In...
Figure 1.12 Bacterial flagellae are spiral structures attached at the cell e...
Figure 1.13 Bacterial fimbriae. Very fine surface appendages.
Figure 1.14 Bacterial endospore structure.
Figure 1.15 Morphology of bacterial endospores formed within a ‘mother’ cell...
Figure 1.16 Transmission electron microscopic image of an endospore formed w...
Chapter 2
Figure 2.1 The phases of bacterial growth in batch culture.
Figure 2.2 The basis of an anaerobe jar to provide an atmosphere free of O
2
....
Figure 2.3 Bottles of commercial bacteriological medium for reconstituting....
Figure 2.4 A culture after plating out and overnight incubation shows separa...
Chapter 3
Figure 3.1 The basic components of a simple autoclave.
Figure 3.2 A simple membrane filter unit for removing microorganisms from li...
Chapter 4
Figure 4.1 Bacterial gene transfer by conjugation through the sex pilus.
Figure 4.2 Generalised transduction.
Figure 4.3 Bacterial transformation.
Figure 4.4 Restriction endonuclease
Eco
R1 recognition sequence and cutting s...
Figure 4.5 Restriction and methylation. The restriction endonuclease recogni...
Figure 4.6 Transposition. An insertion sequence transfers from a plasmid to ...
Figure 4.7 Inverted repeat sequence in DNA.
Chapter 5
Figure 5.1 Specific adhesion of bacteria to a mucosal surface by fimbriae.
Figure 5.2 Chelation of iron by siderophores.
Figure 5.3 Transferrin binding activity found in many pathogens. The pathoge...
Figure 5.4 Surface phagocytosis.
Figure 5.5 Complement‐enhanced opsonophagocytosis.
Figure 5.6 Antibody‐directed complement‐mediated opsonophagocytosis.
Figure 5.7 The effect of streptolysin S around colonies of
Streptococcus equ
...
Figure 5.8 Action of ADP ribosylation of host cell Gs by bacterial toxin cau...
Figure 5.9 (a) Normal antigen presentation; (b) bacterial superantigen cross...
Figure 5.10 The type 3 secretion system of bacteria such as
Salmonella enter
...
Figure 5.11 Lipopolysaccharide (LPS) is composed of lipid A linked through t...
Figure 5.12 Structure of lipid A. The minimal Re chemotype lipopolysaccharid...
Figure 5.13 LPS interaction with macrophage.
Chapter 7
Figure 7.1
Enterobacteriaceae
are intrinsically resistant to penicillin G. A...
Figure 7.2 A bacterial isolate that is highly resistant to ampicillin (AMP2 ...
Chapter 9
Figure 9.1 Biochemical tests to identify enterobacteria. A pure culture of t...
Figure 9.2 The
Salmonella
injectosome apparatus (type 3 secretion system).
Figure 9.3 The genetic basis of flagellar phase change in
Salmonella
.
Chapter 10
Figure 10.1 ETEC: plasmids encode adhesive fimbriae and enterotoxins LT or S...
Figure 10.2 The action of LT on enterocytes causes increased cyclic AMP and ...
Chapter 11
Figure 11.1 Multi‐resistant
E. coli
from urinary tract infection in a six‐ye...
Chapter 12
Figure 12.1 Microscopic appearance of
Campylobacter jejuni
stained by Gram s...
Figure 12.2
Campylobacter jejuni
is carried in poultry but causes no disease...
Chapter 13
Figure 13.1 Under dark‐field microscopy, the unstained
Leptospira
bacteria a...
Figure 13.2 The transmission of
Leptospira
among animals and humans.
Chapter 14
Figure 14.1 Transmission of
Borrelia burgdorferi
.
Figure 14.2 Pathogenesis of Lyme disease following the tick bite.
Chapter 15
Figure 15.1 The morphology of
Brachyspira hyodysenteriae
alongside other Gra...
Chapter 16
Figure 16.1
Pasteurella multocida
colonies showing mucoid colonies coalescin...
Figure 16.2 Porcine atrophic rhinitis. (Left) A section through the snout sh...
Chapter 17
Figure 17.1
Pseudomonas aeruginosa
in culture displaying the characteristic ...
Chapter 18
Figure 18.1 Co‐ordinated transcriptional activation of virulence factors via...
Figure 18.2 BvgAS two‐component signal transduction system.
Chapter 19
Figure 19.1
Brucella abortus
(red coccobacilli) in placental tissue, stained...
Chapter 20
Figure 20.1 Lung lesion of bovine pneumonic pasteurellosis (uncomplicated
M.
...
Figure 20.2 Acute pasteurellosis. Alveolar spaces are filled with pink fibri...
Figure 20.3 Acute pleuropneumonia lesions in the lungs of a pig.
Figure 20.4 Fibrin deposition (yellow) on the surface of the heart, lungs an...
Chapter 21
Figure 21.1 The growth and development cycle of
Chlamydia
. The infective for...
Figure 21.2
Chlamydia abortus
in sheep placenta stained by modified Ziehl–Ne...
Chapter 22
Figure 22.1 The cell envelope structure in pathogenic mycobacteria.
Figure 22.2 Many acid‐fast bacilli (red) in the tissues of an animal with tu...
Figure 22.3 Disseminated tuberculosis in a badger.
Chapter 23
Figure 23.1 Two plasmids encode the genes for pathogenicity in
B. anthracis;
Figure 23.2
Bacillus anthracis
in blood smear from a case of fatal bovine an...
Figure 23.3 Colonies of
Bacillus anthracis
on blood agar.
Figure 23.4 The action of the three‐component anthrax toxin on the mammalian...
Chapter 24
Figure 24.1 The endospores of
Clostridium
species form within the mother cel...
Figure 24.2 Integrated view of key toxin‐based pathogenicity mechanisms invo...
