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Human Microbiology

Simon P. Hardv

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Human Microbiology

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HUMAN MICROBIOLOGY

B Simon P. Hardy

School of Pharmacy and Biomolecular Science,

University of Brighton, UK

London and New York
L/FM.INES

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First published 2002 by Taylor & Francis
1 1 New Fetter Lane, London EC4P 4EE

Simultaneously published in the USA and Canada

by Taylor & Francis Inc,

29 West 35th Street, New York, NY 10001

Taylor & Francis is an imprint of the Taylor & Francis Group
This edition published in the Taylor & Francis e-Library, 2003.

any

electronic, mechanical, or other means, now known or hereafter invented, including photocopying and

recording, or in any information storage or retrieval system, without permission in writing from the

publishers.

Every effort has been made to ensure that the advice and information in this book is true and accurate

at the
time of going to press. However, neither the pubhsher nor the authors can accept any legal

responsibility or
hability for any errors or omissions that may be made. In the case of drug administration, any medical
procedure or the use of technical equipment mentioned within this book, you are strongly advised to

consult
the manufacturer's guidelines.

British Library Cataloguing in Publication Data
A catalogue record for this book is available from the British Library

Library of Congress Cataloging in Publication Data
A catalog record for this book has been requested

ISBN 0-203-16440-7 Master e-book ISBN

ISBN 0-203-27827-5 (OEB Format)
ISBN 0-415-24199-5 (hbk)
ISBN 0-415-24168-5 (pbk)

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CONTENTS

1.1 Bacterial size

1.2 Bacterial structures

1.3 Bacterial movement

1.4 Bacterial genetics

1.5 Bacterial variation

Page V

Series editor's preface viii

Introduction ix

PARTI

INTRODUCTION TO MICRO-ORGANISMS 1

1 Bacterial structure and function 3

1.6 Regulation of gene expression 35

Summary 36

RECOMMENDED READING

Cann, A.J. (2001) Principles of Molecular Virology, 3rd edition, Academic Press, London, UK.
Collier, J. and Oxford, J. (2000) Human Virology, 2nd edition, Oxford University Press, Oxford,
UK.

Dimmock, N.J. and Primrose, S.B. (1994) Introduction to Modern Virology, 4tli edition, Blackwell
Press, Oxford, UK.
White, D.O. and Fenner, F. (1994) Medical Virology, 4th edition. Academic Press, London, UK.

REVIEW QUESTIONS

Question 3.1 How do the growth curves obtained in vitro for viruses and bacteria compare?

Question 3.2 Is viral nucleic acid alone infectious?

Question 3.3 What is the significance of the icosahedron for viruses?

Question 3.4 How does the efficiency of infection correspond to total and viable counts for
bacteria?

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4
FUNGI

Fungi comprise the untidy collection of micro-organisms that includes moulds and yeasts. Toadstools
and mushrooms are actually just particular structures formed by moulds and not separate organisms.
Whilst fungi are of great benefit to mankind and play an essential recycling role in nature, they can
produce disease in humans, hence our interest. Of the micro-organisms considered in this book, fungi
are the only organisms that have forms that are visible to the naked eye. In addition to mushrooms and
toadstools, the filamentous collections of threads that characterise moulds in the bread bin are also
readily seen by eye. Single yeast cells, however, are microscopic despite being up to 10 times the size of
bacterial cells.

The fungal lifestyle can be viewed as two phases:

• the trophic (feeding) phase, mediated through radiating mycelia, and

• the dispersal phase, mediated through production and release of spores.

Whilst termed micro-organisms, fungi attempt to monopolise their potentially large size. If nutrients
exist, they will continue to increase in size, with no restrictions on the final dimensions. In terms of size,
it is estimated that certain fungi, as mycelia radiating in soil, are amongst the largest living organisms
existing and can be considered to represent a primitive multicellular organism. Like true multicellular
organisms, fungi produce specialised structures with distinct functions, notably the production of 'sexual
organs' and large fruiting bodies. Mushrooms are a class of fungus that produce such fruiting body
structures that can be eaten. Other than producing poisons which are toxic to man if eaten, mushrooms
do not directly cause infections of man and will not be central to our discussions. Likewise, the Brewer's
yeast, despite the not insignificant property of converting sugars into alcohol and carbon dioxide, will
not be discussed further.

4.1 FUNGI: MOULDS AND YEASTS

Fungi are eukaryotes, typified by the possession of a nuclear membrane and mitochondria. Unhke
bacteria, fungi are usually multicellular organisms which possess complex hfecycles. Whilst ubiquitous
in soil and many varied environments, fungi are less tolerant of adverse conditions than bacteria and

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prefer warm, moist conditions.

■
4.1.1 MOULDS

Moulds are filamentous fungi; that is, they exist as rigid-walled filaments. These filaments are a mass of
branched tubes called hyphae which collectively are called a mycelium. The mycelia permit great
variation in the structures of the fungus, as

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shown macroscopically in toadstools and mushrooms but equal variety is observed in the microscopic
features. As most fungi are not actively motile, the fungus needs to grow into new areas so as to find
food. The advancing front of the fungus will radiate outwards from a central point so as to maximise the
area covered. The hyphae spread across the soil/tree trunk, etc. and secrete degradative enzymes so as
to break down the substance for nutrients. The hyphae are a series of cylindrical tubes that may possess a
cross wall (septum). The septum itself usually has holes (a pore) within it to allow the passage of
materials (organelles, nuclei, nutrients and solutes) along the length of the hyphum ( Figure 4.1a ). Other
fungi have no cross wall (aseptate) to separate the numerous nuclei. The multinucleate arrangement is
termed coenocytic and represents a more primitive arrangement typical of the lower fungi of the Class
Zygomycetes. The advantage of the variable numbers of nuclei in any one part of the hyphum is
discussed later (pp. 1 14-15). The cytoplasm will stream along the length of the hypha carrying nutrients
to the growing tip such that fungi have been described as amoebae living in tubes ! The hyphae extend
forwards through growth at the tip, continually progressing towards fresh supplies of nutrients and
acting as a tunnelling

• Figure 4.1 (A) The functional zones of the hyphum in a fungus. The apical
growth zone leads, with absorption of nutrients occurring behind the leading
tip. (B) A diagrammatic representation of the apical tip showing the
Spitzenkorper vesicles fusing into the tip to enable the incorporation of new
cell wall. Also shown is the release of the hydrolytic enzymes into the
environment to fuel the growth of the fungus

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fA)

Senescence

Storage;

AbfiOTption

Grmvth

Septum witli pore

(B)

tn^ymtsi

Spitzcnkorpcr fusing inlo apicjl wall

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device. The mycelium is not a homogenous mesh of pipework. Different functions or activities are found
at different points along the length of the hyphae. The leading tip (the growth zone) is concerned
principally with growth so as to advance into new areas. Degradative enzymes are secreted to aid
penetration of the hyphal tip ( Figure 4.1b) . Behind this advancing front is an absorption zone concerned
with absorbing nutrients via active transport processes. Having rigid cell walls, like bacteria, fungi need
to break down food into small molecular weight compounds so that they can be transported into the cell.
Distal to the absorption zone lies a region in which nutrients surplus to requirement are converted into
insoluble forms. This storage zone will provide energy stores for use in leaner times and provide energy
for spore production. In the centre of the mycelial mat the nutrient supply will become exhausted and
toxic products may have accumulated. The hyphae at the centre of the mat will either senesce or die.
This region of the mycelium is called the senescence zone to describe the phenomenon whereby cells
stop dividing but are still alive and metabolically active. The pigments seen in cultures of fungi develop
in old senescent hyphae. The lesions of ringworm are characterised by a circular advancing edge of
mycelia leaving a burnt out centre from which it is rare to culture viable fungi. This pattern of
concentrating energies to the growing edge is also seen in fungi in nature, such as in soil and on plants or
timber.

4.1.2 YEASTS

Yeasts are unicellular fungi which are thought to be moulds that have lost the ability to form true
hyphae and mycelia (although some yeasts can form hyphae). Mostly, yeasts do not form spores or the
speciaUsed structures that house them as seen in moulds (i.e. the fruiting bodies of moulds). Yeasts are
single cells that are usually oval or cylindrical in shape and are mostly haploid. Yeasts mostly divide
clonally (asexually) through binary fission by processes called budding or fission ( Figure 4.2) . Budding
involves

• Figure 4.2 Asexual division in yeasts. (A) Budding. Budding yeasts (e.g.
Candida albicans) produce a small bud that increases in size after the
replicated nucleus is placed in each cell. (B) Fission. Fission yeasts (e.g.
Schizosaccharomyces sp.) replicate the nucleus and then separate the two cells
by producing a septum

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(A) Budding yeasts

(Bj Fiii^iun y(;asls

Co^

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the production of a small bud that protrudes from the main cell body and increases in size until it is
pinched off, leaving a scar on the parent cell. Fission yeasts divide through a binary fission process in
which the yeast divides in two with the production of a septum. The growth of the yeast may not simply
be through multiplication of more and more yeasts but they may produce hyphal outgrowths:

1 . germ tubes, hyphal protuberances that emerge from the yeast cell. The germ tube can be
considered as an immature form of hyphum, i.e. before septa have appeared.

2. Under certain conditions yeast can form pseudohyphae, chains of young (small) yeast cells formed
by budding but still attached. Pseudohyphae appear constricted at the junctions between the
attached cells whereas true hyphae show no constriction at the septal join between cells.

Some yeasts are diploid and reproduce sexually via fusion of different mating types. Those yeasts that
do not reproduce sexually are termed anamorphic yeasts (see Box 4.1) .
Yeasts are different from moulds in several respects.

• The genome is smaller, reflecting a narrower metabolic capacity.

• Yeasts do not form secondary metabolites.

• Yeasts do not form the variety of morphological structures of true fungi.

• Yeasts are less tolerant of adverse conditions (pH, temperature, etc.).

4.2 FUNGAL STRUCTURE AND FUNCTION

4.2.1 CELL WALL

Fungi have rigid cell walls (like that of bacteria) that determine the shape of the fungal structures but the
chemical composition and arrangement is different to bacteria. Fungi do not possess peptidoglycan and
therefore are not susceptible to antibiotics such as penicillins. The fungal cell wall is comprised of a
greater variety of substances and the number of layers is also greater. Most characteristic is the use of
the polysaccharides chitin and glucan in the cell wall. Neither glucan nor chitin are present in
mammalian or bacterial cell walls. Chitin is an extremely tough, linear (homo)polymer of N-acetyl-
glucosamine and is predominant as bundles which strengthen the cell wall. Glucan is a linear
homopolymer of glucose units which are connected by 1-3 and/or 1-6 linkages (the numbers referring
to the carbon number in the carbohydrate molecule). Both are insoluble in water and provide rigidity to
the cell wall. Whereas bacteria cross-link the peptidoglycan with peptide strands, fungi cross-Unk with
the glucan chains. The other typical fungal wall constituent is a variety of mannoproteins. The
composition of

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■ BOX 4.1 SEXUAL AND ASEXUAL FORMS OF REPRODUCTION

Two terms that should be understood are anamorph and teleomorph. Because fungi may have a
sexual and/or asexual means of propagating itself there have been two taxonomic names possible for
the same organism. The anamorphic state refers to the organism named according to its asexual state,
whereas teleomorph is the name of the sexual form. A third term, holomorph, can be used for the
name of a fungus in which both states have been recognised and named. In that case, the name of the
sexual state takes priority.

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• Figure 4.3 A diagrammatic representation of the fungal cell wall. The
cytoplasmic membrane is included so as to demonstrate the presence of a
cytoskeleton. The cell wall itself is essentially of two layers: chitin and cross-
linked glucans, and a mannoprotein layer; that is, covalently bound to the
glucans

Page 107

XX X X XX

ManiioprDtcina

Chimin

1-3 Glucans
1-6 Glucans

Cylopljamic membrane
Cytoskelelon

fungal cell walls is dominated by 80-90 per cent polysaccharide. Figure 4.3 shows a diagrammatic
representation of the key fungal cell wall components. Note that beneath the cytoplasmic membrane (in
the cytosol) the cytoskeleton is indicated. The cytoskeleton, which includes actin, acts as a scaffold
inside the cytoplasmic membrane and helps in the assembly of new cell walls by directing the vesicles to
the apical tip.

The apical growth that characterises growth of mycelial fungi differs from that seen in yeasts and
bacteria ( Figure 4.4 ). Whereas fungi demonstrate apical growth, bacteria grow from the centre of the
bacterial cell. Yeasts grow either by budding or (like bacteria) by fission. Apical growth means that the
tip needs to be sufficiently plastic to permit the insertion of new cell wall material. The new cell wall is
incorporated into the tip via fusion of the dense collection of vesicles at the advancing tip, that
collectively are known as the Spitzenkorper (or apical body). The fungal cell wall becomes more rigid
along the trunk of the hyphum as the 'plastic' tip advances progressively. The trunk becomes rigid
because of increasing cross-linking of the glucan strands and crystallisation, a particular arrangement of
the strands of chitin (Figure 4.5) .

4.2.2 SPORES

The collections of spores produced following sexual fusion of male and female hyphae in different fungi
have characteristic morphological patterns which are useful for classification purposes. Although all
spores themselves are produced by an asexual process, it is not uncommon to find references to sexual
and asexual spores, depending on the sexual pattern observed prior to the production of spores.

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Although the spores produced by fungi are useful to mycologists in the classification and identification
of fungi, their role for the fungus lies in their distinct function:

• asexual spores are produced following mitosis and are produced to increase the mass of fungus
produced (when the spores germinate into hyphae),

• large numbers of small sexual spores are produced following meiosis and produced on specialised
structures that promote the dispersal of the spores on to new environments.

In order to disseminate spores into the environment, fungi employ the wind to carry them. This
distribution mechanism will work best if the spores are elevated in some manner, so as to catch the
wind. The hyphae that can be found extending aerially are

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• Figure 4.4 Patterns of cell growth. Bacteria, yeasts and fungi differ in the
sites at which new cell wall is incorporated. Bacteria (A) deposit new cell wall
along the length of the cell wall. When the cell starts to divide, the new cell
wall is deposited at the site of division (the septum). In contrast, fungi (B) grow
by incorporation of new cell wall only at the tips. Yeasts grow by either fission
(C) or by budding (D). Note that fission is the same process in bacteria and
yeasts

^A) Baclcriiim

(B) Fungal liyplniin
t

(QYcasl (fission)

tD) Ycasi CbudcJing)

• Figure 4.5 The apical growth zone of the fungal hyphum is shown with the
vesicles (circles) supplying the new cell wall material. The structural
arrangements for the cell wall at the tip and along the trunk are given. The tip
is represented as a less ordered structure where the straight lines represent
linear chitin chains and the thicker curved lines are the glucan chains. The
grouped glucan chains in the trunk represent the increased cross-hnking and
concomitant rigidity

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Tnink Tip

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Page 109

used for this purpose. The hyphae will often contain numerous spores at the tip and such a structure is
called a conidiophore (see below, p. 1 16). Conidia are pigmented and impart the distinct colours that
colonies of moulds show when cultured on agar plates. This strategy of releasing spores into the air may
be successful for soil-living fungi and has significance in the airborne acquisition of certain fungi by
humans breathing dusty air containing large numbers of spores.

The environmental conditions that the fungus finds itself in will influence the extent to which a fungus
undergoes sexual or asexual reproduction. The type of nutrient (protein or carbohydrate) and its
concentration are key modulators. In general, if nutrients are scarce, then sexual reproduction is
favoured. The triggers may indeed be the unbalanced nature of the nutrition. Under conditions where
nutrients are plentiful and growth of the hyphae is promoted, then asexual spore formation is usual.

4.3 FUNGAL GROWTH AND METABOLISM

4.3.1 GROWTH

The key characteristic of fungi is the ability to produce mycelia through apical growth.

As the hyphae extend radially (outwards) across a surface, the fungus is able to colonise a large area.

The rate of branching of the hyphae will depend on the concentration of nutrients. If the environment is

nutrient poor, then the hyphae will increase in length rather than produce branches. In nutrient-rich

environments, however, the hypae will branch more frequently in order to maximally utilise the

available nutrients ( Figure 4.6) .

The growth of fungi in laboratory cultures is largely an artefact, in the same way that culturing bacteria

in laboratory culture media has little resemblance to the natural environment. With this proviso, let us

consider some of the features of fungal growth in

• Figure 4.6 Hyphal extension in nutrient-rich and -poor environments. A
fungus will regulate the proportions of hyphal length to number of branches so
as to best find and exploit the available nutrients. This ratio of hyphal length
and tip number is known as the 'hyphal growth unit'

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(A)XntrLciil-paor
cnviruniTL^iiC

(B)Nu[ricnl-ric]i
cnviruniTi^iil:

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vitro. The growth of yeasts and hyphal moulds in liquid batch culture resembles the increase in numbers
and mass seen in bacterial growth. The same phases of growth are seen: lag, exponential, stationary and
decline. The resemblance is best during the early stages of the growth, when the entire fungus is
growing. As hyphal growth occurs at the tips only (apical growth), the centre of the mycelial mat will
autolyse (no viable fungus can be recovered from the centre). The increase in fungal mass occurs only
when all hyphae continue to branch outwards and this occurs in the early stages of the culture only.
When fungal moulds are cultured in broth cultures, they tend to form spherical balls which will restrict
the flow of gases and nutrients into the centre of the fungal ball. Under these conditions the hyphae will
continue to grow and branch at the surface and not in the centre. The mass of the fungus will be
proportional to the volume of the sphere (4/3 Tir^). From this it can be shown that the cube root of the
mass is proportional to time. This is called 'cubic growth' and means that instead of a graph of log mass
versus time, the cube root of the mass of the fungus over time is needed to get the straight line graph.
Fungi will also tend to grow as a mat on the surface of liquid broth media if they are not vigorously
shaken. The increasing thickness of the fungal mat will progressively reduce gaseous exchange in the
broth. Shaking will lead to the formation of fungal balls. In effect, 'problems all round', if you will
pardon the pun.

The mycelium will increase in size until the fungus has depleted the nutrients from its surroundings. The
suboptimal growth rate signals a switch from primary metabolism to secondary metabolism. The
products are termed 'secondary' because they are not involved in the growth of the organism rather than
being produced after the growth phase. As with bacteria, the products of secondary metabolism are not
utilised for growth of the organism but instead serve to aid its survival once nutrient concentrations have
become critical. One of the important functions of secondary metabolites is to trigger the formation of
sporulation in order to shed spores into new territory. Antibiotics are also secondary metabolites and
they function as a survival mechanism by acting on susceptible organisms in the vicinity that may
compete with the organism for the nutrients. You may notice that of the two functions described, one
acts on the host organism whereas the other acts on competitors.

Whilst many of these features mean that many fungi are able to attack a diverse range of substrates
(wood, leather, cloth) the relevance of these attributes to infecting man is not clear. It might be expected
that fungi would cause a significant number of infections in man rather than existing predominantly in
the environment as soil- and plant-associated organisms. The conditions necessary for fungal infection
in man are considered in Chapter 10 .

4.3.2 METABOLISM

Fungi are distinct from plants because they can grow without light. Hence, fungi do not possess
chlorophyll and cannot use carbon dioxide as a carbon source. They therefore need to obtain their energy
through direct uptake of non-living organic matter (heterotrophic or, to be more exact,
chemoorganotrophic). The uptake will need to be of low molecular weight and in a soluble form

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because, unlike most other eukaryotes, fungi bacteria possess a rigid cell wall and therefore cannot
import food via endocytosis. Dependence on pre-formed sources of nutrients means that fungi have
adopted a lifestyle that absorbs food from either

• non-living (dead) organic matter, or

• living organic matter.

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Page 1 1 1

Fungi that obtain nutrients from their host (and at the host's expense) are called 'parasitic fungi'. Some
fungal associations with their host are of benefit to both fungus and host, i.e. symbiotic. For example,
many plants, especially orchids, cannot grow unless the fungi have colonised their root system
(mycorrhizal fungi).

Fungi have limited nutritional requirements. They need carbon and nitrogen in an organic form. Carbon
is abundant in nature but may be present in a form that is unavailable to most other organisms, notably
as polymers such as cellulose, chitin and keratin. Carbon in cellulose is the most abundant atom on the
planet. We on Earth would be overrun with discarded plant material were it not for the ability of fungi to
break down plant cellulose in the cell wall of plants. Different fungi have different nutritional
requirements (reflecting their requirements for nitrogen and carbon in specific chemical forms) and this
will determine where the fungi are to be found. With humans, fungi are unique in their ability to extract
carbon and nitrogen from the keratin in dead skin. The fungi secrete degradative enzymes into the
environment that will hydrolyse the polymers: cellulases, chitinases and keratinases. Fungi will readily
grow with glucose as the carbon and energy source but it is unlikely that in their natural environments it
is readily available. The nutrients freely diffuse across the rigid cell wall but will be actively transported
into the cytosol across the cytoplasmic membrane.

The waste business appears to be profitable. As decomposers and recyclers, fungi appear to be
doing very well. There are tenfold more fungal species currently recognised (c. 90,000) than
bacteria (c.4000) and viruses (c.6000). All three microbial types are thought to be woefully
underestimated and the fraction of possible fungal species currently recognised is possibly as
little as 10 per cent of the real number.

Most fungi are essentially aerobic but are able to tolerate anaerobic conditions. With sufficient oxygen
available, fungi can continue to grow. Without oxygen, growth ceases and aerobic metabolism is
switched to fermentation to maintain sufficient energy for basal metabolic activities. Yeasts are best
suited to dealing with anaerobic fermentation as all beer drinkers are aware. The production of ethanol
and carbon dioxide from carbohydrates by brewing yeasts is likely to be the most well known
biochemical pathway. The anaerobic fungi are grouped in with the lower fungi and do not have any
direct dealings with humans.

Typical conditions for fungal growth are humid atmosphere, damp surface (soil) with a moderate
temperatures (between 4°C and 20°C). Yeasts are able to tolerate situations of low free water (a^) and

they can typically be isolated from fruit. Ripe fruit has high concentrations of solutes such as alcohol
and sugars which reduces the available water.

4.4 FUNGAL GENETICS

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The fungal genome (i.e. the total genetic information) consists of the chromosome and mitochondrial

DNA along with extrachromosomal elements: plasmids. The former two elements are consistent with

other eukaryotes whereas the possession of plasmids is a feature of prokaryotes.

Fungal genomes are not that much larger than those of bacteria and, consequently, are small compared

with other eukaryotes. Fungi do not contain multiple copies of the same genes, nor do they have much

nonsense (i.e. non-coding) DNA (called introns).

Most fungi are haploid (possess one set of unpaired chromosomes, N), although the medically important

yeast Candida albicans is diploid; that is, the chromosomes are in pairs (2N), each pair being

homologous.

Sexual reproduction in moulds is a vast topic because the different classes of fungi reproduce by

different mechanisms and the sexual organs have separate names. In general, lower fungi possess

specialised sexual organs whereas the higher fungi have relatively inconspicuous organs, if at all. Many

of the fungi that infect man are members of the fungi imperfecti and therefore the sexual stage has not

been identified. For those reasons you might find many mycology texts somewhat unforgiving.

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Both sexual and asexual reproduction can be found in most fungi, although many will predominantly use
asexual reproduction and only rarely use sexual means. Some fungi, often those that cause infections in
man, appear to have completely lost the ability to reproduce sexually. The massive numbers of species
of fungi indicates that fungi have benefited from using both sexual and asexual methods of reproduction.
As a mechanism to prevent genetically identical cells from fusing, many fungi have developed mating
systems whereby cells of the same mating type cannot fuse. Such a system resembles the role of male
and female by forcing genetic diversity through recombination of compatible nuclei. Many higher fungi
contain nuclei of two mating types (a male and female equivalent) and are thus able to undergo sexual
reproduction from the same fungal colony, in other words, they are self-fertile (can mate with
themselves). Such fungi are called homothallic. Those that require fusion of nuclei of the opposite
mating type are called heterothallic. Any such restriction on the ability of fungi to mate is called
incompatibility. Incompatibility serves to promote genetic exchange (heterothallism) or to promote the
growth of a particular phenotype by restricting outbreeding (homothallism). The role of mating is also
considered next.

Sexual reproduction involves the cyclical alternation between haploidy and diploidy. The means of
reducing the numbers of sets of chromosomes from diploidy (2N) to haploidy is meiosis. Meiosis
consists of two divisions of the nucleus but the chromosome only dividing once, hence the sets of
chromosomes are halved. For the fungus to produce a diploid cell it needs to fuse two haploid cells
(gametes). The fusion of two sexually compatible cells (be they spores or hyphal cells) is called
plasmogamy. The resulting cell now has two independent and non-identical nuclei, one from each
parent/gamete. The subsequent fusion of the two nuclei is called karyogamy and leaves the cell with a
diploid set of chromosomes ready for reduction by meiosis.

Once the binucleate state has been created by plasmogamy, the fusion of the two nuclei need not occur
immediately, but the cell may multiply further with each of the daughter cells having two separate nuclei
(N-i-N). Each pair of nuclei in a cell is termed a dikaryon (pair of non-identical nuclei).
Thus far we have described three critical and essential events:

• plasmogamy,

• karyogamy,

• meiosis.

Variation in the proportion of time fungi spend as haploid, diploid or dikaryotic cells helps explain the
number of lifecycles that fungi have adopted (and help to explain why most mycology textbooks appear
to describe endless impenetrable lifecycles with no obvious pattern!). We can now predict a number of
lifecycles, which differ in the time spent as haploid, diploid or dikaryotic:

1. Asexual lifecycle. No sexual phase.

2. Haploid lifecycle ( Figure 4.7 ). The vast majority of the cycle is haploid except for the short period
necessary for fusion of two haploid nuclei to generate the diploid state which then promptly
undergoes meiosis.

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3. Diploid lifecycle ( Figure 4.8) . Predominantly the fungus is diploid with only the temporary phase
of haploidy following meiosis.

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Figure 4.7 The sequence of events for a haploid fungus

Page 113

Kar^'oyamy

M^^io^LS

Figure 4.8 The sequence of events in the lifecycles of a diploid fungus

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4. Dikaryotic lifecycle ( Figure 4.9) . The fungus forms dikaryotic cells by immediately fusing the
haploid cells resulting from meiosis.

Further variations are recognised, e.g. haploid-diploid lifecycle, haploid-dikaryotic lifecycle, etc.
Interestingly, lifecycles generally tend to be more compHcated in the higher fungi, and the higher fungi
tend to exist in more specialised ecological environments, although this does not appear to be the case
with many fungi pathogenic to man.

