498q-Essentials of Microbiology For Dental Students (2) - 1-100

Authors: Bagg, Jeremy; MacFarlane, T. Wallace; Poxton, Ian R.
;
Smith, Andrew J.; Bagg, Simon
Title: Essentials of Microbiology for Dental Students, 2nd
Edition
Copyright ÂŠ2006 Oxford University Press (Copyright 2006 by Jeremy
Bagg, T. Wallace MacFarlane, Ian R. Poxton, Andrew J. Smith and
Simon Bagg)
> Fro nt o f Bo o k > Autho rs
Authors
Jeremy Bagg
Professor of Clinical Microbiology
University of Glasgow Dental School, Glasgow, UK
T. Wallace MacFarlane
Emeritus Professor of Oral Microbiology
Ian R. Poxton
Professor of Microbial Infection and Immunity
University of Edinburgh Medical School, Edinburgh, UK
Andrew J. Smith
Senior Lecturer in Oral Microbiology
Simon Bagg
Illustrated by
Authors: Bagg, Jeremy; MacFarlane, T. Wallace; Poxton, Ian R.;
Edition
Simon Bagg)
> Fro nt o f Bo o k > Disclaimer
Disclaimer
Oxford University Press makes no representation, express or implied,

that the drug dosages in this book are correct. Readers must therefore
always check the product information and clinical procedures with the
most up to date published product information and data sheets provided
by the manufacturers and the most recent codes of conduct and safety
regulations. The authors and the publishers do not accept responsibility
or legal liability for any errors in the text or for the misuse or
misapplication of material in this work.
Edition
Simon Bagg)
> Fro nt o f Bo o k > Preface
Preface
The main purpose of this textbook remains unchanged from the first
edition. It sets out to present the science of microbiology in a clinical
context that is relevant to the safe and effective practice of dental
surgery. Many dental school curricula are now changing in line with the
recommendations of the document produced by the UK General Dental
Council, entitled The First Five Years. The two key elements of the
recommendations are that clinical teaching should be brought forward,
ideally starting in Year 1 and that the underpinning basic science,
including microbiology, should be more closely integrated with the
clinical teaching and spread throughout the five years of the course.
When the first edition of Essentials of Microbiology for Dental Students
was prepared, one of our main aims was to produce a book that placed
microbiology firmly in the context of clinical dentistry, an ideal formula
for use in the new style curricula. For this reason, the second edition
has retained the successful format of the first edition, covering the
relevant subject matter in three sections, namely the fundamental
principles of microbiology and immunology, infectious diseases relevant
to dentistry and, finally, oral microbiology. There is extensive cross-
referencing throughout and the book is lavishly illustrated in full colour.
Microbiology is a very rapidly moving discipline. Since the first edition,
a number of infectious diseases such as Creutzfeldt-Jakob Disease and
SARS have emerged as significant clinical problems. There have also
been changes in the epidemiology of certain infections, for example
sexually transmitted diseases. These issues are all covered in this
second edition. The text and lists of further reading have been updated
throughout and changes to antimicrobial drug prescribing regimens
have been included. Major new chapters include those on fungi, human
herpes viruses, infections of the central nervous system, and use of
antimicrobial agents in dentistry. The chapters dealing with
sterilization, disinfection and infection control procedures in dentistry
have been completely re-written, to reflect significant recent changes
in decontamination protocols which have been stimulated by the
problem of prion diseases. In addition to providing a source of further
reading to supplement traditional teaching, the integrated clinical
approach will ensure that the text is valuable to those preparing for
problem-based learning sessions.
Another new feature of the second edition is the inclusion of a
comprehensive glossary of medical, dental and biological terms, all of
which are used in the text itself. It is the authors' experience that
many dental students find the terminology a major problem when they
commence their studies of microbiology. The very clinical emphasis of
the book has dictated that the glossary contains many terms that will
be relevant to other disciplines.
Qualified dentists, particularly those preparing for postgraduate clinical
dental examinations, will also find the book of value. Microbiology is of
immense importance to dentists and it is our hope that the book will
help to make this fascinating subject accessible and relevant to those in
clinical practice.
In summary, we have designed this book to serve as a core reference
text for use throughout all years of the undergraduate dental
curriculum, as a refresher course for postgraduate students and as a
continuing source of reference for dentists in clinical practice. We trust
that our efforts are sufficient to allow these very different groups of
readers to obtain the information they require in a convenient, clear
and interesting way.
J B
T W MacF
I R P
A J S
Glasgow, Edinburgh, 2004
Edition
Simon Bagg)
> Fro nt o f Bo o k > Ackno w ledgements
Acknowledgements
Microbiology is a rapidly moving field and preparation of the second

edition of this textbook has proved a significant task, since considerable
efforts have been made to bring it fully up to date. The authors would
like to thank all those colleagues and students who have provided
advice on the content of the new edition, in particular Drs Claire
Cameron, Andrew Hall and Jim McMenamin. We also wish to thank all
those who have provided illustrative material and who are
acknowledged in the legends to the relevant figures. Finally, our
grateful thanks are due to the staff of Oxford University Press, in
particular Catherine Barnes and Georgia Pinteau, for their support,
encouragement and willingness to set achievable deadlines.
Edition
Copyright ©2006 Oxford University Press (Copyright 2006 by Jeremy
Simon Bagg)
> Table o f C o ntents > Sectio n 1 > 1 - The co ncept o f micro -o rganisms
1
The concept of micro-organisms
Introduction
The history of microbiology and immunology
Before the development of the light microscope all living organisms
could be divided empirically, without too much debate, into two
kingdoms: the plants and the animals. In the 1660s, Robert Hooke,
using a crude compound microscope, was probably the first person to
see cells; a few years later Antonie van Leeuwenhoek, using the first
high-resolution, single lens microscope, saw microorganisms for the
first time. These microscopic living creatures, some of which were seen
to move, were described as small animals or ‘animalcules’. From
Leeuwenhoek's drawings we know them to be bacteria and protozoa. It
is of interest to note that his specimens included material scraped from
his own teeth.
Fig. 1.1 Some key players in early microbiology and
immunology.
There was a gap of almost two centuries before the development of the
first high-quality compound microscope, together with the pioneering
work of Pasteur, Koch and Lister, established the importance of micro-
organisms as agents of disease, and the science of bacteriology (or the
more modern microbiology) was born (Fig. 1.1). Following the discovery
of penicillin by Fleming, the development of antimicrobial
chemotherapy slowly began, and is now the driving force behind a large
and important branch of microbiology. Much of modern immunology had
its roots in the study of micro-organisms by such key players as
Metchnikoff and Erhlich (Fig. 1.1).
Robert Koch was the first person to prove that micro-organisms caused
disease. Absolute proof can be established by fulfilling the following
postulates, though this raises ethical problems in studies of human
disease.
Koch's postulates
1. The specific micro-organism should be isolated from all cases of a
specific disease, and should not be found in healthy individuals.
2. The specific micro-organism should be isolated from the diseased
individual and grown in pure culture on an artificial medium.
3. The isolated micro-organism should reproduce the specific disease
when inoculated into a healthy individual.
4. The specific micro-organism should be re-isolated in pure culture
from the experimental infection.
Although the original Koch's Postulates remain useful, ‘Koch's Molecular

Postulates’, have been introduced recently. These are covered in
Chapter 4.
Fig. 1.2 The relationships between living
organisms.
Fig. 1.3 Diagram of an idealized prokaryotic

cell.
Early studies on unicellular, microscopic organisms established that

there were several different morphological types. Those resembling
animals were referred to as protozoa and those resembling plants were
termed algae. The yeasts, moulds, and other microscopic forms which
resembled plants but did not possess photosynthetic pigments were the
fungi, while the bacteria tended to be smaller agents, simpler than the
other types, and recognized as causing a range of infectious diseases
(Fig. 1.2). The viruses are a special group of acellular structures which
were discovered much later and which cannot be readily termed living
organisms. Viruses are described in detail in Chapter 6.
Prokaryotic and eukaryotic cells

With the development of the electron microscope in the 1950s it was
for the first time possible to explore the internal architecture of cells. It
soon became apparent that in the whole spectrum of living creatures -
from the simplest bacterium to the most complex higher organism -
there are only two basic types of cell, the prokaryotic cell and the
eukaryotic cell. The prokaryotic cell (Fig. 1.3) is small and simple in
structure without any internal membrane-bound structures or
organelles; while the eukaryotic cell (Fig. 1.4) is larger and more
complex, with an obvious membrane-bound nucleus and other internal
organelles such as mitochondria. Bacteria, together with other similar
micro-organisms classified as rickettsiae, chlamydiae, mycoplasmas,
and cyano-bacteria (formerly blue-green algae), are prokaryotic.
Recently a third cell type has been recognized, which is found in
organisms belonging to the domain Archaea (previously
archaebacteria). These are primitive bacteria-like organisms, often
found in extremes of environment. To date none of the Archaea has
been recognized as a pathogen. All other unicellular and all multi-
cellular organisms are eukaryotic. The fundamental differences in cell
structure (Fig. 1.5) have important consequences when considering
antimicrobial action, as many antimicrobial agents target prokaryotic-
specific structures or metabolic pathways.
On examining the structure of mitochondria (and chloroplasts in plant
cells) it soon becomes apparent that they have many similarities to
prokaryotes: size; 70S ribosomes; undergo binary fission; and have
circular DNA. It is therefore assumed that the eukaryotic cell originally
evolved from a symbiotic relationship between a primitive precursor cell
and intracellular prokaryotic micro-organisms.
Fig. 1.4 Diagram of an idealized eukaryotic
cell.
The diversity of micro-organisms and their

habitats
Micro-organisms were the earliest forms of life on the planet, and many
such primitive types still inhabit the harsh environments which must
resemble those which existed when life first began. Virtually every
ecological niche is colonized by micro-organisms, and each species is
adapted to the specific habitat. The vast majority of micro-organisms do
not cause disease and are saprophytic, obtaining their nutrition from
the breakdown of organic matter. Their primary role is in the various
natural cycles such as the carbon, nitrogen, oxygen, and sulphur cycles
which are concerned with the decomposition of living matter and
recycling of elements vital for the well-being of life on earth.
Atmospheric requirements
Many micro-organisms obtain their energy by fermentation (substrate-
level phosphorylation), a process which does not require oxygen. The
smallest amount of oxygen is toxic to some of these micro-organisms,
or at least inhibits their growth, and these are termed strict anaerobes.
Other micro-organisms perform respiration (oxidative phosphorylation),
and are aerobes. A few species of bacteria can respire anaerobically,
utilizing CO 2 or NO3 instead of O2 as the final electron acceptor. Thus,
Pseudomonas aeruginosa, usually considered to be an aerobe, can grow
anaerobically if nitrate is present in the growth medium. Many micro-
organisms can both ferment and respire and are known as facultative
anaerobes. The microaerophilic bacteria, typified by the genus
Campylobacter (see Chapter 15), are unusual in that they require
subnormal levels of oxygen for their growth; while others, the
capnophilic bacteria, which are commonly found in the oral ecosystem,

have a requirement for carbon dioxide.
Fig. 1.5 A comparison of key features of prokaryotic and eukaryotic

cells.
As well as the atmospheric requirements of micro-organisms, other

chemical and physical parameters such as temperature, pH, and
osmolarity are important (Chapter 2) and some species, notably the
Archaea, can exist under extreme conditions (Fig. 1.6).
The normal flora and pathogens

Many sites in the body, for example the skin and the gut, are colonized
by large numbers of micro-organisms in health. This normal human
microbiota, or the normal flora of the human body, is often referred to
erroneously as the commensal flora. The host certainly derives some
benefit from these populations, not least by the colonization resistance
conferred (Chapter 8). Throughout the outside surface and most of the
mucosal surfaces of the human body there are niches of considerable
diversity, ranging from the dry, salty conditions of the skin to the acidic
stomach. In health, micro-organisms exist in a stable equilibrium with
the host, and the innate and acquired immune defences (Chapter 8)
have evolved in parallel with the micro-organisms to maintain this
equilibrium (Fig. 1.7).
Fig. 1.6 Extremes of physico-chemical environment in which

bacteria can grow.
Fig. 1.7 Factors with which normal microbiota must contend in
health. Defects in these factors may result in opportunist infections.
Fig. 1.8 Diagram illustrating the structure of (a) normal and (b)
abnormal (disease-associated) forms of prion protein. The normal
form comprises largely soluble, α-helical protein whereas the
abnormal form contains large elements of insoluble β-pleated sheet.
Those micro-organisms which cause disease are termed pathogens.