Figure 24.3 Action of botulinum toxin at the neuromuscular junction. (a) An ...
Figure 24.4 Action of tetanus toxin at the neuromuscular junction of inhibit...
Chapter 25
Figure 25.1 Coagulase causes proteolytic activation of prothrombin to then c...
Chapter 26
Figure 26.1 Phagocytic uptake of streptococci by neutrophils.
Chapter 27
Figure 27.1 Microscopic appearance of a clinical isolate of
Nocardia
in cult...
Figure 27.2 Microscopic appearance of pus aspirated from a case of lumpy jaw...
Figure 27.3 Dark yellow ‘sulfur granules’ seen macroscopically in the pleura...
Figure 27.4 Microscopic appearance of
Dermatophilus congolensis
in a skin le...
Figure 27.5 A lesion in the skin of a pony is lifted to reveal the pus benea...
Chapter 28
Figure 28.1
Prescottella equi
showing the characteristic pink pigmentation....
Figure 28.2 VapA is a surface‐expressed, membrane‐active lipoprotein encoded...
Chapter 29
Figure 29.1 Thick pus from an abscess which is characteristic of caseous lym...
Chapter 30
Figure 30.1 Colonies of
Listeria monocytogenes
are small and grey. In transm...
Figure 30.2 Transmission routes of
L. monocytogenes
between animals, the env...
Figure 30.3
Listeria
enters host epithelial cells and escapes the intracellu...
Chapter 31
Figure 31.1 Characteristic red, rhomboidal skin lesions are the result of th...
Figure 31.2
Trueperella pyogenes
in pus from a lung abscess in a pig. The ma...
Chapter 32
Figure 32.1 Colonies of
Mycoplasma bovis
growing on solid medium showing the...
Figure 32.2 Lesions of enzootic pneumonia in the lungs of a 17‐week‐old pig ...
Chapter 33
Figure 33.1 A blood smear of a sheep with tick‐borne fever stained by Giemsa...
Chapter 34
Figure 34.1 Formation of arthrospores from septate mycelium.
Figure 34.2 Asexual spores of fungi: conidiospores (left) are produced on th...
Chapter 35
Figure 35.1 Microscopic appearance of
Aspergillus fumigatus
sporing heads pr...
Figure 35.2 Fungal plaque of
Aspergillus fumigatus
growing on the surface of...
Figure 35.3 Nasal aspergillosis; fungus and cellular debris in the nasal cav...
Figure 35.4 Bovine mycotic abortion.
Chapter 36
Figure 36.1 Severe lesions of dermatophytosis in a dog.
Figure 36.2 Septate mycelium of
Microsporum canis
in canine skin scales afte...
Figure 36.3
Microsporum canis
in culture after 10 days at 28 °C on Sabouraud...
Chapter 37
Figure 37.1 Microscopic appearance of yeast cells of
Malassezia pachydermati
...
Figure 37.2 Hyphae of
Candida albicans
in mucosal tissue, stained by Diff‐Qu...
Figure 37.3
Candida albicans
infection in the crop of a chicken – ‘sour crop...
Figure 37.4 Yeast cells of
Cryptococcus neoformans
in nasal tissue from a ca...
Chapter 38
Figure 38.1 The infective, mycelial phase and tuberculate conidia (macroconi...
Figure 38.2 A macrophage with yeast phase cells of
H. capsulatum
.
Figure 38.3 Yeast phase cells of
Histoplasma capsulatum
var.
farciminosum
in...
Figure 38.4 Microscopic appearance of a spherule of
Coccidioides immitis
in ...
Figure 38.5 The characteristic cigar‐shaped appearance of yeast cells of
S.
...
Guide
Cover Page
Title Page
Copyright Page
Table of Contents
Dedication
Preface
About the Companion Website
Begin Reading
Index
WILEY END USER LICENSE AGREEMENT
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Fundamentals of Veterinary Microbiology
Andrew N. Rycroft, BSc, PhD, FRCPath
Professor of Veterinary MicrobiologyRoyal Veterinary CollegeUniversity of London, London, UK
This edition first published 2024© 2024 John Wiley & Sons Ltd
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Limit of Liability/Disclaimer of WarrantyThe contents of this work are intended to further general scientific research, understanding, and discussion only and are not intended and should not be relied upon as recommending or promoting scientific method, diagnosis, or treatment by physicians for any particular patient. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of medicines, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each medicine, equipment, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. While the publisher and authors have used their best efforts in preparing this work, they make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives, written sales materials or promotional statements for this work. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further information does not mean that the publisher and authors endorse the information or services the organization, website, or product may provide or recommendations it may make. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.
Library of Congress Cataloging‐in‐Publication Data applied for
PB: 9780470659472ePDF: 9781119908371epub: 9781119908364
Cover Design: WileyCover Image: © Andrew N. Rycroft
For the four amigos, Simon, Frances, Jennifer and Edward, and for
Genny Moran whose kindness, encouragement and support were always there.
Preface
This book is based on my lectures and the lecture notes for the veterinary microbiology and infectious disease of animals developed at the RVC since 1992. It is intended to focus on pathogenicity and what we know of the host–pathogen relationship. However, it has also been necessary to keep in mind what is relevant for veterinary practitioners (in the widest sense) and the need to give background detail for those wanting to explore further.
Not all the chapters cover the same type of material. Not all the chapters have the same level of detail. The format is deliberately not formulaic, so that each chapter has different sections for different groups of microorganisms, and I hope that is recognised as positive. That is because the level of knowledge differs between diseases, as does the breadth of research and understanding we have on a given pathogen. A part of our knowledge of veterinary pathogens has been inferred from research into human pathogens for which the research funding has always been much better. For example, much of what we know about Bordetella bronchiseptica arises from work on B. pertussis because of its importance as the cause of childhood whooping cough. We have an inferior understanding of some animal pathogens such as Pasteurella multocida or Erysipelothrix rhusiopathiae even though they can have a minor role in human infection. Some pathogens, exclusively seen in animals, have long been neglected.