4.4.1 FUNGAL GENETICS: WHY SO COMPLICATED?

It is interesting to consider why fungi, being eukaryotes, are mostly haploid whereas eukaryotes are
typically diploid, i.e. carry two sets of chromosomes. Diploidy is a mechanism whereby any mutations
that occur in any one gene need not necessarily be expressed. If the mutant gene is recessive, it will not
be expressed because the other, non-mutated copy is dominant. The recombination events that occur in
sexual reproduction via meiosis may result in a mutation becoming expressed, but this will occur only if
a similar recessive mutation is present in the other sexual mate. In contrast, mutations in haploid
organisms will be expressed because there is no second copy to cover the altered gene. The advantage of
the immediate exposure of mutations to haploid organisms is that any advantages the altered phenotype
confers is immediate. The selective pressures on the organism either favours the mutant or eUminates it.
In this way mutation in haploid organisms is of the moment, whereas diploid organisms can accumulate
mutations for variation later. Consider then the fungi that possess coenocytic mycelia. They have more
than one nucleus in the cytosol. If, then, a mutation occurs in one of the nuclei, the variation is hidden
until such times as the fungal

• Figure 4.9 The sequence of events in the lifecycles of a dikaryotic fungus

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a
/I
n

tO;

^ Mciosiis
\ A ^^ Pbsmogainy

/^-—^

xJr

SgxujI cycle:
dikaryotic

w
II

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II
w
//

Hiingui?

ifl^

it
if

Asexual cycle

^*.
1^^

"^.

h
It

^:i— — .^

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mycelia produce uninuclear spores. In this way moulds are haploid but retain some of the features of
diploidy. As mentioned earlier, whilst the yeast Candida albicans is permanently diploid, most yeasts
are haploid but, being separate cells, they cannot exploit the benefits of being coenocytic.
Fungal spores are produced following sexual mechanisms in response to adverse conditions, whereas
asexual spores are used to enable the fungus to quickly multiply and exploit favourable conditions. Why
is sex the reason for the distinct functions of sexually and asexually produced spores? Fungi are
eukaryotes that have sexual and asexual mechanisms of reproduction. There are evolutionary advantages
for both. Asexual reproduction is clonal in effect (more of the same is produced) and quick (mitosis is a
faster process than meiosis and you don't have to wait for a partner); hence, if your existing genotype is
working well in your current environment then you have Httle need to change it. Sexual reproduction in
fungi involves the fusion of + and - mating type nuclei and meiosis. The genetic recombination can
result in the creation of new phenotypes as a consequence of crossing-out errors: different combinations
of alleles from the parental genomes. Meiosis, then, is a cause of genetic diversity and this mix and
match effect on the genotype/phenotype will enable the fungus to produce variants that are suited to
changing environmental conditions.

In addition, you may encounter the term parasexuality when reading about fungal genetics.
Parasexuahty is a cover-all term for further genetic tricks exhibited by fungi which are non-meiotic in
mechanism but their objective remains the same: to generate genotypic diversity. One should note that
most of them have only been observed under laboratory conditions.

4.5 FUNGAL CLASSIFICATION

The taxonomic history of fungi is long and comphcated. This is mentioned so as to warn readers that
older texts are very difficult to cross-reference with each other because the classification varies
considerably. Fungi are eukaryotic (they possess a nuclear membrane) and are placed as a distinct
taxonomic family (Fungi) but historically have been grouped with plants. Fungi differ from plants in two
key features:

• fungi are absorptive heterotrophs and lack chlorophyll and are thus non-photosynthetic, whereas
plants are autotrophic, utilising photosynthesis via chlorophyll,

• fungal cell walls contain chitin but not cellulose.

In 1969, Whittaker rescued fungi from botanists by placing them as one of the five kingdoms (animals,
plants, fungi, protists and monera). The diverse range of forms once considered to be fungi is reflected
by the division of 'Fungi' into three kingdoms:

• Fungi,

• Chromista (water moulds and algae),

• Protozoa (slime moulds and chytrids).

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Of these, the kingdom Fungi contains the organisms that are of interest to medical microbiologists and
will be considered in this book. As discussed in Chapter 6 , the descending ranking order in taxonomy is
as follows: Phylum, Class, Order, Family, Genus and Species, so the three groups (Fungi, Chromista and
Protozoa) are a long way

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from the levels Genus and Species, familiar to bacteriologists. A more selective and strict system of
classifying fungi is emerging that should make a break from old groupings of algae, protozoa, etc.
Indeed, the classification of fungi is likely to see a period of considerable dispute as newer genetic
methods are employed. Traditionally, fungi have been classified according to the older, morphological
character-based ideas. The features of the mycelia, the spores and sporangia and the sexual and asexual
patterns of reproduction have resulted in a classification scheme consisting of six phyla. Fungi are
described as lower and higher fungi in order to reflect evolutionary history, the lower fungi being earlier
and more primitive forms. The lower fungi are those in the phyla:

• Zygomycota,

• Oomycota,

• Chytridomycota.

The higher fungi are represented in the phyla:

• Ascomycota,

• Basidiomycota, and

• Deuteromycota.

'Yeast' is not a formal taxonomic term but a description of a morphology, like the term 'mushroom'.
Yeasts can be produced by fungi of different groups and genera, most commonly under anaerobic
conditions. There are yeasts that do not appear to have any other morphological form because they have
lost the ability to produce mycelia.

Most fungi produce spores that are generated following asexual reproduction (asexual spores) and
following sexual reproduction (sexual spores) and important examples of the names given to the spores
produced in different cycles by different fungi are given in Figure 4.10 . For identification purposes these
spores are critical. The structures that support or house these spores are also used to help identify fungi.
Asexual spores are found:

• in a sac called a sporangium which is supported by a sporangiophore. These spores are called
sporangiospores. This pattern is typical of the lower fungi in the Class Zygomycota.

• unenclosed and simply projecting from a supporting structure called a conidiophore and hence are
called conidia. Conidia are typical of the other (higher) fungi in the Classes Ascomycota,
Basidiomycota and Deuteromycota.

The sexual spores produced following meiosis are identified by the structures in which they are kept.
Each Class of fungus can be identified by the type of sexual spore produced:

• Zygomycota produce zygospores.

• Ascomycota have sexual spores in special sacs called an ascus, hence the term 'ascospores'.

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Page 117

• Figure 4.10 Fungi produce different structures to house spores depending on
whether they undergo a sexual or asexual cycle of division. These are used to
classify and identify the individual organisms

Ascosporcs

Oospores
Zygospores

ConidiospoKS
Sp(>rait5iiLis|u-nfCB

• Basidiomycetes produce basidiospores in which the spores are found on structures called a
'basidium'. Morphologically this resembles the overall structure seen in conidiospore production.

• The Deuteromycota are those fungi that do not produce sexual spores (hence Fungi Imperfecti).

Unfortunately, the more recent grouping of the fungi (sensu latu) has the Kingdom Fungi containing the
phyla Ascomycota, Basidiomycota, Zygomycota and Chytridomycota. The inclusion of organisms with
flagella (Chytridomycota) doesn't help unify the characteristics that constitute a fungus. In addition, the
phylum Deuteromycota, the one collection of organisms of most cUnical relevance, has been lost. The
Deuteromycota, also called the Fungi Imperfecti, is a resting place (a euphemism for dumping ground)
for those fungi in which the sexual stage has not been identified or perhaps does not exist. Of all the
fungal groups it is an important group for medical microbiologists despite its uncertain provenance, in
that several genera that cause human infections are grouped into this Class, in particular the genera
Trichophyton, Microsporum and Epidermophyton. These are the genera that cause skin, nail and hair
infections of man. With the newer molecular phylogenetic studies of micro-organisms, whereby
comparisons can be made on evolutionary conserved gene sequences such as ribosomal subunit genes, it
seems likely that the class Deuteromycota will become redundant. Even for species in which only the
asexual form has been described, it should be possible to identify the sexual form using molecular

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comparisons.

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■ SUMMARY

Fungi are characterised by apical growth through rigid tubular hyphae which accumulate to
form a mycelium. Fungi in the trophic phase are concerned with feeding through the
filamentous myceha but for the colonisation of distinct, new environments fungi use spores
for dispersal (dispersal phase). As saprophytic heterotrophs, fungi are important recyclers of
nutrients from dead and decaying matter through excreting a diverse range of extracellular
enzymes. This lifestyle matches the aerobic existence in the soil. Fungi resist unfavourable
conditions through production of spores. As the centre of the fungal mycelium senesces,
fungi need to periodically undergo recombination events, often sexual, but not always.
Incompatibility systems prevent the co-existence of genetically distinct nuclei but other
fungi possess incompatibihty systems that do the opposite (i.e. force recombination through
incompatibility between genetically identical mating types). All this compUcated genetics
means that fungi possess a variety of patterns in which the haploid and diploid phases of the
cell cycle are organised. Classification and identification of fungi, particularly moulds, is
based on the type of asexual and sexual spore pattern. Nevertheless, as many fungi are able
to form both sexual and asexual spores, the accurate identification of the spore type depends
on knowing whether the spores you see are produced following sexual or asexual processes.
For example, fungi that cause ringworm are members of the genus Trichophyton and are in
the Class Deuteromycota. However, a sexual form of this fungus has been identified and is
classified as a member of the Ascomycota (Arthroderma) because of the ascospores that are
found. The sexual form of a fungus is called the 'teleomorph' , the asexual equivalent the
'anamorph' .

RECOMMENDED READING

Carhsle, M.J., Watkinson, S.C. and Gooday, G.W. (2001) The Fungi, 2nd edition. Academic Press,
London, UK.

Deacon, J.W. (1997) Modern Mycology, 3rd edition, Blackwell Scientific Publications, Oxford, UK.

Griffin, D.H. (1994) Fungal Physiology, 2nd edition, Wiley-Liss, New York, USA.

Guarro, J., Gene, J. and Stchigel, A.M. (1999) Developments in fungal taxonomy. Clin. Microbiol.

Rev. 12; 454-500.

Jennings, D.J. and Lysek, G. (1999) Fungal Biology, 2nd edition, BIOS Scientific Publishers Ltd,

Oxford, UK.

REVIEW QUESTIONS

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Question 4.1 How do moulds differ from bacteria in their growth in broth culture?
Question 4.2 Why are there four functional sections of a fungal hyphum?
Question 4.3 List the differences and similarities between fungi and plants.
Question 4.4 What are the Imperfect fungi?

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5
MICROBIAL DEATH

5.1 INTRODUCTION

As far as is known, bacteria do not die of old age. They replicate themselves by division once they reach
a certain size. If, in the process of duplication of the DNA lethal mutations are made, then the bacteria
will cease to multiply and die. Alternatively bacteria die when conditions for survival exceed the normal
conditions of growth, such as dehydration or exhaustion of nutrients. Bacteria vary in their ability to
resist adverse conditions and the best example of a survival strategy is the bacterial endospore. Bacterial
spores also vary in their resistance to dehydration, heat or freezing but in general they provide a very
efficient mechanism for survival.

An understanding of how to kill microbes is clearly of great value. A few seconds' thought will reveal
that knowledge on how to kill microbes is not just important in trying to treat human infections. The
wider public health arena is important: water supplies and food products, for example. Also implicated
are the veterinary sciences, food manufacture, brewing industries, preservation of materials such as
building timber, and paper.

One difficulty that undermines our confidence in controlling bacteria and viruses is the definition of
death in microbes. Death in bacteria and viruses is a retrospective diagnosis. A bacterium is defined as
dead when it cannot be grown. If an organism fails to grow when cultured in a broth or on a plate,
having previously been successfully cultured, then we can say it is dead. Or we can say that we failed to
grow the organism in the correct conditions. Because it is impossible to prove a negative event (absence
of growth) there is always the worry that we have not killed the organism but simply failed to grow it.
The reasons for organisms not growing in laboratory culture media are considerable. If incubated in
broth for longer, using a different temperature or different culture broth, we might observe growth and
the original conclusion that the organisms was dead would be mistaken.

Of more practical concern to microbiology laboratories is the need to sterilise items. From sterilisation
of culture media, necessary for the culture of microbes in clinical samples and their identification to the
disposal of clinical samples, steriHsation is a prerequisite and demonstrates how microbiologists need to
think in terms of sterility throughout the working day.

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5.2 KILLING IS EXPONENTIAL

A large number of compounds including chemical disinfectants and antibiotics have been identified that
can be used in controlling the multiplication of bacteria. Although the mode of action will differ between
different compounds, it is helpful to distinguish between whether they act by killing bacteria, in which
case they are termed bactericidal or only inhibit bacterial proliferation rather than kill the organism,
when they are described as bacteriostatic (note the different spelling construction).
Figure 5.1 illustrates the growth curve of bacterial cultures in which bactericidal and bacteriostatic
compounds have been added whilst the organisms are in exponential growth. The bactericidal agent
reduces the numbers of organisms, while the bacteriostatic compound only prevents any further
repHcation but does not kill the organisms already present. Whilst it is understandable that bacteriostatic
compounds are useful in circumstances such as preserving foodstuffs and preventing bacterial
multipHcation on the butcher' s workbench, bacteriostatic agents are inappropriate when attempting to
create a sterile medical product. Figure 5.1 also illustrates how the addition of the bacteriostatic
compound still has a dramatic effect simply by preventing the exponential increase in bacterial
numbers. The difference in the slope of the curve demonstrates how useful a bacteriostatic compound
is. In human infections, bacteriostatic antibiotics prevent the increase in bacterial numbers and allow the
immune defences to gain control. The neutrophils will be able to engulf and destroy the bacteria that are
held in check by the bacteriostatic compound, whereas without this assistance the bacterial numbers may
overcome the abiUty of the neutrophils to cope.

If you count the organisms in a culture exposed to a bactericidal agent (heat or chemical biocide) over a
period of time, the numbers will decUne progressively rather than instantaneously. Table 5.1 shows the
numbers of Esch. coli obtained from such an experiment to study the rate of killing when the culture is
heated to 65°C. The results are shown in Figure 5.2 and illustrate how misleading it is to plot the
numbers of organisms in an arithmetic graph ( Figure 5.2a) . The slope of the line appears to be hori-

• Figure 5.1 Effect of bactericidal and bacteriostatic agents (added at the time
point indicated by the arrow) on the numbers of viable organisms in a bacterial
culture. The numbers of bacteria are plotted on a linear scale

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Unlrcjltid contro]
ciillurc

nunibtirs

Bitttciio^^talic

BacLcricidal

Tinru;

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Table 5.1 Data for survivor curve of Esch. coli held at 65°C

Time (mins) Number of organisms % of survivors killed each time interval Cumulative % killed

103

102

101

lOO

10-1

10-2

10-3

10-4

10-5

99.9

99.99

99.999

99.9999

99.99999

99.999999

reas

; the values in Table 5.1 and Figure 5.2b clearly dei

that the slope is the same throughout the 80 minutes. In other words the rate of killing is constant and
does not slow down once the numbers get small. When exposed to a lethal agent not all of the bacteria in
the culture die immediately but, instead, a proportion of the total will be killed per unit time. As the time
course of killing is plotted with a logarithmic Y axis, a straight line is obtained, as shown in Figure 5.2b
and thus, somewhat unsurprisingly, is described as logarithmic, although 'exponential' is more
accurate. (You will notice that this pattern is the same as the description of bacterial growth curve except
the sign of the constant is now negative.) The straight line obtained when the data are plotted on
semilogarithmic paper tells us that the relationship is a first order reaction; that is, the killing is a
constant feature over time and directly proportional to the number of organisms present. This means that
the fraction killed will be the same per successive unit of time. Note that this is not the same as the same
number of organisms killed per unit time. Instead, the exponential nature of the killing means that the
absolute numbers of organisms killed is proportional to the number present at that time point. In other
words, the more organisms there are, the more organisms are killed.

The problem with an exponential decrease in numbers is that the proportional decrease in
numbers means that you cannot obtain zero but simply a smaller fraction of the previous
value.

The rate of killing is the slope of the line and in this case will be negative because we are describing the
reduction in bacterial numbers. This relationship between numbers of surviving bacteria and time can be
described by the mathematical equation:

(5.1)

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where N/N^ is the number of survivors (number at any particular point in time divided by the number at

the beginning (Nq)); e is the natural logarithm; t is time and k is the constant that describes the

relationship between time and bacterial numbers.

Equation 5.2 employs natural logarithms to show that the rate of killing is represented by the slope of the

graph. We can rearrange equation 5.1:

by taking natural logs of both sides:

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Page 122

• Figure 5.2 A survivor curve. The numbers of viable organisms are plotted
against time. In graph (A) the y axis is linear, whereas in (B) the numbers of
bacteria are expressed as logarithms (base 10). Note that the first two points on
the graph represent a 90 per cent reduction in bacterial numbers

fA>

1000-

soo-

600-

40O-

300-

O-i

I—

— [ —
20

— I —

Time

120

(B)

:^3
■E

10^

10-

10'

10"

10-'

10--

10--^

10-*
10-^
10-^'
10 '
10-*-^

60
Tlitic

IPO

120

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which, rearranged, gives:

lriN-|nNo = -Jtl
and further re-arrangement describes the form of a straight line graph (y=ax+ b):

(5.2)

where the inactivation constant {k) is the slope of the line, t is the value at any point in time and Nq is the
number of organisms you started with and, hence, intercepts the y axis at time zero.

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Page 123

Equation 5.2 can be expressed using logarithms to the base 10 because of the following relationship
between logarithms of the different bases:

ill X = 2.iGi logic ^

It is therefore possible to represent equation 5.2 as follows:

2.3Gilog,g(N/N„)= -k

which can be rearranged to:

2.303 (log„oK-log,^N^)=-^i
logi,^N-log,„K„=-i</2.303

and finally:

log,^ M = -it/2 JOJ ■ ( + logio No

(5.3)

The value of calculating the inactivation constant (k) is that it provides a numerical value that can be
used in comparisons of kilUng rates between different temperatures or between different organisms. As
the time interval in equation 5.2 is not defined, k could be misleading because it was calculated from a
small time interval. In order to make more standardised comparisons an alternative parameter, the D
value (decimal reduction time), is widely used in place of k. The D value is the time taken for the
numbers to fall tenfold (or, in other words, by 90 per cent).

The inactivation constant, k (also known as the exponential death rate constant), has the units of
reciprocal time (t~^). This can be seen by rearranging equation 5.2:

In N = -ii + Id N^

In N + it = ill N^

hi = \n K. - In N

k=-

\n ]S"(| — lji N

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(5.4)

D is a time period and has units of time (usually minutes). D will be the inverse of k, and due to the
conversion from natural logarithms to logarithms of base 10,

The lines obtained in killing curves are not always perfectly linear (see Figure 5.3 ). The reasons are not
completely understood but reflect the difficulties in evaluating death in single bacterial cells. Several
explanations have been proposed to account for the deviation from logarithmic death:

• delayed killing (shoulders) may represent poor penetration of the lethal agent into clumps of
bacteria.

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Page 124

• Figure 5.3 Patterns of results frequently obtained from killing curves
(survivor curves) of bacterial populations. Note how the straight lines have
curved shoulders or tails

Log mrvivorjv

(bdciorml

numbcnj]

Time

• the lethal event is the sum of several distinct steps (multi-hit hypothesis) rather than a first order
reaction (single hit).

• the tails may represent a protective effect of dead cells.

• the organisms are not a homogeneous population. (Straight line survivor curves are often obtained
from spore suspensions, which are likely to be less heterogeneous than a population of vegetative
cells.)

Leaving our experimental culture plates on the bench in the sun until all the water has evaporated from
the agar is one way to kill the microbes (and your research project), but it is almost completely without
control or understanding. What controlled means are available to kill organisms? We will consider three
approaches:

• sterilisation,

• disinfection,

• antimicrobial agents (antibiotics).

This disparate group of headings is pragmatic and does not reflect any natural order. Using antibiotics is

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a form of chemical disinfection, although particularly efficacious antibiotics may indeed sterilise a
culture. It is important to remember that micro-organisms vary widely in their susceptibility to
killing. This variation will be seen both between different species of the same genus and different
strains of the same species. Inherent properties of the different genera will account for the large
differences in resistance to heat or drying (for example, Gram positive bacteria resist drying better than
Gram negative bacteria), whereas smaller differences may occur depending on the environmental
conditions. Two strains of the same species may show differences in survival to irradiation depending on
the pH of the culture medium or their stage in the growth cycle. Before meaningful comparisons of
killing between different organisms can be made, rigorous attention must be given to reduce any
variations in culture conditions.

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Page 125

5.3 STERILISATION

Sterilisation is a process whereby an item is freed of any living micro-organism or their dormant stage.
In theory sterility is an absolute state (despite mathematical predictions), hence it is nonsense to use
phrases such as 'nearly sterile' or 'almost sterile'.

If we had a large volume of fluid that we wanted to sterilise, the point of absolute sterility can never be
obtained because the numbers of organisms remaining continues to reduce by the same fraction per unit
time. You will appreciate that this means the number never reaches zero, and absolute sterility cannot be
achieved. This may seem a pedantic mathematical artefact but it has implications when you wish to
sterilise large volumes. Take, for example, a pharmaceutical company that manufactures 100 ml volume
bags of saline solutions for intravenous injection. What duration of time should the bags be sterilised
for? The saline solutions start out with 10^ organisms in 100 ml it takes 40 minutes to reduce the
numbers of organisms to less than one per bag. To be exact, 0.1 organisms per 100 ml. This means that
in every 10 bags there will be one bag that contains one organism. If they sterilised 100 bags then
potentially there will be 10 bags with a surviving organism present. You would not take the chance that,
if you were the patient needing a saline drip, the bag you were given was not sterile. To avoid this
problem manufacturers of 'sterile' medical products set the steriHsation time such that the probability of
finding one organism alive is 1 in a million (10~^). This is called the 'sterility assurance limit'. The time
taken to reach 1 in a milUon is extrapolated from a survivor graph of bacterial spores (being heat
resistant, the use of spores creates a worst case scenario) as illustrated in Figure 5.4 . In practice, the
solutions have less than one spore per ml at the outset so that, after steriUsation, the chances of a product
being non-sterile are vanishingly small.

There are different strategies for steriUsing items, each having advantages and disadvantages. The
methods are: the use of moist or dry heat, filtration, chemical treatment or irradiation.

• Figure 5.4 D values and sterility assurance. The D value is the time taken for
the numbers of organisms to reduce tenfold. For the organism shown here, the
D value will be the time difference (usually in minutes) between point A and
point A'. The sterility assurance value will be the time taken (from the start) to
reach point SA. This represents the time taken for one organism to have been
reduced a further six logarithmic reductions

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■c

C<-i

Tims;

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Page 126

5.3.1 MOIST HEAT

Most non-sporing bacteria (i.e. the vegetative form) are killed when heated to 60°C. Yeasts and fungi
need temperatures over 80°C. Bacterial endospores, however, are only killed to any significant extent
when held at temperatures above 100°C for over 5 minutes. Bacterial endospores, therefore, pose the
greatest problems in obtaining sterility. Because water acts as a better conductor of heat than air, the
transfer of energy into the microbe is achieved more efficiently when the organisms are heated in moist
or wet conditions (i.e. in suspension) rather than on dry material. The resistance of microbes to 100°C
as boiling water or dry heat differs considerably.

In order to obtain temperatures above 100°C (i.e. raise the boiling point) of water it is necessary to heat
water under pressure. This process is achieved in a pressure cooker at home and, when constructed for
sterilisation purposes, is called an autoclave. Heating water under pressure raises the boiling point and
the released steam is able to transfer the heat (latent heat of vapourisation) rapidly into any colder
surface material, which in this case will be the micro-organism as the steam condenses. This will then
draw more steam into the target (see Box 5.1) .

The relationship between temperature and pressure is shown in Table 5.2 . The temperatures most
commonly employed to sterilise culture media or sterilise biological waste are 1 15°C, 121°C or 126°C.
At first glance it would seem that any increase in temperature is going to have only beneficial effects on
the efficacy of sterilisation. After all, the higher the temperature the greater the rate of killing of bacteria.
However, if moisture is driven out of steam you are left with hot air ( Box 5.1) and are now simply using
dry heat, which is much less efficient at killing micro-organisms, as discussed below.
In practice the biggest problem of autoclaving samples is the complete removal of air. Where air
remains, the temperature will be prevented from rising to that obtained

■ BOX 5.1 PROPERTIES OF STEAM Steam is the gaseous form of water released
once the boiling point is reached. The steam that is required for sterilisation is dry
saturated steam. It has no water droplets contained within it but is the same temperature
as the water from which it is driven. Above this equilibrium point, also termed a 'phase
boundary', the steam becomes superheated steam, a 'dehydrated' form of steam that
is no longer saturated with water vapour. The steam will need to cool to the appropriate
temperature before it can release the latent heat of vapourisation. Beneath the phase
boundary line, the steam becomes 'wet' steam. This is too clogged up with water
droplets rather than gaseous steam and consequently has lower latent heat energy and
the transfer of energy is reduced.

Table 5.2 Relationship between temperature of steam under differing pressures

Temperature (°C) psi* kPascals

Tl5 10 69

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121 15 103

126 20 138

134 30 207

* psi: pounds per square inch.

Pressures given are above standard atmospheric pressure.

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Page 127

by steam under pressure ( Figure 5.5) . Also, the penetration of steam into objects is not always
guaranteed. A closed container such as a bottle or closed bag will not permit entry of the steam and
remain 'dry'. Again, dry heat is far less efficient in its killing potential.

Autoclaves are usually employed to sterilise items that are not adversely affected by moisture, such as
culture media. The sterilised items will emerge from autoclaving wet. This is a problem if the items to
be sterilised are powders or woollen blankets!

5.3.2 DRY HEAT

Sterilisation by dry heat takes higher temperatures for longer periods than wet heat. Steam at 121 °C will
sterilise a spore suspension in 15 minutes, whereas at 120°C in dry heat you will need 8 hours! For this
reason hot-air ovens are usually held at 160-180°C for 2 hours.

Heat can be applied to a material in various forms. Incineration or passing through a Bunsen flame are
two forms of applying dry heat, but usually the end product is not in much of a fit state to use. Any
means of holding the items at a raised temperature for long enough will coagulate proteins in the
microbes but the risks of damaging the item itself usually limit the range of applications of dry heat
sterilisation. Hot-air ovens are used to sterilise products that must not get wet and are heat stable. These
include powders, certain surgical instruments and glassware. The transfer of heat is a slow process and
insulated and/or large objects that heat slowly need to be accommodated for in the duration of the heat
cycle.