These may be primary pathogens which, as part of their normal life
cycle, cause harm to the host, and are examples of true biological
parasites. Many pathogens, however, are part of the normal microbiota
where they exist in a symbiotic or commensal relationship, or are
environmental saprophytes. These may, if the opportunity presents,
become opportunist pathogens. Typically, it is when the normal host
defences break down and the individual becomes immunologically
compromised (for example in AIDS) that the host becomes susceptible
to diseases caused by these opportunist pathogens. Pathogenesis will be
covered in detail in Chapter 4.
Emerging and re-emerging pathogens

Interactions between pathogens and their hosts are dynamic. This
means that over time new pathogens can emerge, and pathogens that
were considered conquered, or no longer harmful can re-emerge;
Mycobacterium tuberculosis is a good example of the latter. Sometimes
emerging pathogens are genuinely new, such as human
immunodeficiency virus (HIV) and severe acute respiratory syndrome
(SARS) virus, or are a new especially virulent variant of a relatively
harmless organism, such as E. coli O157. Other pathogens are simply
recently recognized, such as Helicobacter pylori, the cause of peptic
ulcers. Agents such as Legionella pneumophila, the cause of
Legionnaires' disease, have existed for millennia, but have only
recently become apparent as a
human disease, in this case because air-conditioning systems have

permitted their transmission. Influenza virus, an agent well known for
change, is currently causing concern because of the threat of an avian
strain moving into Man. Generally, infectious agents moving from an
animal species to Man (zoonotic spread), especially if this has not
occurred before, are likely to result in a severe infection. In subsequent
chapters many of these emerging and re-emerging pathogens will be
described more fully.
Prions and transmissible spongiform

encephalopathies (TSEs)
Prions, from the term ‘proteinaceous infectious agent’, are thought to
cause variant Creutzfeldt-Jacob Disease (vCJD), bovine spongiform
encephalopathy (BSE: mad cow disease) and other TSEs such as scrapie
in sheep. The theory suggests that the infectious particle is composed
entirely of protein; it does not contain nucleic acid and is therefore not
a conventional microorganism or virus. Prion proteins normally occur
on the surface of many different cell types, but when an abnormal form
(Fig. 1.8) accumulates in the brain, disease results - this abnormal
form is the infectious agent. As these agents are not typical cells, their
removal by conventional sterilization procedures and their transmission
pose special problems for hygiene and prevention of cross-infection.
Prions will be covered in more detail in Chapter 17.
Key facts
The main groups of micro-organisms are bacteria, fungi, protozoa,

algae, and viruses.
Most are microscopic (<10 µm) and unicellular.
All living cells have a cell type that places them in one of three
domains: Prokaryotes (the bacteria), Eukaryota (higher
organisms) or Archaea (primitive, mainly extremophilic micro-
organisms).
Prokaryotes are simple cells with no internal membranes or
organelles, for example bacteria.
Eukaryotes are complex cells with a nucleus, internal organelles,
and extensive internal membranes, for example protozoa, fungi,
and human cells.
Micro-organisms are found in extremely diverse habitats.
Most bacteria are saprophytic, involved in recycling elements, but
some cause disease (pathogens).
The human microbiota (normal flora) is found throughout the skin
and mucosal surfaces of the body and is in equilibrium with the
host defence mechanisms.
Micro-organisms that cause disease are either primary pathogens
or opportunist pathogens.
Emerging and re-emerging infections are a constant threat.
Prions - infectious proteins - pose special problems for infection
control of these agents.
Further reading
Mims C, Dockrell HM, Goering RV, Roitt I, Wakelin D, Zuckerman M
(2004). Microbes as parasites. In Medical Microbiology, 3rd edn,
Chapter 1. Elsevier Limited, London.
Edition
Simon Bagg)
> Table o f C o ntents > Sectio n 1 > 2 - Bacterial structure and physio lo gy
2
Bacterial structure and physiology
Bacterial cell structure

Size and shape
Most bacteria are either spherical (coccus) or rod-shaped (bacillus), and
are usually arranged in a manner characteristic of the genus, growing
either singly, in clusters or in chains. The dimensions of a bacterium
are typically in the order of 1µ (Fig. 2.1).
The Gram stain

As described in the previous chapter, bacteria are typical prokaryootes.
Most bacteria can be divided into one of two types, Gram-positive and
Gram-negative, by means of the Gram stain, which is summarized in
Fig. 2.2. Although developed empirically by Christian Gram, a Danish
microbiologist, in the 1880s as a convenient method for classifying
bacteria, it also gives a great deal of information on the structure of
the bacterial cell envelope.
Gram-positive bacteria have a relatively thick amorphous wall (F ig.
2.3) which retains the fixed violet dye within the cell. Gram-negative
bacteria have a layered appearance due to the presence of two
membranes: the inner or cytoplasmic membrane and the outer
membrane (Fig. 2.4). The organic solvent used in stage four of the
Gram-staining process disrupts this membranous envelope and the stain
is washed out of Gram-negative bacteria.
Fig. 2.1 The various shapes and arrangements of bacteria (not
drawn to scale).
Fig. 2.2 The method for Gram staining a bacterial

film.
Fig. 2.3 (a) Diagram of the envelope of Gram-positive bacteria. PL,
phospholipid; P, protein (b) Electron micrograph of the envelope of
Gram-positive bacteria.
Fig. 2.4 (a) Diagram of the envelope of Gram-negative bacteria.
LPS, lipopolysaccharide; LP, lipoprotein; PL, phospholipid; P, protein
(b) Electron micrograph of the envelope of Gram-negative bacteria.
P.11
Some groups of bacteria cannot be considered typical Gram-positive or

Gram-negative organisms, as they either do not take up the Gram
stain, or they have a different type of envelope entirely. The
mycobacteria, such as Mycobacterium tuberculosis and M. leprae, the
causative agent of tuberculosis and leprosy respectively, are perhaps
the best-known examples. These mycobacteria have a waxy envelope
containing complex glycolipids, which renders them impervious to the
Gram stains. Traditionally, myco-bacteria are stained by the Ziehl-
Neelsen stain, which involves driving a carbol (phenolic) fuchsin stain
into the bacteria with
heating, and then decolourizing the background with dilute acid in

alcohol (3% HCl in 95% ethanol), giving rise to the term acid-fast
bacilli or AFB; AFB stain bright red.
Fig. 2.5 Diagram of a typical membrane (fluid mosaic

model).
Structure and function of the bacterial cell

envelope
The bacterial cell envelope provides a structural and physiological
barrier between the cytoplasm (inside) of the cell and the external
environment. The cytoplasmic membrane is a typical membrane
consisting of a bilayer of phospholipid into which are embedded
proteins, some of them crossing the whole width of the membrane: the
fluid mosaic model (Fig. 2.5). It is a semi-permeable membrane and
osmosis takes place across it. The result is that the inside of the cell is
at a high osmotic pressure compared to the outside environment. One
of the major functions of the layers outside the cytoplasmic membrane
is therefore to withhold this extreme osmotic pressure, thus giving the
cell its strength and shape. The functions of the bacterial envelope are
listed in Fig. 2.6. Many of these will be covered in detail in later
sections of the book.
Fig. 2.6 Summary of functions of the bacterial cell

envelope.
The Gram-positive bacterium possesses a structure outside of the

cytoplasmic membrane that could be termed a wall. This consists of two
major polymers: peptidoglycan and a secondary wall polymer which is
often a teichoic acid. Many Gram-positive bacteria also often have a
protein layer on the surface of the wall. These proteins are sometimes
arranged as fibrils perpendicular to the wall and are involved in
adhesion, or they may also be present in crystalline surface layers (S-
layers) or regular arrays with more of a structural function.
The Gram-negative surface layers are best described as an envelope
rather than a wall, as the outer membrane (OM) structure does not
readily equate with the wall of Gram-positive bacteria or plant or fungal
cells. In the electron microscope the OM appears as a classical
membrane, but in chemical terms is it very different from the typical
structure: it is asymmetric. The inner leaflet consists of the usual
phospholipids, but the sole lipid component of the outer leaflet is the
lipopolysaccharide (LPS) molecule, resulting in a relatively non-fluid
membrane and a major permeability barrier. There are a small number
of major outer membrane proteins (OMPs), fewer in number than in the
cytoplasmic membrane. Some of the OMPs form transmembrane
diffusion channels and are termed porins.
Outside the envelopes of many Gram-positive and Gram-negative
bacteria there is often a capsule or slime layer, which is described later.
Macromolecules of the envelope

Peptidoglycan
Peptidoglycan is the major structural molecule of the envelope and is
responsible for the shape and strength of the cell. It can be
considered a single molecule which covers the whole three-dimensional

surface of the bacterium. Peptidoglycan consists of long, parallel chains
of N-acetylated amino sugars (alternating N-Ac glucosamine and N-Ac
muramic acid) which are cross-linked by short peptide chains (Fig. 2.7).
There is a greater amount of peptidoglycan in the Gram-positive wall
compared to the Gram-negative envelope.
Fig. 2.7 The chemical structure of
peptidoglycan.
Some species, notably Cblamydia spp., appear Gram-negative in the

electron microscope but they do not have any peptidoglycan layer
between the cytoplasmic (inner) and outer membrane. Instead they
have their major outer membrane proteins crosslinked through
disulphide bridges, giving the OM a degree of strength.
Teichoic acids and other secondary cell wall

molecules
In the strictest sense, teichoic acids are polymers of poly (ribitol
phosphate) or poly (glycerol phosphate) which are found in the cell
walls of Gram-positive bacteria (Fig. 2.8). These polymers may have
sugar or amino acid substituents, either as side chains or within the
chain of the polymer. The phosphate group confers an overall negative
charge to the polymers, and one of the main functions of the
macromolecule is thought to be in the scavenging of divalent cations,
particularly calcium and magnesium, from the growth environment of
the bacterium. These polymers are also the substrate for autolytic
enzymes, and they are also exploited as antigens for serological
classification of certain genera.
Some Gram-positive bacteria have analogous, negatively charged wall
polymers which are not true teichoic acids, since they do not contain
glycerol or ribitol phosphate. They do, however, contain charged groups
such as uronic acids or phosphates and functionally they are the same
as teichoic acids.
Fig. 2.8 Chemical structure of typical teichoic
acids.
Fig. 2.9 Diagrammatic representation of

lipopolysaccharide.
The membrane-bound analogues, the lipoteichoic acids and other
membrane-bound lipocarbohydrates, are anchored in the cytoplasmic
membrane through a glycolipid. They also have a role in sequestering
divalent cations and channelling them down to the membrane, together
with a possible role in bacterial adhesion.
Lipopolysaccharides
Lipopolysaccharides are complex, amphipathic molecules located as the
sole lipid in the outer membrane of Gram-negative bacteria.
Structurally, LPS molecules are divided into three main sections: the
lipid A, the core oligosaccharide and the O-polysaccharide or O-antigen
(Fig. 2.9). The O-polysaccharide is made up of a chain of repeating
oligosaccharide units of heterogeneous length. This normal
configuration of LPS is usually referred to as smooth-form LPS (S-LPS),
since wild type bacteria possessing this molecule produce colonies with
a ‘smooth’ appearance. Mutant bacteria lacking the O-polysaccharide
appear ‘rough’ and have a rough-form or R-LPS. Some species of
bacteria naturally produce an R-LPS and these molecules are sometimes
termed lipo-oligosaccharides (LOS) (Fig. 2.9).
The lipid A molecule consists of a disaccharide backbone (usually bis-
glucosamine) which is substituted with five or six fatty acids and two
phosphates. Between different species of Gram-negative bacteria, there
is little variation in structure of the lipid A part of the molecule, with
identical structures between related species. In the core region, there
are several unusual sugars which are found only in bacteria, for
example 3–deoxy-D-manno-2–octulosonic acid, which is still more
commonly known by the initial letters of its previous name, KDO (keto-
deoxy-octonic acid). The inner part of the core is, like lipid A, highly
conserved within related species, but some variation can occur in the
outer part. However, it is in the O-polysaccharide that the real variation
occurs. The many different possible combinations of sugars and the
configuration of the linkages between them give rise to an infinite
number of different oligosaccharide-repeating units. In the species E.
coli alone there are approximately 160 different structures recognized,
giving rise to the distinct O-serogroups.
LPS is an essential component of the Gram-negative bacterium. Its
functions include a structural role, where the fatty acid chains pack
tightly in the OM resulting in a relatively non-fluid structure, which
forms an impervious barrier. As described earlier for teichoic acids, LPS
molecules are negatively charged, and the long sugar chains confer a
degree of hydrophilicity on the bacterial surface. In pathogenic
bacteria, LPS is considered to be one of the most important virulence
determinants. It has a role in protecting the organism from the effects
of serum complement, and the lipid A region is extremely biologically
active, giving rise to the synonym for LPS of endotoxin. These
properties of LPS/endotoxin will be covered in more detail in Chapter 4.
Capsule/slime
Many bacteria, both Gram-positive and Gram-negative, possess a gel-
like layer outside of the envelope. This may be in the form of a discrete
capsule, which can extend from the envelope for a distance several
times the diameter of the cell. Alternatively, this gel material may not
be firmly attached and forms a slime, which is released into the
surrounding environment. Most of these materials are polysaccharide in
nature, and are often referred to collectively as exopolysaccharides.
However, some species, for example Bacillus spp, produce a capsule
consisting of poly-amino acids. The capsule of Bacillus antbracis, the
causative organism of anthrax, has a capsule of poly(D-glutamic acid).
Exopolysaccharides are sometimes neutral homopolysaccharides, for
example the glucans and fructans of many oral streptococci, or
negatively charged heteropolysaccharides, where uronic acids are
common constituents. The poly(D-glutamic acid) capsule with its
negative charge is probably physiologically analogous to the negatively
charged polysaccharide capsule.
The capsule is a highly hydrated gel and in the microscope, with both
living and stained bacteria, it is invisible. The simplest way to
demonstrate the capsule is to mix a suspension of bacteria with an
equal volume of Indian ink on a slide, cover with a coverslip, press it
down firmly to create an extremely thin film, and then view it in the
microscope. The capsule is ‘negatively’ stained,
appearing as a clear zone around the cell where the microscopic Indian
ink (carbon) particles have not penetrated (Fig. 2.10).
Fig. 2.10 Indian ink stain to demonstrate bacterial capsule.
Courtesy of Sheila Patrick.
The capsule has various functions and perhaps the most universal of
these is its involvement in adhesion to surfaces. Many bacteria colonize
both inert and living surfaces, and grow in microcolonies, or as a
consortium in a biofilm. Dental plaque is an excellent example of this.
Other functions can be considered as protective and in the pathogen
include protection from, and evasion of, the immune system. These will
be covered in detail in Chapter 4.
Surface appendages
The surface appendages can be divided into two main types: those
involved in motility (flagella), and those involved in adhesion
(fimbriae/pili).
Flagella
Many species of bacteria can move actively through an aqueous
environment. This motility can be governed by several different
mechanisms, including the poorly understood gliding motility of
Cytophaga spp. However, the mechanisms involving flagella (singular:
flagellum) are the most common and the best understood. The
arrangement of flagella varies between different species, but is the
same for a given species. Flagella can be found singly (monotrichous)
or in bundles (lophotrichous) at one or both poles of the cell, or they
can be arranged over the whole cell surface (peritrichous) (Fig. 2.11).
The flagellum consists of a long thin filament projecting for up to
several micrometres from the cell surface. The main part of the
filament is made up of protein subunits (flagellin) arranged in several
helices around a central hollow core. At the base of the flagellum, a
‘motor’ is located in the cell envelope. It consists of a series of rings
through which the base of the filament can rotate in either a clockwise
or an anti-clockwise direction. Above the base of the filament there is a
short bent region, which produces a propeller-like propulsion from the
revolving flagellum (Fig. 2.11). A motile bacterium is able to perform
chemotaxis or phototaxis, that is, move towards or away from stimuli
such as chemicals or light respectively. Receptors that are sensitive to
such stimuli are located on the bacterial surface and respond by making
the flagellum rotate in one or other direction. The resultant motility,
which varies between a tumbling and a directional mode, depending on
the direction of rotation, allows the bacterium to swim by means of a
‘random walk’ towards or away from the source of the stimulus.
Fig. 2.11 Diagram illustrating the various arrangements of bacterial
flagella, fimbriae and the structure of the flagellar motor.
Spirochaetes move in a corkscrew manner by means of an axial