I have tried to simplify the taxonomic issues of bacteria of veterinary importance. Remembering names is hard enough without them changing every few years and I know how it infuriates veterinary students when names change! Modern analysis of whole‐genome sequences has allowed a further proliferation of bacterial species names. We don't need that for most practical purposes and so I have attempted to walk the line in taxonomy between being correct yet not complicating the subject with dozens of new names for minor pathogens.
I have also tried to refer to human disease agents that are related to the animal pathogens. So much of the quality research that has informed us of the pathogenic mechanisms in human pathogens has been relevant to pathogens of animals. Of course, the medical pathogens attract the research funding. Nevertheless, there have also been good opportunities to investigate animal pathogens that have translated into better understanding of human pathogens. It is also interesting and useful to understand the relationship of the human disease agents to those causing disease in animals.
While I have included antimicrobial drugs and discussed bacterial resistance mechanisms, I have generally not attempted to give suggestions for antimicrobial treatments. That is another subject area altogether and it is open to very different views, pressures, geographical factors and ideas. Only occasionally, where it is relevant to the microbiology or history, have I ventured into mentioning antimicrobial treatment.
There are very many people who should be thanked for their contribution to this book through their careful research into the pathogens of animals. There are others I wish to thank, particularly the pathologists (Hal Thompson, Lawson Macartney, Irene McCandlish, Os Jarrett, Sonja Jeckel), geneticists (Simon Baumberg), bacteriologists (Peter Kite, Peter Taylor, Steve Hammond, Harry Smith, David Taylor, John Smith, Werner Goebel, Niels Friis) and mycologists (Glyn Evans, Christine Dawson) who kindly taught or unknowingly inspired me over the years.
Finally, I must offer my gratitude to the many undergraduate, postgraduate and doctoral students of microbial pathogenicity and veterinary medicine who were obliged to spend time exploring aspects of microbiology with me in their research projects. These addressed so many research questions and led to discussions that forced me to strive to become au fait with the stream of new ideas, pathogens and techniques.
I hope you find the book helpful.
Andrew N. Rycroft
Northaw, February 2023
About the Companion Website
This book is accompanied by a companion website:
www.wiley.com/go/veterinarymicrobiology
The website includes:
Figures
Case studies
1The Bacterial Cell
Bacterial Structure
Bacteria are prokaryotic cells. The term ‘prokaryote’ includes the bacteria, the Archaea and blue‐green algae. The distinguishing feature of a prokaryote is that its nucleus is not surrounded by a nuclear membrane, but the nuclear material (DNA) is free in the cytoplasm of the cell. In addition, there is no nucleolus, mitotic spindle or (usually) any separate chromosomes. Bacterial cells are distinctively smaller in size than eukaryotic cells of plants, animals and fungi (Figures 1.1 and 1.2).
Shape
Individual bacteria have characteristic shapes. The cells may be spherical (coccus), rod shaped (bacillus), comma shaped (curved rod), spiral (spirochaete) or filamentous. Bacterial shape differs to some degree with the growth conditions (e.g. whether in the body or artificial medium of one kind or another). In some species, therefore, a bacterium may appear as long rods in lab culture, but as short rods or coccobacilli in the body when causing disease. Nevertheless, the shape of most bacteria can be seen in the light microscope and is an important clue to their identity (Figure 1.3).
Anatomy of the Bacterial Cell
The bacterial cell consists of the protoplast containing numerous organelles, which is bounded by a thin, elastic, semi‐permeable cytoplasmic membrane supported by the porous, relatively permeable rigid cell wall which bears a number of other structures (Figure 1.4).
Cytoplasmic Structures: Ribosomes, Nuclear Body
The cytoplasm is a gel containing organic and inorganic solutes, enzymes, ribosomes and the nucleic acids DNA and RNA. The ribosomes of prokaryotic cells are smaller than those of eukaryotic cells (plants, animals, fungi). They are known as 70S rather than the 80S ribosomes found in eukaryotic cells. This reflects a size difference because the Svedberg unit (S) is a unit of sedimentation and 80S ribosomes have a greater sedimentation rate than 70S. Both prokaryotic and eukaryotic ribosomes function to synthesise peptides (proteins), but they are sufficiently different organelles in the two groups for them to respond differently to inhibitors of protein synthesis such as some antibiotics which selectively disrupt the function of the ribosome.
The nuclear material (the DNA) is not a true nucleus. It is sometimes referred to as the nuclear body in bacteria because it is effectively free‐floating in the cytoplasm. Bacterial cells are haploid (one copy of each gene) and the DNA is arranged in a single closed circular molecule of about 1000 μm in length. The bacterial chromosome is not bound to protein histones as it is in eukaryotic cells, and it does not stain like a mammalian chromosome. In a section through a bacterial cell in the electron microscope, it appears as complex folds. Two or even four nuclear bodies may be seen in a bacterial cell as DNA replication and segregation occurs before cell division.
Figure 1.1 Bacterial cells stained by Gram stain and seen by light microscopy. The shape and arrangement are clear, but the resolution of the cells is limited by the light microscope.
Figure 1.2 Transmission electron microscope picture of a bacterial cell. The small granular organelles scattered throughout the cell are ribosomes; lighter regions are due to nuclear material.
Figure 1.3 Bacteria show different shapes: cocci, rods and curved rods.
Figure 1.4 Prototypic bacterial cell to show the common subcellular features.