5.3.2.1 Controls

In the use of autoclaves and hot-air ovens it is essential that the process is monitored. The temperatures
quoted are never obtained instantaneously and, likewise, glassware in hot-air ovens will take a long time
to cool. Hence both operate via automated

• Figure 5.5 Effect of incomplete removal of air on autoclave temperature. The
autoclave chamber was held at 15psi pressure but varying amounts of air were
removed prior to starting. Note the slow rise and final temperature when no air
is removed

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121 —

ton —

50 —

Purt: biXizam i 1 00% air rcirHJVtd )
50% air ncmovcd

^oairn;nK?v«l

Timt

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Page 128

control panels that can be programmed to take into account the heating up and the cooling down phases
of the cycle and, most importantly, the holding time, the period at which the load is kept at the required
temperature. Thermocouples are placed inside mock large objects that represent the point of least
penetration by the steam. Although measurements of the temperature are important, biological controls
offer a direct measure of the sterilising power of the system. For this purpose suspensions of bacterial
spores (commonly Bacillus stereothermophilus spores) are placed inside the autoclave and then tested
for growth once the cycle is finished. This will measure the effectiveness of the sterilisation, albeit
taking a further 24-48 hours and rigorous aseptic technique. Chemical dyes, known as Brownes tubes,
can also be used. These change colour once a certain temperature has been reached but give no idea of
the length of time the temperature was maintained.

5.3.3 IRRADIATION

The waves of energy carried by electromagnetic radiation can kill micro-organisms by a number of
mechanisms, depending on their frequency. Ranked from lowest to highest frequency, the different types
of radiation that are useful in sterilisation are infrared, ultraviolet. X-ray and y-rays.

5.3.3.1 Infrared radiation

Infrared radiation raises the temperature of objects rapidly (like microwaves!) so that its sterilising
ability acts as dry heat sterilisation. Under vacuum the temperatures rise further.

5.3.3.2 Ultraviolet radiation

UV radiation ranges between 210-330 nm, but those around 260 nm are efficiently absorbed by nucleic
acid and cause dimer formation between adjacent thymines, with lethal, mutagenic effects. The biggest
drawback of UV light is the very poor penetrative power, such that it is ineffective through thin layers of
dust or glass.

5.3.3.3 Ionising radiation

Ionising radiation such as X-rays and y-rays are of short wavelength (<1 nm) and possess much higher
energy and greater penetrating power than non-ionising wavelengths. As the rays pass through the cell
they remove electrons from molecules thus creating ionised (charged) molecules. When water is ionised
it forms hydroxyl radicals that initiate destructive free radical chain reactions which are damaging to
most nucleic acid and proteins.

Sterilisation by irradiation is not readily available in laboratories but will be used by the pharmaceutical
industry to sterilise single-use medical devices.

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5.3.4 FILTRATION

Temperature- sensitive substances such as carbohydrates and amino acids that will be destroyed by heat
sterilisation need to be sterilised by filtration. The pore size (0.2 |im pore diameter) will prevent bacteria
passing through the filter. Filtration does not involve killing organisms but rather simply trapping them
on a filter, small enough in effective 'pore' size that the organisms do not pass. Whilst filtration has no
destructive effect on organisms or the product, the released products such as Gram negative cell wall
(LPS) or even secreted toxins will not be retained by filtration. This is a potential risk in solutions for
medical administration such as saline drip solutions. Similarly,

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Page 129

viruses will not be retained by filters of 0.2 |am pore size. Special ultrafiltration membranes are needed
to filter viruses.

5.3.5 CHEMICAL STERILISATION

Most chemicals cannot be relied upon to sterilise items, but rather act as disinfectants. There are two
chemical compounds that are employed as sterilising agents: ethylene oxide and formaldehyde.
Ethylene oxide ((CH2)20) is a colourless gas that when used as a gas is able to kill both vegetative cells

and spores. It is toxic and potentially inflammable but is used in pharmaceutical and veterinary
applications.

Formaldehyde is a toxic gas (formalin is the commercial solution of 40 per cent formaldehyde) which
whilst able to sterilise exhaust cabinets and rooms at low concentrations (1 per cent) should not be
inhaled, for obvious reasons. Formaldehyde is also used in combination with low temperature steam
steriHsers for sterilising heat-labile items such as medical instruments (e.g. fibre-optic bronchoscopes).
The heat-sensitive instruments are kept at 73 °C for 2 hours and Mycobacterium tuberculosis will be
killed by this procedure. The related aldehyde, glutaraldehyde, is also an effective sterilising solution.
In their favour, chemical methods of sterilisation overcome the limitations posed by heat-labile items but
unfortunately leave a potentially toxic chemical residue.

5.4 DISINFECTION

Disinfection is defined as the destruction or removal of organisms capable of giving rise to infection.
This partial selectivity means that disinfection reduces microbial numbers to below the infectious dose
and does not imply sterihty. Disinfection is often a description of the use of chemical agents but physical
methods such as heat treatments are also included. Where sterilisation is impossible (e.g. human skin) or
practically unreaHstic (sterilising toilets and hand basins) disinfection is of great practical importance.
Care and thought is needed to obtain successful results.

As a general rule chemical disinfectants work well and are used at high concentrations but are
toxic, whereas antiseptics are less efficacious, used at low concentrations and are non-toxic.

Antiseptics are chemical disinfectants that have suitably low toxicity such that they can be appUed to
mammaUan tissues in order to prevent infection.

Disinfection need not mean the addition of toxic chemicals! Strictly, disinfection will include heating or
simple washing with detergent. Both procedures will remove significant numbers of organisms so that
the risk of infection is virtually ehminated. Nevertheless, disinfection with chemical disinfectants means

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that you will also kill a proportion of the organisms irrespective of whether you remove them in the
bucket along with the soap and water. And, to be exact, soaps will also inhibit organisms.
Figure 5.6 presents a diagram representing the route by which organisms are

• Figure 5.6 Route of transmission. An infection can be viewed as the
movement of an organism through four stages. Disinfection aims to interrupt
the flow between stages 2 to 3

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Page 130

Table 5.3 Chemical disinfectants

Group

Example

Concentration Inactive against

Alcohols

Halogens

Phenolics

Biguanides

Quaternary ammonium compounds (QACs)

isopropanol
iodine chlorine
cresol, hycohn
chlorhexidine
cetrimide

90% v/v
2% w/v Ippm
0.5-3% v/v
1-4% v/v
0.1%

spores/viruses

spores/ mycobacteria

transferred in the sequence of events that result in an infection. The usual sequence of events follows the
diagram from left to right. Disinfection aims to break the connection between the reservoir and the route
of spread. In the context of a hospital ward the source of a micro-organism is usually unknown, but
application of antibacterial hand cream following hand washing by the hospital will help minimise the
risk of transfer of organisms from the wound of patient X (reservoir) to the next patient (susceptible
host). The concept of reservoir and source will be examined further in Chapter 7 .

5.4.1 CHEMICAL DISINFECTANTS

There are a large number of chemicals that exert antimicrobial activity. Unlike antibiotics, they need
relatively high concentrations to work and all are inactivated by dirt or organic matter. The target sites
are often not clearly defined but include disrupting cell wall and cell membrane integrity or oxidising/
denaturing proteins within the cytoplasm.

The major groups of disinfectants that are used in hospital and laboratory settings are given in Table 5.3 .
There are two particular challenges to the use of disinfectants in killing microbes. These are the i)
variation in susceptibility of different micro-organisms to disinfectants, and ii) the inactivation of
disinfectants in their working environment.

(i) Perhaps not surprisingly, the diversity in structure of micro-organisms manifests itself in a

spectrum of tolerance to disinfectants, ranging from susceptible through to resistant. The sequence
of decreasing resistance is shown in Figure 5.7 .
(ii) The environment in which the disinfectant is expected to work can vary enormously and often has
a number of features that inactivate or impair their bactericidal action. One of the most
problematic features is the presence of organic matter. Organic matter will compete with the
microbes as the target for the disinfectants and either exhaust or neutralise the active component
of the disinfectant. The problem is easily demonstrated by considering what to do about
disinfecting any body fluid spill (sputum, urine, blood) onto a floor or uniform. The organic
nature of the spill poses a serious challenge to any effective killing by disinfection of any
microbes present with this predominantly organic cocktail.

Before addressing this problem, other factors that alter the efficacy of disinfectant action need to be

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discussed:

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• Figure 5.7 Spectrum of resistance of different micro-organisms to chemical
disinfectants MOST RESISTANT

MOST RESISTANT

Bficteria] spores

Ncin-cn^ic1ci|Kd viruses

E-ungi (yeasts and niouEds)

Vcgcliitivc fomis of bacteria

EnvclupcJ viniiics

LEAST RESISTANT

• duration of exposure. It appears all too easy to assume that having sprayed the worksurface with a
disinfectant (e.g. 70 per cent alcohol) and then wiped it down with paper cloth the surface is
suitably disinfected. Disinfectants do not achieve their desired effect instantaneously but need time
to act.

• population size. As discussed earlier, the concept of exponential killing dictates that the larger the
initial population of microbes the longer it will take for the number to be reduced to an acceptable
level.

• disinfectant concentration. Within limits, an increase in concentration will hasten the disinfection
process.

• temperature. Raising the temperature will also hasten the reduction in microbial numbers as the
speed of reaction increases. In fact, the relationship between the two is not linear. The rate of
microbial killing goes up geometrically with linear increases in temperature. Clearly, extremes of
temperature may confound the disinfection process by either inactivating the chemicals themselves
or promoting the growth of the microbes.

The guiding principle for use of disinfectants is 'strong enough for long enough'.

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Is there then any hope for disinfectants achieving any significant reduction in the microbial load?
Disinfectants are prepared at recommended concentrations following testing under experimental
conditions in which the above variables are examined. In hospital wards and laboratories recommended
procedures will be in place which should provide a level of protection that is acceptable for most
encounters. Fortunately, the risk of infection from spills is usually low due to the unlikely chances of an
infectious dose being transmitted in an appropriate manner.

If, by cleaning with warm soapy water, you remove most interfering organic matter along with a
proportion of the microbes why use disinfectants? Disinfection of surfaces like walls and floors or
equipment that pose negligible risk of infection to staff will be a waste of expensive disinfectant. Under
such circumstances the question is entirely

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self-justifying; however, where instruments or surfaces are contaminated with pathogenic microbes
disinfection is necessary prior to cleaning. For example, sputum from a patient with tubercle bacilli will
need effective disinfection, i.e. reduction in the numbers of viable organisms before the spillage can be
removed. In contrast, disinfectants are usually used following cleaning on hospital baths and kitchen
surfaces so as to prevent cross-contamination of patients and food respectively.

5.5 TESTS OF DISINFECTANT EFFICACY

In microbiology laboratories dangerous organisms are grown in large numbers. Particular attention must
be given to disinfection pohcy so as to permit safe working practices. What are the relevant and
important needs in the testing of disinfectants? There are three questions that should be asked when
deciding to use a disinfectant in the laboratory.

1. How does this disinfectant compare with others?

2. What is the appropriate concentration for use?

3. How does the disinfectant stand up to the daily laboratory needs?

There are numerous methods for evaluating disinfectants. The differing tests can be used to answer the
three questions raised above.

Questions 1 and 2 can be addressed by testing the new disinfectant alongside existing disinfectants and
comparing the concentrations that are effective in kilhng the added bacteria. Use-dilution tests test the
efficacy of disinfectants under defined conditions in which the disinfectant is tested at increasing
dilutions and the number of survivors are counted after defined exposure times. Such methods are
quantitative and therefore can yield percentage kill (99 per cent, 99.9 per cent, 99.99 per cent, etc.).
With capacity tests, the disinfectant is repeatedly challenged with known numbers of organisms at
timed intervals. The result is quahtative in that the disinfectant passes or fails the test.
Two technical details of critical importance need mentioning. It is important to inactivate the
disinfectant once the samples are removed from the tubes otherwise the disinfectant will simply continue
to kill the bacteria and the element of samphng at time points is lost. Having stressed that organic matter
inactivates disinfectants, the disinfectant needs to be tested both in the presence and absence of organic
matter if it is to have any relevance to question 2. This is usually attempted by adding known
concentrations of killed yeast extract/albumin. The extent to which this mirrors the rigours of a daily use
in the working laboratory can be assessed by the in-use test. In-use tests sample the disinfectant pot that
is on the bench during the working day. The samples are inoculated onto agar plates and incubated
overnight. If the numbers of bacteria are low or absent then the disinfectant appears to be effective and
of real value.

Thus far we have considered the kilhng of micro-organisms outside of the host. We need to shift our
emphasis from preventing infections through killing bacteria in the environments to treatment of

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individuals with existing infections.

5.6 ANTIBIOTICS

Surgeons taunt their medical colleagues that surgery cures people of their illnesses whereas medical
treatments just alleviate symptoms without curing the underlying pathology. The best rebuttal to this
taunt has been the treatment of infectious disease with antibiotics. In the time before antibiotics (and it
was not that long ago), relying on the host immune responses to combat the infection was the only hope
of survival.

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Patients with pneumococcal pneumonia were simply observed, waiting for the critical period (crisis) at
around 2 weeks after becoming ill. If the patient did not die at this point then the fever and sweats would
then slowly resolve as the host immunoglobulins catalysed the clearance of the circulating Streptococcus
pneumoniae.

Antibiotics are probably thought to be wonderfully lethal to bacteria by most people who take them. The
reality is a little less dramatic. The benefits are undisputed; antibiotics have undoubtedly reduced the
mortality rate. What has been sobering is the finding that antibiotics have had little effect if any in
reducing the incidence of infectious disease. The reasons are complex and are further discussed in
Chapter 1 1 . Nevertheless, a partial explanation can be offered here.

Antibiotics can be lethal (bactericidal) but many simply inhibit bacterial growth and replication
(bacteriostatic). Even so, bactericidal antibiotics at low concentrations are often bacteriostatic rather
than lethal. Their efficacy relies on the effective contribution of the immune response of the host. This
limitation is seen in the treatment of patients with heavily impaired immunity such as neutropenia (very
low numbers of white cells). In these cases patients need combinations of antibiotics for longer periods
to try to effect a cure. Antibiotics also are often unable to prevent a systemic infection such as
septicaemia from killing someone. Again this is testament to the value of the host defences; antibiotics
alone are not as efficient as people believe.

Antibiotics are secondary metabolites produced by bacteria and fungi that inhibit the growth of other
micro-organisms at low concentration.

Such a definition is useful but problematic. Bacteriocins and coUcins fall into this definition but are not
called antibiotics. Producing an antibiotic is linked to the ecology of the organism. Antibiotics are
secondary metabolites ( Table 5.4 ). Such compounds have no role in the growth of the organism
(mutants unable to produce antibiotics are able to grow normally), but are produced when one or more
nutrients are hmiting to growth and metabolism becomes unbalanced. Bacteria that produce antibiotics
need to be resistant to their effects for obvious reasons, but it is common that antibiotics inhibit closely
related bacteria (of the same genera and species). Figure 5.8 shows how antibiotics, Uke all secondary
metaboUtes, are produced during stationary phase (in fact their synthesis is repressed during growth). As
most bacteria are resistant to the damaging effects of antibiotics when in stationary phase, this may
explain why antibiotic-producing organisms gain benefit. The inhibition of competing organisms occurs
only after growth and multipHcation of the antibiotic-producing cells. The temporal production also
permits the bacterium to produce antibiotics even if it is susceptible itself.

Strictly, one distinguishes between true antibiotics (i.e. compounds that are synthesised by other micro-
organisms as secondary metaboUtes (compounds synthesised by bacteria that are not essential for its
growth in comparison to primary metaboUtes

Table 5.4 Primary and secondary metabolism

Primary metabolites Secondary metabolites

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Distribution Found in all organisms Restricted

Production Lag and exponential phase Late exponential phase

Role in growth Essential Not directly involved

Effect on other organisms No harmful effects Mostly inhibitory

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• Figure 5.8 Antibiotic production (typical of secondary metabolites) is shown
in relation to the growth phase of the organism (solid line). The antibiotic
(dotted line) is produced during late exponential growth phase

Numhcr of

(Log scale)

AiUibLQlic
- prodjc'Cion

Time

which are essential for their own growth)) and compounds that act as metabolic analogues (i.e.
compounds that resemble normal metaboHtes or substrates but are antagonistic to growth through
inhibition). The latter are termed 'antimetabolites' or 'chemotherapeutic agents' rather than antibiotics.
Metabolic analogues are chemically synthesised and are not produced by micro-organisms.
The number of different organisms that the antibiotic works against is called the spectrum of activity.
This varies for different antibiotics, with some having a broad spectrum of activity (many different
organisms inhibited) whereas others have a narrower range of action. The spectrum of activity can be
altered by chemical modification of the antibiotics, notably the penicilUns.
The ideal features of an antibiotic for human use are as follows:

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(i) selective toxicity. This term describes a property of the antibiotic in which the target site is
present in the bacterium but not the host. Selective toxicity can be expressed numerically as the
selective index, the ratio of 50 per cent value of toxic dose to 50 per cent inhibitory dose (TD50/

IC50). When assessed in vivo the therapeutic index can be assessed as the ratio of toxic dose to the

effective therapeutic dose.

For peptidoglycan in bacterial cell walls, the selective toxicity is obvious as humans lack such a
structure. However, many antibiotics rely on more subtle differences. For example, the differences
in ribosomal subunit structure between prokaryotes and eukaryotes. The targets consequently
become more problematic still when targeting fungi and viruses,
(ii) appropriate pharmacology. Does the antibiotic reach the site of infection? Having found a
suitable target with your new antibiotic, it is essential that the pharmacological properties are
suitable for use in patients if there is any hope of bringing the compound to market. Variation in
pharmacokinetics are useful. Some antibiotics are not absorbed across the intestinal tract and need
to be injected intravenously. Then you might find that the drug concentrates in liver but you wish
to treat a urinary tract infection and need an antibiotic that is excreted via the kidney.

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(iii) retain efficacy. The antibiotic needs to avoid inactivation by neutralising agents (if urea in urine
interferes with the antibiotic then treatment of urinary tract infection will be compromised).
Conversely, the route of elimination of the antibiotic from the body can help direct the drug to the
appropriate site (e.g. renal excretion is suited to the treatment of urinary tract infection) but many
antibiotics are eliminated from the body very quickly, necessitating repeated dosages.

5.7 ANTIBIOTIC TARGETS

The variety of target sites exploited for developing drugs for use in treating human infections are given
for antibacterial, antiviral and antifungal compounds. The antibiotics discussed are chosen on the basis
of their selective toxicity and are summarised in Table 5.5 . In other words, the important points to
consider are the targets within the microbes that are sufficiently different from mammaUan cells to
minimise toxicity to humans but still inhibit the microbes. The events that actually kill the bacterium are
surprisingly hazy in most cases despite apparent understanding of the molecular targets. Appropriate
texts on antibiotic action should be consulted for details.

5.7.1
ANTIBACTERIAL COMPOUNDS

The target sites of greatest value to date are as follows:

• bacterial cell wall,

• cytoplasmic membrane,

• protein synthesis,

• nucleic acid synthesis and replication.

5.7.1.1 Bacterial cell wall

Peptidoglycan provides the bacterium with a rigid cell wall to confer strength and resistance to turgor
pressure from the cytosol. P-lactam antibiotics (penicilHns and cephalosporins) target the assembly of
the peptidoglycan. The polymers of NAG-NAM molecules are cross-Hnked by peptide chains. The
controlled insertion of new

Table 5.5 Antibiotic targets and mode of action

Structure Target Effect

CELL WALL

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P-lactams

Penicillin binding proteins
PROTEIN SYNTHESIS
Aminoglycosides, e.g. 30S ribosomal subunit
Streptomycin

Tetracycline

Erythromycin

Chloramphenicol

Fluoroquinolones

Sulphonamides

Trimethoprim

SOS

NUCLEIC ACID

DNA gyrase

Dihidropteroate synthetase

Dihydrofolate reductase

Impair cell wall synthesis, trigger autolysis

Misreading

Inhibit tRNA

Inhibit translocation

Inhibit peptidyl transferase, thus transpeptidation

Inhibit DNA replication
Inhibit folate synthesis
Inhibit thymidine synthesis

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NAG-NAM units is essential if the peptidoglycan integrity is not to be compromised. There are several
different types of enzymes that facilitate this process. Functionally, the enzymes are transpeptidases and
carboxypeptidases, collectively called penicillin-binding proteins (PBPs) as they are the targets for the
P-lactams. There are between five and seven different PBPs per species and different PBPs bind
different P-lactams. The P-lactams impair the extension of the cell wall as the organism attempts to
grow. The exact reasons for the cell death induced by P-lactam antibiotics is not clear but the triggering
of bacterial autolysins is suspected. Such enzymes may be PBPs themselves in that they regulate the
transient disassembly of the peptidoglycan whilst new peptidoglycan is inserted. Perhaps when bound by
penicillins they are irreversible in their action. Unrestricted, the osmotic pressure will burst the
cytoplasmic membrane if the rigid peptidoglycan is breached. Certainly, P-lactam-treated bacteria are
able to survive if they are kept in osmotically balanced solutions the bacteria form spheroplasts and
protoplasts, viable bacterial forms that have only a fraction of the normal complement of cell wall
(spheroplasts) or no cell wall at all (protoplasts).

The location of the PBPs in the bacterium has significant impact on the efficacy of P-lactam antibiotics
between Gram positive and Gram negative bacteria. With Gram positive bacteria the target
(peptidoglycan) Ues outside the cytoplasmic membrane and the P-lactams, being water soluble, have free
access to their targets. Gram negative bacteria will have the outer membrane external to the cytoplasmic
membrane and, being a permeability barrier to aqueous molecules, it will limit the rate of entry of P-
lactams to the peptidoglycan. The hydrophilic P-lactams pass through the outer membrane via water-
filled porins.

5.7.1.2 Cytoplasmic membrane

Interior to the bacterial peptidoglycan is the cytoplasmic membrane. Of the differences that exist
between mammahan and bacterial cytoplasmic membranes, of most relevance is the overall surface
charge. Mammalian phospholipid membranes are predominantly zwitterionic (possessing both negative
and positive charge and the major component is phosphatidylcholine) whereas prokaryotes contain a net
negative charge through the dominance of anionic phospholipids. One antibiotic that preferentially binds
to negatively charged membranes is polymixin, representative of the class of cyclic peptide antibiotics.
Being polycationic, polymixin will bind to negatively charged phosphoUpids and disrupt the membrane
integrity with subsequent osmotic perturbation (leakage of intracellular contents). You will recall that
the outer leaflet of the outer membrane of Gram negative bacteria does not contain phospholipid, but,
negatively charged lipopoly saccharide (LPS). Polymixin will also bind to LPS but this binding will not
result in disruption of the cytoplasmic membrane.

A similar affinity for negatively charged phospholipids underlies the action of epithelial antibacterial
peptides, compounds secreted by mammalian epithelial cells in response to microbial infection. These
peptides form part of the innate response to infection and are expressed in plants, and insects as well as
animals, and are under investigation for use as therapeutic agents. The peptides (and there are a growing
number identified) are cationic and small (less than 100 amino acids) and have a broad spectrum of

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antibacterial action. The peptides bind to and disrupt both the LPS and cytoplasmic membranes. The
selective toxicity lies both in the affinity for anionic bacterial membranes and because they are inhibited
by the cholesterol present in mammalian membranes. Whilst not true antibiotics, they are hoped to
provide a new line of treatment. Optimism might be considered justified because these peptides have a

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number of advantages: selective toxicity, broad spectrum of activity, rapid killing time through acting at
more than one site. The sites and mechanisms of action mean that the bacteria cannot develop resistance
very readily.

5.7.1.3 Protein synthesis

A number of antibiotics act by inhibiting aspects of protein synthesis (chloramphenicol, erythromycin,
tetracycline and the aminoglycoside group of antibiotics). Protein synthesis occurs at the ribosome and
selective toxicity arises from differences between eukaryotic and prokaryotic protein at the ribosome.
This is reflected in the fact that eukaryotic ribosomes are larger than those in prokaryotes (see Figure
1.13) . These differences have a number of implications for their cell biology.

Size of ribosome

• Considerably more associated proteins and cofactors are required for eukaryotic protein synthesis
(e.g. approximately 10 initiation factors compared with 3 in bacteria). In terms of making the
ribosome and associated factors, there are metabolic savings to be made if fewer proteins are
required and the size of the ribosome is smaller.

Speed

• Protein synthesis is faster in many bacteria than eukaryotes (e.g. humans). The spatial restriction of
the mammalian genome inside the nuclear membrane means that mRNA travels further to reach the
cytosoUc ribosome. No such nuclear membrane exists in prokaryotes, hence turnover is
approximately tenfold greater.

• Bacterial mRNA is polycistronic, i.e. numerous different proteins are coded for on the same mRNA
(often as an operon).

Output

• mRNA is translated by several ribosomes simultaneously (polysome), thereby increasing the rate of
production of proteins.

Examples of antibiotics that target protein synthesis (see Figure 5.9) are as follows.

5.7.2 ANTIBIOTICS THAT TARGET THE 30S SUBUNIT

5.7.2.1 Aminoglycosides

Streptomycin and gentamicin are the most representative members of this large group of antibiotics.

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Streptomycin was discovered in the 1940s and became the first antibiotic to be used against
Mycobacterium tuberculosis. Unfortunately, resistance readily develops against streptomycin and it is
nephrotoxic and ototoxic. Gentamicin is used for serious infections and is preferred because of its broad
antibacterial spectrum (which is considered to be bactericidal rather than just bacteriostatic), although it
is still toxic.

The reason why aminoglycosides are bactericidal remains unclear despite considerable research.
Aminoglycosides have two effects: the misreading of the mRNA chain and prevention of initiation of
protein synthesis. These two events may appear contradictory. How do you cause misreading if you have
prevented the process from starting? It is possible that the two events are induced by differing
concentrations of antibiotic.

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• Figure 5.9 Sites of action for the antibiotics that target protein synthesis at
the ribosome

Page 138

Grnwang

pcpiidc
chain

t.lietraciiclln&
3. efytlvonnycln

2. aininoglycosldes
4. ctnlamnn^enlcDJi

At low concentrations, streptomycin causes misreading of pre-existing and active processing in the
abundant polysomes; but it is only at higher concentrations that the antibiotic is able to prevent the
initiation of new ribosomal activity.

5.7.2.2 Tetracyclines

(Tetracycline, minocycline, doxycycline) are broad spectrum antibiotics. Resistance has developed and
they are less used than previously, although newer derivatives have been developed. Tetracyclines block
the binding of tRNA to the A (acceptor) site on the 308 subunit.

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5.7.3 ANTIBIOTICS THAT TARGET THE SOS SUBUNIT

5.7.3.1 Chloramphenicol

Chloramphenicol is readily absorbed from the gut and distributes itself widely throughout the body. This
made it useful for treatment of infections in relatively inaccessible organs such as the central nervous
system (meningitis) and the gall bladder (typhoid), but is not now widely used because of bone marrow
suppression (aplastic anaemia) and widespread resistance.

Chloramphenicol prevents the transfer of the growing polypeptide chain from the P (peptidyl-tRNA) site
to the next aminoacid-tRNA in the A site. The enzyme that catalyses the transfer is called peptidyl
transferase and it is this process (transpeptidation) that is inhibited. Note that the peptide chain is the
effective target rather than the ribosome.