filament. This consists of a bundle of flagellum-like structures, which
lies between the cell surface and an outer sheath and connects one end
of the cell to the other (Fig. 14.3, p. 149).
Fimbriae/pili
The terms fimbriae and pili (singular: fimbria/pilus) (Fig. 1.3) are
synonymous for most purposes. These appendages tend to be found
only on Gram-negative bacteria, and are shorter and straighter than
flagella (Fig. 2.11), although the basic structure is the same: helices of
the protein (always called pilin) arranged around a hollow core, but
without a motor. The term fimbria originated in the UK where it was
first recognized and studied in enterobacteria, while pilus was coined in
the USA for the appendage on the gonococcus. Because of the
abundance of American textbooks, pilus is perhaps more commonly
used, though many microbiologists would like to have some consensus
as to when each term should be used. There is a view that fimbriae
should be used for those structures having a role in adhesion, the
commonest property of these appendages, while pilus be restricted to
the sex pilus, the appendage which connects mating cells and through
which the DNA passes during the process of conjugation (Chapter 5).
Fimbriae usually bind to a surface through a specific recognition
mechanism. The end of the fimbria carries a molecule (peptide) which
recognizes a specific receptor on the surface to which it binds. The host
cell receptor is usually a sugar residue, and the recognition molecule on
the bacterium is termed a lectin.
Gram-positive bacteria do not have classical fimbriae, but many possess
a fine fibrillar arrangement of proteins on their surface, sometimes
termed fibrillae, which bind to host molecules. This is best exemplified
by M-protein of Streptococcus pyogenes.
The cytoplasm
The cytoplasm of bacteria contains all the biosynthetic machinery
required by the bacterium for growth and cell division, together with
the genetic material. The latter consists of a double helix of DNA
arranged in a supercoiled circular structure. It is not enclosed in a
nuclear membrane, but is often referred to as the ‘bacterial
chromosome’ because of the analogy with the eukaryotic structures. In
growing bacteria the DNA can account for up to 20% of the volume of
the bacterium. Smaller circles of ‘extra-chromosomal’ DNA are often
present in bacteria and these are termed plasmids (Chapter 5). They
carry genes which may confer the bacterium with properties such as
antibiotic resistance or the capacity to produce toxins or enzymes.
The ribosomes used for protein synthesis are smaller than their
eukaryotic counterpart, with a sedimentation coefficient of 70S
(compared with 80S in eukaryotes). They consist of two subunits of 30S
and 50S, giving a net 70S. They are the site of action of several
antibiotics such as the aminoglycosides, macrolides and tetracyclines
(Chapter 10).
Many bacteria possess inclusion granules within their cytoplasm, and in
some species they are utilized as characteristics which can serve as
markers for identification purposes. Their main function is thought to
be one of storage, being produced when their main constituent element
is present in excess in the culture medium. Metachromatic granules, or
volutin are deposits of inorganic polyphosphate, which can be dyed with
methylene blue and are characteristic of Corynebacterium diphtberiae.
Starch and glycogen granules are commonly revealed in many bacteria
when stained with iodine. Lipid inclusions are found in mycobacteria
and are visualized with Sudan Black dye. Some bacterial toxins
characteristically form as crystalline cytoplasmic inclusions.
Sporulation
Bacterial cells, unlike all higher and many lower eukaryotic cells, are
unable to differentiate, or undergo morphogenesis. However, some
bacterial species, notably the aerobic Bacillus spp. and the anaerobic
Clostridium spp. can perform a primitive process of differentiation
termed sporulation. The bacterium, on encountering stressful conditions
such as dehydration or nutrient limitation, can convert to a resting or
dormant endospore. This endospore is then highly resistant to many
harsh physical and chemical conditions such as heat (some can
withstand boiling), dryness, toxic chemicals, and radiation. From a
practical point of view, spore-forming bacteria can be very difficult to
eradicate during sterilization procedures.
During the process of sporulation, before the spore is free of the
‘parent’ cell, it often has a characteristic appearance within the
bacterial cell. This can be defined on the basis of the spore's location in
the cell (terminal/subterminal/central), its diameter
(smaller/same/greater than vegetative cell) and its shape
(spherical/oval) and can be used to assist in diagnosis.
On returning to favourable conditions, spores can germinate and form
new vegetative cells.
Bacterial physiology
Nutrition
The nutritional requirements of bacteria vary enormously. At the
simplest extreme, Escherchia coli and related enterobacteria can grow
in a simple synthetic medium consisting of the inorganic salts which are
necessary to supply the major essential elements of carbon, hydrogen,
oxygen, nitrogen, phosphorus and sulphur, together with a source of
energy, the simplest being glucose. Trace elements are not usually
added specifically since they typically occur at adequate levels as
contaminants in commercial chemicals. Some bacteria release
extracellular hydrolytic enzymes into the environment for nutritional
purposes and such enzymes, for example proteases, may play a role in
disease production.
Conventionally, bacteria are cultured in liquid media (broths) or on the
surfaces of solid media which have agar added as a gelling agent. Agar,
a polysaccharide extracted from seaweed, is relatively inert (is not
metabolized by most bacteria), gels below 50°C (heat-labile ingredients
can be added after sterilization) and melts at 100°C (does not melt
when incubated). Agar media are usually contained in flat, circular
plates (Petri dishes).
Fig. 2.12 Summary of energy production and associated metabolic
pathways in bacteria.
Many bacteria require certain amino acids, vitamins and other growth
factors, which can be supplied by adding yeast extracts and meat
digests. These are available commercially as various forms of nutrient
broths and nutrient agars. Other bacteria require more extensive
additions, and for many clinically important bacteria, these
requirements can be fulfilled by the addition of blood or serum (from
horses).
Some bacteria require living cells for their growth, and these can be
supplied by tissue culture methods. However, a few species, notably
Mycobacterium leprae and Treponema pallidum, the causative agents of
leprosy and syphilis respectively, can only be cultured when inoculated
into a living animal.
Energy production
Production of energy can be considered as the processes by which
bacteria produce adenosine triphosphate (ATP) by phosphorylation of
adenosine diphosphate:
Adenosine – P – P + energy + P → Adenosine – P – P – P
As described briefly in Chapter 1, this is mainly by fermentative or
respiratory processes. The large group of photosynthetic bacteria
produce ATP by harnessing energy from light, but these will not be
discussed any further in this book.
The anaerobic bacteria, which produce energy only by fermentation,
use substrate-level phosphorylation, and can utilize carbohydrates or
amino acids as fermentable substrates. Aerobic and facultative
organisms utilize both substrate-level and oxidative phosphorylation,
producing much more ATP per mole of substrate if the oxidative process
is used (Fig. 2.12). Glycolysis is the central pathway for carbohydrate
metabolism, but many bacteria have alternative pathways such as the
pentose phosphate and Entner–Doudoroff pathways, which are outside
the scope of this book. The end products of fermentation are typically
ethanol, carbon dioxide, lactic acid and volatile, short-chained fatty
acids, the latter giving some anaerobic bacteria their characteristic foul
smell.
Biosynthesis of macromolecules
The various macromolecules (nucleic acids, proteins, carbohydrates and
lipids) required for the structure and biosynthetic machinery of the
bacterial cell must be assembled from the nutrients supplied from the
growth medium. As described previously, the nutritional requirements
of different bacteria range from extremely simple to complex. There is
consequently a range of biosynthetic capabilities in different bacteria.
Some, such as E. coli, can synthesize their macromolecules from simple
inorganic molecules, while other bacteria have requirements for amino
acids, peptides, vitamins and growth factors. Although bacteria have
some pathways peculiar to themselves, the basic pathways which they
possess for their biosynthetic processes are similar to their eukaryotic
counterparts. The reader is referred to a standard biochemistry
textbook if more information is required.
Fig. 2.13 Diagram illustrating bacterial multiplication by binary

fission. Each circle represents a bacterial cell.
Growth of bacteria
Growth can be defined as the orderly increase of all the chemical
components of the cell. Bacteria fulfil this definition, and when they
have grown to a certain size (length) they undergo cell division to
produce two daughter cells identical to the parent cell. The process is
termed binary fission (Fig. 2.13). Bacterial growth can be equated to
cell number: one bacterium divides into two, these two produce four,
and then eight and so on. The growth rate of a bacterium is therefore
measured by measuring the change in bacterial number per unit time.
The time between cell divisions can be as short as 10–20 minutes for
many common bacteria growing in laboratory media.
The number of bacteria at a given time can be estimated by taking a
small sample and counting the number of organisms directly by use of a
counting (haemocytometer-type) chamber and a microscope, or by
diluting a sample and spreading small volumes over the surface of an
agar plate and counting the number of colonies after a suitable
incubation time.
If bacterial number is plotted against time the shape of the curve will
show an exponential increase (Fig. 2.14). If however the number is
plotted as a log value, a straight-line graph is the result (Fig. 2.14).
The bacterial growth curve

If a broth culture of bacteria is set up from a small inoculum, the
population size increases following a classical pattern (Fig. 2.15).
There are three distinct phases:
1. The lag phase: the inoculated bacteria are becoming acclimatized to

the environment, switching on enzymes, adjusting to the
temperature and atmospheric conditions.
2. The logarithmic, or exponential phase: there are no restraints on

the bacteria, and they are dividing exponentially: 1 to 2 to 4 to 8 to
16….
3. The stationary phase: an essential nutrient becomes limiting, or
there is a build-up of a toxic metabolite (for example an acid or
alcohol) which stops the growth. At this stage some bacterial
species die and there is a decline phase, while other bacteria may
remain viable for an extended time. Sporeformers often sporulate at
this time.
Fig. 2.14 Arithmetic and logarithmic graphs illustrating changes in
bacterial number per unit time.
Fig. 2.15 The classic bacterial growth curve for a batch

culture.
This is the type of culture that is typically set up in the laboratory to
investigate the identity of a bacterium, or its susceptibility to an
antibiotic. It is termed a batch culture and has some parallels in nature,
for example the contamination of a sterile fluid or the formation of an
abscess or lung infection. Obviously many other parameters must be
considered, not least the response of the host, when the ‘culture’ is an
active infection.
Another way of culturing bacteria, which is commonly used industrially,
is by means of continuous culture, a process whereby medium is
constantly added with the excess medium plus culture draining away.
The growth rate can be controlled by altering the dilution rate of the
culture. This type of culture also has parallels in nature, for example
the normal flora of the mouth and gastrointestinal tract, diarrhoeal
disease, and urinary tract infections.
Key facts
Shapes and arrangements of bacteria: cocci, rods/bacilli, vibrios

and spirals. These may grow singly, in clusters or in chains.
Gram-positive bacteria are dark violet and Gram-negative
bacteria are pink. The Gram reaction is related to the structure of
the cell envelope. Gram-positives have a cytoplasmic membrane
and wall; Gram-negatives have inner and outer membranes
(latter with LPS and porins). Other examples: waxy envelopes of
mycobacteria (acid-fast bacilli)
The structure and function of typical membranes: lipid bilayer and
fluid mosaic model. The process of osmosis.
The structure and function of peptidoglycan: long parallel chains
of alternating N-acetyl glucosamine and N-acetyl muramic acid,
cross-linked through peptide side-chains and cross bridges.
Responsible for shape and strength of the bacterium.
Teichoic acids in Gram-positives: negative charge, scavenging
divalent cations.
Lipopolysaccharides in Gram-negative outer membrane: S- and R-
forms, non-fluid membrane, endotoxin.
Capsules, slime, exopolysaccharides: role in adhesion/biofilm and
in protection.
Surface appendages: flagella for movement, fimbriae/pili for
attachment.
Bacterial cytoplasm: DNA supercoiled, circular; 70S ribosomes for
protein synthesis; inclusion/storage granules.
Spoculation in Bacillus and Clostrdium, and resistance of bacterial
(endo)spores.
Nutrition: simple to complex.
Energy production: fermentation and respiration pathways.
Binary fission and bacterial growth: exponential. Batch and
continuous culture.
Further reading
Mims C, Dockrell HM, Goering RV, Roitt I, Wakelin D and Zuckerman
M (2004). The bacteria. In Medical Microbiology, 3rd edn, Chapter 2.
Elsevier Limited, London.
Edition
Simon Bagg)
> Table o f C o ntents > Sectio n 1 > 3 - Bacterial taxo no my
3
Bacterial taxonomy
Taxonomy is the term given to the systematic classification, naming,