Multiplication of bacteria is by simple growth and fission, not by mitosis. It is now recognised that bacteria have a cytoskeleton, which is needed for successful cell division. When a bacterial cell grows to sufficient size, the FtsZ protein forms a ring structure in the middle of the cell, known as the Z‐ring. This apparently constricts or contracts to make a pinch point or septum for cell division. FtsZ also acts to organise other cell division proteins at the site of septum formation, so it is likely to have a complex role. Other cytoskeletal proteins are necessary for positioning of the septum, involved in the shape of the bacterial cell and in the successful partitioning of the daughter chromosomes into separate ends of the cell following DNA replication (Egan et al. 2020).
The cytoplasmic membrane (or plasma membrane) limits the cytoplasm. It is a typical fluid‐mosaic model lipid bilayer about 9 nm across. It is composed of phospholipid and protein. The phospholipids are primarily phosphatidyl ethanolamine, with smaller proportions of phosphatidyl glycerol and cardiolipin (Figure 1.5).
Sterols are absent in almost all bacteria, but some Mycoplasma species, which have no cell wall, require sterols for growth and incorporate these into their membrane where they are essential for membrane stability. The membrane is flexible and is usually supported by a cell wall to maintain its integrity. The cytoplasmic membrane is the site of active transport via specific permease proteins. Its integrity is also essential for the maintenance of the proton gradient which is the driving force of electron transport and hence oxidative phosphorylation. Electron carriers of the respiratory chain, and ATPase, are located on the cytoplasmic membrane.
The Bacterial Cell Wall
The structures external to the cytoplasmic membrane constitute the bacterial envelope. One of these, the bacterial cell wall, provides the characteristic shape of the organism and prevents osmotic lysis of the cytoplasmic membrane. If the wall is ruptured, the cytoplasm expands through the gap and the cytoplasmic membrane bursts, killing the organism. This breakdown process is called lysis and can be caused by a number of agents: the enzyme lysozyme, some antibiotics, enzymes produced by bacteriophages (bacterial viruses) or enzymes produced by bacteria themselves (Figure 1.6).
Figure 1.5 The cytoplasmic membrane: phospholipid bilayer embedded with protein molecules.
When the cell wall is weakened or lost due to one of these agents in a situation where osmotic lysis does not occur (hypertonic solution), the shape of the organism may change. Spheroplasts (from Gram‐negatives) and protoplasts (from Gram‐positives) are formed. If bacteria lose their cell wall in vivo (in the body of an animal), they are known as L‐forms which may be a means by which some bacteria persist in the body during infection.
The cell wall of bacteria is a crucial structure as it is the site of action of some important groups of antimicrobials and the location of certain important antigens utilised both in identification of bacteria and in the immune response of the body to bacterial infection.
Figure 1.6 The cell wall peptidoglycan surrounds the cytoplasmic membrane as a tough sack‐like structure.
Peptidoglycan
Bacterial cell walls are quite different from those of eukaryotic cells, and they contain substances unique to bacteria. Peptidoglycan, formerly known as mucopeptide or murein, is the most important component of the cell wall. It is common to both Gram‐positive and Gram‐negative cells. It surrounds the cell, external to the cytoplasmic membrane, as a single bag‐like molecule. It is composed of linear glycan chains of alternating residues of N‐acetylglucosamine and N‐acetyl muramic acid. These are linked together by short peptide bridges to form a cross‐linked insoluble polymer (Rohs and Bernhardt 2021).
The peptide bridges vary between different organisms, but they are known to contain biologically exotic substances including meso‐diaminopimelic acid, D‐alanine and D‐glutamic acid. The peptidoglycan is a rigid structure which gives shape and strength to the bacterial cell wall.
Peptidoglycan forms the basic structure of the bacterial cell wall and similar peptidoglycan is found in the unconventional, obligate intracellular bacteria: Chlamydia and Rickettsia (Figure 1.7).
Figure 1.7 Peptidoglycan: long, linear glycan chains cross‐linked by short peptide bridges to form the tough cell wall polymer.
Figure 1.8 Simplified Gram‐positive cell envelope structure: thick peptidoglycan layer.
Figure 1.9 Simplified Gram‐negative cell envelope structure: thin peptidoglycan and a second membrane – the outer membrane.
In addition, other accessory polymers are also found in most bacteria. Gram‐positive organisms, such as staphylococci, contain teichoic acids composed of either poly‐glycerol phosphate or poly‐ribitol phosphate. These occur both within and on the surface of the cell wall and may account for 20–50% of the dry mass of the cell wall. On the streptococci, teichoic acids are sometimes the Lancefield group ‘carbohydrate’ antigens used in their classification and identification (Figure 1.8).
In Gram‐negative cells, the peptidoglycan is much thinner than in Gram‐positive organisms. Outside the peptidoglycan lies a second lipid bilayer membrane, the outer membrane (OM). This is a similar membrane to the inner, cytoplasmic membrane but it contains different proteins and, in addition to the phospholipids of the cytoplasmic membrane, a component unique to Gram‐negative bacteria: lipopolysaccharide (LPS). This is located in the outer leaflet of the outer membrane. The outer membrane functions to protect Gram‐negative bacteria against a harsh environment. It acts as a barrier and yet it allows molecules through via general porins (outer membrane proteins which act as a diffusion pore for small molecules) and substrate‐specific porins (Figure 1.9).
Lipopolysaccharide
Lipopolysaccharide has three regions to the molecule. The lipid region (lipid A) is relatively invariable and contains 3‐hydroxy fatty acids linked to a diglucosamine backbone. These fatty acids are hydrophobic and intercalate into the phospholipid bilayer of the OM (Putker et al. 2015) (Figure 1.10).