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5.7.3.2 Erythromycin

This antibiotic represents the class called macroUdes, which have a broad spectrum of activity. Typically
erythromycin can be used in patients allergic to penicilUns. Erythromycin prevents the translocation of
the peptide chain and tRNA (peptidyl-tRNA) from the A to the P site on the 50S subunit.

5.7.4 NUCLEIC ACID FUNCTION

There are a number of compounds that bind directly to DNA either as intercalators (ethidium bromide),
cross-linking agents (mitomycin C) and alkylating agents (nitrogen mustards). Intercalators insert in
between the base pairs in DNA. Usually flat, planar molecules they distort the reading frame such that
frame shift mutations occur in the copying of the DNA. One of these, acridine orange, is fluorescent and
used to detect bacterial DNA. All three types of compounds are useless as antibiotics as a result of the
absence of selective toxicity. More important are the compounds that follow.

5.7.4.1 DNA replication

Quinolones (naUdixic acid, ciprofloxacin) inhibit the function of DNA gyrase (DNA topoisomerase II).
These enzymes catalyse the unpacking of the supercoiled DNA ready for DNA replication or
transcription by RNA polymerase. Once DNA packaging is disrupted, DNA synthesis ceases. You will
recall that when unravelled, bacterial DNA is around 1000 times longer than the bacterial cell body. Any
interference with such a critical operation makes sense as a good target for antibiotic action.
Unfortunately, it is not entirely clear what the lethal events are in the action of quinolones. Selective
toxicity derives from differences in subunit composition of the DNA gyrases between man and bacteria.

5.7.4.2 DNA synthesis

Sulphonamides and trimethoprim are not true antibiotics but chemotherapeutic agents as they are
synthetically devised and manufactured. They target two distinct steps in the bacterial synthesis of
folate, a precursor of the purine base thymidine. Selective toxicity derives from the fact that man cannot
manufacture folate but instead absorbs it entirely from the diet, hence humans lack the relevant enzymes
that these antibiotics target. Sulphonamides are homologues of para-aminobenzoic acid (PAB A) and act
as competitive inhibitors, thereby blocking the manufacture of dihydrofolate.
Figure 5.10 shows that trimethoprim acts on an enzyme further down the folate pathway. The
significance of this is of some interest. In the laboratory, if the two antibiotics are used together the
inhibition of growth is synergistic (the inhibition of the two is greater than the addition of the effect of
the individual antibiotics). The reasons for the synergy have been debated for many years. It is possible
that this synergy is due to the combined action on two different targets of the same pathway. However, it

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has been argued that if the pathway is hnear, any inhibition of an enzyme at one point in the pathway
will be overcome because of the build up of substrate (because of the build up of substrate prior to the
step catalysed by the affected enzyme). The synergy occurs only if the pathway is cyclical rather than
linear because then all components will be affected when one enzyme in the cycle is inhibited.

5.7.5 ANTIVIRAL COMPOUNDS

The exploitation of the mammalian host cell machinery by viruses poses numerous challenges to the
development of antiviral compounds that exert a selective inhibition

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• Figure 5.10 Site of action of folate synthesis inhibitors, sulphonamides and
trimethoprim. Both antibiotics act as competitive inhibitors of the appropriate
enzymes (shaded arrows). Enzymes are given in italics

Sulphtmamide

Paia-aminubcnzoic; aciJ
(PABA)

+
ptcridine

i^fhyiimpsertiiilti
sviiihase

Dihydroptcroatc

DihydrorolLitc
fFAM.)

Tctrdtiydiorolalc

Trirnti!th4>priiii

Tlwmidtjie Purines Amino acids
(DKA) (RNA.DNA) (proteins)

of vims multiplication and not mammalian cell processes (protein synthesis, nucleic acid replication and
cell growth). Although this predicts that antiviral compounds will likely be toxic to humans, drugs have

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been developed with remarkably good selective and therapeutic indices. A more thorny problem occurs
with the pattern, seen with many viruses, of short and asymptomatic incubation periods. The infection
may need to develop beyond this stage before the patient becomes aware that they have an infection.
This means that the delay in diagnosis prevents antiviral treatment at an early stage in the infection and
thereby preventing virus production. Commencing treatment after the incubation period will do nothing
to limit the spread of the virus and is likely to have little effect on symptoms. It follows that the slower
viral infections offer greater chances for antivirals to exert an effect that will be significant. The
argument leads to the prediction that prophylactic antiviral treatment will have the greatest impact in
limiting viral infections both to the patient and the community. The problems of cost.

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toxicity and common sense over-ride such an approach from becoming widespread practice.

In comparison to bacterial infections, viruses will follow a theoretical replication cycle against which

antivirals can be targeted. At various stages in this cycle the viral particles may be susceptible to

antiviral compounds. Understanding the cycle offers, at least in principle, the benefit of theoretical

predictions of target sites.

You will recall the steps in a viral repUcation cycle are:

• the attachment of the virus to the cell receptor,

• virus entry and uncoating to release nucleic acid within the cell,

• nucleic acid transcription and replication,

• virus assembly,

• virus release.

The early steps on the course of an infection are not suitable targets because most viral infections have a
silent incubation period during which the host is shedding large numbers of virus. Treatment will be too
late as the early steps in the attachment and uncoating of the virus has already occurred. An additional
concern is the susceptibility of latent virus to antiviral chemotherapy. Whilst no new virus is being
manufactured the virus is shielded from attack by the antiviral agent.

The viral targets that have proved suitable for attack are those that occur late in the course of replication
of virus. Successful antiviral agents have been developed against virally encoded enzymes involved in
the synthesis of new virus. Three enzymes are mentioned as examples:

• thymidine kinase/DNA polymerase (in Herpes viruses),

• retrovirus reverse transcriptase,

• viral proteases in retroviruses, in particular HIV.

5.7.5.1 Thymidine kinase/DNA polymerase

Acyclovir is a nucleoside, a molecule consisting of the sugar ribose with a base attached, e.g. adenine,
thymine. The possession of a nuclear base in the structure tells us that acyclovir must act by interfering
with viral DNA chain synthesis. Acyclovir is actively transported into the infected cell which then
becomes a target for the Herpes virus enzyme called thymidine kinase (TK). This enzyme catalyses the
first in a series of three phosphorylation (addition of a monophosphate group) events which converts
acyclovir into acyclovir mono-, di- and triphosphate. The viral DNA polymerase enzyme is inhibited
competitively by acyclovir-triphosphate so that any acyclovir triphosphate that is incorporated into the
growing viral DNA causes chain termination ( Figure 5.11) . These events prevent further synthesis of
progeny virus. Other viruses do not synthesise TK, instead relying on host kinases, hence acyclovir is
specific for Herpes viruses. Thus, the selective toxicity of acyclovir lies in two features: its susceptibility
to phosphorylation by thymidine kinase produced by Herpes viruses but not mammalian nucleoside
kinases and the selective activity of phosphorylated acyclovir in inhibiting viral DNA polymerase over
eukaryotic DNA polymerase. Other nucleosides are being developed (e.g. cidofovir, a cytosine

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derivative) which also act as competitive inhibitors of viral DNA polymerase and prevent DNA chain
elongation. However, because they do not rely on the prior activity of viral thymidine kinase but are
phosphorylated by host kinases, they have a broader spectrum of antiviral activity.

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• Figure 5.11 Action of acyclovir. Acyclovir acts as a substrate for the viral
thymidine kinase (TK). The viral TK, but not human kinases, phosphorylate
the acyclovir such that when incorporated into new viral nucleic acid, it
prevents DNA replication

-cr

New DNA cliain

Vira] DNA

DNA (cmivtate

l_l

ill

5.7.5.2 Reverse transcriptase inhibitors

Reverse transcriptase (RT) catalyses the synthesis of DNA from RNA. The inhibitors of retroviral RT
such as AZT are also nucleoside analogues and thus work using the same process as acyclovir, namely
inhibition of DNA chain elongation following incorporation into the new chain. Retroviruses do not
synthesise their own thymidine kinase so AZT is phosphorylated by the host cell kinases. This means
that uninfected cells will metabolise AZT. The selective toxicity has to exploit the unique possession of
RT by retroviruses but not eukaryotes. The AZT must inhibit RT but have nominal effect on host DNA
polymerase. This is the difference between acyclovir and AZT: acyclovir targets viral TK whereas AZT
targets viral RT, although the end result is the same.

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5.7.5.3 Protease inhibitors

That viruses use host cell machinery for synthesis of viral proteins makes the identification of specific
antiviral agents that target protein synthesis very challenging. One key difference that has been
identified is the processing of proteins after synthesis (post-translational modifications). Eukaryotic
mRNA is monocistronic; that is, it codes for only one protein. Viral mRNA by contrast is polycistronic.
The different proteins coded for on the single mRNA are synthesised as a large polyprotein and need to
be cleaved into the different proteins by specific, virally encoded proteases. One such protein that has
been targeted is the HIV protease that releases viral proteins from the precursor protein, thereby
arresting the production of a new virus.

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Analysis of the structure has identified potent inhibitors that target the active site of the protease.
Unfortunately, the high mutation rate seen in RNA viruses results in mutations in the protease that
reduce the affinity of the inhibitors. This has forced the use of multiple protease inhibitors in treatment,
reminiscent of the use of multiple antibiotics against single target sites.

These three target sites have yielded only two distinct strategies for antiviral agents. More ambitious
strategies to inhibit viral replication have been proposed, namely the use of antisense oligonucleotides.
Theoretically, a sequence of nucleotides can be made that will hybridise to viral mRNA and interfere
with normal RNA processing. The antisense nucleotides need to exhibit increased hybridisation affinity
(i.e. raise thermal dissociation, T^) and be chemically modified to reduce degradation by host cell

nuclease activity. An alternative new development is the use of ribozymes, RNA molecules that
hybridise with the target RNA and then catalyse a break in the target RNA strand.
There are other possible targets (release of new virus via budding, for example) that are under
investigation, but numerous specificity and drug delivery issues need to be resolved.

5.7.6 ANTIFUNGAL COMPOUNDS

Being eukaryotes, fungi possess a number of features in common with mammalian cells and this poses

restrictions on target sites when devising a selective antifungal agent. Fortunately a number of suitable

targets can be identified that are unique to fungi. Most antibacterial antibiotics do not work against fungi

and this highlights the successful targeting between prokaryotic and eukaryotic cell machinery/

biochemistry.

The most useful clinical antifungal antibiotics have two targets:

• cell membrane,

• nucleic acid function.

5.7.6.1 Nucleic acid function

Chemically, 5-flucytosine is a fluoropyrimidine, i.e. a pyrimidine base with a fluoride group attached.
This description indicates that 5-FC works by mimicking nucleotide bases. 5-FC is biologically inert but
converted (deaminated) to the 'biologically active' 5-fluorouracil which is incorporated into RNA in
place of the normal base uracil. Toxicity correlates with the extent to which 5-FC accumulates in mRNA
and tRNA, presumably by interfering with DNA replication (5-FU interferes with thymidine production)
and protein synthesis (malfunctioning RNA).

The selective toxicity lies in the fact that most fungi actively transport 5-FC across the cell membrane
but mammalian cells cannot as they lack the transporting enzyme (cytosine permease). Cytosine
permease transports other nucleosides, adenine and cytosine. There are no transporters for 5-FU in either
fungi or mammalian cells. This is important because 5-FU is toxic to mammalian cells (5-FU can be
used as a chemotherapeutic agent for treating cancer).

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5.7.6.2 Cytoplasmic membrane

Two classes of antifungal drugs (Polyenes and Azoles) exert their effects by interfering with the sterol
ergosterol present in the cytoplasmic membrane. Fungi use ergosterol whereas mammals use the related
compound cholesterol. These membrane steroids act to stabilise the bilayer structure and modulate
membrane bound enzymes ( Box 5.2) . Selective toxicity arises because fungi synthesise ergosterol
whereas

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■ BOX 5.2 FUNCTION OF ERGOSTEROL IN FUNGAL CYTOPLASMIC
MEMBRANE

Membrane integrity.

Membrane fluidity.

Function of membrane bound enzymes.

Necessary for progression through cell cycle ('sparking').

mammalian cells simply incorporate exogenous cholesterol (from the diet), plus the affinity of drug-
binding to ergosterol is higher than for cholesterol. Polyenes (nystatin for topical use and amphotericin
B for systemic use) have a broad spectrum of activity against fungi possibly because they target
ergosterol itself rather than its synthesis and ergosterol is found widely in many fungi. The polyenes

bind to ergosterol ( Figure 5.12 ) and aggregate to form pores in the membrane of 8 A in diameter

(hydrated sodium ions are 2 A in diameter). This breach in membrane integrity results in osmotic

disruption and leak of cytosolic components (of appropriate size).

Azoles are totally synthetic antifungals, classed as imidazoles or triazoles depending

• Figure 5.12 Polyene action on fungal plasma membranes

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tA)

... J.u

AnipbDlcricin monomers approach cytoplasmic
nicmlnranc and liind to gir^goMeroL in Inlaysr (grey
rtctanjjlcs.)

"Iwo harnel-sfiapcd
porcii UTC formed by
oHgDjtic?rJsatioit nf
[jmpbutoricin
mcmoiTH^rSr Two
separate barrels are
fumtd, one; on lop of
the oilier itiortlef to
[rjvunic; ihi^trlirc
nieiviHrane hihvcf

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Page 145

on the number of nitrogens in an azole ring present in the structure. The target site is an enzyme
involved in the modification (oxidation) of the precursor steroid (lanosterol) earlier in the biosynthetic
pathway for ergosterol. The accumulation of precursors of ergosterol cannot replace the functions that
ergosterol fungus and the membrane function suffers in terms of loss of membrane integrity and
impaired functioning of membrane bound enzymes.

■ SUMMARY

Efforts to control bacterial numbers consist of three broad strategies: sterilisation,
disinfection and the use of antibiotics. A spectrum of selectivity exists with the first of these
processes being completely non-selective in the targets (all organisms) through to
antibiotics being used to target single species. Sterilisation is a probability rather than a
certainty because of the exponential nature of death in microbial populations. The rate of
killing of bacteria can be described as exponential decay in which the numbers of organisms
fall proportional to how many are left. If plotted as logarithms, the numbers decrease
linearly with time. Sterilisation uses physical (e.g. heat) or chemical (e.g. formaldehyde)
agents to kill all living microbes from a material. Filtration may also sterilise a solution by
physical removal of micro-organisms.

Disinfection is the destruction or reduction in numbers of organisms that may cause disease.
As sterility is a theoretical concept (in that bacterial numbers will never reach zero), the
probabihty of sterility is chosen (sterility assurance) which is considered the best balance
between theory and practice. Antibiotics are microbial products that act preferentially on
bacterial targets. Their therapeutic value lies in the selective toxicity. The inactivation of a
suspension of virus particles is quite straightforward, and greater ingenuity is needed to
target viruses within human hosts. As with antifungal agents, the selective toxicity of
antiviral agents is of greater importance in order to avoid toxicity to the eukaryotic host.

RECOMMENDED READING

Challand, R. and Young, R.J. (1997) Antiviral Chemotherapy, Spektrum Academic Publishers, Oxford,

UK.

Franklin, T.J. and Snow, G.A. (1998) Biochemistry and Molecular Biology of Antimicrobial Drug

Action, 5th edition, Kluwer Academic Publishers, Dordrecht, The Netherlands.

Hugo, W.B. and Russell, A.D. (1998) Pharmaceutical Microbiology, 6th edition, Blackwell

Scientific Publications, Oxford, UK.

Russell, A.D., Hugo, W.B. and Ayliffe, G.A.J, (eds) (1998) Principles and Practice of Disinfection,

Preservation and Sterilization, 3rd edition, Blackwell Scientific Publications, Oxford, UK.

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REVIEW QUESTIONS

Question 5.1 What are the proposed explanations for the deviations in hnearity for a survivor
curve?

Question 5.2 What is the basis for selective toxicity for polyenes?

Question 5.3 What is the D value for antibacterial agents?

Question 5.4 What is the proposed explanation for why wet heat has greater antimicrobial activity
than dry heat at equivalent temperatures?

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6
MICROBIAL TAXONOMY

The importance of correctly identifying and naming five, ten and twenty pound notes has never been
questioned, yet the idea that microbes should be treated with the same attention to detail comes as a
surprise to many students. Microbial taxonomy may seem a touch irrelevant to a book on the
mechanisms of human infections with micro-organisms, but the elements of taxonomy are critical in
understanding the evolutionary development of microbes. In addition, taxonomy is needed to help
identify, name and classify organisms that infect humans. Just as any chef needs to know the names and
relationships of his foodstuffs in order to work, so microbiologists need to organise rules for
systematically organising microbes. With increasing recognition of the value of biodiversity within the
planet, microbial taxonomy will play an essential part in defining and documenting huge numbers and
types of bacteria that exist as well as prevent confusion in their identification. All students and perhaps
especially those of biological and biomedical sciences face an enormous new vocabulary. To memorise
the names is important but an understanding of the grammar, that is, the rules governing microbial
taxonomy, will be of greater benefit in the long run.

6.1 SYSTEMATICS OR TAXONOMY?

'Systematics' is the term used to define the study of the diversity of hfe and their relationships, whereas
'taxonomy' tends to be restricted to the theory and practice of classifying organisms. Classification
attempts to group organisms according to their similarity. In view of the importance of evolutionary
pressure on all hfe, classification of organisms should reflect their evolutionary history. The big
questions concerning the evolutionary history of ah hfe forms are being addressed through a form of
taxonomy called phylogenetic systematics. Phylogeny investigates the relationships between organisms
according to the evolutionary distance between them (a measure of the ancestral development, hence
evolutionary developments) and the results are presented as trees, as with family histories (see Box 6.1) .
Thus phylogenetics seeks to provide a classification that reflects the 'natural' relationship between
organisms and has explanatory power for the development of this order.

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■ BOX 6.1 MOLECULAR CLOCKS

Gene sequencing provides the molecular data for phylogenetic studies to compare the relationships
between prokaryotes and eukaryotes. One particular concept that relates molecular data to
evolutionary development is the idea of the molecular clock. The accumulation of mutations in the
genes of certain marker molecules (notably ribosomal RNA) is assumed to accumulate at a fixed
rate. If so, and this idea is not without its problems, then the number of mutations provides a
molecular chronometer, an indication of the time elapsed. By comparing the number of mutations
between different organisms it is then possible to estimate when the two organisms diverged (the
'evolutionary distance' between them). The greater the extent of mutation in the gene sequence
places the organism as a more recent derivative. Conversely, the greater similarity in rRNA sequence
between two organisms reflects a shared evolutionary history.

The rRNA molecule has been chosen because it is widely distributed across all organisms where it
performs the same function and is also large enough to contain sufficient information for
comparative analysis (see Box 6.2) .

Figure 6.1 shows how the results of phylogenetic studies of molecules such as rRNA indicate that all life
is encompassed within three domains:

Bacteria, Archaea and
Eukarya.

This contrasts with the traditional view of the five kingdoms of life:

Bacteria (or Monera), Plants, Animals, Fungi and
Protists

where only the first is prokaryotic, the rest being eukaryotes. Not surprisingly, such fundamental
changes in the origins of life have led to some debate between biologists and microbiologists because
the new three-domain system places bacteria as the ulti-

• Figure 6.1 The phylogenetic tree of all life, based on comparison of rRNA
sequences. The three Domains are shown in capitals. Note how animals, fungi
and plants are all recent divergences and that Bacteria and Archaea are distinct

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liUKARYA
Aniuials

ARCrJARA

iiACrt^RlA

FLEinl!>

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mate ancestor and all eukarya as late descendants. Most biologists will not take kindly to being told that
they are a sub-discipline of microbiology! More pragmatic problems have arisen. Viruses have not been
included since they do not code for rRNA. Nevertheless, to ignore such a large group of organisms is
seriously misleading. Incorporation of viruses and related agents has been suggested at the level of a
fourth Domain representing acellular infectious agents.

Phylogenetic classifications cut across conventional bacterial classifications. The rearrangements
necessary to bring both systems of classification into line will result in tears as breaking with the old
conventions will be difficult. So, far from dull regulations concerning name changes, taxonomy
(systematics, if you prefer) has emerged as a catalyst for change with many intellectual challenges.
Conventionally, taxonomy comprises three topics:

• classification,

• characterisation/identification and

• nomenclature.

Classification is the theory and practice of ranking units (bacteria, viruses, fungi) into groups that can
be ranked according to increasing inclusiveness (i.e. Species are grouped into a higher rank of Genus.
Genera are included in a Family, etc.). Classification seeks to provide an ordered summary of the state
of objects with predictive power. This means the properties of any one particular species or genus (etc.)
will apply to your newly identified organism. Everyone expects a kilogram to contain 1000 grams, and
in the same way one should be able to predict the properties of the new member of the group.
Characterisation/identification is the comparison of unknown units with previously recognised units/
groups. True taxonomists consider identification to be a separate, distinct process that is not strictly
taxonomy. In taxonomic terms, the process of obtaining information about organisms is called
characterisation. The difference may appear slight but the characterisation process is carried out with a
taxonomic objective, whereas identification is usually for a specific purpose in an applied setting such as
the need to identify organisms recovered from clinical samples obtained from patients. The tests carried
out in order to characterise an organism for taxonomic purposes will differ in scope and number quite
considerably from those used for identification purposes. The characters tested are either phenotypic or
genotypic. Phenotypic traits are observable physiological or biochemical properties that can be
measured without any reference to its genetic basis. Hence, shape, number of flagella, ability to ferment
sucrose, etc. are all phenotypic properties. Genotypic properties are molecular comparisons of nucleic
acid such as gene sequences, base ratios, etc.

Nomenclature is the labelling of units into appropriate taxonomic positions as defined by classification.
This somewhat unfriendly series of definitions only formalises the actions that are carried out routinely
in sorting out our books, clothes and weekly food stocks.

6.2 CLASSIFICATION

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Classification brings order to what otherwise would be a bewildering collection of objects. Now, some
may argue that the constant revision of the classification of microbes does little to establish order but
merely continues to destabilise; but it is the objective of classification to bring order and perspective. If
such restructuring were to

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Page 149

be avoided, one could just give a new number to each subsequent organism thereby simply 'cataloguing'
organisms as we describe them. With such a system one could arrange them alphabetically or
numerically.

As many different classification schemes have been described as there are characters. Cars can be
classified according to size, colour, make, hub cap diameter, etc., but micro-organisms suffer in lacking
many obvious morphological features. In addition, the embryological development of higher organisms
can be used to help in their classification but again bacteria lack such clues. Nevertheless, ordering of
microbes into basic groups can be carried out according to criteria such as biochemical properties or
genome analysis. To help set a framework for comparison, type strains are held in reference collections
of bacteria. These strains represent the typical organism that all species so named must closely
match. All strains deemed to be sufficiently similar to this can be assigned to the same species and have
the same name.

Classifications will always be artificial constructions because they are attempting to divide the spectrum
of organisms that is actually a continuum. Fortunately, bacteria tend to fall into groups that can be
distinguished from each other. The organisms themselves will evolve and change and this muddies the
distinctions between them. This variability is in complete contrast to the ideas of Linnaeus who
considered species to be distinct and unchanging, just like the stuffed animals in boxes in museums.
Classification orders organisms into species and then arranges species into increasingly larger taxonomic
ranks such as genus, family, order, etc. From this process it is easy to view organisms inherently existing
as distinct species. This idea is, however, mistaken. The concept of classifying organisms is a
theoretical, abstract idea. Whilst a particular organism exists as a real phenomenon, classifying it as a
particular species within a genus is an entirely conceptual event.

All biological classifications are comparative. The extent to which organisms differ determines the level
at which they are grouped into taxa, such as families, genera or species. The species is the basic unit of
division. What constitutes a species is another matter entirely and a source of endless discussion for
taxonomists. Traditionally, it was possible to define a species by two ways:

• a morphological species concept (based solely on morphological characters), or

• a biological species concept (organisms able to breed (sexually) only amongst themselves. Higher
organisms acquire their genetic material, in roughly equal proportion, from each parent. The
genetic reassortment that occurs in meiosis will create slightly different genotypes for each
individual. In addition, animals often are limited to geographic areas).

Both have obvious limitations when dealing with bacteria and viruses. Fungi are more amenable to such
concepts and great emphasis has been placed on morphological structures in fungal taxonomy, but the
complicated and varied sexual/asexual reproduction of different fungi make the biological species
concept unworkable. Likewise, difficulties arise with bacteria because of genetic exchange. Even with
parasexual events such as conjugation, genetic exchange can occur across genera as well as species.
Hence, a third concept has been proposed: the phylogenetic species concept. As discussed above, the
evolutionary history of micro-organisms has become one of the most important defining characteristics
upon which the term 'species' has been built. The phylogenetic species concept views organisms as

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lineages and orders organisms according to the relatedness in protein and gene sequence patterns over
evolutionary timescales. The

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number and type of differences in the sequences of certain marker proteins such as ribosomal RNA give
an indication of how related the strains are, in evolutionary terms. The tracking of the rate of evolution
in primary sequences means that the term 'phylogeny' is often interchanged with 'genealogy' because of
the interest in the ancestors. Given the significance in variation in the primary sequences of the tracked
proteins, it is necessary to make sure that the chosen proteins are common to all bacteria and perform the
same function. 16S and 23S rRNA molecules fulfil these criteria for bacteria, 18S rRNA are used for
fungi (see Box 6.2) .

Whilst phylogeny seeks to uncover the universal tree of life, as sought by Darwin, bacteriologists are
more often interested in less high-minded desires, driven instead in the search for order amongst the
spectrum of properties exhibited by bacteria. These differences highlight how the concept of the species
has dual function, depending on the user. For phylogeneticists, the species is the unit of evolution and
therefore a variable entity, whereas for taxonomists, the species is a unit of classification that, to be of
value to all biologists, needs to be as stable as possible.

For bacteriologists, a bacterial species is considered to be a group of strains that share many common
features but differ significantly from other strains. The increasing analysis of microbial genomes has
led to the concept of bacterial species as strains with high similarity in genomic relatedness (>70 per
cent DNA sequence) as well as phenotypic characteristics. This reflects the idea that genes are
exchanged in the natural world, hence species share a common gene pool. Ideally, the bacterial species
should be based on the results of as many characters as possible and include phenotypic and genomic
data. This inclusive approach is called polyphasic. A related concept is the polythetic species. Rather
than choosing one particular property of an organism to be the defining property of the species,
polythetic species have a set of characteristic properties that are shared amongst the species. The
traditional view is to define a particular property that must be present in all members of that species,
whereas polythetic classification permits inclusion of members that do not have one defining
characteristic but a pool of shared characteristics ( Figure 6.2) . In this way individual organisms are not
excluded on the basis of one result.