and identification of living organisms. Bacterial taxonomy is often
thought of as a complex but ‘necessary evil’, that is uninteresting to
learn and overwhelms many students. It is, however, at the very root of
biological communication and is essential for describing an organism,
whether newly discovered or well known. A working knowledge of
taxonomy is also useful for diagnostic microbiology and for studies in
epidemiology and pathogenicity. The intricacies of the rapidly moving
field of modern taxonomic research and its methodologies are largely
beyond the scope of this book. However, sufficient detail will be given to
enable the reader to acquire adequate skills in the communication of
bacterial names, and to illustrate the reasons why taxonomy is so
important.
Traditionally, all living organisms were classified into different
hierarchical groups or taxa (singular, taxon), based on a system
originally devised by Linnaeus (a Swedish botanist) in 1735. The term
kingdom is at the top of the hierarchy, and there are five kingdoms:
Prokaryotae (bacteria), Protista (algae and protozoa), Myceteae (the
fungi), Plantae, and Animalia. This is followed by phylum (animals and
protozoa) or division (plants, algae, fungi, and bacteria), class, order,
family, genus, and finally species. This system is still the most widely
used for the classification of plants, animals, and most other living
organisms, and remains the main method for micro-organisms, with the
exception of viruses (Chapter 6).
The following scheme is illustrated for the human being:
Kingdom: Animalia
Phylum: Chordata
Class: Mammalia
Order: Primates
Family: Hominoidea
Genus: Homo
Species: sapiens
The genus and species names are used together to give the scientific
name (or Latin binomial) of the organism (although Greek and
sometimes modern European words are commonly used and are
Latinized). This binomial is printed in italics with the genus name
beginning with an upper case (capital) letter, and the species name with
a lower case (small) letter, for example Homo sapiens. In a piece of
writing this can be abbreviated to the form H. sapiens after it has been
written out initially in full.
Bacterial classification and nomenclature

The system described above is commonly abbreviated for bacterial
classification. The kingdom Prokaryotae was divided into three
divisions: Eubacteria, Archaebacteria, and Cyanobacteria. However, with
the Archaebacteria being separated into its own domain (kingdom), the
Archaea, it is perhaps easier just to think of the
bacteria (or Prokaryota) as a single entity. Class and order are usually
ignored, being replaced instead by morphological divisions based on cell
shape, Gram reaction, and nutritional properties. The next important
level is family, followed by genus and species. The use of these terms
will now be illustrated for two important medical pathogens, Escherichia
coli and Staphylococcus aureus.
The organism Escherichia coli is a Gram-negative, rod-shaped,
facultatively anaerobic organism which is tolerant to bile. This places it
in the family Enterobacteriaceae. A series of metabolic tests, including
the fermentation of various sugars and the production of indole from
tryptophan, separates it from other genera of Enterobacteriaceae such
as Salmonella and Proteus and places it in the genus Escherichia and
the species coli. It is commonly abbreviated to E. coli.
Fig. 3.1 Phenotypic methods used in bacterial
taxonomy.
Staphylococcus aureus is a facultatively anaerobic Gram-positive coccal

organism growing in clusters, belonging to the family Micrococcaceae. It
is tolerant to 10% sodium chloride, resistant to lysozyme, but sensitive
to lysostaphin, and thus belongs to the genus Staphylococcus. As it
possesses Protein A on its cell surface
and produces the enzyme coagulase this places it in the species aureus.
This can be abbreviated to S. aureus.
In writing, where two different species are being discussed which both
have the same initial letter for the genus, for example Staphylococcus
aureus and Streptococcus mutans, it is common to use the termsStaph.
and Strep. as the abbreviations, to avoid ambiguity. When bacterial
names are used as adjectives, for example staphylococcal toxin or
clostridial spore, or are used collectively, for example salmonellae or
streptococci, the names are not italicized and do not begin with a
capital letter.
Different approaches to taxonomic methods

Traditionally, morphological and biochemical characters have been used
in taxonomy. These characters are referred to as phenotypic, and tend
to group organisms into structurally and physiologically related
organisms. In bacterial taxonomy the shape of the organism, the Gram
stain, and its metabolic processes are phenotypic characters of prime
importance. Figure 3.1 summarizes the most important phenotypic
methods used in bacterial taxonomy. This form of taxonomy can
therefore be thought of as a natural system which shows the degree of
relatedness of organisms. In order for the organisms to be arranged in
a classical taxonomic tree it is necessary to devise a hierarchy of
characters, with some characters being more important than others.
The relative importance of a character is often very subjective with
little or no scientific objectivity. For example, is the shape of an
organism more important than its reaction to the Gram stain or its
ability to ferment glucose?
Numerical, or Adansonian, taxonomy is significantly different from the
classical approach described above and was originally
devised about 200 years ago to avoid inherent bias in the classical
methods. Every observable character of an organism carries an equal
weight and they are considered equally in calculating similarities or
differences between organisms. A similarity coefficient can be
calculated between two or more organisms. The results of such methods
do not give rise to the classical genus and species, but to mathematical
relationships which are often represented as a dendrogram
demonstrating similarities and differences. Modern computer programs
can be used to analyse numerical data and construct dendrograms (Fig.
3.2).
Fig. 3.2 Numerical taxonomy and construction of

dendrograms.
Fig. 3.3 Genotypic methods used in bacterial taxonomy. Full details
of the methods are beyond the scope of this book, but see Chapter 5
for more information.
Evolution is an important concept to consider in taxonomy, as

genetically similar organisms are certainly related and are also likely to
be morphologically related. Evolutionary relatedness and similarities in
structure and physiology have given rise to the term phylogenetic or
natural taxonomy. In the past 10 years the sequencing of ribosomal
RNA (rRNA) and the genes coding for rRNA have revolutionized our
views on bacterial phylogeny.
The classical and numerical systems could conceivably result in the
grouping of organisms which are structurally and physiologically
related, but which are genetically distinct, within the same taxon. Again
in the last 10 years or so, this problem has led to the wide-scale use of
genetic or molecular approaches. Genotypic taxonomy uses the
organism's genetic characteristics, which are considered by many to be
more important than phenotypic characteristics. Figure 3.3 lists the
main genotypic methods in current use. It appears that this modern
approach to taxonomy is being led by the rapid advances in nucleic
acid-based technology and has placed taxonomy into the ‘hi-tech’ end of
biology, regaining a status which has been absent for the best part of a
century. Further details of genotypic methods are given in Chapter 5.
Fig. 3.4 A simplified scheme for bacterial identification - major
steps and a summary of techniques.
The definition of a species can be described in genotypic taxonomy as ‘a

group of strains sharing 70% or greater DNA:DNA relatedness, with 5°C
or less difference in melting points by stepwise denaturation between
homologous and heterologous hybrids formed under standard
conditions’ (see Chapter 5 for further explanation). Phenotypic
taxonomic methods should agree with this definition.
As stated earlier, one of the prime uses of taxonomy is in
communication. For example, the diagnostic laboratory must be able to
communicate with the clinician in terms of bacterial nomenclature. If
the system is so confused and rapidly changing then this will be
unworkable. This means that there should perhaps be two levels of
bacterial classification and nomenclature: one at the research level
which is detailed and progressive, the other at the pragmatic level
which is changed less frequently. There is an inevitable dichotomy, but
there must also be compatibility between the two systems, with the
latter being adaptable to change as development proceeds at the
research level.
Fig. 3.5 Clinically important Gram-positive
bacteria.
Fig. 3.6 Clinically important Gram-negative
bacteria.
A final term to be introduced here is polyphasic taxonomy. It was
coined in 1970, but has recently been reintroduced as a ‘Consensus
approach to bacterial systematics’. In principle, this method uses all the
phenotypic, genotypic, and phylogenetic information available.
Databases can be constructed of genotypic, phenotypic, and numerical
results and, assuming that the technology produces reproducible data,
access should allow universal use of this data. It is likely that for the
foreseeable future, however, nucleic acid sequence data will be the
most useful of these facilities.
Bacterial identification
The steps taken in the laboratory to identify a bacterium follow a series
of routine procedures which are summarized in Fig. 3.4. This is a
practical topic and there are many textbooks and laboratory
manuals dedicated to the subject of bacterial identification. As

demonstrated in Fig. 3.4, the major techniques employed are
traditional, based on culture and identification by classical methods
such as microscopy, colonial appearance, growth characteristics, and
biochemical/metabolic tests. For some bacteria, simple Gram staining
and colonial appearance on a selective medium are sufficient for
presumptive species identification by an experienced microbiologist. For
other species, a more extensive battery of tests would be required. A
myriad of commercial test kits is now available for the rapid
identification of many clinically important bacteria. However, they
should be used with caution, and a skilled technician is still required to
ensure that pure cultures are being used and that correct
interpretations of the tests are made.
Fig. 3.7 Miscellaneous clinically important
bacteria.
Fig. 3.8 The future of bacterial identification? See Further reading
at the end of this chapter and Chapter 5 for an explanation of
terms.
Semi-systematic lists of clinically important bacteria are given inFigs.

3.5, 3.6, and 3.7.
Identification beyond species level

For most known bacteria it is possible, using a few key tests, to
establish the genus and the species to which a particular bacterium
belongs. However, it is often advantageous to go beyond the species
level to give important extra information. The terms subspecies, race,
and variety, which are used in other classification schemes, are not
commonly used in bacterial taxonomy. It is more common to use the
suffix ‘-type’. This can be serotype (based on the presence of certain
antigens), biotype (based on certain biochemical reactions which can be
used to subdivide a species), phage- or bacteriocin-type, or toxin type.
These are phenotypic characters and can give important information in
some epidemiological investigations.
An example would be the investigation of an outbreak of food poisoning
caused by E. coli. If E. coli serotype O157 were identified it would
predict the risk of the haemolytic uraemic syndrome, a major cause of
acute renal failure in children suffering from diarrhoea due to this
organism. It would then be important to find its source and prevent
infection of others.
Another way of showing identity between strains of the same species in
epidemiological investigations is to ‘fingerprint’ them. This usually
means genetic fingerprinting, using one of the methods listed in Fig.
3.3. Restriction enzyme digests of whole cell DNA or polymerase chain
reaction (PCR)-amplified genes are the usual methods (see Chapter 6).
Phenotypic fingerprinting is also possible using polyacrylamide gel
electrophoresis (PAGE) of whole cell or envelope proteins, or
lipopolysaccharide patterns.
Bacterial identification: the future

Some laboratories are already moving away from the classical approach
to bacterial identification and utilizing a wholly molecular approach.
The procedure outlined in Fig. 3.8 is an attempt to illustrate what the
future may bring. Those laboratories that are already pursuing such a
scheme at present tend to do this in parallel with traditional methods.
It is perhaps in oral microbiology, where so many of the indigenous
bacteria are difficult or impossible to culture, that these molecular
approaches may be most useful. Good communication is essential
between laboratory scientist and physician and, finally, experience is
paramount.
Key facts
Taxonomy is the systematic classification, naming and

identification of a living organism.
Bacteria belong to the kingdom Prokaryota.
The scientific name of an organism is given as a Latin binomial
and is printed in italics. The genus name is given first with the
initial letter a capital, and the species name in small letters, for
example Escherichia coli.
Taxonomic methodologies include phenotypic, numerical,
phylogenetic, genotypic and polyphasic schemes.
Bacterial identification to species level is performed by following
a series of defined steps which may include culture, microscopy,
morphological, biochemical, immunological and genetic tests.
At the level below species, typing or fingerprinting may be
performed to give extra information for epidemiological or
taxonomic studies.
Further reading
Collier, LH (ed.) (1998). Topley and Wilson's Microbiology and
Microbial Infections, 9th edn. Edward Arnold, London.
Fredricks DN and Relman DA. (1996). Sequence-based identification

of microbial pathogens: a reconsideration of Koch's Postulates.
Clinical Microbiology Reviews 9, 18–23.
Edition
Simon Bagg)
> Table o f C o ntents > Sectio n 1 > 4 - Bacterial patho genicity and epidemio lo gy
4
Bacterial pathogenicity and
epidemiology
The relationships between bacteria and the

host
A bacterial pathogen is simply a bacterium that causes harm to a host,
resulting in disease, whilst pathogenicity is the process by which this is
brought about. Before describing bacterial pathogenicity, it is useful to
consider some of the biological relationships that occur between
bacteria and the human body.
The resident microflora