Linked to the lipid A is a short oligosaccharide which is variable between bacterial types, and which contains very unusual sugar residues. This is known as the LPS core region that protrudes into the environment. In some bacteria, the LPS stops at this point, and they are known as R‐form or ‘rough’ bacteria. Such bacteria will auto‐agglutinate in saline (unless other polysaccharides are external to the LPS) and these bacteria are often of low virulence. However, in many bacteria there is a third region, the O‐side chain. This is a repeating oligosaccharide (perhaps five or six sugars linked together in the same pattern) with as many as 50 or 80 repeat units of this extending into the external environment of the bacterium. This makes the surface of the bacterium hydrophilic, and the O‐side chain is highly antigenic, being the O or somatic antigen of Gram‐negative bacteria. With a full O‐side chain, bacteria are termed S‐form or “smooth”, and virulent bacteria are often of this type.
The LPS also has biological properties. The lipid A part of the molecule was thought to disrupt mammalian cell membranes. It is now known to act in a more subtle way to cause a host of biological effects upon the body, including pyrogenicity (raised body temperature) and the manifestations of Gram‐negative septicaemia and circulatory collapse. The LPS is therefore also termed ‘endotoxin’ because it is toxic and yet a part of the bacterial cell and not secreted. In fact, we can think of the LPS as a signal, alerting the body to the presence a foreign invader. LPS becomes associated with the LPS binding protein. It then interacts with tissue macrophages through the CD14 protein, Toll‐like receptor 4 (TLR4) and an associated protein called MD‐2. Through cell signalling pathways, this leads to the release of potent cytokines such as IL‐1 and TNF‐α. In turn, these act on the hypothalamus to cause increased body temperature (fever) and other proinflammatory biological effects including vasodilation and hypotension.
Figure 1.10 Lipopolysaccharide structure.
In mycobacteria, which are Gram‐positive organisms, wax‐like mycolic acids are covalently linked to the peptidoglycan. These make the bacteria ‘acid fast’, resistant to desiccation, some disinfectants and most of the body's immune defence mechanisms.
Figure 1.11 Negatively stained image of capsules of E. coli. Particles of India ink are excluded from the clear polysaccharide capsule which shows up as a bright area surrounding the dark bacteria.
Capsule
Many bacteria are surrounded with a layer of polysaccharide material known as the capsule (Figure 1.11).
Capsules may be either homopolysaccharide, being composed of a polymeric form of a single sugar type, or heteropolysaccharide, having two or sometimes more sugar residues. Very unusual sugar residues may be found quite often. In some cases, the capsule may be thick and visible in the light microscope (using special stains) and makes the bacterial colony viscous and slimy. In others, only a very thin layer, known as a microcapsule, is present. This means that the polysaccharide can be detected only by chemical or serological means or by electron microscopy (Orskov et al. 1977).
Most capsules are antigenic but some are relatively non‐antigenic, presenting a surface which the body and the biochemical components of the immune system fail to recognise as foreign. Furthermore, the capsular antigen may mask other, deeper antigens in the cell envelope such as the teichoic acids or LPS which are easily ‘seen’ as foreign.
The function of capsules, at least in the body, is to evade phagocytosis and some of the most important pathogens have capsules which are essential for their ability to cause disease. One such organism is Bacillus anthracis. This has a most unusual capsule in that it is not polysaccharide but poly‐amino acid: poly D‐glutamic acid. This capsule is essential for B. anthracis to survive in the body and cause the disease anthrax. It is also important in the identification of B. anthracis in the blood of a fallen animal. The capsule shows a characteristic pink or mauve colour surrounding the bacterial cell (M'Fadyean reaction) when stained with polychrome methylene blue. This is diagnostic. It is also an important statutory examination before the removal and disposal of the carcass of a sudden death case in a large animal can be carried out.
Flagellae
Bacteria also carry surface appendages. Flagellae are protein structures which function in the movement of bacteria – motility (Figure 1.12).
They are composed of protein subunits (flagellin), and these are important antigens in the identification of some bacteria (e.g. the H antigens of Salmonella). Bacterial flagellae are carried as a polar flagellum at the end of the cell (such as on Pseudomonas aeruginosa) or they may be present over the surface of the bacterium which are referred to as peritrichous flagellae (such as on Proteus mirabilis). The flagellae of spirochaetes are specialised in being located not externally, but within the periplasmic space (between the inner and outer membranes). Here, they are termed ‘axial filaments’ but serve the same function.
Flagellae cause bacteria to be motile by rotating (Bardy et al. 2003). Because it is curved, the filament of a flagellum acts as a propeller driving the organism through the medium. The energy for this rotation is derived from the proton motive force (PMF) or proton gradient at the cytoplasmic membrane by a molecular electric motor. In this, protons pass through the mechanism across the membrane and bring about a part turn of the flagellum.
Figure 1.12 Bacterial flagellae are spiral structures attached at the cell envelope.
Figure 1.13 Bacterial fimbriae. Very fine surface appendages.
Many bacteria are chemotactic, reacting positively to some chemical stimuli by moving towards them and/or negatively to others.
Fimbriae
Fimbriae are also protein surface appendages. They are sometimes referred to as pili and are composed of subunits of pilin. They are thinner and shorter than flagellae and can only be seen by electron microscopy (Figure 1.13).
With a few exceptions, fimbriae are only present on Gram‐negative bacteria. Their function in nature is to adhere to surfaces but different bacteria carry different fimbriae that adhere to different surfaces – some through extremely specific interactions. In some cases, this is a mucosal surface such as the small intestine or the urinary tract of an animal. Fimbriae in pathogenic bacteria may function as colonisation factors without which they would not cause disease. Antibody to fimbrial antigens can be protective in preventing attachment of the pathogen. Thus, fimbrial protein is the basis for a number of new or experimental vaccines against a surprising variety of diseases.
Sex pili are specialised fimbriae involved in the process of conjugation or transfer of plasmid genes between bacterial cells.