If the concept of species is woolly then the next taxa up is worse still. Species are collected into a genus
(plural, genera). A genus is the collection of species that form a group sufficiently similar in themselves
but sufficiently distinct from other (unrelated)

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■ BOX 6.2 CHOOSING A MOLECULAR CLOCK

Genomic analysis of microbes has one clear advantage over traditional methods: the study of
organisms that have not been grown in culture. Nucleic acid hybridisation will still occur in the
organism and will be considerably faster in obtaining an identification. The choices of probe
sequence has great importance and the target of choice is usually dominated by the ribosomal RNA
(rRNA) sequences, either the genes coding for the rRNA (rDNA genes) or the ribosomal RNA itself.
The bacterial ribosomes consist of the small 30S and large 508 subunits, which have 16S rRNA and
23S rRNA strands respectively. The rRNA is abundant in bacterial cytoplasm, are of appropriate
base length and stretches are conserved within species (i.e. minimal mutational events can occur
without serious detriment to the organism). Lateral transfer of these genes has not been a frequent
event in evolutionary terms, thus they make a good target for estimating bacterial diversity and
evolution.

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• Figure 6.2 Polythetic concept of taxonomy. Four bacteria are shown, each
with their particular set of characters named A to E. If the species has been
defined by the five properties A to E, then all four bacteria are members
because, despite the fact that they do not possess all five properties, no one
property defines the species. In this way individual strains that show variability
are not excluded

A R c r>

B C D E

AC DR

ABDE

strains. The other ranks of taxa (Family, Order, Class) are broader, more inclusive levels of similarity.
The order that different taxonomic ranks are arranged (in descending order towards species) is as
follows:

Domain (Bacteria, Archaea,

Eukarya)

Kingdom

Phylum/Division

Class

Order

Family

Genus

Species

Examples of three different micro-organisms are given in Table 6.1 .

Microbiologists will talk mostly of species and genera. The higher ranks are usually only discussed by
pure taxonomists who wish to make major rearrangements of the classification of organisms. With the
increasing understanding of evolutionary history of microbes, discussions of this sort might become a
more frequent event but at this stage we will stick mostly to genera and species. With fungi the taxon
'Class' is a useful tool, but somewhat fmstratingly, viral classification has not strictly followed the
binomial nomenclature, and genus and species names are not necessarily adhered to. Indeed, viruses are
not represented in the highest taxa (Domain, Kingdom, Phylum or Class) but are only ranked from Order
downwards.

Table 6.1 Examples from the three micro-organisms representing bacteria, viruses and fungi

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Escherichia coli

Poliomyelitis virus

Cryptococcus neoformans (sexual state:
Filobasidiella neoformans)

Domain: Bacteria
Kingdom: Bacteria
Phylum/Division:
Proteobacteria
Class: Zymobacteria
Order: Enterobacteriales
Family: Enterobacteriaceae
Genus: Escherichia
Species: coli

Order: Nidovirales
Family: Picomaviridae
Genus: Enterovirus
Species: poliomyelitis
virus

Domain: Eukarya
Kingdom: Fungi
Phylum/Division: Basidiomycota

Class: Basidiomycetes
Order: Tremellales
Family: (not applicable)
Genus: Filobasidiella
Species: neoformans

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Classification systems in microbiology can be grouped into three types:

(i) phenetic classification: the natural grouping of organisms according to the maximum observable

similarity of their characteristics (phenotype).
(ii) phylogenetic classification: the grouping of organisms according to the closeness of their

evolutionary history,
(iii) special purpose classifications that are created for particular areas of microbiology such as food,
medical or plant microbiologists.

Problems arise through conflicting interests of the different users which results in a lack of consensus of
a single unifying classification system. Whilst a 'true' classification scheme is an arbitrary concept, the
different systems are continually under revision through the continual fine tuning of the methods and
results, thereby perpetuating a sense of uncertainty.

6.3 CHARACTERISATION/IDENTIFICATION

The clumsy dual subtitle recognises that the identification of organisms from patients is not a true
taxonomic process. Taxonomists will characterise isolates that are previously unrecognised in order to
assign them to taxonomic groups (taxa) and not so as to help identify the cause of a patient's fever.
Likewise, an organism can only be identified if it has previously been studied and given a taxonomic
position. A semantic point perhaps, but worth consideration.

Two approaches can be adopted when identifying organisms, both illustrated in Figure 6.3 . Sequential
identification employs a series of different tests that determine the next course of action and are called
dichotomous steps. The term 'dichotomous' should serve to indicate that, according to the result of one
test, the identification can proceed one of two ways, only one of which is truly correct. A dichotomy that
some might feel is too much like Russian roulette. The practice of proceeding on the basis of one test at
a time has not been adopted in laboratories. The construction of suitable contrasting parameters is
difficult and the step by step procedure prolongs the time taken to reach an identification as any one or
more of the tests may require overnight incubation.

The simultaneous identification concept involves the use of a combination of several tests performed
simultaneously. The results of all the tests are then checked against reference tables. By using a set of
tests at once, the time involved is considerably reduced as is the emphasis given to each individual test
by weighing up the total pattern of reactions. Simultaneous identification systems is more in keeping
with a polythetic concept of a species than dichotomous key methods.

In practice, bacteriologists for example make a compromise between the two approaches in that a
number of simple tests (e.g. Gram stain, motihty, catalase and oxidase reaction) are carried out before
setting up a more complete set of tests. This way the identification is optimised in terms of speed and
cost.

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For a list of tests that are used in the identification of bacteria see Barrow and Feltham (1993). The wide
range of phenotypic tests measure a range of characters which include physiological (e.g. whether the
organism is motile or not) or biochemical (e.g. the presence or absence of particular enzymes)
characters. Table 6.2 lists the broad range of categories that can be used. Unfortunately, phylogenetic
studies have shown that many of the traditional properties of bacteria that are used to create the current
classifications (biochemical tests, gaseous requirements. Gram reaction, spore formation) are very poor
markers of 'true' phylogenetic history.

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• Figure 6.3 (A) shows the procedure by which an organism can be identified
using dichotomous keys. Depending on the result of the test, a particular series
of tests are carried out until sufficient information is acquired to identify the
organism. (B) shows a table where four organisms can be distinguished after
testing six tests simultaneously. The letter V means that a variable proportion
(between 20-80 per cent) of strains possess this property

(A) Soqucnlial identification

Ttst I

If positive, proceed lo Icsl 2
irncgalivc. pri^tucd to tchit !!■

Test 2

Teal 3

]f jiDfiilivc, proceed Ed tcsls 4 and 5

Test 4

(B) .S iniultancniis idicntiftcation

Test 1 Test 2 Test 3 Test 4 Test 5 Test 6

Organism t

Oj;g4jnisni 2
Organisms
Ora^ism 4

+
V

+
+

-H

For special purpose classification schemes, identification is usually the prime objective. The experienced
microbiologist will determine which characters are of greatest discriminatory and predictive value, i.e.,
will give unequal weighting to different tests. The tests used to identify the organism will be very

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heavily weighted so as to optimise speed with accuracy. It cannot be overemphasised, however, that an
accurate identification requires that the culture is pure.

6.4 NOMENCLATURE

The name of someone imparts a certain amount of information simply through the agreed convention of
carrying the father's surname down the male hne. The names themselves can reveal something of the
person's origins. Smith, Cheng, Gonzalez and Goldstein can suggest a certain country of origin. This is
unhkely to be correct all the time, but it is a useful starting point. Similarly, the names can tell
something of their function, for example, surnames derived from the person's occupation.
The naming of organisms is regulated by an international body which pubhshes the

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Table 6.2 Characteristics used to characterise bacteria

RECOMMENDED READING

Balows, A., Tmper, H.G., Dworkin, M., Harder, W. and Schleifer, K.H. (1991) The Prokaryotes. A
Handbook on the Biology of Bacteria, 2nd edition, Springer, Berlin.

Barrow, G.I. and Feltham, R.K.A. (1993) Cowan and Steel's Manual for the Identification of Medical

Bacteria, 3rd edition, Cambridge University Press, Cambridge, UK.

Cowan, S.T. (1971) Sense and nonsense in bacterial taxonomy. J. Gen. Microbiol. 67, 1-8.

Garrity, G. (2001) Bergey's Manual of Systematic Bacteriology (No.l), 2nd edition. Springer- Verlag

New York Inc., New York, USA. (See the updated introductory chapters.)

Holt, J.G., Kreig, N.R., Sneath, P.H.A., Staley, J.T. and Williams, S.T. (1994) Bergey's Manual of

Systematic Bacteriology, 1st edition, Williams and Wilkins, Baltimore, USA. (See the introductory

chapters on bacterial classification.)

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Page 160

Lengeler, J.W., Drews, G. and Schlegel, H.G. (1999) Biology of the Prokaryotes, Georg Thieme

Verlag, Stuttgart, Germany.

Mayr, E. (1998) Two empires or three? Proc. Natl. Acad. Sci. USA 95, 9720-3.

Rossello-Mora, R. and Amann, R. (2001) The species concept for prokaryotes. FEMS Microbiol.

Rev. 25, 39-67.

Woese, C.R. (1987) Bacterial evolution. Microbiol. Rev. 51, 221-71.

Woese, C.R. (1998) Default taxonomy: Ernst Mayr's view of the microbial world. Proc. Natl. Acad.

Sci. USA 95, 11043-6.

REVIEW QUESTIONS

Question 6.1 What are the problems when trying to define the term 'species' in microbiology?

Question 6.2 Why does taxonomy matter?

Question 6.3 Compare the different types of classification systems.

Question 6.4 What are the key features of a bacterial typing system?

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II
MICROBIAL INFECTIONS OF

HUMANS

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7
TYPES OF ASSOCIATION

Ignoring a low rate of mutation as a source of heterogeneity, bacterial division results in clonal
expansion, with the daughter cells considered to be similar if not identical to the parent cells.
Collectively, these cells are called a population. However, bacteria rarely exist as a single species
within any one habitat but, instead, are usually found as collections of different species called
communities, where each particular species will exist in a particular niche but may well contribute to
the maintenance of the entire community (e.g. syntrophism).

Interactions between microbial communities may have a negative (e.g. competition) or positive (co-
operation) outcome. The interactions will be between both a single population (i.e. between members of
the same species) or between members of different populations. Typically, co-operation will occur at
low population densities, whereas competition will dominate at high population density. The net effect
will regulate the population size to an optimum, depending on the selection pressures.
It is of interest that bacteria that infect humans proliferate in a clonal manner seemingly independent of
other organisms. The invading organism is thus growing as a population and not a community. Whilst
there are examples of synergistic infections, where more than one species of organism is needed to
initiate the infection, this appears to be the exception rather than the rule. As we shall see later, rather
than depending on the microbial community in the host, an invading pathogen is often inhibited from
proliferating by the presence of microbial populations residing normally in the host.
The interactions between microbes and humans will be the focus of this chapter. The terms employed to
describe the interactions between organisms are defined below and summarised in Table 7.1 . We will
view humans as the host in these definitions.

7.1 INTERACTIONS BETWEEN ORGANISMS

7.1.1 NEUTRALISM

Neither organism benefits, but neither is harmed. It is suspected that such a state is rare in nature.

■
7.1.2 COMMENSALISM

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Literally 'eating at the same table', cohabitation is the association of two different organisms where
neither is disadvantaged but, unlike symbiosis, the mutual dependence is

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Table 7.1 Interactions between microbes and man

Population 1 Population 2

Neutralism

Commensalism +

Mutualism (symbiosis) + +

Synergism + +

Competition - -

Parasitism - +

Population 1 represents the human as the host and Population 2 are microbes that interact with humans. Note that

there are three possible effects:

= no effect

+ = ebeneficial effect

- = edetrimental effect.

minimal. Whilst neither organism has any strict dependency on the other (e.g. no nutritional needs),
commensalism usually benefits one of the partners whilst having no impact on the other.

7.1.3 MUTUALISM (SYMBIOSIS)

Mutually-dependent association of two different organisms, conferring benefit on both. The interaction
is obligatory for both parties. The association may be temporary or permanent but usually implies the
latter as the interaction confers very specific benefits to each population that are not likely to be seen
with other combinations of organisms. See also Box 7.1 .

7.1.4 SYNERGISM

Both populations benefit from the interaction. Unlike mutualism, however, synergistic interactions are
not obligatory for both populations. Synergistic interactions permit activities that could not be carried
out by either organism alone. Syntrophism (see Chapter 2) is an example of a synergistic interaction.

7.1.5 COMPETITION

The antithesis of symbiotic interactions, competitive interactions result in both populations incurring
detrimental effects. Both populations will be competing for the same resource (typically a nutrient),
therefore by definition both cannot occupy the same niche (competitive exclusion). Competition
between two bacterial strains has been discussed in Chapter 2 and represents a good example of

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competition where the detrimental effect is the impaired growth of the organisms, either in rate or in
maximum yield. In chemostat studies the competition eventually results in one population dominating
such that the other does not survive.

7.1.6 PARASITISM

Parasitism represents the interaction between two different organisms, with the parasite exploiting the
host to its own advantage. The state of parasitism is not necessarily harmful to the host but usually the
interactions are specific and the parasite is smaller than the host. Both of these conditions are fulfilled
when humans represent the host. Some definitions include the requirement for metabolic dependency of
the parasite on the host.

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■ BOX 7.1 SYMBIOSIS

The term 'symbiosis' has also been used to simply describe the association of two different
organisms without any inference of benefit or harm to either organism. Symbiosis is just an
association that is subdivided into the three categories as follows:

As with all attempts at definitions, the above descriptions tend to divide the associations between
organism into very distinct groups. This in turn ends up highlighting the types of association that do not
fit neatly into any one category; for example, the microbial flora that colonise the human body (see later,
p. 178). It is therefore more appropriate to consider the above terms as points along a spectrum of
associations ranging from mutually beneficial (symbiotic) through to an entirely one-sided relationship
where the parasite derives all the benefits at the expense of the host (parasitism).
Bacteria are found in most sites on the planet from hot springs and thermal sea vents to the human
stomach at pH 1 and dust in the floor of our houses. What is important to consider is the sites at which
bacteria can replicate. Bacteria found on floors may simply be there because they have fallen from our
skin and are unable to repHcate there. The Helicobacter species that live in the stomach are able to
multiply under such extreme hydrogen ion concentrations. Likewise viruses can be found in inanimate
situations (kitchen surfaces, bed sheets, etc.) but they are unable to multiply in such conditions. Those
organisms that are able to repHcate outside of a host cell are termed free-living in contrast to those that
are obligate intracellular parasites such as all viruses and certain genera of bacteria such as Rickettsia

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and Chlamydia. Not surprisingly there are bacteria that are able to do both, i.e. replicate inside and
outside host cells and these are termed 'facultative intracellular parasites'. Salmonella typhi is an
example: it replicates within human epithelial cells and macrophages during the course of human
infection but is also able to multiply in the environment if nutrients are available.
It is easy to visualise how birds that eat the insects that attack the skin of the elephant are mutualistic.
There are also examples of bacteria that exist in mutualistic associations

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with higher hosts. The bacterium Buchnera sp. exists as an intracellular parasite in the aphid (Greenfly
and Blackfly). The bacteria have an obligate intracellular existence but in return synthesise essential
amino acids that are not present in the sap of the plants that the aphids colonise. Bacteria that exist in a
strict intracellular mutualistic existence with their host are called endosymbionts.
In being useful to the host cell endosymbionts can be viewed as intermediates between free-living
organisms and the intracellular organelles that are thought to derive from bacteria: mitochondria in
animals and plastids in plants. Free-living organisms need to utilise sufficient nutrients from an
environment that may not provide regular nutrient supplies. Choosing to exploit the intracellular
environment of a host guarantees nutrients and, what's more, there are not hundreds of other organisms
to compete for their share of the spoils. The host has evolved numerous defence mechanisms in order to
prevent this bacterial pillaging. Thus, it is not surprising that intracellular parasites (either obligate or
facultative), in their attempts to avoid or resist the host defences, have become pathogenic.
In adapting to an intracellular existence, the microbe has two options: simply become increasingly
dependent on the host for certain nutrients or provide a function that benefits the host. In other words,
become mutualistic or simply exploit the host as a parasite.

What are the consequences of becoming an intracellular parasite or an endosymbiont and how do they
differ from the intracellular organelles (likely to have once been bacteria)?

7.1.7 INTRACELLULAR PARASITES

The genera Chlamydia and Rickettsia both contain species that cause human disease. Chlamydia
trachomatis causes trachoma and Rickettsia prowazekii causes typhus fever. Studies of the genomes of
these intracellular bacteria have yielded interesting patterns. By utilising host nutrients, these organisms
have lost the corresponding biosynthetic pathways; the amino acid synthesis pathways are absent with
the organisms choosing to utilise those of the host cell. Similarly the enzymes for synthesis of purines
and pyrimi dines are largely absent. Energy requirements of these bacteria are still paramount but they
have lost the need for anaerobic glycolytic pathways as they now live in an aerobic environment. The
reduction in requirement for such enzymes results in the reduction in size of the genome of the bacteria.
The striking feature of these bacteria is the small genome size, around 1 megabase (1 Mb), roughly
equivalent to 800-900 genes, compared with free-living bacteria such as Esch. coli with a genome of 4.6
Mb in size. There are, however, pressures. The genomes of these intracellular parasites exhibit mutation
rates higher than free-living bacteria. One of the reasons is the loss of DNA-mismatch repair enzymes.
Another reason is the reduced opportunities for recombination or horizontal gene transfer with other
bacteria when choosing an obligate intracellular replication strategy. A similar problem is seen with
viruses. The intracellular existence will be dominated by the considerably larger host genome so it is
economical to lose any duplicated bacterial genes. Finally, the asexual, clonal replication with this
reduced genome repair kit will result in the accumulation of neutral and deleterious mutations, which
cannot be removed by selective pressure. Such a scenario might suggest that the organism has not got
much hope of survival. However, the organism can devote a dominant proportion of its genome to code

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for features that favour persistence in the face of host defences.

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7.1.8 ENDOSYMBIONTS

As with obligate intracellular parasites, the asexual, clonal strategy results in a reduced genome with a
high proportion of mutated code. The residual genome codes for essential cell functions such as growth
and division. In contrast to obligate intracellular parasites, the host benefits from housing endosymbionts
such as Buchnera spp. because they synthesise amino acids that are available to the host as well. This
contrasts with Chlamydia and Rickettsia, both of which have lost the amino acid biosynthetic capability
and therefore compete for amino acids with the host. The gene loss has consequences for other cell
functions. An endosymbiont has no need for genes that code for antigenic variation and other defensive
host protection mechanisms. Likewise the organism has no need for mechanisms of entering and
leaving the host cell as it has become a stable partner (see also Box 7.2) .

The conflict of interest between the organism and the host will result in a reciprocal exploitation that
may result in mutualism or parasitism. One of the advantages of becoming an endosymbiont is efficient
transmission from host to offspring (vertical transmission) and this benefits the host as well as the
symbiont, whereas the genetic isolation and increased mutation rate might be seen as a disadvantage.
The accumulation of deleterious mutations in a genetically isolated population (with almost no
opportunities for recombination) is known as MuUer's ratchet. Endosymbionts will suffer from this
gradual loss of fitness through Muller's ratchet. Conversely, the obligate intracellular parasite is usually
spread between unrelated hosts (horizontal transmission) and increases the chances of recombination,
and thus conflict between host and bacterium.

7.2 THE TERMS

From here on we will concentrate on microbial associations with humans, especially those that are
detrimental to health. It is necessary to provide some further definitions of the common terms.

7.2.1 PARASITE

The parasite can be quahfied further as an ectoparasite, where it exists on the outer surfaces of the host,
in contrast to an endoparasite, one that is found inside the host. Multicellular parasites such as flukes
and worms are usually referred to as macroparasites and can be considered separately from the
microparasites we have introduced above (viruses, bacteria). Macroparasites have adapted some of
their structural features such that they have either disappeared or reduced in size, e.g. organs involved in
movement, organs for storing food, compHcated nervous systems, lengthy intestinal tract (in fact,
tapeworms have no intestinal tract). With bacteria there are no such structural differences between strict

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parasitic species and harmless species.

■ BOX 7.2 ORIGINS OF EUKARYOTIC INTRACELLULAR ORGANELLES

Mitochondria appear to he hacteria that have undergone such drastic genome reduction that they are
now obligate intracellular partners in the host; in fact, genes are donated to the host genome.
Whereas endosymbionts and intracellular parasites infect only certain cells, mitochondria are present
in virtually all cells of the body. Mitochondria still provide benefits for the host cell: they are net
exporters of energy but cannot replicate independently of the host cell.

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7.2.2 PATHOGEN

This is a micro-organism that can cause disease. All organisms can cause disease under appropriate
circumstances but for some micro-organisms this is much more Ukely. These organisms are called
'primary pathogens', whereas those that need special circumstances are called 'secondary pathogens'. It
might seem unnecessary to distinguish between parasite and pathogen but they are not synonymous. An
organism may be one but not the other. Whilst it is not difficult to imagine that a parasite may cause no
overt (cUnically apparent) disease, i.e., is not pathogenic, it is possible that organisms are pathogenic but
not parasitic. Consider food poisoning caused by Staphylococcus aureus and Clostridium botulinum.
The toxins are released by the organisms when they multiply in the food. Thus humans ingest pre-
formed toxin and develop symptoms of vomiting and botuHsm respectively. In this instance both
organisms are pathogenic but not parasitic to man.

7.2.3 INFECTION

Someone is infected if the microbe has successfully colonised or at least estabhshed itself, albeit
temporarily, on a host. If they develop symptoms of disease they have a cUnical infection. Alternatively,
if the infection does not result in symptoms the person is infected but asymptomatic.

7.2.4 COLONISATION

This describes the presence of micro-organisms, pathogenic or non-pathogenic, in large numbers, on a
host without tissue damage or clinical disease.

7.2.5 DISEASE

This is a condition of disordered well-being. In terms of infection, this means the pathogen is able to
cause pathological damage, that is, tissue damage.

7.2.6 INFECTIOUS DISEASE

Also referred to as a communicable disease, an infectious disease is an infection that can be transmitted
between humans (or organisms/animals, etc.). The organism is able to spread from one host to another
and is therefore infectious. The term contagious means the same but is less frequently used. Not all

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infections are infectious, i.e. are not naturally transmissible from one person to another (e.g. brain
abscess, osteomyelitis, infective endocarditis).

7.3 A LITTLE MORE ON PARASITISM

For the purposes of a text on the microbiology of humans, the above terms can be viewed/amended in
terms of humans and micro-organisms. Rather than a series of distinct definitions, the above terms
represent a spectrum of association. Where humans represent the host environment the relationship
between the host and the normal microbial flora is mostly mutuahstic, but under certain circumstances
when members of the normal flora are able to cause disease, the relationship will be parasitic, and often
harmful. With viruses, it is clear that they are obligate intracellular parasites. They utilise the host
synthetic machinery to reproduce. The association between virus and host cell is absolute and the virus
will only replicate at the expense of the host.

The extent of the association between parasite and host may be determined by the biochemical
dependence of the parasite on the host. For bacteria found on humans, they are able to multiply outside
of the host when suitable nutrients are provided.

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With bacteria parasitic to iiumans, the organisms have evolved such that they obtain essential nutrients
directly from the host rather than from the environment or de novo synthesis. There are indications that
organisms with a restricted metabolic capability (for example, within the genus Salmonella) are those
that cause the more severe infections. This may also account for species specificity as exemplified by
Salmonella dublin which infects cattle whereas Salmonella choleraesuis infects pigs.

7.4 HUMAN INFECTIONS

The history of mankind is regularly punctuated with adverse deahngs with microbes, both good and bad:
the great famine in Ireland; the consequences of smallpox on the native peoples of North and South
America; the influenza pandemics and the march of AIDS across the world. To help explain the variety
of different human infections, an understanding of their epidemiology is essential. This is dealt with in
greater detail in Chapter 1 1 but an introduction is necessary. Whilst this catalogue of disasters is
sobering, it is worth noting that most infections are asymptomatic. The patient seen in the doctor's
surgery with a clinical infection represents a minority of the total number who will have been exposed to
that organism.

Most people learn to avoid dangerous situations. Most try to avoid sitting next to someone sneezing and
blowing their nose. This behaviour is based on the recognition that germs are present. All infections
arise as organisms are transferred from a reservoir to a source and then transmitted to a susceptible
host. The transmission of microbes between humans is critically important in the successful spread of
the organism within a population. The often-called chain of infection is as follows:

Reservoir: Source: Mode of transmission: Host
The reservoir is the primary habitat of the organism. The differing habitats illustrate the variety of
reservoirs, e.g. in humans Salmonella typhi, measles virus are the only known reservoirs of the organism
(hence they are strict human pathogens). Animal reservoirs exist for all zoonoses, e.g. rabies virus.
Salmonella enteritidis. Environmental reservoirs include soil, maintaining Clostridium botulinum, and
water from which Legionella spp. are acquired.

The source of infection is the actual site from where the infection is acquired. The source is not
necessarily the same as the reservoir. Salmonellae are commonly found in the intestinal tracts of
chickens. When you develop food poisoning, the meal in the restaurant is the source. When the infection
is transmitted from an inanimate object it is often called the 'vehicle of infection' but the chicken flock
is the reservoir. The contaminated chickens can be distributed to more than one restaurant. When an
infection is acquired via an inanimate (non-human) source such as water or food it is often termed the
'vehicle'. With infections that develop from the patient's own flora, e.g. urinary tract infections with
Escherichia coli, an organism that normally colonises the intestine, the host is both the reservoir and the
source!

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Certain infectious diseases of humans are restricted to humans only. No other animal or environmental
reservoir appears to exist. Examples of organisms that are strict human parasites are listed in Table 7.2 .
It is clear that a number of human infections have no other reservoir or host than humans themselves. It
also is worth noting that fungi that are strict human pathogens are not well represented. Most fungi that
cause infections in humans have a free-living equivalent in the environment, such as soil or plants.
Candida albicans is often found as

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Table 7.2 Strict human pathogens

Organism

Disease

Viruses

Measles
Rubella virus
Polio virus
Mumps virus
Bacteria
Salmonella typhi
Shigella spp.
Treponema pallidum
Neisseria gonorrhoea
Neisseria meningitidis
Bordetella pertussis
Fungi

Pityrosporum furfur
Trichophyton spp.
Epidermophyton spp.

virus measles
German measles
poliomyelitis
mumps

typhoid

dysentery

syphilis

gonorrhoea

meningitis, septicaemia

whooping cough

tinea versicolor

tinea

part of the normal flora in humans. The fungi that cause infections called tinea (or ringworm) are
thought to be those that have lost the ability to reproduce in the environment but have become strict
parasites of humans. In the absence of finding the equivalent sexual form (the teleomorph, able to exist
in the environment) they will be included in the Table.