The body is naturally colonized by a vast and complex microflora or
microbiota, which consists mainly of bacteria. These bacteria are
generally considered harmless and are found over the whole outer
surface of the body and in much of the gastrointestinal tract, the
urogenital tract, and the oropharynx. Thus, most epithelial surfaces,
with the exception of the lungs and the upper small intestine, have a
resident microflora. This association of bacteria with the host is a
relatively stable symbiotic relationship. The
bacteria gain obvious benefit from the relationship by being in a fairly

constant environment, to which they are adapted.
Commensal and symbiotic relationships

If it is considered that the host does not benefit from the relationship,
but is unharmed, the association would be termed a commensal
association. However, if the human host also gains from such an
association (and this is perhaps more usually the situation), then it is
more accurate to describe the relationship as mutualistic or truly
symbiotic. This normal microflora, however, is often termed the
commensal flora, which is an erroneous description in most cases. It is
likely that some of the symbiotic bacteria produce nutrients and
vitamins for the host and break down harmful chemicals, but one of the
most important benefits that the host derives is protection from
harmful micro-organisms. The harmless bacteria that naturally colonize
areas prevent access by pathogens, a feature often referred to as
colonization resistance. For example, if the normal bacterial flora of the
mouth is disturbed as a result of treatment with a broad-spectrum
antibiotic, a possible consequence might be the development of a
candidal infection such as thrush (see Fig. 27.5, p. 277). This is
because the yeast Candida albicans - a eukaryotic fungal pathogen
which is naturally resistant to antibacterial agents – is allowed access
to sites normally colonized by harmless bacteria.
Parasitism
The final type of biological association that must be considered is
parasitism, where the host is harmed by the relationship but the
parasite gains. In biological terms an obligate parasite requires a host
for all or part of its life cycle. This is summarized in Fig. 4.1. For the
parasite to be successful in biological terms it must be able to complete
this life cycle, even if the host is ultimately killed. Bacterial parasites of
humans are, by definition, pathogens, since they harm the host and
cause disease. Furthermore, they are termed obligate or primary
pathogens, since causing harm is a necessary part of their life cycle.
They are also infectious and can be transmitted from a single individual
to one or many others. The organisms Vibrio cbolerae and Neisseria
gonorrboeae, which cause cholera and gonorrhoea, respectively, are
both good examples of obligate parasites, that is to say infectious
agents requiring a human host. Other bacteria, including some which
produce food poisoning, for example Salmonella enterica, have their
main reservoir in animals where they live parasitically, but they can
cause disease in humans following transmission via food or drink. A
disease that is transmitted from an animal to man is known as a
zoonosis.
Fig. 4.1 A simplified life cycle of a parasite. There may be more
than one host species, and the parasite may have a free-living
stage.
Not all bacterial pathogens are true parasites, because some do not
depend on a host for their life cycle:
Some bacterial pathogens normally live in the environment, existing

saprophytically (gaining their nutrition from dead organic matter)
and only causing disease when they inadvertently enter the host.
Clostridium tetani (which can cause tetanus when it enters a wound
from the soil) and Legionella pneumophila (which causes
Legionnaires' Disease but lives naturally in water) are good
examples of pathogens that are not parasites of humans.
The components of the normally harmless microflora may also
become pathogenic. Thus, a bacterium which lives normally and
harmlessly on the skin may become a pathogen if it enters a wound,
for example Stapbylococcus aureus. Similarly, oral streptococci,
which are harmless in the mouth, can cause infective endocarditis
following a bacteraemia in patients with rheumatic heart disease.
Such infections are known as endogenous infections, compared to
exogenous ones that can come either from another host or the
environment.
Opportunistic infections
A bacterium which normally lives harmlessly with the host, and is non-
pathogenic because of a host's normally functioning immune system,
may become pathogenic if the immune system is compromised in some
way. Such organisms are termed opportunist pathogens. This type of
infection has assumed mounting importance in recent years, following
the use of powerful immunosuppressive drugs in modern medicine and
the emergence of AIDS. A good example of a versatile opportunistic
pathogen is Pseudomonas aeruginosa, which can cause serious
infections of burns or contribute to severe lung disease in cystic fibrosis
sufferers, both conditions where innate immune mechanisms have been
compromised. Many of the bacteria of the normal oral flora can cause
opportunistic infections when they enter sites in which abscesses can
develop, both within the mouth (Chapter 25) and elsewhere in the
body, for example the brain (Chapter 17).
Transmission of bacteria
Bacteria may be transmitted to a new host in four different ways:
Direct spread In these cases, there is direct spread to the host

from the microbial source, for example drinking contaminated water.
Indirect spread Indirect spread occurs if a secondary inanimate

microbial source (fomite) is involved in the transmission of microbes
to a new host. This may occur when dental instruments (secondary
source) used in the patient's mouth (primary source) are not
properly sterilized before being used on another patient.
Airborne spread The third mode is airborne spread, also called
droplet infection. For example, if an individual with influenza
sneezes, another host may inhale some of those contaminated
droplets or aerosols, and become infected.
Vector-borne spread The last mode of spread is vector-borne
spread, which involves insects. This may be external, in which
microbes contaminate the outside of the insect, for example flies
crawling on faeces, followed by contamination of food which is then
ingested. Internal vector-borne spread occurs when the microbe is
ingested by the insect, for example rat fleas ingesting Yersinia
pestis and then deposited on the skin of a new host. As the flea
bites the new host, it frequently defaecates or regurgitates and the
host scratches the irritation, thus rubbing the contaminated
material into the skin.
Fig. 4.2 The stages in bacterial pathogenesis. Parasites, by
definition, complete all these stages, but this is not necessary for all
pathogens.
Pathogenesis of bacterial disease

All pathogens, whether parasites or not, go through a series of stages
during the pathogenic process. These steps are referred to
as pathogenesis and are summarized in Fig. 4.2. Most pathogens
possess several different pathogenic mechanisms which operate in
concert, and the whole process is often referred to as being
multifactorial. It is most unusual for only a single pathogenic factor to
be involved in a bacterial disease, though an example is botulism,
where the ingestion of a potent toxin produced in food by Clostridium
botulinum results in a potentially lethal poisoning.
Virulence
An important term, which is used in connection with pathogenesis, is
virulence. Virulence is a measure of how harmful a pathogen might be
to a host. The determinants that confer on the bacterium the potential
to cause harm are termed virulence factors. These may either be
associated with the cell, usually the cell surface, or be produced
extracellularly as exotoxins. Virulence factors may be involved in one
or more of the stages of pathogenesis such as adherence, invasion,
nutrient acquisition, or evasion of immune mechanisms, as well as
contributing to the disease process. Many virulence factors have a role
in the primary physiology or structure of the bacterium and their role
in virulence may be purely coincidental.
Virulence factors
Surface-associated virulence factors
These factors are summarized in Fig. 4.3. Full details of their structure
and physiological function are given in Chapter 2. Many of these
surface structures act by allowing the bacterium to evade the immune
response by three main mechanisms (see Chapter 8 for details of the
host defence mechanisms mentioned below):
Fig. 4.3 Surface-associated bacterial virulence factors. LPS,
lipopolysaccharide; LOS, lipo-oligosaccharide.
The long O-polysaccharide chains of lipopolysaccharide (LPS,

endotoxin), or capsular polysaccharides, help to protect against
complement-induced lysis by activating complement at a distance
from the bacterial cell membrane, thus preventing insertion of the
complement-membrane attack complex.
Bacteria with capsules are often resistant to phagocytosis. The
capsule is usually a large hydrated gel with hydrophilic properties
surrounding the bacterium. During growth, capsulate bacteria
frequently stick together, producing a microcolony, which is often
attached to a substratum as a biofilm. This is resistant to phagocytic
cells simply because of its large size. Capsulate pneumococci
(Streptococcus pneumoniae) are the classic example of this type of
evasion mechanism.
Certain bacteria ‘hide’ from the immune system by having their
outer surface covered by a material which mimics host (self)
material. This is termed antigenic mimicry or camouflage. There are
several well-known examples of this phenomenon, but the most
striking is that of the group B capsular type of Neisseria
meningitidis, the type of meningococcus responsible for most cases
of bacterial meningitis in temperate regions of the world. The group
B capsular polysaccharide is made up of sialic acid (poly-N-acetyl
neuraminic acid), a substance commonly found on the cell surface of
mammalian cells. Its structure is identical to that
of an embryonic neuronal cell coat. Apart from it hiding the

bacterium from the immune system, presenting as a self-antigen, it
is also non-immunogenic, and thus of no use in vaccine formulation.
Lipopolysaccharide and to a lesser extent peptidoglycan, teichoic acids,

and some other cell-surface carbohydrates, act in pathogenesis as
potent stimulators of the immune system. The resulting responses –
which include inflammation following the release of cytokines such as
interleukin (IL)-1, IL-6, and tumour necrosis factor (TNF), recruitment
of leucocytes, and triggering the complement and clotting cascades –
are beneficial when produced at an optimal level. However, if they are
overproduced, because of excessive stimulation, this can result in
severe immunopathology (Chapter 18). Endotoxic shock, septic shock,
severe sepsis, and the systemic inflammatory response syndrome
(SIRS) (Chapter 18) are terms used to describe this condition. It is a
major cause of death in severely ill patients.
Bacterial exotoxins
In contrast to endotoxins (LPS) – which are cell-associated during
normal growth of the Gram-negative bacterium and heat-stable –
exotoxins are extracellular products. They are produced by both Gram-
positive and Gram-negative bacteria. Exotoxins are proteins, often
enzymes, usually of high molecular mass and are heat-labile. They
cause damage to cells and tissues or can affect the whole animal.
Fig. 4.4 Classification of bacterial exotoxins by their action at the
cellular level.
Several different methods are available for the classification of

exotoxins. A simple method is to classify them by their site of action
within the body. Thus, neurotoxins act on nerves, enterotoxins act on
the gastrointestinal tract, and cytotoxins act on cells. Another approach
is to describe them by their biochemical mode of action, for example
proteases, phospholipases, collagenases, ADP-ribosylating (an ADP-
ribose is transferred from NAD+ ), and bipartite (two parts: A – active
and B – binding). The latter two properties are common to several
types of exotoxin. A further method of classification is to group them
into their action at the cellular level (Fig. 4.4).
The major benefits endowed on a pathogen by exotoxins include aiding
its spread through the body (tissue destruction), killing phagocytic cells
(diphtheria toxin, and some staphylococcal toxins), and contributing to
transmission (diarrhoea, coughing). Others such as the neurotoxins, for
example tetanus and botulinum toxins, seem to have no beneficial role
for the pathogen.
Some bacterial metabolites can act as virulence factors and play a role
in disease production. These include acids, which are key factors in
dental caries.
Genetic regulation of virulence factors

Bacterial pathogenicicy is often multifactorial, with several virulence
factors working together to cause disease. Some bacteria always
(constitutively) produce their full battery of virulence
factors in all growth conditions. However, for many bacteria, especially

those that can exist in an environment where they are not being
pathogenic, it is unnecessary (energetically wasteful) for them to
produce virulence factors which would only be required during their
pathogenic phase. Many of these bacteria have evolved genetic systems
for regulating the phenotypic expression of virulence factors. The
expression is often regulated by environmental stimuli and is controlled
in a coordinated manner, a single stimulus resulting in the coordinated
expression of two or more virulence factors. An example is the toxR
gene of Vibrio cholerae. A single protein, with both sensing and kinase
activity, coordinately regulates the expression of both cholera toxin and
the ‘toxin co-regulated pilus’ (which is used for adherence), following
stimulation brought about by a change in temperature, pH, amino acid
availability, or in osmolarity.
Many genes which code for virulence are carried on specific regions of
the bacterial chromosome termed pathogenicity islands. They often
code for several virulence factors and appear to have been acquired
from other bacteria by horizontal gene transfer. They are identified as
‘foreign’ as they often have a different proportion of the bases GCAT
than the rest of the chromosome. E. coli O 157 is a good example of an
organism with a pathogenicity island. It possesses the locus of
enterocyte effacement (LEE) pathogenicity island, which codes for the
machinery involved in intimate adherence of the organism to the gut
wall and subsequent pathogenicity. Other virulence genes are more
obviously horizontally transferred as they are carried on plasmids or
lysogenic bactehophage (prophage) within the bacterium. The bacteria
become non-pathogenic if they lose their plasmid or prophage.
Examples of bacteria carrying virulence plasmids are the
enterotoxigenic E. coli (diarrhoea-causing E. coli) which have plasmid
genes encoding both their colonization factors (fimbriae) and
enterotoxin. Diphtheria toxin, E. coli shiga toxins and some botulinum
toxins are encoded by lysogenic bacteriophages (Chapter 5).
Molecular pathogenesis
In recent years this new term has been coined for the investigation of
pathogenic mechanisms using a largely genetic approach. As the
genomes of infectious agents and their hosts are determined it is
possible to knock out key genes, both of putative virulence factors and
of host defences (Chapter 8), to see the outcome. This field is still in its
infancy but it has a massive potential.
Fig. 4.5 Establishment that a bacterium is the cause of a disease

and identification of virulence factors by fulfilment of molecular
Koch's Postulates.
As mentioned in Chapter 1, the aetiology of an infectious disease has

traditionally been investigated through the establishment of Koch's
postulates. However, for many reasons, not least those of ethics,
inability to culture an organism and the preponderance of opportunist
infections arising from the endogenous microbiota, they have been
impossible to fulfil. As a result of this, molecular Koch's Postulates have
been created and are shown in Fig. 4.5.
Epidemiology
Epidemiology is the study of the occurrence, spread, and control of
disease. It relies upon the collection of detailed statistical information
recording the diseases affecting a population and, in the case of
infectious diseases, may help to identify their causes and modes of
transmission. It may also predict the future likelihood of infection,
identify risk factors, and help in the planning of control measures
involving chemotherapy and vaccination.
An understanding of how and why infectious diseases occur requires
consideration of not only the specific determinants themselves, but also
how they interact. The three basic determinants are:
1. the number of microbes entering the body (dose);