Spores
Bacterial spores are produced by only two genera: Bacillus and Clostridium. They are correctly known as endospores and are formed inside the mother cell in response to adverse conditions. They are not reproductive, only one spore being produced per bacterium and only one bacterium being produced by the germination of a spore. Spores are able to tolerate heat, desiccation, cold, radiation and chemical treatments that vegetative bacteria cannot survive (Nicholson et al. 2000).
Bacterial spores comprise the genomic DNA of the cell, surrounded by the cytoplasmic membrane and a layer of ‘normal’ peptidoglycan. External to this is the cortex, a specialised thick layer of peptidoglycan that has a much looser, less cross‐linked structure. It is known to be responsible for the dehydration of the spore's core and is probably the structure which confers resistance to heat, desiccation and radiation. The coat protein is a keratin‐like, very thick, highly resistant protein stabilised by disulfide (─S─S─) bonds. It probably confers chemical resistance on the spore (Figure 1.14).
Bacterial spores may remain viable for many years. They are reawakened by favourable environmental conditions. However, some spores require an activation step such as heat‐shock or boiling to trigger germination. Germination is the resynthesis of metabolic enzymes, and the degradation and removal of spore‐specific components and outgrowth is the return to life as a vegetative bacterial cell.
Figure 1.14 Bacterial endospore structure.
Spores are important because they are the microorganisms that are most difficult to destroy and because they are formed by a number of very important veterinary bacterial pathogens. These include the agents of clostridial diseases of sheep, tetanus, botulism, blackleg of cattle and also anthrax.
The position of a spore within the mother cell tends to be characteristic of a particular species. Spores are produced at the end of a bacterium (terminal spore), as by Clostridium tetani, or within the centre of the cell (central spore). Similarly, a clostridial spore will bulge the mother cell and distend it considerably outside the normal bounds of the bacterium; the spores of Bacillus are more confined to the bounds of the bacterial wall (Figures 1.15 and 1.16).
Figure 1.15 Morphology of bacterial endospores formed within a ‘mother’ cell. These vary according to the species of bacteria producing them.
Other Forms of Bacteria
L‐forms of bacteria are sometimes generated during infection by adaptation of pathogens such as streptococci. These are produced during treatment with antibiotics which damage cell walls. They lose their cell wall peptidoglycan and remain viable in protected areas of the body. This may allow infection to persist in spite of antibiotic treatment, and by reverting to the normal (cell‐walled) form of the pathogen they can produce relapses of infection. While L‐forms may be responsible for some cases of chronic or unexplained recurrent infection, in practice they are rarely detected in veterinary infections.
Figure 1.16 Transmission electron microscopic image of an endospore formed within a vegetative ‘mother’ cell.
References
Bardy, S.L., Ng, S.Y.M., and Jarrell, K.F. (2003). Prokaryotic motility structures.
Microbiology
149: 295–304.
Egan, A.J.F., Errington, J., and Vollmer, W. (2020). Regulation of peptidoglycan synthesis and remodelling.
Nature Reviews Microbiology
18: 446–460.
Nicholson, W.L., Munakata, N., Horneck, G. et al. (2000). Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments.
Microbiology and Molecular Biology Reviews
64: 548–572.
Ørskov, I., Ørskov, F., Jann, B., and Jann, K. (1977). Serology, chemistry, and genetics of O and K antigens of
Escherichia coli
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Putker, F., Bos, M.P., and Tommassen, J. (2015). Transport of lipopolysaccharide to the gram‐negative bacterial cell surface.
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Rohs, P.D.A. and Bernhard, T.G. (2021). Growth and division of the peptidoglycan matrix.
Annual Review of Microbiology
75: 315–336.
2Metabolism, Growth and Culture of Bacteria
Multiplication of bacteria is by binary fission. As the bacterial cell grows in size, the bacterial chromosome replicates by the semi‐conservative replication of DNA, beginning at a site known as the origin. New cell wall forms at the poles of the cell and at the septum or cross‐wall where the cell will divide. If separation of the daughter cells is delayed after cell division, then the characteristic arrangements of bacteria (chains of streptococci, bunches of staphylococci) are produced, depending on the plane of cleavage.
Although bacteria do not have a sexual cycle, the exchange of genetic material may occur naturally between individuals of the same or related genus by the process of conjugation: self‐directed transfer of plasmid DNA (see Chapter 4).
The speed of bacterial growth is measured as the generation time. The generation time is the interval from completion of septum formation to septum formation, or from one bacterial cell to two cells at the same stage. This may be as short as 20 minutes for Escherichia coli and as long as 16 hours (even under optimised conditions) for Mycobacterium bovis. The generation time for bacteria growing in the body may be quite long as they may be deprived of certain important nutrients.
A starting culture of bacteria in a fixed quantity of culture fluid is termed a ‘batch culture’ and it goes through a predictable series of growth phases. The first of these is lag phase when the organism is adjusting to the new environment and synthesising new enzymes. No increase in bacterial numbers takes place during this time. The bacteria then enter log (or exponential) phase in which a period of sustained growth and division takes place. With bacteria dividing to become 2, 4, 8, 16, 32 and so on, the increase in numbers or turbidity (cloudiness) is exponential. When a nutrient is exhausted or a toxic metabolite begins to inhibit growth, the bacteria enter stationary phase. No further increase in numbers then occurs. Decline phase is the final stage in which bacteria die at an exponential rate (exponential decay) (Figure 2.1).
In industrial culture, batch culture is often used for the production of antibiotics. Secondary metabolites are only formed in late exponential or stationary phase. Some processes use continuous culture, in which a population is grown by constantly supplying fresh medium to the culture and removing culture at the same rate. This allows a controlled steady state to be achieved whose growth rate can be manipulated at will. Growth in the body is closer to continuous culture than batch culture, but the analogy is not simple.