The implications of organisms being strict human pathogens are intriguing. We will discuss the periodic
epidemics of infectious diseases like measles (see Chapter 11) . The question remains: where does the
organism reside during the years when the illness is not epidemic? Either there are enough infections
(presumably most of which are asymptomatic) in the community to maintain persistence or there could
be other reservoirs, as yet unidentified. An interesting development has arisen on this idea. Vibrio
cholerae is the causative agent of cholera. V. cholerae has been identified in river waters in Kent in
England. It is also known that organism produces the toxin responsible for the diarrhoeal disease when
the organism itself becomes infected with a bacteriophage. The strains isolated from human disease
therefore may not have a strict human reservoir but switch between reservoirs, the difference being
whether the bacterium is infected with the appropriate phage. If so, then, the Vibrio is able to invade and
infect humans.

7.5 VARIETIES IN TYPES OF INFECTION

Acute infections will be those that arise quickly (e.g. tonsilhtis) and progress rapidly, in contrast to
chronic infections which have a longer course (e.g. tuberculosis), lasting for weeks up to years without

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resolution. Latent infections are those in which the microbe is able to persist for years within a site in
the host and cause minimal clinical disease for most of the time the organism is present. Examples of
latent infections are Herpes simplex virus infections that result in periodic episodes of cold sores, but, in
the absence of suitable triggers, remain silent most of the time. The virus resides within

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the nerve cells that supply the lips. The vims cannot be recovered from these infected cells but is able to
maintain its genome within the cell. Varicella zoster virus, the aetiological agent of chicken pox, also a
herpes virus, is likewise able to reside in nerve cells for years until it is reactivated to cause shingles
(zoster).

Other terms are useful in the description of types of infections. The extent to which an infection spreads
throughout the body can be described by the terms localised or generalised infections. It is easy to see
that generalised infections are often more severe. The involvement of numerous organs throughout the
body will lead to more complications than simply infecting a site on the skin. Another term for systemic
infection is 'multiorgan infection'. Generalised infections will arise from infections that enter the
bloodstream and will be distributed around the body in the bloodstream by the host. Spread through the
body is not, however, limited to passage in the bloodstream. Streptococcus pyogenes can cause a
cellulitis where the organism spreads through the skin. In this particular instance the movement of the
organism through the tissue can be surprisingly quick. Spreading cellulitis can be fatal within hours if
left unchecked.

It does not follow that a localised infection is going to pose no significant threat because it is contained
to one focal point. Several infections due to toxin-producing bacteria result in serious life-threatening
infections. Diphtheria is a localised infection of the throat but the release of the diphtheria toxin into the
surrounding tissues spreads throughout the body. The toxin is thought to be responsible for the cardiac
changes, the paralysis and general toxicity.

As mentioned above, a distinction can be made between primary and secondary pathogens. A primary
pathogen is a micro-organism that can establish itself in a new healthy host and cause disease unaided. A
secondary pathogen is one that requires a breach in the normal host defences before it can proliferate in
the host. Secondary pathogens are commonly referred to as 'opportunists' but this term can equally
apply to both primary and secondary pathogens. In humans, secondary pathogens usually infect people
with illness or other predisposing conditions that allow the organisms to gain a foothold and colonise the
site. Salmonella typhi, the aetiological agent of typhoid is a primary human pathogen; you simply need
to ingest sufficient numbers. Whereas, with the secondary pathogen Pseudomonas aeruginosa, there will
need to be a breach in the host defences for the organism to establish an infection, e.g. burn injury to the
skin, unchanged contact lens use or diabetic ulcer on the foot, before the organism can establish an
infection.

Some organisms are able to cause numerous types of infections, others are not ( Table 7.3) .
Staphylococcus aureus is able to cause a disarmingly diverse list of infections, yet

Table 7.3 The variation in number of infections caused by microbes

T. pallidum

Mumps virus

S. typhi

St. aureus

Ps. aeruginosa

boils

keratitis

abscess

otitis

piieuiiiuiiid
septicaemia

enteritis

syphilis

mumps
meningitis

typhoid

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endocarditis

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others are limited to a particular infection. In general, those organisms that can establish themselves and
cause illness (primary pathogens) tend to cause one particular disease. The pathogenesis of typhoid
reflects the particular sequence of events that occurs as Salmonella typhi progresses through the body in
a set pattern. Organisms like Staphylococcus aureus and Pseudomonas aeruginosa are found in a wider
range of infections because they are able to exploit breaches in the host defence (hence the term
'opportunist'). Instead of following a particular route through the body like Salmonella typhi,
Staphylococcus aureus will multiply at most body sites once given the opportunity: a breach in the skin
(cuts, abrasions, surgical wounds) or invading the lungs to cause pneumonia following an infection with
the influenza virus. The greater diversity in infections reflects the increasing number of opportunities in
the patient as a result of modern medicine.

7.5.1 SPECIFIC OR NON-SPECIFIC INFECTIONS

Some diseases result in typical features that are termed pathognomic: the features are characteristic of a
particular infection or disease. The lockjaw characteristic of tetanus or the rash of measles are
pathognomic signs. Tetanus and measles are two infections that are caused by a particular organism. No
other organism is responsible for measles except the measles virus. These are specific infections, with
characteristic signs, symptoms and cellular pathology. Robert Koch demonstrated that the bacterium
Mycobacterium tuberculosis was responsible for a specific human disease (tuberculosis) and, with the
work of Louis Pasteur, the microbiological revolution had begun. Numerous diseases were found to
have bacterial causes. From the list of diseases caused by single organisms (tetanus, botulism,
tuberculosis, anthrax, rabies ...) it is understandable that microbiologists believed the 'one germ, one
disease' principle must be the only model. It was felt that infections are caused by a single species, and
if more than one species was cultured from the infected tissues then it was contaminated. This idea
stems from Koch's postulates in which the organism must be found in the diseased tissues in pure
culture (see Chapter 8 ). It is now recognised that infections may be polymicrobial in origin (dental
caries, anaerobic abscesses), although there are not that many.

Signs and symptoms Medical doctors distinguish between signs and symptoms. Signs are
features observable by the medic in the patient whereas symptoms are what the patient feels
and complains of. A sore throat is a symptom and the reddening throat and enlarged lymph
glands seen on examination are signs.

Most bacteria that cause cUnical disease in humans are able to cause the host to respond with rapid
accumulations of white cells and other inflammatory cells and compounds. This accumulation of
immune cells, bacteria and necrotic tissue is what everyone recognises as pus. Bacteria able to induce
pus formation are called pyogenic, a term not to be confused with pyrogenic, the ability of certain
organisms to induce a fever.

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Micro-organisms can infect any tissue in the body. Bacteria can infect most. Viruses will only infect
living tissues and fungi are particularly good at extracting nutrients out of dead tissue (surface skin and
nails and hair). It is of value to memorise a few examples of microbial infections of the different sites
because they highlight the different paths taken by microbes in the course of an infection. The diagnostic
microbiologist needs to be alert to the likely causes of infections in particular tissues and organs.
Table 7.4 lists a few examples of human infections and the corresponding route of transmission of the
aetiological agents. Note how the site of infection leads directly to that organ and the organ then
subsequently develops pathology. The exception is with meningitis. The central nervous system is not
directly infected by either Herpes simplex virus or Neisseria meningitidis; rather, they are acquired from
respiratory secretions (saliva, kissing, etc.) and infection of the meninges is an unusual consequence
following bloodstream invasion or direct entry through the nerves. This is a dead-end in terms of
transmitting the organisms to new hosts. In other infections, organisms might infect more than one organ
at once. Alternatively many viral infections will show lesions on

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Table 7.4 Bacterial and viral infections of the body systems and the route of transmission

Body system

Disease

Aetiological agent

Route of transmission

Central nervous system

Gastrointestinal

Genito-urinary

Respiratory

Skin

meningitis Neisseria meningitidis

Herpes simplex
diarrhoea Salm. enteritidis

hepatitis A virus
gonorrhoea Neisseria gonorrhoea

Herpes simplex
tuberculosis, Mycobacterium tuberculosis
pneumonia influenza virus

cellulitis, Streptococcus pyogenes

warts papillomavirus

airborne

food-borne

sexually transmitted

airborne

direct contact

the skin but these are consequent to blood-borne spread through the body and multiplication in several
body organs.

7.6 MODE OF TRANSMISSION

The ability to reproduce will be the ultimate driving force for all organisms. For bacteria that infect
humans there are several routes that can be employed to infect new hosts. The various means by which a
micro-organism may be transmitted from the source to the new host are given in Table 7.5 . Note that
some organisms can be spread

Table 7.5 Routes of transmission

Type

Mechanism

Example

Direct contact

Respiratory/airway transmission
(airborne)

Faeco-oral transmission
(food-, water-borne)

Parenteral/B lood-bome
(percutaneous)

direct contact

inhalation

ingestion

inoculation

Streptococcus pyogenes

Dermatophytes

Papillomavirus

Measles virus

Mumps virus

Influenza viruses

Mycobacterium tuberculosis

Salmonella typhi

Shigella dysenteriae

Vibrio cholerae

Hepatitis A virus

Poliomyelitis virus

HIV

Hepatitis B virus

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Venereal/sexually transmitted direct contact Neisseria gonorrhoea

Treponema pallidum

Vector-borne inoculation Yellow fever virus

Plasmodium spp.
direct contact Yersinia pestis

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by more than one mode of transmission. Measles virus is likely to be spread by airborne and direct
contact. The number of times people touch their faces with their hands is enough to almost guarantee
that virus in the nose or saliva will be transferred to the hands. You will notice other flaws in the
scheme. The table distinguishes between sexually transmitted infections and those obtained by direct
contact. It could be argued that they are the same mode of transmission. The example of dermatophytes
as directly transferred infections also needs qualifying. In the latter cases the fungi are often transmitted
through shared, inanimate objects like towels or combs. This is not direct transmission in the strictest
sense. Perhaps 'indirect transmission' is a more appropriate term.

Two important terms are vertical transmission and horizontal transmission. Rather than referring to
positions of the host when they came into contact with the infectious agent (!) they are used to
distinguish between infections transferred from mother to child (vertical) and those acquired from other
sources (which in this context means predominantly people) and are called horizontal. Two of the most
important examples of vertical transmission are rubella virus and Treponema pallidum (syphilis). Such
infections of the foetus can have serious developmental consequences. Congenital rubella, for example,
may lead to microcephaly (small head), impaired vision and impaired intellectual development.
The significance of vertical or horizontal transmission for the microbe lies in the number of people that
can be infected. With vertical transmission only one new host is infected and that is the child (or
children). With horizontal transmission there is the opportunity to infect significantly greater numbers of
people. It follows then that if one person is able to infect five others, these five will then infect 25 and so
on.

The exact point at which infections of the newborn baby stop being vertical transmission and become
horizontal is debatable. Certainly vertical transmission can be subdivided into:

• germline transmission (spread of virus integrated within the genome),

• prenatal transmission (infection of the foetus in the uterus),

• perinatal transmission (infection during birth),

• postnatal transmission (infection following birth).

Strictly, only infections of the foetus in utero are vertically transmitted. Perinatal infections (those
acquired by the baby in the vaginal tract during the process of delivery) and postnatal infections (e.g.
acquired via breast milk) are horizontally transmitted.

Vector-borne infections are those that are transferred by a vehicle/carrier, which are insects. The term
vector is (most usefully) restricted to living animals such as lice, fleas or mosquitoes. Combs and
hairbrushes are thus not usually called vectors, but fomites. The transfer of dermatophytes via
hairbrushes is best described as 'direct transfer'. Vectors serve to transmit organisms from the
bloodstream of infected patients to new susceptibles. The organisms (notably arboviruses: arthropod-
bovnt viruses and protozoan parasites such as malaria) are not designed to withstand any time outside of
a host. The transfer of the organism is dependent on the distances the vector can travel in its lifetime. A
wide range of insects transmit infectious microbes, as shown in Table 7.6 .

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Table 7.6 Insects that transmit infectious micro-organisms

Insect

Disease

Microbe

Trypanosoma sp. (protozoa)
Yersinia pestis (bacterium)
Rickettsia sp. (bacterium)
Borrelia sp. (bacterium)
sand fly virus
yellow fever virus

Bugs

Chagas' diseas

Fleas

Plague

Lice

Typhus fever

Ticks

Lyme disease

Flies

Sand fly fever

Mosquito

Yellow fever

7.7 ZOONOSES

Zoonoses are infections that can be transmitted between vertebrate animals and humans (singular:
zoonosis). The natural host is the animal. Often a human infection represents a dead-end in terms of
perpetuating the spread of the organism because we are unlikely to pass the organism to another animal.
Having mentioned malaria as an arthropod-borne infection, one should note that malaria in man is not a
zoonosis. There are numerous (hundreds) of animals that are infected with different Plasmodium spp.
but the Plasmodia that commonly cause disease in humans appear to have evolved so that humans are
the 'natural' hosts, thereby disqualifying themselves as zoonoses.

SOURCES OF ZOONOSES

Zoonoses have arisen mostly through the need of humans to collect food. Humans have
been able to domesticate animals both as livestock (cows, pigs, etc.) and as helpers
(sheepdogs) as well as deforesting areas for crops. These activities bring man into close
contact with the micro-organisms that infect the animals. Tuberculosis, anthrax and
brucellosis are caught directly from farmed animals. Others arise from those animals we
encounter whilst disturbing them in their natural habitats (yellow fever, Lyme disease,
Ebola vims). Not that zoonoses are only caught by venturing into dark jungles! Pets are a
common source of various microbes that can cause human disease, especially fungal
dermatophyte infections. More important in terms of national public health is food
poisoning. Increasing in incidence in the Western world. Salmonella enteritidis and
Campylobacter jejuni are the leading causes of food poisoning in developed countries.
Mostly as a result of battery farming, both organisms are frequently isolated from uncooked
chickens but inadequate precautions in food hygiene fails to reduce their numbers to below
the infectious dose necessary to induce food poisoning. Despite being a preventable
infection, food poisoning is a national public health concern and burden in lost manpower
to industry.

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The transmission from the animal to humans (and it is only this direction that concerns us here!) can be
separated further, depending on the natural lifecycle of the micro-organism in question.

1. Direct transmission (direct zoonosis): transmission occurs via direct contact from the original
animal (e.g. rabies transferred through the saliva of the dog). The microbe does not undergo any
developmental change between the two hosts (rabies, brucellosis).

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2. The micro-organism has an intermediate (or secondary) host in its complete life-cycle and humans
can only become infected with the infective stage of the organism at the appropriate stage of the
micro-organism's lifecycle (plague, schistosomiasis).

3. The organism infects humans having spent a period in a non-living site. The organism may develop
into the infective form (e.g. hookworm larvae hatch from the eggs present in the soil contaminated
with faeces. When someone sits on the soil, the larvae penetrate the skin). Other infections are
caught from the non-living site without any developmental change in the organism (e.g.
cryptococcal infections, other invasive mycoses, leptospirosis).

Staphylococcus aureus food poisoning is due to the consumption of preformed toxin in the
food. The enterotoxin triggers vomiting which occurs quickly (5-8 hours after ingestion of the
toxin). Salmonella food poisoning, however, is due to ingestion of large numbers of
organisms, at least lO^/gm. The diarrhoea results from the multiplication in the small intestine
and this takes 18 hours before the patient develops symptoms. This highhghts the inaccuracy
of the term 'food poisoning' because the poisoning implies chemical- or drug-induced
toxicity.

By considering the natural reservoir of the organism, one is able to plan what precautions can be taken to
prevent a recurrent infection or an outbreak. Knowing the route of transmission offers the possibility of
breaking the flow of organisms. A consideration of food poisoning organisms illustrates this idea.

7.7.1 FOOD-BORNE INFECTIONS (FAECAL-ORAL TRANSMISSION)

Food-borne disease due to microbes can be split into two pathogenic mechanisms:

• intestinal infections,

• intoxications.

Intestinal infections cause diarrhoea following the multiplication of bacteria within the intestine,

whereas intoxications cause diarrhoea and/or vomiting after the ingestion of a toxin present in the

contaminated food. Intoxications occur because the organism has multiplied in the food and released

enterotoxins that are ingested (if they are not inactivated by cooking).

The mechanisms of staphylococcal and salmonella food poisoning (see margin note) illustrate how

preventing food poisoning can be achieved by preventing multiplication either in the food or in the

intestine. Complete elimination of the organism from the food is not necessary; instead, preventing the

organism from multiplying will prevent the required number for the infectious dose or producing toxin.

Note that viruses that cause food-borne disease will not be able to multiply in food or water but simply

utilise them as the vehicle for transmission.

One mechanism that attempts to maximise the opportunities for transmission is the carrier state. A

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carrier is a person who is passing an organism into the environment without showing any symptoms or
recognised pathology. Salmonella typhi is an organism that can infect people and maintain itself in the
gallbladder (bile duct) so that it is released in low numbers into the intestine. The person may or may not
have a symptomatic illness prior to becoming a carrier. 'Typhoid Mary' serves to illustrate the condition.
Mary Mallon lived and worked in the catering business, against the strict instructions from the New
York State that forbade her to handle food. One can imagine the confusion in her mind that an
apparently healthy woman was causing deaths through outbreaks of typhoid. Finally, imprisonment was
the only recourse left to the authorities to prevent further cases of typhoid. In general, the reasons for
why some people become carriers and others do not remains a mystery. With typhoid carriers, it is of
interest to note that gallstones are usually present and it is suggested that they predispose to carriage.
The transmission of microbes from host to host is the key parameter in the persistence of an obligate
human parasite within a community. What features help the efficiency of transmission?

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7.7.2 THE NUMBER OF INFECTIOUS PARTICLES SHED

Evolutionary success means that the organism continues to propagate. As with the number of offspring
produced by all animals, the numbers produced depend on the likelihood that they will survive for
sufficient time to produce new offspring. With microbes, the more organisms that are shed, the greater
the chances of infecting a new host. Sexually transmitted infections are organisms that do not tolerate
drying and the route of transmission is quite demonstrably one in which the organisms are transmitted
without any time spent outside of the body. Correspondingly, there will be less need to release as many
organisms as would those organisms that risk desiccation during transmission. The stability of the
infective particle is thus another parameter of relevance.

7.7.3 STABILITY OF THE MICRO-ORGANISM IN BETWEEN HOSTS

Airborne transmission runs both the risk of the organisms drying out and failing to not encounter a new
host whilst the particles are suspended in the air. Gram positive organisms resist desiccation more
readily than Gram negative bacteria, but the most stable infective form is the endospore. Tetanus,
anthrax and botulism spores are all known to be stable in the environment (typically, soil) for years, if
not decades.

7.7.4 THE INFECTIVE DOSE

Obviously, it suits the micro-organism to have a low infectious dose so as to efficiently infect new hosts.
This number may be reduced if there are breaches in the host defences or other compromising events
that lower the resistance of the host. A well-recognised example is the reduction in infectious dose for
salmonellosis in patients with achlorhydria (absent or reduced gastric acid production).

7.7.5 TRANSMISSION AND VIRULENCE

From these three factors, the reproductive fitness of the organism (the efficiency with which the
organism is maintained in the population) is directly linked with its transmission between hosts. In
evolutionary terms, natural selection will favour the organism that is able to produce sufficient infectious
offspring. To do this the organism needs to multiply and thus may inflict damage on the host (termed
virulence). The relationship between transmission and virulence is unresolved but it can be argued that
the route of transmission will influence the numbers shed. Vertical transmission will require fewer
infectious particles than horizontally transmitted infections because the vertically transmitted microbe

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will depend on the host and the host' s reproductive success to maintain the organism. In horizontally
transmitted infections the numbers of potential new hosts will be much greater and the invading microbe
does not need to concern itself with the host' s ability to reproduce, therefore the organisms can be more
virulent so as to produce more infective particles.

Since all parasites will exploit their hosts so as to best maintain their own reproductive fitness, the
transmissibility of an organism may be coupled to its virulence. When the organism is able to multiply
within its host it will produce more infectious offspring and increase the chances of infecting a new host.
It is possible that this will damage the host. People with cholera will excrete millions of Vibrio cholerae
bacilli in the fluid stools. If the microbe is transmitted by a vector (e.g. yellow fever virus and mosquito)
then the immobilisation of the host is probably beneficial. It is easier to get bitten lying ill in bed than
being healthy and mobile! If organisms can replicate sufficiently and maintain opportunities for
transmission, then the microbe and host may maintain a stable relationship but, if the host seeks to
eliminate the microbe or more than one

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microbe is competing for the same niche (more correctly, both microbes have the same resource
requirements), then an increase in virulence is required to overcome the competition. Thus evolutionary
pressures will force the microbe to evolve to a level of virulence that trades transmission opportunities
with keeping the host alive (see Section 7.10) .

7.8 THE NORMAL FLORA

One instructive area for assessing the interactions between microbes and humans is the populations of
microbes that inhabit the various econiches ( Box 7.3) on our body. This collection of cohabitants is
collectively termed the normal flora. The word 'flora', borrowed from the botanists, describes the
collection of plants in an area. In microbiological terms, it helps cover the fact that many, if not most, of
the members of this flora have not been identified or grown in vitro. Nevertheless, the different types of
association are richly illustrated when the normal microbial populations that occupy various parts of the
human body are discussed.

The normal flora refers to all the microbes present on sites on the body. In terms of number and
diversity, the normal flora is a rich collection of microbes. Essentially, all external surfaces are colonised
to some degree with microbes. The skin obviously is a large site that houses organisms, but the mucous
membranes of the respiratory tract, genito-urinary tract and gastrointestinal tract (and others) must be
viewed as involuted external surfaces that lead to the body surface at some point. The mucous
membranes harbour the largest number of organisms. Estimates of the numbers of bacteria in the colon
are in the order of 10 ^^/g of faeces.

The association of the bacteria with the host surface will vary. Some appear virtually permanent whereas
others may be only transient occupants. Several mechanisms exist that regulate the density of the normal
flora. The flushing action of urine flowing over the mucous membranes of the urethra is going to remove
organisms that have not developed appropriate means of attachment. Similarly, the continual shedding
of skin and epithehal cells will also act as a means of eUminating organisms. The sites that organisms
occupy are highly sought after and therefore organisms adopt various mechanisms to secure their
attachment.

The organisms that make up the normal flora is not a clearly defined list but large-scale studies of people
have provided a core of organisms that are routinely isolated. The cast Ust will vary depending on the
body site. Clearly, organisms occupying the skin will have a number of characteristics suited to that
environment and these will be different to those found in the throat or the colon. It is not unusual to find
that certain people may harbour organisms that are considered pathogenic. Neisseria meningitidis may
be found in the throat of at least 1 per cent of the population without any associated disease. Not
surprisingly, the normal flora will be affected by the activities of the host. People Hving in tropical
countries, eating diets that are predominantly vegetarian will have an intestinal flora that varies from
people hving in northern hemispheres and eating a packaged diet. Whilst there will be variations in the

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exact number of different species present, there will be a core of bacteria that are almost universal; that
is, which are present all of the time. In addition, there will be a number of species that appear
temporarily (transients), some of which may well be pathogenic.

The development of the normal flora starts immediately the foetus is delivered. The passage through the
genital tract provides the child with its first encounter with large numbers of organisms. The mother will
also provide a steady supply of intestinal skin and respiratory tract-derived organisms that will begin to
establish themselves in the

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■ BOX 7.3 ECONICHE

The skin covering the face of humans is a normal habitat for certain bacteria. The 'habitat' is simply
that particular area. The conditions across the surface of the face differ. For example, there will be a
gradient of humidity, ranging from low on the forehead but increasing under the chin and nose.
Bacteria have preferences for the different conditions on the face because they offer different
resources. The term 'econiche' (or niche) is not a location but represents the particular set of
resources that are utilised by organisms within that habitat. In Chapter 2 we examined the
preferences bacteria have for temperature, a^, gaseous conditions, etc., which tend to give bell

shaped curves. If the preferences (hmits) for two particular components are known for an organism it
is possible to plot a graph outhning the zone within which the organisms will grow ( Figure 7.1) .
When three parameters are known, then a three-dimensional graph can be plotted to show the pattern
formed by this particular niche. Since the niche is a property of the organism (and not the
environment) there is no such thing as an empty niche.

growing child. The two main sites that are colonised with normal flora are those that are exposed to the
outside world: the skin and the mucous membranes.

7.8.1 THE SKIN AND THE MUCOUS MEMBRANES

The skin is the largest organ in the body and offers a variety of conditions which microbes can exploit.
The skin provides moisture in varying degrees, as exemplified by the groin and the forehead, as well as a
surprisingly varied range of nutrients such that organisms are easily recovered from most skin sites.
Sweat will provide a significant number of aqueous nutrients and minerals, which supplement those
recoverable from sebum, the lipids and fatty acids. The cells on the surface of the skin are composed of
mostly keratin and, as dead squamous epithelia, will provide very little nutrient for most bacteria. The
flora is not entirely bacterial as yeasts such as Pityrosporum spp. are frequently found in healthy skin.
The variation in temperature over the body will clearly affect the flora that can colonise any particular
site. This has implications for the temperature at which you attempt to grow the organisms in the
laboratory. For example, numerous respiratory viruses prefer 33°C to 37°C.

The other sites are the mucous membranes: the gastrointestinal tract, the upper respiratory tract and the
geni to-urinary tract. Both the density and absolute numbers of

• Figure 7.1 The figure shows the niche defined by temperature and humidity
for a particular organism. Within the inner ring represents the optimal
conditions for growth, whereas the outer ring represents the outer limits for
growth, thus the organism will not be found outside the outer ring (except
transiently). A different organism will have its own particular zone of
preferences that may partly overlap (niche overlap). The important principle is
that no two species can coexist in the same niche; the competition between the

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two species will result in the exclusion of the organism that is least fit
(competitive exclusion)

Tcrnpcralurc

Humidity

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organisms found in these sites is considerably higher than on the skin, probably due to the degree of
available water. The mucous lining covering the epithelia of these body cavities and tubes promotes a
deep layer of organisms that arrange themselves according to the extent to which they are aerotolerant.
At the surface of the epithelial cell, the Ej^ will be highly negative (that is, very reduced, very anaerobic).

The organisms will layer themselves so as to mirror the oxygen tension. The most oxygen sensitive will
be closest to the tissues, and the more aerotolerant organisms will layer themselves in the outer, more
oxygen-rich zones closer to the outer surface.