2. the ability of the microbe to cause disease (v irulence);
3. the ability of the body to defend itself against the specific microbe
(resistance).
The occurrence of disease or maintenance of health is determined by

the interaction of these three factors. High dose, high virulence, and
low resistance result in the greatest chance for developing disease.
Thus, prevention of disease focuses on lowering the dose of the
microbes that contaminate our bodies (for example by wearing gloves,
masks, eyewear, and gowns at the dental chairside) and increasing body
resistance by vaccination.
It should be stressed that many factors other than mere exposure to a

pathogenic microbe must be satisfied for clinically evident infection to
result. Thus, there are several stages in the development of an
infectious disease, starting with the source of a microbe and ending
with a harmful infection in a new host. These have been described
earlier in this chapter.
Occurrence of diseases
Infections may occur as occasional cases at irregular intervals (sporadic
occurrence), as a low number of cases at regular intervals (endemic
occurrence), or as a high number of cases appearing suddenly
(epidemic occurrence). A pandemic is an epidemic that has spread
between continents.
The prevalence of a disease refers to the proportion of the population
affected, or to the numbers of cases that are active at a particular time.
This differs from the incidence of a disease, which is a rate, indicating
the number of cases within a specific population in a defined time
period.
Epidemiological studies
There are two main analytical epidemiological methods available. In a
case-controlled study, the investigation starts with a clinical effect and
attempts to identify the cause of that effect. This is also referred to as
a retrospective study – going back in time to determine what caused a
specific effect. In a cohort study, also referred to as a prospective
study, a population exposed to a presumed cause is followed to identify
if and when disease supervenes and the outcome.
An additional method is a cross-sectional study, which looks at the
population at a specific time point to describe the prevalence
relationships outlined above.
Identification and typing of micro-organisms are crucial in most
epidemiological studies and are described in Chapters 3 and 5. Indeed,
epidemiology is advancing rapidly with the advent of molecular
techniques to identify and type infectious agents.
Conclusions
Bacterial pathogenicity is a rapidly growing area of microbiology. It is
very amenable to investigation by modern molecular techniques, the
cloning of virulence factors, and determination of the DNA sequence of
their genes, therefore allowing greater insight into their roles in
virulence and to their control.
It should be emphasized that bacterial pathogenicity has to be viewed
from two aspects, from that of the pathogen (its virulence) and of the
host (the immune system). The host-pathogen relationship is in a state
of equilibrium, and pathogenicity is sometimes referred to in terms of
the host-pathogen equation. The outcome of the association, whether in
favour of the host or the pathogen, can be strongly influenced by
clinical intervention. Thus, understanding of pathogenicity forms the
basis of many of the strategies employed for the prevention and
treatment of bacterial diseases. Bacterial genetics, the immune system,
and intervention strategies are described more fully in later chapters.
Key facts
Bacterial pathogenicity is the process whereby the bacterium (the

pathogen) causes harm or disease to the host. It is usually a
multifactorial process.
Three natural associations can exist between bacteria and host:
symbiosis (or mutualism) – both gain; commensalism – bacterium
gains, host is unharmed; and parasitism – bacterium gains, host
is harmed.
Pathogens can be primary/obligate pathogens or they can be
opportunists.
There are several stages in pathogenesis, including entry or
attachment, evasion of the immune system, cause of harm,
release and spread.
Virulence is a measure of the potential the bacterium has to
cause harm. Virulence factors can be bacterial cell surface-
associated, or extracellular exotoxins.
Harm is brought about by a process which is often multi-factorial,
involving both bacterial factors and the host response.
The expression of many virulence factors is under environmental
and genetic control. Some virulence genes are transferred
horizontally and are carried on pathogenicity islands, plasmids or
prophages.
Molecular pathogenesis, including molecular Koch's Postulates, is
clarifying many previously unknown pathogenic mechanisms.
The host-pathogen relationship is a state of equilibrium which can
be altered by clinical intervention.
Epidemiology is the study of the occurrence, spread, and control
of diseases.
The three factors determining whether an infectious disease is
established are the dose and virulence of the organism and the
host resistance.
Most human pathogens are contracted from other humans, but
some are from animals (zoonoses) and some from the
environment.
Diseases may be sporadic (occasional cases at irregular
intervals), endemic (low number of cases at regular intervals), or
epidemic (large number of cases appearing suddenly).
Prevalence refers to the proportion of the population affected, or
the number of active cases at a particular time. Incidence is a
rate, indicating the number of cases in a specific population over
a defined time.
Further reading
Coggan D, Rose G and Barker DJP (1997). Epidemiology for the
Uninitiated, 4th edn. BMJ Publishing Group, London.
Mims C, Nash A and Stephen J (2001). Mims' Patbogenesis of

Infectious Disease, 5th edn. Academic Press, London.
Mims C, Dockrell HM, Goering RV, Roitt I, Wakelin D and Zuckerman

M (2004), Pathologic consequences of infection. In Medical
Microbiology, 3rd edn, Chapter 17 Mosby/Elsevier: London.
Salyers, AA and White, DD (2001). Bacterial Pathogenesis: A

Molecular Approach, 2nd edn. American Society for Microbiology,
Washington DC.
Ala'Aldeen D (2002). Bacterial pathogenicity. In Greenwood D, Slack

RCB, Peutherer JF (eds) Medical Microbiology, 16th edn, Chapter 8.
Churchill Livingstone, Edinburgh.
Edition
Simon Bagg)
> Table o f C o ntents > Sectio n 1 > 5 - Bacterial genetics and mo lecular bio lo gy
5
Bacterial genetics and molecular biology
Genes and DNA

Genes are the elements of the hereditary material of living cells that
carry the information to code for all the necessary components and
reactions of life. At cell division the genes are replicated and a copy
goes to each daughter cell. It was not until the 1950s that
deoxyribonucleic acid (DNA) was recognized as being the substance of
genes. During the past half century the subject of molecular biology has
developed into a major discipline, underpinning many of the other fields
of biology and medicine. Molecular biology had its roots in bacteriology,
and many of the continuing advances in the subject still depend on
bacteria. Molecular biology is a vast and rapidly developing subject,
much of which is beyond the scope of this book. This chapter will give a
brief introduction to the basics of the subject, describe some of the
important aspects of bacterial molecular genetics, and demonstrate
some of the uses to which molecular biological techniques are being
directed.
Fig. 5.1 The structure of DNA. (a) The double helix: two anti-
parallel strands of deoxyribose nucleotides, linked along their
length with phosphodiester bonds, and cross-linked through
hydrogen bonding between complementary bases. (b) A detailed
diagram demonstrating base pairing.
The structure of DNA

DNA consists of the famous ‘double helix’, two antiparallel strands (3′ to
5′ and 5′ to 3′) of a sugar-phosphate backbone substituted
with purine and pyrimidine bases. Complementary base pairs are cross-
linked through hydrogen bonds, guanine (G) pairing with cytosine (C)
and adenine (A) with thymine (T) (Fig. 5.1). The four bases represent
the letters in a four-letter alphabet that are read linearly in three-
lettered ‘words’.
DNA replication
During cell growth the DNA replicates by a process known as semi-
conservative replication (Fig. 5.2a). This takes place at a replication
fork, where the separate helices of the DNA act as templates for the
new, daughter strands. Deoxyribonucleotides are added sequentially, a
phosphodiester bond forming between the deoxyriboses while a guanine
pairs through hydrogen bonds with a cytosine, and an adenine with a
thymine, and so on. However, as the DNA polymerase enzyme can only
add bases to the 3′ end of a growing strand, and the two parent strands
are antiparallel, the replication processes at the division fork are
asymmetrical (Fig. 5.2b). The initiation of a replication fork and the
subsequent DNA replication is performed by a series of enzymes
coordinated with the DNA polymerase. These include a topoisomerase
(DNA gyrase) to relieve helical winding and tangling problems, a DNA
ligase to join together newly synthesized fragments, and initiator
proteins which bind to specific DNA sequences and initiate a replication
fork.
Fig. 5.2 The replication of DNA. (a) Semi-conservative replication:

on replication each daughter cell receives an original strand plus a
newly synthesised strand. (b) A detailed diagram showing the
asymmetric synthesis of DNA at the replication fork. Both strands
are synthesised from 5′ to 3′ by the DNA polymerase. The lower
strand is replicated in short sections which are subsequently joined.
Mutation
Although the process of DNA replication is highly efficient and accurate,
with built-in repair mechanisms, mistakes can occur. For example, one
or more nucleotides can be omitted, or extra ones can be inserted, a G
can be added instead of an A, or a T instead of a C. These mistakes
occur in fewer than 1 in 10 9 nucleotides added. Such mispairings are
one of the methods by which mutations occur. Other methods by which
mutations occur include direct damage to the DNA by radiation or
chemicals. These errors in the DNA can be passed on to future
generations.
The result of a mutation can vary, depending on the site and its extent.
The transcription and subsequent translation (see later) of the mutant
genes produces variations in the amino acid sequence in the proteins,
which may have properties so different that they do not function as
well, or they may be inactive and result in cell death. Some
transcription errors of DNA may be silent because the change in the
resultant protein is insignificant. Some mutations can even be better
than the original – this is the basis of ‘natural selection’, where
beneficial mutations can be selected for better survival of the organism.
The genetic code and protein synthesis

The processes by which the base sequence of the DNA is converted to
proteins are termed transcription (Fig. 5.3) and translation (Fig. 5.4).
The DNA sequence must first be transcribed to a complementary

messenger ribonucleic acid (mRNA), which is then read as a three-letter
code – the genetic code. RNA polymerase makes a complementary copy
of one of the strands of DNA from the 5′ end to the 3′ end. There is
only one type of RNA polymerase in bacteria, but three in eukaryotes.
Note that the mRNA has uridine (U) in place of the thymine (T) of DNA.
The RNA polymerase binds to the DNA at a starting point termed the
promoter. A length of mRNA is transcribed for a specific protein, the
transcription stopping when the polymerase reaches a stop or
termination signal, which in bacteria (E. coli) is when a series of U's is
added from a complementary series of A's on the transcribed DNA.
Fig. 5.3 Transcription. The mRNA is transcribed from the DNA
template producing a complementary copy of the DNA strand from
which it is being synthesized.
Fig. 5.4 Translation of mRNA at the ribosome - protein synthesis.

(a) The ‘empty’ ribosome showing binding sites for transfer RNAs.
(b) The incoming tRNA carrying an amino acid enters the A-site, the
anti-codon matching to the three-base codon of the mRNA. A
peptide bond is formed and the ribosome moves along the mRNA so
that the aminoacyl tRNA moves to the P-site where it becomes the
peptidyl tRNA.
In the cell, each amino acid has one or more of its own transfer RNA
(tRNA) molecules, which are specific short chains of folded RNA. One
end of the tRNA binds an amino acid, while the other end has a three-
base recognition sequence (anticodon) which binds to a complementary
three-base sequence in the mRNA – the codon. The genetic code is a
series of codons (three-base sequences) that determines the sequence
of the amino acids in the primary structure of the protein. Protein
synthesis takes place by a ribosome moving step-wise along the mRNA
chain (Fig. 5.4). Each ribosome has two binding sites, one which holds
the tRNA attached to the growing peptide, the P site, and the other the
incoming tRNA with the amino acid attached, the A site. Peptide bond
formation occurs here. Special codons code for the start and stop of the
peptide. In bacteria, formyl methionine, coded for by the sequence
AUG, is the initiator tRNA which always starts the peptide chain.
Genetic recombination
This is a basic mechanism by which genetic variation can occur, and is
an important phenomenon in survival and evolution. In simple terms,
recombination is the rearrangement of DNA that
involves the breaking and rejoining of two homologous DNA double

helices and is guided by base-pairing interactions between
complementary strands. This is shown simply in Fig. 5.5.
Fig. 5.5 Genetic recombination. (a) Pairing berween homologous
lengths of DNA. (b) The upper strand is nicked which permits partial
exchange of strands. (c) The lower strand is nicked. (d) The
recombinant molecules are resolved by further nicking and joining.
So far, the basic synthesis of nucleic acids and proteins has been
described, and this forms much of the fundamental knowledge required
for understanding the mechanisms of genetic transfer and genetic
techniques that are the subject of the remainder of this chapter.
Genetic transfer mechanisms in bacteria

One of the earliest observations in bacterial genetics was that bacteria
could easily transfer genes from one organism to another. This was
most dramatic when it was realized that resistance to antibiotics was
readily transferred from one bacterium to another.
There are three general methods for genetic exchange in bacteria:
transformation, transduction, and conjugation. The key points of each
are summarized below, and the latter two mechanisms are represented
diagrammatically in Figs. 5.6 and 5.7. Genetic transfer from one
bacterium to another is often referred to as horizontal gene transfer.
Transformation
DNA is released from a bacterium on lysis.
DNA is ‘naked’ - it is sensitive to the action of DNA-degrading
enzymes (DNase).
The recipient bacterium must be competent.
Transferred genes are inserted into the genome of the recipient cell
by recombination (Fig. 5.5).
Transduction (Fig. 5.6)