Atmospheric Requirements of Bacteria
Many bacteria grow well in air. Some, such as Bordetella bronchiseptica and M. bovis, cannot do without the oxygen present in air. They are said to be obligate aerobes.
Others that grow in air can tolerate conditions in which there is no O2. These, such as E coli, are termed ‘facultative anaerobes’: they have the capacity to metabolise anaerobically but they prefer to utilise molecular oxygen as the terminal electron acceptor, when it is available, for respiratory metabolism. This is because respiratory metabolism generates a far greater amount of energy in terms of ATP than does fermentation.
Microaerophilic bacteria prefer a reduced oxygen tension. They will not grow or grow very poorly in the absence of O2 or in the open atmosphere. Campylobacter species are microaerophilic.
Other organisms are unable to tolerate molecular O2. They are termed ‘obligate anaerobes’. The degree of ‘strictness’ varies between organisms such that some clostridia will tolerate and grow in a few per cent O2 (aerotolerant anaerobes) while others will be killed by even brief exposure to oxygen. Strict anaerobes are thought to be sensitive to O2 because they possess a respiratory chain (electron transport chain) which, through partial reduction of O2, generates free radicals such as superoxide (O2−) and hydrogen peroxide (H2O2). These highly reactive compounds are normally decomposed very rapidly by enzymes (superoxide dismutase and catalase) and cause no harm. Strict anaerobes do not possess these enzymes and are damaged by them. For example, when O2− and H2O2 are allowed to combine, they yield the extremely toxic hydroxyl radical (.OH). Strict anaerobes include Dichelobacter nodosus (the agent of ovine foot rot) and Brachyspira hyodysenteriae (swine dysentery).
Figure 2.1 The phases of bacterial growth in batch culture.
To have an anaerobic environment in the clinical laboratory and enable the culture of anaerobic bacteria, an anaerobic jar has been traditionally used. This was an airtight sealable jar, with pressure gauge and valves to allow gas in and gas out. Culture plates or broths were placed in the jar and the lid closed. Part of the air was removed with a vacuum pump and then hydrogen gas (often mixed with CO2 for safety) was run into the jar. Using the accelerating effect of the palladium catalyst attached to the inside of the lid, the H2 combines with the residual O2 until all the O2 is consumed and the atmosphere is free of any oxygen. This was incubated in the 37 °C incubator (Figure 2.2).
To replace gas cylinders, gas packs able to generate H2 were introduced. More recently, gas packs with their own catalyst have become popular and, for large numbers of clinical cultures or for research, an anaerobic cabinet is used. An indicator system, often just some methylene blue solution to indicate a chemically reduced atmosphere, is used to verify that anaerobic conditions have been achieved.
Figure 2.2 The basis of an anaerobe jar to provide an atmosphere free of O2.
Despite their sensitivity to O2, some strict anaerobes such as Clostridium species can still respire but they use elements other than oxygen as a terminal electron accepter. Some use NO3− and release NO2− or N2. These are denitrifying bacteria, and the process is anaerobic respiration.
Bacteria which do not have the oxygen‐detoxifying enzymes but possess no respiratory chain to generate free radicals are aerotolerant. This includes the lactobacilli (usually harmless commensals and also used to make yoghurt) and many of the streptococci which include species that are important animal pathogens. These organisms are indifferent to the presence or absence of oxygen and generate their energy solely by non‐respiratory metabolism (fermentations).
Nutritional Requirements
Bacteria may be autotrophic, making their own nutrients from carbon dioxide and inorganic salts together with a source of nitrogen, iron, calcium, phosphorus and other mineral nutrients. Other organisms, which require a source of preformed organic nutrients, are heterotrophic and these include all the pathogens of man and animals. Some, such as E. coli, will grow in a solution of glucose in phosphate buffer and ammonium sulfate together with a minimal source of inorganic metal salts. Others, such as streptococci, require complex preformed organic compounds as nutrients and building blocks together with co‐factors and vitamins which they are unable to synthesise themselves (Markey et al. 2013).
Bacteriological media, for the routine isolation of pathogens in a clinical laboratory, reflect the complex requirements of pathogens in general. This is so that one or two standard complex media can be used which will be expected to grow the majority of cultivable pathogens when the population of bacteria in a clinical sample is not known. They are therefore prepared from digested meat and extracts of yeast, etc. These complex preparations are then supplemented where necessary with blood, serum, etc. to further enrich them.
pH Requirements
Most of the bacteria of veterinary importance grow best in the pH range 7.2–7.6, but many can tolerate conditions outside this range and still grow satisfactorily.
Among the acidophiles (growing under relatively acid conditions) are the lactobacilli; the alkophiles include Vibrio spp. including V. cholerae, the cause of the human disease cholera.
Temperature Requirements
Most pathogenic bacteria produce optimum growth at approximately 37 °C. Some can grow at temperatures between 15 °C and 45 °C. Those only able to grow at low temperature (less than 20 °C) are called psychrophiles and those only able to grow at high temperature are called thermophiles. Because these are so far from the body temperature of mammals, they are not pathogens of animals. Growth at low temperature (10–15 °C) is a characteristic of Listeria spp. This is known as a psychrotroph because it can also grow at normal body temperature.
Culture Media
All bacteria that may cause disease or live on a body are heterotrophic. They require a preformed source of carbon for building new molecules and for energy. Among these, some have very diverse metabolic capability. They can degrade complex organic molecules and build complex molecules from nothing more than glucose, inorganic nitrogen, inorganic phosphate and some trace elements. E. coli is one such species that will grow on a solution of ammonium salts, phosphate buffer and glucose.
Defined media are a mixture of substances whose chemical properties can be exactly reproduced and defined. Nevertheless, many bacteria of veterinary and medical importance have evolved to require preformed complex organic molecules for their nutrition, and these cannot be exactly defined.