Hopefully the discussions of bacterial growth in Chapter 2 are beginning to ring some bells. The
different sites of the host that are colonised with the normal flora can be seen to provide the
requirements for growth. What provisions, then, does the body take to limit itself from becoming
overwhelmed by proHferating microbes? Those people with poor dental hygiene amply provide an
example of the balance between growth and growth limitation being lost. Several mechanisms are used
by humans to control the numbers of organisms carried. The body loses squamous epitheha from the
skin along with the associated bacteria. Coupled to regular shedding, the skin will dry out, thereby
reducing favourable growth conditions. The skin also has several antibacterial agents. Lysozyme is
secreted into sweat and the acids produced by certain skin bacteria (propionibacteria) are inhibitory to
certain organisms, not least through dropping the pH. Rapid utilisation of the available nutrients by the
flora will be a major Hmiting factor for bacterial growth in all sites. In the intestine, this is likely only to
be a temporary starvation. To get to the intestine, bacteria pass through the stomach but stomach acid
will help kill most bacteria. The numbers of bacteria in the proximal small intestine are low but increase
the further you go down the intestine. Highest numbers are found in the colon. Tolerance to bile also
helps. The differences between Gram positive and Gram negative bacteria in resistance to membrane
destabilising agents such as bile salts was discussed in Chapter 1. Within the small intestine the epithelia
are continually replaced as the tips of the villi and adherent bacteria are released into the lumen, to be
carried out with the faeces.

Factors that Hmit bacterial proHferation are not all host derived. The competition for nutrients and space
by other bacteria are also recognised. Certain bacteria produce bacteriocins when they are in late
exponential/stationary phase of growth. These secreted proteins act with varying specificity to inhibit or
even kill other bacteria. The ehmination of competing species is clearly an advantage when the food
returns. The space left by the killed organisms is also then available for occupation.
The normal flora does not appear to be simply parasitic or even cohabiting as commensals because there
are a number of functions and benefits that humans derive from the normal flora. Again, the GI tract
provides several examples of benefits to humans, with the essential digestive functions of the flora in the
rumen of herbivores as a famous example.

• The synthesis of vitamin K and vitamin B 12 by the intestinal flora. Germ-free animals (gnotobiotic
animals) suffer from vitamin K deficiency until the intestinal flora is allowed to estabUsh.

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» The short chain volatile fatty acids (VFAs) produced by anaerobes in the colon are utihsed by the
epithelial cells lining the colon (colonocytes) as their primary metabolic fuel (over glucose). This
makes sense in light of the absence of glucose that far along the intestine. VFAs as nutrient
supplies to the colon is highlighted when the colonic bacteria are reduced in number in patients
with a colostomy that

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develop a severe colitis in the defunctioning length of colon (from stoma to anus). When the
terminal length of colon is flushed with saline in an attempt to keep it clean, the starvation of the
colonocytes results in colitis (called diversion colitis) which is reversed by passing a solution of
the VFAs into the lumen. The substance that imparts the distinctive odour of faeces is the very stuff
that is used to feed the intestinal epithelia.
► A third example of benefit is the finding that gnotobiotic animals have poorly developed lymphoid
tissue compared with the normal animals. The continued exposure to bacterial products (cell wall
especially) acts as a continual low-level stimulus for antibody production in the gut associated
lymph tissue.

7.8.2 PROTECTION AGAINST PATHOGENS

The normal flora offers some protection against invading pathogens. The pattern resembles the problems
of getting seeds to root in an estabhshed field of grain. The space is Umited, the water and soil nutrients
are all being taken by estabhshed root systems. This protection, when applied to the intestine, is called
colonisation resistance, although the principle appHes to any situation on the human body where the
normal flora Hmits the ability of an invading organism to gain a foothold. The efficacy of this protection
is highlighted by the disease pseudomembranous colitis (PMC). Patients with PMC develop severe
diarrhoea and have a characteristic membrane over the surface of the colon visuaHsed by
sigmoidoscopy. PMC is caused by the multiplication and colonisation in the colon by the anaerobic
Gram positive rod Clostridium dijficile. When the normal intestinal flora is disrupted by treatment with
poorly absorbed antibiotics, patients can become readily colonised by Clostridium difficile from sites
vacated by the depleted normal flora. Clostridium difficile can produce at least two protein toxins that
contribute to the diarrhoea that develops. The organism can be isolated from healthy people at low
frequency, implying that the organism either exists in very low numbers in the gut and only multipHes
when given the opportunity following antibiotic treatment, or is ingested at the appropriate time (in
hospital, for example).

Colonisation resistance applies to all sites where the normal flora is present. Within the vaginal tract,
glycogen is present in relatively high concentrations due to the resident lactobacilU that produce lactic
acid as the principle by-product of fermentation of glycogen. This production of acid lowers the pH to
acid levels as low as pH 5, an environment that is antagonistic to many bacteria.
Harbouring a normal flora is not without risks. Under normal circumstances the normal flora will most
Ukely not present a problem. As soon as new and unusual circumstances arise, however, some microbes
will seek to exploit a new potential site for growth and colonisation. Anyone who has cut themselves
will recognise that often the cut becomes infected. Most Ukely it will be a Staphylococcus aureus
infection, the organism originating from the nose. Urinary tract infections by Escherichia coli will arise
from the organisms that have passed through the gastrointestinal tract. Such organisms are thus termed
'opportunists'; they require a breakdown in the normal protective mechanism (e.g. cut in the skin). It is
also possible to culture organisms that cause serious infections from a person who is asymptomatic. We

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can conclude thus far by saying that the relationship between host and the normal flora is in equilibrium
until a perturbation of normal flora or host tissue occurs. Then the balance shifts in favour of the
bacteria, rapidly multiplying in the new environment, at least in the short term.
Knowledge of the dominant types of bacteria from the different sites of the body is essential when
looking for pathogens in clinical samples. The use of appropriate

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selective culture media can be employed to help isolate any pathogens in a sample that may be
dominated by normal flora (e.g. looking for salmonellae in faeces). The diagnostic microbiologist needs
to learn how to recognise and distinguish the normal flora and the potential pathogen, not least because
it is not always possible to obtain a specimen from the patient without sampHng the normal flora as well
as the diseased tissue. A patient with a chest infection needs to produce a sample by coughing. The
difficulty is trying not to contaminate the sputum (from the lungs) with saliva and organisms from the
normal flora of the mouth. These procedures are possible because the organism you are looking for is
known and identifiable. Dental bacteriologists have not had it so easy. It is likely that tooth and gum
decay is caused by the collective action of members of the normal mouth/gum flora. The problem arises
because of the vast excess of glucose and other refined sugars that the modern diet provides. The marked
acidity resulting from the fermentation of the sugar erodes the tooth enamel and enables a proliferation
of anaerobic (hence, foul smelling) bacteria. It has been not been possible to convincingly identify a
single culprit responsible for dental caries.

You will have noticed that the normal flora has been contained to bacteria and yeasts that live on the
external faces of the body (where the respiratory tract, genito-urinary tract, etc. are considered external
faces). Internal organs are considered sterile. It is tacitly accepted that the presence of viruses is
abnormal (i.e. an infection). The sequencing of the human genome has unearthed numerous stretches/
fragments of past virus infections. Whether viral genome in human tissues will ever be considered
normal flora is up for debate.

One important area of concern to hospital microbiologists is the problems of infections transmitted in
hospitals. The topic brings together both the transmission of microbes and the state of the health in the
host. Does illness cause a shift in the balance between the host and the microbes they encounter?

7.9 NOSOCOMIAL INFECTIONS

Florence Nightingale said that going into hospital should at least do the patient no harm. Today, people
often acquire an infection during their stay in hospital and these are called nosocomial infections. The
reasons are not hard to find. Patients will likely have one or more of their natural protective mechanisms
breached. This can mean inserting an intravenous Une or undergoing surgery, thereby breaching the
intact skin barrier. Alternatively, if not in addition, they might have a urinary catheter inserted, thereby
compromising the normal flushing by passing urine via the urethra. The opportunities for opportunistic
microbes to establish an infection are provided. To further increase the risk, the patient will be visited by
numerous staff and they themselves get the opportunity to touch and collect bacteria from all the other
patients. Despite rigorous hand washing it is not always possible to remove all the bacteria. Nosocomial
infections are opportunistic infections, considered separately from those infections acquired in the
community. The distinction is usually made because the compromised condition of the patient leads
them to be become infected with micro-organisms that, in healthy people, present little problem/risk.
Not that hospitals are protected from ordinary infectious diseases. The infectious diseases that infect

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healthy people outside of hospitals can easily be brought into hospitals via the staff.
Infections that are acquired from external sources, i.e. are not normally resident within the host, are
termed exogenous infections. Those infections that originate from microbes present in the host normally
are called endogenous. The terms 'exogenous' and 'endogenous' infection have particular value when
investigating noso-

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comial infections because many infections arise from the patient' s own flora. If the infecting agent can
be shown to be an exogenous infection it points to a problem of cross-infection. The term iatrogenic
infection applies to those infections that arise as a direct consequence of a medical or surgical procedure
(e.g. infection of a new hip joint, infections following insertion of a urinary catheter or intravenous line).
Iatrogenic infections, for example an infected intravenous line, can be either endogenous or exogenous
in origin. The implications for control are obvious, the cost is another factor altogether.
The sources of nosocomial infections are other people (staff or patients) or the patient' s environment.
This will include such diverse sources as organisms growing in ventilator tubes, or inadequately
disinfected endoscopes, through to organisms in the dust, air or food. In addition to the compromised
defences of the patient, the bacteria that cause nosocomial infections are increasingly resistant to the
action of antibiotics. Often, the organism is able to tolerate a range of different antibiotics (multiple
antibiotic resistance), posing greater problems in the treatment of the infection.
It is interesting that bacteria are the most common causes of nosocomial infections. However, more
recent trends show an increasing incidence in fungal infections. It is suggested that the advances in
medicine are paralleled by an increase in the number of infections by organisms of low virulence. The
people who previously would have died, who now are being kept aUve, are liable to acquire infections
with organisms correspondingly 'weak'. Whilst viral and fungal infections do occur, bacteria appear to
be the dominant problem. Why?

The reasons may be the source of nosocomial infections: humans and contaminated objects. Humans
carry vast numbers of microbes as normal flora, and bacteria are able to quickly grow to large numbers
in a wide variety of sites. Providing moisture and nutrients are available, bacterial growth will occur.
Being microscopic, they will not necessarily be dealt with. Fungi may be able to grow on a wider range
of substrates with less moisture (a^) but they form visible colonies of myceha. Once noticed, they will

be eliminated. As seen in Chapter 3 , viruses will have an obligatory requirement for living hosts if they
are to multiply. Viral infections might induce illness and this may limit their spread, as suitable
precautions can be taken to prevent further infections.

The most frequent types of infection in hospitals occur in the urinary tract, bloodstream, the chest and in
surgical wounds. The risk of developing a nosocomial infection is related, mostly, to the severity of the
underlying disease. Patients who have impaired defences, whether they are inherited diseases of
impaired immunity or secondary to other diseases or the treatment are all called 'immunocompromised
hosts'. The efficiency of treating the infection corresponds to the resolution of the original pathology. In
addition to the primary disease predisposing a patient to nosocomial infections, the treatment itself can
often have an effect. Drug treatment to immunosuppress patients following transplantation or treatment
of cancer with cytotoxic drugs will impair the host immune response. Antibiotic treatment itself will
disturb the normal flora (and therefore the colonisation resistance) and can select for bacteria that are
resistant to that antibiotic.

The interactions between populations defined at the start of the chapter tends to imply that they are
stable in nature. More correctly, these states will continually be challenged by new circumstances which
result in changes in the interactions. The dynamic nature of population interactions is highlighted by

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nosocomial infections where the condition of the host, the patient, is temporarily (we hope) altered. The
medical and surgical improvements have created a greater variety of situations for

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microbial exploitation with tremendous financial costs. The challenge for medicine is to attempt to treat
disease with as minimal disruption to the patient's physiology and normal host flora as possible.
From such pragmatic issues we conclude this chapter by revisiting the biology of the micro-organism
and the reasons for parasitic infections in humans. What are the costs and benefits in choosing to infect
humans?

7.10 WHY ARE MICROBIAL PATHOGENS DAMAGING TO THE

HOST?

For a long time it was thought that infections are damaging to the host because of insufficient time for
adaptation by the microbe to the new host. In evolutionary time, the association between the microbe
and the host has been too short to allow the organism to gradually reduce its virulence. The underlying
rationale being that the microbe does not want to damage its host; in other words, all organisms are
striving for a state of commensahsm. The problem with this argument is that evolutionary pressures do
not worry about the host; the primary importance is the propagation of the self, whatever the cost to the
infected party. Current thinking favours the trade off between reproductive fitness of the organism and
the survival of the host. The organism may damage the host in the quest for obtaining nutrients, but the
long-term yields will be curtailed if the host dies. The trade off means that a balance will be estabhshed
between being too damaging or not damaging enough ( Figure 7.2 ) rather than just becoming
harmless. There are a number of proposed theories that offer new views on the driving forces for
virulence in microbes (and parasites in general). Note that 'virulence' here simply means damage to the
host.

7.10.1 VIRULENCE IS A RESULT OF THE ORGANISM INFECTING THE

WRONG (UNINTENDED) HOST

The devastating effects of Ebola virus outbreaks are considered an example of such a hypothesis. Ebola
virus is thought to be a zoonosis and the accidental infection of humans is a result of the virus straying
from its natural host (as yet unknown). The virulence observed in the accidental host is considered
irrelevant because the infection is not part of the normal path of transmission, e.g. Cryptococcus
neoformans meningitis. You should note that such an infection yields almost no value to the parasite in
terms of enhanced reproduction or transmission. By using Ebola virus infections, the hypothesis
artificially suggests that all infections in accidental hosts will have high morbidity and/or mortaUty. This
impression results from the high impact of 'virulent' infections such as Ebola virus. The reaUty may be
that most infections in unnatural hosts fail to

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• Figure 7.2 The trade off between virulence and transmission for a micro-
organism that can kill its host. The optimal level of virulence will be balanced
at the apex of the curve to maximise both mortality and transmission

Rait ol"

Host mortal ily

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establish themselves and those that do are asymptomatic. We are left with wondering why strict human
pathogens are virulent in humans, e.g. measles or typhoid. The hypothesis does not readily explain their
virulence.

7.10.2 BETTER TO BE VIRULENT THAN HARMLESS

Evolutionary pressures will favour virulence in a parasite if it results in increased reproductive success,
albeit at the expense of the host. So, if any damage to the host is going to help the reproduction or
transmission of the parasite (e.g. causes diarrhoea or coughing) then virulence is better than being
harmless.

7.10.3 VIRULENCE IS A TRADE OFF AGAINST OTHER

CHARACTERISTICS

In this scenario, the organism causes harm to the host 'against its own wishes' because it is a necessary
evil if the organism is to survive in the host. The virulence trait is offset by the advantage gained in the
host. In the need to acquire nutrients, for example, the organisms need to lyse host cells to release the
cell contents. This provides nutrition but causes tissue damage which, in turn, induces a host immune
response.

7.10.4 VIRULENCE IS DETERMINED BY THE ARMS RACE BETWEEN

HOST AND PARASITE

This hypothesis arises from a view that both the host and the parasite are evolving in response to each
other. If the parasite did not adapt then the virulent strains would be eliminated by the host and harmless
strains would dominate. Virulence will decrease but the parasite might lose fitness, hence the parasite is
proposed to counter the efforts of the host in eliminating it by developing new virulence traits to
maintain its own fitness.

Much debate has arisen concerning the evolution of virulence. At this stage the evidence supports the
idea that the interplay between parasites and their hosts is going to vary in each case. The interplay
between damage and host response will result in a trade off between harmless and damaging depending
on what is optimal for the parasite. If damage results in greater evolutionary fitness, greater reproductive
success then being nasty is best. The study of the introduction of myxoma virus (causing myxomatosis
in rabbits) into Australia has provided a useful case study. In 1950 the vims was introduced to control
the burgeoning rabbit population. Over the next ten years, rather than become harmless, the virus
evolved to an intermediate degree of virulence, presumably sufficient to maintain itself within the rabbit

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population. The outcome of parasite-host interactions will vary according to the circumstances, but
evolutionary pressures mean that the parasite may adopt a harmless, virulent or intermediate strategy
depending on the reproductive fitness.

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■ SUMMARY

Bacteria can be seen as either generalists or specialists. Generalists will occupy a broad
niche, where niche refers to the requirements of an organism rather than physical location.
Specialists will be more exacting and Hmited in their capabilities and habitats. It is likely
that the extent to which micro-organisms adopt mutuahstic or parasitic modes of existence
will depend on whether they are generahsts or specialists. The communities of microbes
that are resident on or in the human host (the normal flora) provide a useful casebook for
studying the balance in human host-microbial parasite equilibrium. Benefits (e.g.
colonisation resistance) and costs (endogenous infections) are implicit in maintaining a
normal flora. The relatively few primary pathogens that infect humans are usually
exogenously acquired and only transient parasitic episodes. CHnical disease often only
develops in a fraction of the total number of people infected. The outcome will depend on
the virulence of the organism and its infectious dose along with the condition of the
host. The modes of transmission are varied, as are the sources and reservoirs of infection,
but the majority of encounters will be via mucous membranes. Where it was previously
considered that disease results from the organism having inadequate time to adapt to a
lower level of virulence, recent evolutionary theory proposes micro-organisms adopt a level
of virulence that is optimal for reproductive success and this includes efficient transmission
between hosts.

RECOMMENDED READING

Atlas, R. and Bartha, R. (1998) Microbial Ecology: Fundamentals and Applications, 4th edition,
Benjamin Cummings, California, USA.

Balows, A., Truper, H.G., Dworkin, M., Harder, H. and Schleifer, K.-H. (1991) The Prokaryotes. A

Handbook on the Biology of Bacteria, 2nd edition. Springer, Berlin.

Berg, R.D. (1996) The indigenous gastrointestinal microflora. Trends Microbiol. 4, 430-5.

Ebert, D. and Herre, E.A. (1996) The evolution of parasitic disease. Parasitology Today 12, 96-101.

Mason, T.G. and Richardson, G. (1981) Escherichia coli and the human gut; some ecological

considerations. J. Appl. Bacteriol. 1, 1-16.

Mims, C, Playfair, J., Roitt, I., Wakehn, D. and Williams, R. (1998) Medical Microbiology, 2nd

edition, Mosby International Ltd, London, UK.

Moran, N.A. and Wernegreen, J.J. (2000) Lifestyle evolution in symbiotic bacteria: insights from

genomics. Trends Ecol. Evol. 15, 321-6.

Vollaard, E.J. and Clasener, H.A. (1994) Colonization resistance. Antimicrob. Agents Chemother. 38,

409-14.

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REVIEW QUESTIONS

Question 7.1 What is 'colonisation resistance'?

Question 7.2 To what extent does the normal flora of humans provide examples of commensalism,
symbiosis, synergism, competition and parasitism?

Question 7.3 List the benefits and costs of humans housing a normal flora.

Question 7.4 List examples of microbes that are transmitted by 1) water, 2) soil, 3) food, 4) air
(borne) and 5) blood.

Question 7.5 Distinguish between a source, a reservoir and a vehicle.

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8
BACTERIA AS PARASITES

Fortunately, only a small fraction of the number of bacteria recognised are able to cause disease in
humans and animals. What is it about this select group of organisms that confers the ability to cause
disease in humans? This chapter will examine some of the properties of these bacteria.
Before discussing how pathogens behave, let us examine the criteria by which organisms are classified
as pathogens or not. A patient has a small abscess on her hand. The lesion is red and inflamed and pus is
draining from it. You culture the pus and you isolate an organism. How do know whether it is
responsible for the inflammation? Did the organism cause the lesion or simply arrive and start
multiplying after the lesion appeared? This sort of problem is conventionally addressed by reference to
Koch's postulates.

8.1 KOCH'S POSTULATES

In the last decade of the 1800s, the bacteriologist Robert Koch demonstrated that Mycobacterium
tuberculosis was the aetiological agent of tuberculosis. Having grown the organism from human cases of
tuberculosis on his newly created culture media, he succeeded in reproducing a tuberculosis-Uke disease
after inoculating the organism into rabbits. Although never directly stated by Koch, a number of
necessary steps have been outhned which are to be followed when one wishes to see whether a particular
bacterium is the cause of a particular disease. They are called Koch's postulates and can be summarised
as follows:

• the organism should be found in all cases of the disease, and in the affected lesions,

• the organism should be grown from the lesion in pure culture,

• inoculation of the isolated organism should reproduce the disease in a susceptible animal.

Koch's postulates were later amended to the following:

• the microbe must be present in every case of the disease,

• the microbe must be isolated from the diseased host and grown in pure culture.

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• the disease must be reproduced when a pure culture is introduced into a susceptible host,

• the microbe must be recovered from an experimentally-infected host.

The logic cannot be faulted and in the late nineteenth century Koch was setting admirable standards with
both the theoretical principles and practical methods for investigating infectious diseases. Today,
however, they need qualification. Some of the postulates cannot be fulfilled because:

• we cannot culture certain organisms in culture media. To the embarrassment of medical
microbiologists everywhere, the causative agents of syphilis {Treponema pallidum) and leprosy
(Mycobacterium leprae) cannot be grown outside animals, i.e. on culture media in vitro.

• certain diseases are caused by combinations of bacteria. Dental caries is widely believed to a
polymicrobial condition.

• many specific infectious diseases of humans have no appropriate or equivalent animal counterpart,
hence the human disease cannot be reproduced. Examples include the sexually-transmitted diseases
syphilis and gonorrhoea, for which no suitable animal models exist that mimic the human infection.

Furthermore, there are other issues that need comment.

8.1.1 SITE OF ENTRY

The site and method by which an animal is infected with the test organism is not accounted for.
Intraperitoneal injection is the standard site for transmitting Mycobacterium tuberculosis in guinea pigs.
Whilst this method is very efficient in the transfer of the organism in these animals it bears little
resemblance to the respiratory route of transmission in man. Likewise, mice are unaffected by inhalation
of aerosols of encapsulate Streptococcus pneumoniae but very quickly succumb to intraperitoneal
injection.

8.1.2 INFECTIONS DUE TO SECONDARY PATHOGENS

Infections by organisms that are are unable to cause clinical disease in healthy people with no
predisposing factors (immunosuppression, catheters and intravenous lines, etc.) were not studied by
Koch. Koch's postulates were conceived whilst identifying what we now call 'primary pathogens' and
the problems of identifying opportunist infections were not appreciated at the time. Since then we now
appreciate that certain organisms are able to exist as commensals but at certain times proliferate when
certain opportunities arise. The results obtained from injecting organisms into healthy animals give little
clear indication as to whether the organism is acting as a primary or secondary pathogen.
Staphylococcus aureus can readily be isolated from the noses of healthy people but has the potential to
cause a huge variety of specific infections. Thus, simply isolating the organism from patients is not
enough to decide whether it is responsible for any particular disease without considering the context.

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8.1.3 RISE IN ANTIBODY TITRE

With appropriate methods, one can measure quantitative increases in levels of specific antibody
(increasing titre) against the pathogen, strong evidence for infection by that organism. The increase in
antibody titre is important because the qualitative presence of antibodies alone does not distinguish
between previous or current infection.

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8.1.4 CARRIER STATE

It has become clear that certain organisms are able to induce a carrier state. Salmonella typhi causes
typhoid fever but in a very small proportion of cases persist in the gall bladder of people for years. They
need not have developed the full clinical illness yet still excrete the organism in the faeces. The
existence of asymptomatic carriers does not argue against Salmonella typhi causing typhoid but does
complicate the interpretation that it is a primary pathogen.

8.2 HOW DO YOU DEFINE THE ABILITY TO CAUSE DISEASE?

8.2.1 BACTERIAL PATHOGENICITY

The ability of an organism to cause disease in a host is referred to as the pathogenicity of an organism.
The process by which the disease is caused is termed the 'pathogenesis' (composed from the phrase
'genesis of pathology'). The disease will be the result of the destructive effects of the bacterium itself
and the effects of the host as it attempts to deal with the invader. Pathogenicity is therefore the sum of
bacterial and host effects and not the sole action of the organism. Table 8.1 helps illustrate the concept
of pathogenicity. Using the same inoculum size for both animals, the results show that Organism X is
pathogenic for mice but not rats. To examine the role of the host in greater detail we will look at two
organisms in the same host: humans. In Table 8.2 we compare the outcomes of exposing the same
animal with two different bacteria. Staph, aureus is shown to be the more pathogenic of the two
organisms tested because it was able to establish itself on the abraded skin and cause an infection,
whereas the Staph, epidermidis was unable to do so under the same conditions. For Staph, epidermidis to
establish itself, the presence of a foreign body was required (the IV line catheter). This illustrates the
importance of the condition of the host. The condition of the skin had a large influence on the outcome
following exposure to two different organisms but the invading organisms differed in their ability to
exploit the abraded skin. Such an experiment tells us nothing about the mechanisms that account for the
virulence of the organisms. The properties of the organism that were responsible for the difference
between the two organisms are called virulence factors and further experiments need to be devised to
test for virulence factors.

Table 8.1 Effect of route on bacterial virulence

Organism Animal Route

Outcome

Organism X rat inhalation
Organism X mouse inhalation

no effect
pneumonia

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Table 8.2 Outcomes of inoculation onto skin

Organism Conditions Outcome

Staph, aureus healthy skin no infection

Staph, aureus abraded skin infection

Staph, epidermidis healthy skin no infection

Staph, epidermidis abraded skin no infection

Staph, epidermidis normal skin + intravenous line infection

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8.2.2 VIRULENCEA^IRULENCE FACTORS

Virulence factors can most simply be defined as the character(s) that are directly involved in the
development of disease. The term virulence is used to grade the ability of an organism to cause disease.
It is used without reference to the host but as a comparative term of the extent to which strains of the
same species cause disease. The measurement of virulence is made by comparing the numbers of
organisms necessary to cause disease in a suitable model. Often this involves the use of animals but
wherever possible in vitro models are used (e.g. tissue culture systems). Figure 8.1 shows an example of
an experiment comparing the virulence of two strains of the same species. Strain 2 is more virulent than
strain 1 because the lethal dose 50 (LD50) is lower for strain 2 (2 x 10^ organisms/animal) than strain 1

(3 X 10^ organisms/animal).

If the ability of an organism to cause disease resides in its possession of one or more virulence factors, it
follows that the removal of this character results in a strain/mutant that has a reduced ability to cause
disease. If you delete an enzyme in a central metabolic pathway that is necessary for the growth of the
organism, e.g. an amino acid auxotroph, the result may be that the organism is unable to grow at all or
does so at a very reduced rate compared to the wild type strain. The auxotroph is unable to cause disease
in the experimental model. Does this mean that the particular enzyme is a virulence factor? To get
around such awkward issues of definitions, it is necessary to distinguish between characters that are
involved in basic cell physiology (as reflected in growth rate of an organism), called 'housekeeping'
characters, and those that determine virulence

• Figure 8.1 Estimation of lethal dose 50 per cent (LD50) for two strains of the
same bacterial species

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kJ^

T.og inoculiinfi !5i7c

■ BOX 8.1 WHY MEASURE LD50?