Bacterial viruses (bacteriophages), during their normal lytic cycle,
package a short length of DNA from the bacterial chromosome into a
virus ‘shell’.
DNA is transferred by the bacteriophage to a recipient bacterium.
Only short lengths of DNA (a few genes) can be transferred in each
virus particle.
The integrated bacteriophage genome is now termed a prophage.
Conjugation (Fig. 5.7)

This is only possible between closely related species.
A sex pilus produces a bridge between conjugating cells in Gram-
negative bacteria (in Gram-positives, mating cells lie side by side
with no visible connecting pilus).
Long lengths of DNA can be transferred.
Conjugative plasmids are most commonly transferred.
A whole chromosome can be transferred if it contains an integrated
F plasmid (Hfr donor: high-frequency recombination).
Genes can be mapped (in minutes) on the chromosome by
performing interrupted mating at set times and observing the genes
that have been transferred up to that time.
Resistance transfer
This is the term given to the genetic transfer of antibiotic-resistance
genes from one bacterium to another. The resistance genes are carried
on plasmids known as R-plasmids and are transferred by conjugation to
other, usually closely related, bacteria. R-plasmids of E. coli can be
transferred to such species as Salmonella, Shigella, Proteus, and
Haemophilus. R-plasmids often carry several different genes coding for
resistance to different antibiotics.
Transposons and insertion sequences

The fact that several different resistance genes could be carried on the
same R-plasmid has been recognized for many years. However, the
reasons why this was so common were not understood until the
existence of mobile genetic elements was discovered. Transposons are a
type of mobile DNA of 2000–20000 base pairs. They do not exist
independently but have the characteristic of jumping from one part of a
chromosome to another or to a
plasmid, or from one plasmid to another, or back to the chromosome. If

they are plasmid-located they can easily transfer from one bacterium to
another. Characteristically they carry several different antibiotic-
resistance genes. They are identified by having palindromic sequences
of bases (inverted repeats) at their ends (Fig. 5.8). Shorter lengths of
mobile DNA are termed insertion sequences which, by moving from one
site to another, can create genetic variation and are also believed to
control various cellular processes.
Fig. 5.6 Transduction. (a) A lytic bacteriophage attaches to a
susceptible bacterium and injects its DNA. (b) The bacteriophage
DNA codes for new bacteriophage coats and DNA. (c) The bacterial
DNA is disrupted and, during assembly of new bacteriophage
particles, some of the bacterial DNA is packaged inside
bacteriophage coats. (d) The bacterium lyses, releasing both normal
bacteriophage and those carrying some bacterial DNA. (e) A
bacteriophage carrying bacterial DNA can infect a new bacterium,
carrying into it genes from the donor bacterium. (f) This DNA can
integrate into the chromosome of the recipient by recombination
and be transferred to daughter cells on cell division. The integrated
bacteriophage genome is referred to as a prophage.
Manipulation of DNA and recombinant DNA
technology
DNA denaturation (melting)
On heating DNA the strands separate. On cooling, the strands renature
by complementary base pairing. The slower the rate of
cooling, the more accurate is the process of renaturation (Fig. 5.9a).

The melting temperature gives an indication of the proportion of G/C
and A/T in a sample of DNA.
Fig. 5.7 Bacterial conjugation. Demonstration of plasmid transfer.
(a) The donor (F + ) cell is carrying a plasmid which carries a motility
or F factor. It also codes for a specialized pilus, the F or sex pilus,
which is used to bind this cell to a recipient (F - ) cell. (b) The phage
replicates, and one strand of DNA moves through the sex pilus to
the recipient cell. (c) The incoming DNA is copied in the recipient
cell to form a new double-stranded DNA plasmid. (d) The recipient
cell now carries the plasmid with the F factor and has become a F +
cell.
Restriction endonucleases
These enzymes (commonly, simply termed restriction enzymes) occur
naturally and cut DNA at specific sites. They recognize a short sequence
of base pairs (bp), usually 4–6 in number, and cut at that site. The
enzyme recognizes a palindromic sequence of bases and cuts in a
straight or staggered manner, resulting in blunt or sticky ends,
respectively (Fig. 5.9b). Sticky ends are perhaps more common, and
are certainly more useful in DNA technology where they can be used for
grafting other pieces of DNA cut by the same restriction enzyme. Pieces
of DNA with sticky ends are also able to circularize readily, a form of
event which may be how plasmids originate.
Gene cloning
Gene cloning is simply the artificial introduction of a gene, or a series
of genes, from one source into a new host cell where they are
incorporated by genetic recombination into that cell's genome. Here
they can be expressed and passed to daughter cells on cell division,
resulting in millions of cells producing the same product. Multiple copies
of the gene can be made which can then be transferred, allowing
hyperproduction of the gene product. Many biotechnological processes
now depend on gene cloning, for example genetic engineering and the
promised revolution in gene therapy. Thus, one insulin preparation used
to treat humans with diabetes mellitus is produced by bacteria which
have been cloned with human insulin genes. Also the hepatitis B virus
vaccine is produced in yeasts cloned with genes encoding part of the
coat of the hepatitis B virus.
A generalized scheme for DNA cloning is shown inFig. 5.10. The DNA to
be cloned is usually extracted from the cells and purified. It is then cut
into short fragments by means of a restriction enzyme - leaving sticky
ends. These can then be inserted into vector DNA by first cutting the
vector with the same restriction enzyme, and then allowing
complementary sticky ends to pair up, treating finally with a DNA ligase
which joins the breaks.
Specific genes can be amplified from minute amounts of DNA by the
polymerase chain reaction (PCR) (see p. 50). Also, RNA can be the
source of specific genes by using reverse transcription: complementary
DNA is produced from an RNA template when the enzyme reverse
transcriptase and DNA bases are incubated together.
The vector used for the transfer is usually a plasmid or a virus. When
on a plasmid, the gene does not need to integrate into the host genome
as the plasmid will self-replicate at cell division. If it is an R-plasmid it
can move into other bacteria and be selected by antibiotic resistance. A
virus vector can carry more genes than a plasmid, and a lysogenic
bacteriophage - which has the property of being integrated into the
host chromosome as part of its normal life cycle - is usually used. For
example, bacteriophage lambda (λ) is often used when E. coli is being
cloned.
The vector containing the DNA has to be inserted into a cell by one of
several means:
Fig. 5.8 Diagrammatic representation of a transposon,

demonstrating palindromic sequences of base pairs (inverted
repeats) characteristic of insertion sequences and other mobile
genetic elements.
Fig. 5.9 Manipulation of DNA. (a) Melting and renaturation of DNA.
(b) The action of Eco RI, a restriction endonuclease producing sticky
ends.
Fig. 5.10 Gene cloning. (a) DNA is extracted from cells containing
the desired gene (the gene may be amplified by PCR - see later),
and it is cut into fragments by a restriction endonuclease. (b) A
suitable vector is prepared, in this case a plasmid. (c) The plasmid
DNA is cut by the same restriction enzyme as the DNA containing
the gene to be cloned, and the two DNA preparations are mixed and
treated with DNA ligase to produce recombinant DNA molecules. At
this stage there will be many different recombinant plasmids, most
not containing the particular DNA to be cloned, as well as normal
plasmids not containing any extra DNA. (d) The recombined DNA is
inserted into a population of the recipient bacteria, and again the
bacteria will contain many different lengths of DNA. These
recombinant bacteria are the gene library. (e) Bacteria expressing
the particular gene(s) must be selected (see text for methods). (f)
Once selected, all progeny of the cloned bacteria will express the
selected gene.
Transformation - the easiest, if cells can be made competent, but

may need treatment, e.g. E. coli is treated with weak calcium
chloride solution.
Electroporation - uses an electric current to make pores in the cell
membrane.
Gene gun - minute tungsten or gold particles are coated with the
DNA to be inserted, and are propelled into cells by a burst of
helium.
Microinjection - the vector can be injected directly into an animal
cell by means of a glass micropipette.
As in all genetic experiments, selection of the clone with the required

gene(s) is the final crucial step. Antibiotic resistance is one of the main
methods. For example, the plasmid vector might carry the genes for
resistance to two antibiotics: A and B. The restriction site into which
the foreign DNA is inserted is chosen to be in the middle of the gene for
resistance to antibiotic B. Therefore bacteria containing a plasmid into
which foreign DNA has been inserted will be resistant to A, but
sensitive to B, since the gene for resistance to B will be inactivated by
insertion of the DNA. This permits bacteria with cloned foreign DNA to
be selected - these are termed a gene library.
Identification and selection must then be made from this library for
clones producing specific genes. This may be done by probing for the
gene using specific gene probes (see below) if available, or the gene
product (protein) may be detected by a specific antibody using a
method similar to Western blotting (Fig. 8.20, p. 89).
Gene probes
DNA or gene probes are simply pieces of labelled, single-stranded DNA
or RNA from a known source that can be mixed with an unknown single-
stranded (melted/denatured) DNA. If complementary base sequences
are recognized the probe will hybridize, thus labelling the unknown
DNA. The label can be radioactive or fluorescent. The technique is
summarized in Fig. 5.11. DNA probes are highly sensitive and are a
powerful method for diagnostic microbiology. Probes may be made from
whole genomic DNA, or more specifically by cloning, direct chemical
synthesis (if the base sequence is known), or polymerase chain reaction
(PCR). The method can be used to probe DNA purified from whole cells
in a test tube, DNA applied directly to a nitrocellulose or nylon support
(dot blotting), or DNA restriction fragments separated on an
electrophoresis gel (Southern blotting). ‘Blots’ of bacterial colonies can
be made directly on to nitrocellulose membranes, and following lysis of
the bacteria and denaturation of the DNA, can be probed for
identification purposes.
Polymerase chain reaction

In recent years this extremely powerful technique has revolutionized
many nucleic acid-based procedures. In simple terms, the technique
allows the amplification of DNA sequences over a million-fold in a
matter of 2–3 hours by repeating a cyclic procedure of DNA
denaturation, annealing of short primers (specific short sequences of
DNA at either side of the required section to be amplified - one for each
strand), followed by DNA synthesis. The technique is summarized in Fig.
5.12. The major discovery that made the technique possible was the
isolation of a heat-stable DNA polymerase, the most widely used being
the Taq polymerase, isolated from Thermus aquaticus, a thermophilic
bacterium isolated from hot springs. This polymerase is stable at the
denaturation temperature of the DNA (which repeatedly reaches 92–
94°C), so that the enzyme and nucleotides need only be added to the
reaction mixture once at the beginning. Theoretically, if a single target
molecule of DNA is amplified through 30 cycles, typically in 2–3 hours,
it will be amplified 2 30 times, i.e. 109 -fold. However, in practice, the
procedure is not 100% efficient, but 10 6 -10 7 -fold amplification is
typical. Purpose-made pieces of apparatus known as thermal cyclers are
available which can be programmed to run the whole series of cycles at
the required temperatures and times. The product of the PCR is
detected by electrophoresis on agarose gels, stained with ethidium
bromide, and visualized in ultraviolet light or by Southern blotting, or
it can be
done without the electrophoresis step by dot-blot hybridization using a

standard DNA probe.
Fig. 5.11 Gene probing. (a) The DNA is bound to a nitrocellulose or

nylon membrane filter. (b) The DNA is denatured (melted). (c) The
labelled probe is added. (d) The reaction mixture is cooled to allow
hybridization of the probe with complementary sequences. Any
unhybridized probe is removed and the radioactivity or fluorescence
is measured.
Fig. 5.12 The polymerase chain reaction. (a) The DNA containing
the target sequence to be amplified is heated to 94°C to melt the
DNA. (b) This is cooled to 60°C and the reaction mixture is added.
This contains the heat-stable DNA polymerase, the four bases ATP,
GTP, CTP and TTP, and a pair of short primer sequences which are
known to be complementary with base sequences on each strand at
either side of the target DNA. (c) The mixture is incubated for a few
minutes at 60°C to allow synthesis of complementary strands. (d)
This produces two copies of the target sequence. (e) The
temperature cycle of heating to 94°C for a few minutes followed by
cooling to 60°C is repeated 30–40 times, giving rise to over a
million copies of the target DNA in 2–3 hours.
The standard PCR method described here is constantly being modified

and made more sophisticated to increase specificity and to extend its
uses. For example, reverse-transcription (RT)-PCR can be used to detect
RNA in RNA viruses, or it can detect mRNA to indicate transcription of a
specific gene, i.e. gene activity.
The extreme sensitivity of PCR is also its main drawback. To prevent

amplification of contaminating DNA - giving rise to false-positives – it is
essential that all PCR equipment and reagents, and the environment in
which they are used are kept free from any extraneous DNA. This is
crucially important in laboratories where the organisms from which the
DNA is being detected are being worked on.
Real-time PCR
Although exquisitely sensitive, traditional PCR methodologies have not
been quantitative, and although rapid, the time it has taken to get a
result has depended on how quickly the PCR products can be run on a
gel and analysed. However, recently both of these problems have been
overcome simultaneously by the advent of real-time PCR. Real-time
PCR depends on detection and quantitation of a fluorescent reporter
signal that increases directly in proportion to the amount of PCR
product that is formed. Interested readers are referred to the relevant
reference at the end of this chapter.
Techniques for the molecular ‘typing’ of micro-

organisms
These techniques are used primarily for taxonomic, diagnostic, and
epidemiological purposes in microbiology. Some of the basic principles
have been described earlier in this chapter, while some of the uses in
taxonomy have been described in Chapter 3.
Restriction enzyme analysis