Media for routine use are prepared from digested meat (e.g. peptone or brain heart infusion) together with extracts of yeast, and often supplemented with blood or serum. These are known as complex media. The basic material for bacteriological medium is prepared as freeze‐dried powder to be reconstituted with water and sterilised by heating (Figure 2.3).
A complex liquid medium is termed a broth, e.g. nutrient broth or tryptone soya broth. In order to be able to grow colonies separated from each other on the surface of a medium, the broth must be solidified. For this purpose, agar is used at a concentration of 1.0–1.5%. In the past, gelatine was used to solidify a medium but at temperatures much above 28 °C, gelatine melts so it was unsuitable for cultures incubated at 37 °C. Also, some bacteria digest gelatine and liquify it. Instead, agar, an inert complex galactan polysaccharide extracted from seaweed, melts at 95 °C and then re‐solidifies at 45 °C. This is perfect: it allows the addition of heat‐labile substances to different growth media before they are allowed to solidify and yet there is no chance of the agar melting when incubated even at temperatures above 37 °C. Agar is not digested by bacteria, nor used as a nutrient.
Figure 2.3 Bottles of commercial bacteriological medium for reconstituting.
Common complex media used for routine clinical microbiology include blood agar (a rich meat extract and peptone base supplemented with 7% horse, sheep or calf blood when cooled to 55 °C) and heated blood agar (known as chocolate agar) which is prepared as blood agar but heated to 85 °C to coagulate and denature blood proteins before allowing to cool. The characteristic colour provides the trivial name. These are poured into petri dishes at about 50 °C.
Selective and Differential Media
Some complex media contain substances which deliberately inhibit the growth of particular organisms while allowing others to grow. MacConkey's agar is the most widely used of these. It contains peptone as the main nutrient, bile salts (a mild detergent) which have a weak suppressive effect on non‐intestinal bacteria, and lactose and neutral red which together detect lactose fermentation. Because it differentiates lactose fermenters (pink colonies) from non‐lactose fermenters (yellow colonies), it is also a differential medium. Other selective media, which often have a similar differential system incorporated, include XLD (xylose, lysine, desoxycholate) medium and DCA (desoxycholate citrate agar) although there are many others that have been devised for the selective isolation and recognition of specific pathogens (Markey et al. 2013).
Enrichment Broths
Enrichment broths increase the number of the organism sought relative to the normal bacteria present. These are used primarily in the isolation of salmonellae from faecal material where a very large number of commensal organisms are present compared with the pathogen. The most important examples are selenite F broth and tetrathionate broth for the enrichment of Salmonella. After incubation, samples are taken from the cultured enrichment broth and plated onto a suitable selective, differential medium which will allow salmonellae to be distinguished.
Culture of Bacteria on a Solid Medium
Bacterial growth appears as a haze in liquid medium and as discrete colonies on solid media. Each bacterial colony is the descendant of one bacterial cell. If the inoculating material contains many bacteria, colonies will develop closely to give confluent growth. If separated or diluted, this inoculum can give rise to discrete colonies. To achieve single colonies from a mixture or a large number of bacteria, material is diluted on a plate; the process is called ‘streaking’ or ‘plating out’ (Figure 2.4).
Figure 2.4 A culture after plating out and overnight incubation shows separated colonies (each is a clone derived from a single bacterial cell). The colonies all look the same so this is likely to be a pure culture.
Dilution of an inoculum can also be done by serial 10‐fold dilution in tubes of saline. A pure culture must be obtained before the properties of a bacterial isolate can be investigated, and this is normally done by selecting a single colony and subculturing it.
Each stage (set of streaks) is a further dilution because the loop should be sterilised by flaming. In this way, the bacteria in the inoculum are reduced in number until they become separated enough to form individual colonies and the components of a mixture (e.g. a mixed infection) can be seen. When the properties of a bacterial isolate are investigated, perhaps to identify it, one colony is selected and subcultured. The properties of the clone of cells derived from it are then studied as a pure culture.
Identifying Bacteria in Culture
Bacterial cultures are identified by comparing the morphological, cultural, biochemical and antigenic properties of an unknown isolate with those of known bacterial species (Barrow and Feltham 2009). Many of the bacteria regularly isolated from disease in animals can be identified provisionally by an experienced bacteriologist from their appearance on standard media supplemented by the use of one or two appropriate tests.
The growth medium is important in determining the characteristics of a bacterial colony. Bacterial isolation is usually carried out on a rich medium which allows the growth of most bacteria (e.g. blood agar) together with a selective medium (MacConkey's medium) which inhibits some bacteria while allowing growth of others and distinguishing lactose fermenters from non‐lactose fermenters.
Once isolated in pure culture and the basic growth characteristics recognised, a pathogen is identified by a variety of directed tests and investigations. The skill of taking an unknown isolate in culture and applying the correct tests to identify it take some time to develop. In recent years, methods based on matrix‐assisted laser desorption/ionisation‐time of flight (MALDI‐TOF) mass spectrometry (MS) have become widely used for the rapid and accurate identification of bacterial and fungal pathogens in clinical microbiology (Clark et al. 2013; Angeletti 2017). Nevertheless, well‐prepared cultures are still needed to separate and provisionally categorise bacterial pathogens from a clinical sample.
References
Angeletti, S. (2017). Matrix assisted laser desorption time of flight mass spectrometry (MALDI‐TOF MS) in clinical microbiology.
Journal of Microbiological Methods
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Barrow, G.I. and Feltham, R.K.A. (2009).
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Clark, A.E., Kaleta, E.J., Arora, A., and Wolk, D.M. (2013). Matrix‐assisted laser desorption ionization‐time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology.
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Markey, B.K., Leonard, F.C., Archambault, M. et al. (2013).
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