This simply utilizes restriction enzymes to cut the DNA into small
fragments (Fig. 5.9b). The fragments are run on an agarose
electrophoresis gel, and the fragments viewed under ultraviolet
illumination after staining with ethidium bromide. The banding patterns
(‘fingerprints’) produced tend to be extremely complexthey resemble
massive barcodes - but similarities and differences can be seen between
different strains.
Fig. 5.13 Examples of some clinical applications of molecular

biology.
Restriction fragment length polymorphism

analysis (RFLP)
This technique is very similar to the above method; in fact the first
stages are identical. However, after the restricted DNA is separated on
the agarose gel, it is transferred by blotting onto a nitrocellulose or
nylon membrane, and probed with DNA probes that react with specific
DNA sequences (Southern blotting). Restriction length polymorphisms
are commonly occurring short deletions or insertions in the DNA, which
will change the size of restriction fragments between different
organisms but will be identical in clones from the same organism, or in
different tissues from the same organism. This forms the basis of DNA
fingerprinting in forensic science. A commonly used probe is for genes
coding for ribosomal RNA. These genes are highly conserved, and in
bacteria the sequences can be readily detected by probes produced to E.
coli rRNA genes. This technique is known as ribotyping.
PCR-RFLP
If the source of DNA to be probed is present in extremely small
amounts, e.g. viral DNA in tissue from an infected individual, or in
minute amounts of tissue or body fluid collected at a crime scene, it
would be insufficient for the above sort of fingerprinting. In such
situations the DNA can be amplified by PCR, and then analysed as
above.
Pulsed-field gel electrophoresis

This technique is similar to restriction enzyme analysis. However, the
restriction enzymes used are selected to cut the chromosomal DNA into
large fragments. By conventional agarose gel electrophoresis these
fragments would be too large to be resolved in
the gel. The large molecules become stretched out and work their way
snake-like through the gel, and are not well separated. However, with
the advent of a special electrophoresis power supply, where the polarity
of the current is regularly reversed, these large fragments are
sufficiently distorted so that they can be resolved by the system.
Clinical applications of molecular biology

Clinical applications of recombinant DNA technology range from the
relatively simple detection and identification of an infecting organism -
especially those that cannot be cultured, e.g. the hepatitis viruses - to
the techniques which are verging on science fiction, such as gene
therapy (using genetic techniques to replace defective mutant genes) or
cloning animals. Most of the applications are still in their infancy and
many are still at the research laboratory stage. However, this is an
extremely rapidly developing field and commercial kits for a variety of
applications, based on molecular biology, are becoming increasingly
available. In Chapter 3, a totally molecular approach to bacterial
identification, that can be used in the twenty-first century diagnostic
laboratory, is summarized in Fig. 3.8, p. 30. Some of the other
applications of molecular biology to clinical problems are summarized in
Fig. 5.13.
Key facts
DNA consists of two anti-parallel strands of deoxyribose

phosphates cross linked through purine and pyrimidine bases.
The bases of DNA are guanine (G), cytosine (C), adenine (A) and
thymine (T). G pairs with C, A with T.
DNA replicates by a process known as semi-conservative
replication. New bases are added asymmetrically at the
replication fork.
Mutations are faults in the DNA that are result of errors in DNA
replication or damage caused by chemicals or radiation.
Consequences can be malfunction of cellular processes, lethality,
or sometimes even beneficial changes. They can be passed on to
future generations and are the basis of natural selection.
DNA is transcribed to messenger RNA which is translated to
protein. The sequence of the amino acids in the protein is
determined by a three-base code - the genetic code - which is
organized linearly on the mRNA. Transfer RNAs carry the amino
acids and recognize specific three-base sequences - the codon -
by part of their structure termed the anti-codon at ribosomes -
the site of protein synthesis.
Genetic transfer in bacteria occurs by one of three methods:
transformation (naked DNA), transduction (via bacteriophage)
and conjugation (directly from one bacterium to another -
transferring a plasmid or part of, or the whole, chromosome).
Resistance transfer is when antibiotic resistance is transferred by
a plasmid.
Transposons and insertion sequences are mobile lengths of DNA
able to jump from one site in the chromosome or plasmid to
another. They are characterized by having palindromic base
sequences (inverted repeats) at their ends. Transposons
commonly carry several different antibiotic-resistance genes.
DNA strands separate on heating - DNA melting - and reassemble
on cooling.
Restriction endonucleases cut double-stranded DNA at specific
sites leaving blunt or sticky ends.
Gene cloning, the introduction of foreign DNA into another cell
where it can replicate and be expressed, is the basis of
biotechnology.
Gene probes, or DNA probes, are small pieces of labelled DNA
that can be used to detect specific sequences of DNA by pairing
with complementary bases following melting of the target DNA
into single strands.
Polymerase chain reaction (PCR) is a system for amplifying the
number of copies of a sequence of DNA over a million-fold in a
few hours by repeating a cycle of denaturation (melting), primer
binding and DNA synthesis. It was made possible by the discovery
of a heat-stable DNA polymerase that is stable to the high
temperature used for the denaturation step.
Restriction analysis can be used to fingerprint organisms. It can
be made easier to analyse by combining it with Southern blotting
and hybridization.
The clinical application of recombinant DNA technology is in its
infancy but advancing rapidly.
Further reading
Alberts, B, Johnson A, Lewis J, Raff M, Roberts K and Walter P
(2002). The Molecular Biology of the Cell, 4th edn. Garland, New
York.
Fredricks DN and Relman DA (1996). Sequence-based identification

of microbial pathogens: a reconsideration of Koch's Postulates.
Clinical Microbilogy Reviews 9, 18–23.
Mackay, IM (2004). Real-time PCR in the microbiology laboratory.

Clinical Microbiology and Infection, 10, 190–212.
Edition
Simon Bagg)
> Table o f C o ntents > Sectio n 1 > 6 - Viruses
6
Viruses
Viruses are an important cause of human disease. They are relevant to

dentists because they may cause disease either directly (for example
herpes simplex virus) or indirectly (for example HIV) in the oral and
perioral regions. Furthermore, the prevention of cross-infection with
viruses during dental treatment (Chapter 30) is an important element
of safe dental practice.
Characteristics of viruses
Viruses differ from other groups of micro-organisms, such as bacteria,
in many key respects. The important characteristics of viruses are
summarized in Fig. 6.1. They are very small agents with a simple
chemical composition comprising the viral genome and an outer protein
coat known as the capsid. The capsid is composed of multiple building
blocks known as capsomeres, and each particle has a strict geometric
structure. The genome may be either DNA or RNA, but never both, and
may be single or double-stranded, linear or circular.
Viruses are unable to synthesize proteins from the information in their
genome: they have no ribosomes or otganelles such as mitochondria.
For this reason they are strict intracellular parasites and can only
repiicate after infection of a living host cell. Viruses are resistant to
antimicrobial agents used to treat bacterial and fungal infections but
are very sensitive to interferon.
In addition to the nucleic acid and protein capsid, some viruses have an
outer coat known as an envelope. This is lipoprotein in structure and is
derived from either the plasma- or nuclear-membrane of the infected
cells. In general, non-enveloped viruses are more resistant than
enveloped viruses to environmental factors such as drying, gastric
acidity, and bile.
Symmetry of viruses
The complete unit of capsid and nucleic acid is termed the nucleo-
capsid. Viral nucleocapsids have a strict geometric structure and can be
divided into two main symmetrical forms. Cubic or icosahedral
viruses, for example the herpes viruses, comprise an outer icosahedral

protein shell which houses the nucleic acid (Fig. 6.2). Orhers, for
example influenza viruses, have a helical symmetry, in which the
capsomeres are arranged intimately around the spiral of nucleic acid
(Fig. 6.3)- Most helical viruses are enveloped. A small number of
viruses, for example poxviruses, have neither icosahedral nor helical
symmetry and are described as complex.
Fig. 6.1 Summary of the important biological characteristics of

viruses. Classification of viruses
Viruses may be classified into families on the basis of a number of

features including symmetry, presence or absence of an envelope, type
of nucleic acid in the genome, the number of nucleic acid strands and
their polarity. None of these methods permit an ideal classification and
a useful ‘supergrouping’ of viruses has been proposed known as the
Baltimore classification (Fig. 6.4). This classification is based upon the
type of genome, its polarity and the route by which messenger RNA
(mRNA) is produced within the infected cell.
Fig. 6.2 Diagram illustrating generalized structure of icosahedral
viral particles. Redrawn from Taussig MJ, 1984; with permission
from Blackwetl Scientific Publications.
Detection of viruses
The laboratory detection of viruses is important both for diagnostic and
research purposes. The methods available are summarized in Fig. 6.5.
More comprehensive details of methodology are given in Chapter 12.
Viral particles may be detected by electron microscopy (F ig. 6.6),
allowing rapid detection of viruses directly in clinical specimens. The
specificity of the method is poor, but can be improved by adding specific
immune serum that agglutinates a particular virus.
Since viruses are stria intracellular parasites they must be provided
with living cells if they are to be cultured in the laboratory. Most viral
culture is now performed in tissue culture systems (Fig. 6.7), although
for some viruses such as hepatitis B no cell lines are available which
wilt permit viral culture in the laboratory. The specimen is collected
into virus transport medium and inoculated into the cell culture tube or
flask. The cells are then incubated and observed for development of a
cytopathic effect. This appears within 48 hours for some viruses, but
may take as long as 14 days to develop. The changes in cell morphology
which constitute a cytopathic effect may be characteristic of a particular
virus. Some viruses induce no cytopathic effect and must be detected
by other means.
Detection of antiviral antibodies (serology) may be useful in diagnosis,
although for common viral infections this requires the
collection of two blood samples, 10–14 days apart, to permit detection

of a rising titre of IgG antibodies. For some infections, such as rubella,
a more rapid diagnosis can be made on the basis of detection of speci6c
IgM ciass antibodies in a single specimen. For certain viral infections
such as HIV, hepatitis B and hepatitis C, serology is the main method of
diagnosis.
Fig. 6.3 Diagram illustrating generalized structure of helical viral

particles. Redrawn from Taussig MJ, 1984; with permission from
Blackwell Scientific Publications.
Fig. 6.4 The Baltimore classification of viruses. Messenger (m) RNA
and strands of the same polarity as mRNA are designated ‘+’. Viral
DNA and RNA strands complementary to mRNA are designated ‘-’.
Double-stranded nucleic acid is denoted‘+’. Arrows indicate the
route of arriving at mRNA. Redrawn from Taussig MJ, 1984; with
permission from Blackwell Scientific Publications.
Fig. 6.5 Summary of methods available for diagnosing viral

infections.

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Authors: Bagg, Jeremy; MacFarlane, T. Wallace; Poxton, Ian R.

Oxford University Press makes no representation, express or implied,

Microbiology is a rapidly moving field and preparation of the second

Although the original Koch's Postulates remain useful, ‘Koch's Molecular

Fig. 1.3 Diagram of an idealized prokaryotic

Early studies on unicellular, microscopic organisms established that

Prokaryotic and eukaryotic cells

The diversity of micro-organisms and their

capnophilic bacteria, which are commonly found in the oral ecosystem,

Fig. 1.5 A comparison of key features of prokaryotic and eukaryotic

As well as the atmospheric requirements of micro-organisms, other

The normal flora and pathogens

Fig. 1.6 Extremes of physico-chemical environment in which

Those micro-organisms which cause disease are termed pathogens.

Emerging and re-emerging pathogens

human disease, in this case because air-conditioning systems have

Prions and transmissible spongiform

The main groups of micro-organisms are bacteria, fungi, protozoa,

Bacterial cell structure

The Gram stain

Fig. 2.2 The method for Gram staining a bacterial

Some groups of bacteria cannot be considered typical Gram-positive or

heating, and then decolourizing the background with dilute acid in

Fig. 2.5 Diagram of a typical membrane (fluid mosaic

Structure and function of the bacterial cell

Fig. 2.6 Summary of functions of the bacterial cell

The Gram-positive bacterium possesses a structure outside of the

Macromolecules of the envelope

considered a single molecule which covers the whole three-dimensional

Some species, notably Cblamydia spp., appear Gram-negative in the

Teichoic acids and other secondary cell wall

Fig. 2.9 Diagrammatic representation of

Spirochaetes move in a corkscrew manner by means of an axial

Fig. 2.13 Diagram illustrating bacterial multiplication by binary

The bacterial growth curve

1. The lag phase: the inoculated bacteria are becoming acclimatized to

2. The logarithmic, or exponential phase: there are no restraints on

Fig. 2.15 The classic bacterial growth curve for a batch

Shapes and arrangements of bacteria: cocci, rods/bacilli, vibrios

Taxonomy is the term given to the systematic classification, naming,

Bacterial classification and nomenclature

Staphylococcus aureus is a facultatively anaerobic Gram-positive coccal

Different approaches to taxonomic methods

Fig. 3.2 Numerical taxonomy and construction of

Evolution is an important concept to consider in taxonomy, as

The definition of a species can be described in genotypic taxonomy as ‘a

manuals dedicated to the subject of bacterial identification. As

Semi-systematic lists of clinically important bacteria are given inFigs.

Identification beyond species level

Bacterial identification: the future

Taxonomy is the systematic classification, naming and

Fredricks DN and Relman DA. (1996). Sequence-based identification

The relationships between bacteria and the

The resident microflora

bacteria gain obvious benefit from the relationship by being in a fairly

Commensal and symbiotic relationships

Some bacterial pathogens normally live in the environment, existing

Direct spread In these cases, there is direct spread to the host

Indirect spread Indirect spread occurs if a secondary inanimate