The slope is greatest at the mid point, reflecting the biggest increases in the proportion of animals
dying per dose. This means that the difference between the LD30 and LD70 will be reasonably close

to that of the LD^q, but values of the LDgg may be indistinguishable between 10^ and lO^O

organisms.

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but not growth of the organism. Thus loss of virulence factors should not reduce the general fitness of
the organism for growth. With the molecular biology skills now available to researchers, a twentieth-
century set of Koch's postulates have been proposed to examine the role of individual genes and their
products as virulence factors. The so-called Koch's postulates for genes are:

• the gene or product should be found in the strain causing disease but not in avirulent strains,

• disruption of the gene will result in reduced virulence,

• introduction of the cloned gene into an avirulent strain will increase its virulence,

• the gene is transcribed during infection,

• specific immunity to the product is protective.

It is argued that the term 'virulence factors' should only apply to those microbial products that
directly interact with the host rather than any product that affects the ability of an organism to
damage the host. In this way any essential microbial metabolite or product essential for
general microbial growth can be excluded as virulence determinants.

To summarise thus far, virulence is a quantitative estimate whereas pathogenicity is generally used as a
qualitative term. Staph, aureus, strain 1 may cause disease in rabbits more effectively than Staph,
aureus, strain 2. Therefore, strain 1 is more virulent than strain 2. This latter comparison is based on the
differing abilities of the organism and not the host. Descriptions of pathogenicity (e.g. Table 8.1 ) should
take the effect of the host into account. Organism X may cause disease in mice but not rats. Organism X
is therefore pathogenic for mice but non-pathogenic for rats. Likewise, Table 8.2 examines the effect of
two different organisms in the same host but this time with differing circumstances. We could conclude
that Staph, aureus was more pathogenic than Staph, epidermidis. An organism that is completely non-
pathogenic for humans would not be able to cause an infection even when the host defences are
breached, e.g. the presence of an IV line. For details of the role of the host immunity in the interplay
between pathogenic organisms and humans, the reader is referred to an accompanying text in the series
(Infection and Immunity, Davies et ah).

8.2.3 INFECTIVITY

In Chapter 7 we saw that it is important to distinguish between the ability of an organism to cause
disease and the ability to infect. Infection is not the same as disease. It can occur in the absence of
disease and, indeed, many infections only result in a fraction of the total number infected becoming ill or
showing any signs of disease. This may be memorised as:

Infection -i- signs & symptoms = disease

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We can distinguish between the organisms that infect humans but just become part of the normal flora of
man and those that result in illness. Infections describe an acute and transient process that is progressive,
whereas when organisms infect a host but become members of the normal flora in stable association
without progression to pathology, the process is called colonisation. The term 'infectious dose' is
familiar to most people and is a measure of the number of organisms required to cause an infection/
disease. It can be measured for any particular organism in the same way as shown in Figure 8.1 but
using different parameters. In determining infectivity, the end point is the number of organisms per
animal rather than the number of animals that die. Having chosen a suitable time point, samples are
taken from the animals and the number of organisms from each animal is counted. The results are
plotted on a semi-log graph and the 50 per cent values are estimated to yield the infectious dose 50

(IDso)-

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It is necessary again to distinguish between infectivity, pathogenicity and virulence. A harmless
organism may be highly infectious. Alternatively, a pathogenic organism may need a high infectious
dose. Infectivity therefore only describes the ability of an organism to establish itself in a new host and
this is defined by the ID^q- The LD50 is used in the measurement of virulence between two strains of the

same organism. The comparison of pathogenicity between two different species may also use LD50

values but this is problematic for a number of reasons. Death as an end point is not well defined because
it is the net effect of the invading organism and the host. We know that many virulent micro-organisms
do not usually kill people but nevertheless cause considerable morbidity (e.g. salmonella food
poisoning). For organisms that cause food poisoning it is more appropriate to measure a related
phenomenon in the experimental model (fluid output, i.e. diarrhoea, following ingestion of the
organism) but this also measures a host response. Another problem with LD50 comparisons between two

species is that the shape of the curves will often be different; that is, the slope will be different. It is not
appropriate to compare the values from curves with different slopes. The fact that the slopes are different
reflects the fact that the systems are not the same. This is not the case with isogenic mutants (the two
organisms differ only in the possession or absence of one particular virulence factor, e.g. a capsule)
which should yield curves of similar shape, differing only in their position along the x-axis. It is a
problem in the use of any experimental system that the results obtained reflect the conditions that were
used/measured. In other words, it is evidence, not proof.

Pathogenicity

• The ability to cause disease.

• A quahtative term.

• Viewed from the host's perspective.

Virulence

• The degree or ease with which an organism is able to cause disease.

• A quantitative term of comparison between strains.

• Independent of the perspective of the host.

8.3 THE PROCESS OF AN INFECTION

The sequence of events that occur after a pathogen enters a new host can be grouped into a series of
steps:

• attach,

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invade,
multiply,
spread,

persist/evade host defences,
• exit.

Before describing these steps in more detail, one should note that these events do not necessarily occur
in the order they are listed but can occur simultaneously. An infection may be viewed as a race between
the organism and the host. The principal advantage of

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the microbe is the rephcation rate. For the microbe, one can see how the advantages of dividing as often
as every 30 minutes pays off when attempting to estabhsh itself in a new host. Whereas, if the host
response is quick and efficient, the establishment of infection is prevented and disease is avoided.

Important exceptions to the six- step rule are microbial intoxications, where the ingestion of
pre-formed toxins results in clinical illness but the organism is not present or replicating
within the host. Examples of such are botulism and Staphylococcus aureus food poisoning.

The first three steps listed suggest that the only difference between a pathogen and a non-pathogen that
only colonises a new host without causing disease (a commensal) is in the ability to invade the host (step
2). For this reason, pathogenicity can be defined as the ability to gain access and multiply and/or persist
at sites within the host that commensals cannot. The emphasis being that commensals do not invade host
tissues.

8.3.1 STEP 1. ATTACH

Much interest is shown in the mechanism of attachment of microbes to surfaces, and not only human
surfaces. The accumulation of bacteria on inert structures submerged in water such as stones in rivers is
a perfectly natural occurrence, but consider also the effect on cooling system vents blocked by a mass of
organisms (biofilm). Immersed structures such as ship hulls, pipes and oil rigs are all at risk from
possible corrosive activity of the accumulating biofilm. The adhesion and accumulation of bacteria on
human surfaces is of primary importance in the course of infecting humans and has stimulated research
to target the attachment process with prophylactic compounds.

Bacteria employ specific adhesins to bind to host receptors. As mentioned in Chapter 2 , the adhesins
may be located on fimbriae, flagella, or the cell surface structures such as the capsule, or cell wall (outer
membrane, or peptidoglycan). The specificity of this adhesin to receptor will target the bacterium to the
appropriate host cell/tissue/organ, from which the organism may then multiply and possibly invade. The
location of the receptor also varies. In addition to integral membrane protein of the host cell, some
bacteria choose to bind to receptors present in the extracellular matrix, a structural network of proteins
such as fibronectin, elastin and collagen. Third, epithelia secrete mucous to act as a surface lubricant.
Mucous contains various glycolipids, proteins and carbohydrates, all of which may act as possible
receptors.

Adhesion to a host surface, whilst a prerequisite for pathogenicity, does not always lead to disease. The
continuous presence of the normal host flora tells us that not all bacteria that adhere are pathogenic.
Adhesion is just a necessary step in maintaining an existence on a mucous membrane or skin, both of
which are continually attempting to expel organisms. The flow of urine, the movement of mucous on the
cilia, shedding of skin squamous cells, the coughing and sneezing reflexes all try to eject non-adherent
bacteria. Adhesins are present on pathogenic and non-pathogenic strains alike and thus should be seen as

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essential components simply for colonising a site.

Having adhered to the appropriate host cell surface, several bacteria are able to induce disease without

invading cells. Corynebacterium diphtheriae appears to be localised to the oropharyngeal epithelium yet

is able to cause a devastating illness by releasing the exotoxin which then circulates in the body

(bacterial exotoxins are described below, p. 198). When the course of an infection involves

multiplication at different sites in the host, i.e. an invasive infection such as typhoid, the organism will

need to express different adhesins at the appropriate points in the course of the infection.

Numerous studies have examined the effects of adhesin-deleted bacterial mutants on virulence and often

find that they are impaired compared with their wild type counterparts. Strictly, defects in adhesion are a

measure of infectivity rather than pathogenicity per se where infectivity is defined as the capacity to

establish a primary lodging on arrival at the body surface.

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8.3.2 STEP 2. INVADE

Invasion may simply reflect the attempts of a pathogen to exploit the nutritional resources that are held
on the other side of the plasma membrane. Additionally, invasion may be the means by which the
organism can reside for a longer period in the host. The obstacles to invasion are predominantly the skin
and the epithelia lining the mucous membranes. Skin appears to be a more difficult route to penetrate
unaided, therefore epithelia are the usual route of entry.

The primary function of epithelia is to provide a selective barrier between the host and the external
environment. The uptake of nutrients, water and oxygen is required but the entry of bacteria is not.
Bacteria move (translocate) from surfaces to tissues beneath the epithelia either via transcellular
(entering via the cell) or via paracellular routes (passing through the cell to cell junctions). Bacteria may
cross via a paracellular route by first entering mobile cells such as macrophages. Macrophages have
their own mechanisms for traversing epithelial monolayers as part of their normal role of immunological
surveillance. A distinction can be made between bacteria that target phagocytic cells such as
macrophages and neutrophils and those that enter non-phagocytic cells such as epithelia. The research
on enteric pathogens has yielded many insights into the mechanisms of entry in epithelia. Specific
adhesins called invasins trigger the host cell membrane to engulf the attached bacterium such that the
organism enters without having to disrupt and thereby kill the host cell. It is clear that such a tidy
mechanism avoids the release of compounds that will attract the host immune cells. One of the most
surprising elements in the strategies of bacterial pathogens is the extent to which the bacterium controls
the situation. It is now evident that bacteria such as salmonellae secrete regulatory molecules that control
events in the host cell using the various protein secretion systems. Many bacterial virulence factors are
proteins that need to be excreted so as to exert their action on the host cells. Examples include
haemolysins, proteases, lipases and nucleases, along with the classic excreted exotoxins. Of the four
currently recognised secretion systems, type III is of particular interest in that it mediates the transfer of
a bacterial signalling molecule into the host cell. Viewed as a syringe with which to inject the molecules
into the recipient host cell, it requires physical contact (contact dependent or contact mediated) with the
host cell for it to work. Examples of the activity of these signalling molecules (effector proteins) are the
Sip proteins injected by salmonellae, which cause rearrangement of the enterocyte membrane and a
subsequent ruffling appearance. These events facilitate the uptake of the salmonella into the host cell.

8.3.3 STEP 3. MULTIPLY

Little is known about the multiplication of bacteria in human or animal tissues during infections.
Obviously, the conditions in which bacteria are cultured in the laboratory do not accurately mimic those
inside cells or tissues. One of the recognised differences is the concentration of free (available) iron.
Unlike culture media, animal tissues hold iron at very low concentrations (10~9 M) by use of iron-

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binding protein carriers. In the extracellular fluids transferrin and lactoferrin are used, whereas inside
mammalian cells iron that is not complexed in specific proteins (e.g. heme proteins) is held in ferritin.
For bacteria to multiply in human tissues the production of siderophores to extract iron from the iron-
binding proteins is an essential and rate- limiting step (see Chapter 2 for details of iron limitation).

Other variables include gaseous conditions (O2, CO2), redox potential, pH and osmolarity, all of which
will change depending on the location within the body. This

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must be compared with the closed conditions within a batch culture which can be predicted (and
measured). Further complications in the measurement of growth rates in vivo and in vitro include the
problems of bacterial lag times (which may vary between site within the host) and the numbers of
organisms killed by the host defences (e.g. the numbers multiplying may balance the numbers killed).
Such problems mean that measured doubling times in culture media are likely to be artificial.
Furthermore, it is often assumed that increasing growth rate in vivo equates with virulence and that rapid
multiplication in vivo is preferred. However, the slow-growing Mycobacterium tuberculosis provides an
example of an alternative plan.

The ability of the pathogen to utilise nutrients that are particularly abundant at the site of infection
makes sense, even if specific examples are few. The ability to break down urea by urease in the urinary
tract would be a good example, but unfortunately the most frequent cause of human urinary tract
infections is Esch. coli, which does not possess ureases. Proteus spp., which do produce urease, are less
common. Obtaining sufficient nutrition is an important limitation on growth in vivo and of particular
interest is the uptake of nutrients of intracellular pathogens. Virulence factors that are used to obtain
nutrients from sites have been termed pabulins. One proposed mechanism for obtaining scarce and
limiting nutrients is to lyse the host cell with cytolytic toxins. The regulation of the diphtheria toxin by
concentrations of free iron is a strong argument in favour of the centrality of nutrient acquisition driving
the virulence of any pathogen.

The three cardinal features of pathogenic bacteria discussed (attach, invade and multiply) are those
considered to be the minimum needed to create an infection. Additional characteristics can be found in
some but not all pathogens. These include:

• the ability to spread within the host,

• the ability to persist within the host, and

• the means with which to exit the host.

8.3.4 STEP 4. SPREAD WITHIN THE HOST

The variety of clinical infections outlined in Chapter 7 tells us that not all infections follow identical
paths. Options open to pathogenic bacteria are to multiply only at the initial site of entry
(Corynebacterium diphtheriae) or multiply at a site distinct from the site of entry. Alternatively, bacteria
may spread at both the entry site and elsewhere. The extent to which the infection spreads gives rise to
the terms 'local' or 'generalised' infections. The spread to other organs/tissues may simply be passive, i.
e. the infection spreads into adjacent tissues (e.g. staphylococcal abscess) or spills over into the
bloodstream, and the organisms are carried to other sites in the host. In such cases the infections in the
distant sites will only be seen in a proportion of cases and therefore are considered as complications. In
systemic infections such as typhoid fever, the organism (Salmonella typhi) takes a route through the host
that encompasses several distinct organs/tissues in the host. The ordered sequential multiplication in
different organs is what defines the disease in humans. The attachment and entry via the M cells in the

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intestine is followed by migration in macrophages to the lymph nodes and subsequently to the spleen,
liver and bone marrow. From here the organisms multiply and then subsequently invade the
bloodstream, the intestine and gallbladder. This staged course of events taken by the organism in the
course of its life in humans could not occur by accidental spread of the organism through the host.
The mechanisms of spread throughout the host can vary. The routes are as follows:

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• via lymph/bloodstream,

• via nerves,

• through tissue.

In contrast to viruses, the migration of bacteria along nerve fibres has not been shown. Bacterial
infections of nerve cells is also rare. The only bacterium that primarily infects nerve cells in humans is
Mycobacterium leprae, the causative agent of leprosy. The other famous bacterial infection involving
nerve cells, this time the nerve fibres themselves, is tetanus where the movement of the secreted toxin
occurs along the nerve fibres from the site of infection towards the motor neurones in the spine.

8.3.5 STEP 5. ABILITY TO PERSIST WITHIN THE HOST (EVADE THE

HOST IMMUNE RESPONSE)

Whilst the normal bacterial flora is able to colonise the host, these bacteria are technically on the host,
not within it. Certain pathogens are able to persist at sites on the host along with the other members of
the normal flora (Neisseria meningitidis can be isolated from the throat), whilst other pathogens can
persist at sites inaccessible for harmless commenals, i.e. within the host. These organisms often cause
long-term, persistent infections. Mycobacterium tuberculosis is a good example of a pathogen that has a
long-term strategy for maintaining itself within a host (and consequently the population). The organism
is able to grow (slowly) intracellular ly; that is, within vacuoles of the host cell. Most people vaccinated
with the BCG vaccine are able to contain the infected cells within a wall of calcifying tissue. Only if the
immunity of the host fails (as seen most devastatingly in AIDS patients) does the organism resume
multiplication and spread throughout the lung tissue and beyond. Prior to AIDS, the organism usually
had to wait until the immunity of the host waned, typically when the patient became old. (The
mechanism of this failing immunity with age is largely hypothesis with proof, as yet, lacking.) With
tuberculosis infecting approximately one-third of the world's population, this patient waiting game
clearly works.

To persist unharmed in an immune-competent host depends on a mechanism of evading or resisting host
defences, hence some people consider this to be a defining feature of a pathogenic micro-organism. The
ability to evade host defences can apply to the resident normal microbial flora that colonises the host
without evident harm and appears to evoke minimal immune defence. For this reason it can be argued
that resistance to host defences is not a prerequisite for pathogenicity. However, the argument does not
end there. Pathogens invade host tissues whereas the resident flora do not (under normal circumstances).
The immune system has evolved such that the response is graded to the level of invasion, meaning that
entry into the body evokes a vigorous response but the arrival of a new organism on the skin is unlikely
to evoke any immune response. Hence, the normal flora will have little need to develop strategies to
thwart any immune responses because they do not invade.

Pathogenic bacteria such as Corynebacterium diphtheriae and Clostridium tetani are toxigenic
organisms that produce toxins that are antigenically invariant (and, being protein in nature, are highly

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immunogenic and good vaccines). As far as is known the organisms do not possess any special
properties designed to avoid or minimise their immunogenicity. For several viruses an infection confers
immunity for life (measles, chickenpox) because the virus exists as a single antigenic type. For most
bacterial infections there are multiple types (strains) of the organism and immunity to one does not
always confer protection against the others. Hence, one can get repeated infections with the 'same'
organism.

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Having argued that pathogenicity does not demand that the organism is able to resist immune responses
recognition, such an attribute is clearly of great value in the business of invading immune competent
hosts. The property of invading tissues may itself be an attempt to evade the host defences as the
intracellular life provides a good method for escaping attention (at least in the short term). Several
mechanisms have been identified by which the organism minimises recognition by the host immune
response (notably viruses, see Chapter 9) .

8.3.5.1 Capsules

Consisting of polysaccharides, capsules are poorly immunogenic and they confer protection from
engulfment by neutrophils by resisting phagocytosis. The capsules of numerous pathogens (e.g.
Streptococcus pneumoniae. Neisseria meningitidis, Haemophilus influenzae) have been the subject of
much research in attempts to make vaccines. The three organisms listed can all be isolated from the
throat of a small proportion of the normal healthy population yet all are able to cause meningitis when
encapsulate. Protection against these three can be conferred by the presence of anticapsular antibody
which permits efficient phagocytosis and intracellular destruction in phagolysosomes.

Not all bacterial capsules that act as virulence factors are polysaccharides. The capsule of
Bacillus anthracis consists of D-isomers of the amino acid glutamic acid.

8.3.5.2 Outer membrane proteins

According to the environmental conditions. Gram negative bacteria express a range of proteins within
the outer membrane of their cell wall. They are collectively termed 'outer membrane proteins' as a
generic term as their functions vary. One example of their function is illustrated by the upregulation of
particular outer membrane proteins when Gram negative bacteria are cultured in the presence of serum.
The increased production of this particular outer membrane protein confers resistance to lysis by the
terminal membrane attack complex of complement components C5-C9, a property termed serum
resistance. Clearly, this is an important protection against the humoral arm of the immune system for
invading pathogens.

8.3.5.3 IgAses

IgA has been called 'antiseptic paint' in recognition of the protective role it plays on the epithelial
surfaces of humans. As a secreted immunoglobulin it is one of the first line defences against invading
pathogens that enter the host via the mucous membranes. A number of bacteria have been shown to
secrete proteases that target and degrade secretory IgA, the theory being that pathogens such as
Neisseria gonorrhoea and Haemophilus influenzae will need to produce IgAse at the mucosal site of the
infection, although non-pathogenic bacteria have also be shown to express IgAses.

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8.3.5.4 Residence in an immunologically privileged site

Immunologically privileged sites are those that are relatively sheltered from the immune surveillance of
roving macrophages. These relatively protected sites may be inside host cells or general locations such
as the central nervous system and the skin.

8.3.5.5 Antigenic variation

Surface structures will be those exposed to the host, and therefore these are the products that pathogens

have chosen to vary antigenically: capsules, flagella, pili, S-layers and outer membrane proteins. The

genetic mechanisms responsible are either random or controlled in response to environmental signals.

Two types of variation of surface antigens should be considered: phase variation and antigenic

variation.

The former is the (apparently) random expression of surface antigens on pili and

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flagella brought about by the inversion of a part of the appropriate promoter region. In the 'on'
configuration the transcription can occur. However, the promoter region has the abihty to excise and re-
insert itself (integrate). When the region re-integrates the other way round (the 'off configuration), and
with this region of DNA inverted, transcription can no longer occur and the pilus/flagellar antigen is not
expressed. Phase variation is reversible and the switching occurs more frequently than random
mutational events.

Antigenic variation is not a random event but a co-ordinated effect to buy time in the host. The
production of specific antibodies takes time (approximately 2 weeks) and so switching antigens keeps
the organism one step ahead of the specific host response. The genetic mechanisms are varied but
include small deletions/insertions/point mutations of base pairs which cause shifts in the reading frame.

8.3.6 STEP 6. EXIT: SHEDDING

For many pathogenic bacteria multiplication within the human body provides a means of generating
large numbers of progeny, thus increasing the numbers that can be shed into the environment. The host
may inadvertently assist; for example, diarrhoea is both a mechanism for shedding the organism from
the host and a suitable vehicle for spreading large numbers of the organism into the environment.

8.4 SPECIFIC EXAMPLES OF VIRULENCE FACTORS: BACTERIAL

TOXINS

8.4.1 PROTEIN EXOTOXINS

Many of the most dramatic infectious diseases of man (for example, anthrax, botuhsm and tetanus) are
the result of protein exotoxins released by bacteria. The chnical disease can be reproduced almost
entirely by the injection of the pure toxin in these three examples, thereby confirming that the exotoxins
are virulence factors that can act at sites distant from the organism. Bacterial exotoxins exert their action
either at the plasma membrane or cytosoUc (intracellular) targets.

8.4.1.1 The plasma membrane

Toxins acting on the plasma membrane either form transmembrane pores (pore-forming toxins, e.g.
Staphylococcus aureus a-toxin) or enzymatically attack the membrane phospholipids (phospholipases,
sphingomyelinases). The fluxes of ions that follow will result in the disruption of the ionic balance with
subsequent influx of water sufficient to burst the cell.

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8.4.1.2 Cytosolic (intracellular) targets

Toxins with intracellular targets include diphtheria, cholera and tetanus toxins. The intracellular targets
can themselves be split into three categories:

• toxins interfering with signal transduction (e.g. cholera toxin, CT, activates adenylate cyclase
which, by raising cAMP, causes chloride channels to open. This loss of chloride pulls water into
the gut which results in diarrhoea).

• toxins inhibiting protein synthesis (diphtheria toxin (DT)).

• toxins which interfere with the cell cytoskeleton (Clostridium difficile toxin A and B).

Both DT and CT work by catalysing the addition of ADP-ribose (from the intracellular NAD) to the
appropriate target enzyme. For DT an elongation factor involved in protein synthesis is ribosylated.

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Whilst the intracellular targets vary with each toxin, they all need to cross (translocate) the plasma
membrane in order to reach the intracellular site of action. Translocating toxins are therefore called 'AB
toxins', reflecting the two functional domains within the protein: the A (active) domain which contains
the enzymatic activity and the B domain which is the region of the protein that Z^inds to the cell receptor
prior to translocating the A subunit into the cell. Translocating AB toxins exist as two forms:

• simple AB toxins (one of each domain) such as DT, or

• oligomeric AB5 toxins (e.g. CT) where five B subunits are coupled to one active subunit.

A key feature of protein exotoxins is their amphipathic nature, the presence of both hydrophobic and
hydrophilic regions. This dual behaviour is required because the toxin needs to be water-soluble whilst
being carried through aqueous environments such as blood or tissue, but hydrophobic when it inserts
into the lipid interior of the plasma membrane ( Figure 8.2) .

• Figure 8.2 Two mechanisms by which protein toxins interact with
membranes. (A) shows an AB5 toxin such as cholera toxin using the five

binding subunits to form a ring through which the active A subunit is inserted
through the membrane. (B) shows how a protein toxin monomer (e.
g. staphylococcal alpha toxin) forms oligomers on the membrane. Once bound
to the membrane, the hydrophobic sections of the toxin monomers (black line)
are able to collectively form the barrel of the channel that is sufficiently
hydrophobic to act as the transmembrane pore

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Toxins can be grouped according to the general sites of action; iience toxins can be:

• neurotoxins: tetanus toxin and botulinum toxin,

• enterotoxins: cholera toxin acts on intestinal epithelia,

• haemolysins: Staph aureus a- toxin is haemolytic to red cells,

• leucotoxins: toxins that attack leucocytes (white blood cells).

Note that toxins do not necessarily kill the target cell. If the toxin is damaging to the cell then it can be
called a cytotoxin, but toxins do not always kill cells (e.g. CT raises intracellular second messengers
(cyclic AMP)) which in turn opens chloride channels on the apical membrane of the enterocyte. The loss
of chloride ions from the cell draws water into the lumen of the gut, hence the diarrhoea. These events
may kill the host because of profound dehydration but the affected cells themselves are not killed!
Alternatively, toxins can be grouped according to the mode of action:

• enzymatic membrane lysis: phospholipases, sphingomyelinases,

• pore-forming toxins: Staph, aureus a- toxin,

• ADP-ribosylating toxins: diphtheria toxin, cholera toxin,

• inhibitors of neurotransmission: tetanus and botulinum toxin.

A number of exotoxin genes are held within bacteriophages (CT, DT) or other mobile genetic elements
( Table 8.3) . The diphtheria toxin (DT) actually resides on a particular bacteriophage. Only when
Corynebacterium diphtheriae becomes infected by the phage is it toxigenic. Hence, cultivating the
organism from a patient by itself does not mean that the patient had clinical diphtheria. Instead the
organism needs to be tested to see if it is toxigenic or not. This forces us to consider the idea that
diphtheria is a disease caused by a phage. The toxin in the phage happens to use the bacterium as the
vehicle for expression. In more general terms, the mobility of toxins occurs as part of a package of
virulence-factor coding genes, known as pathogenicity islands (PAIS, see later, p. 204) which, together,
have the ability to move between organisms (via transduction, conjugation or transformation). The
acquisition of such an 'island' will have dramatic consequences in conferring numerous virulence
factors. Thus, evolution of pathogenicity in bacteria occurs both by small gene changes (mutational
events, phase switching, etc.) and more substantial alterations in gene content by acquiring pathogenicity
islands.

'Doctor, I've been infected with a bacterium that has been infected with a phage.'

Table 8.3 Location of virulence genes

Organism Virulence factor Mobile element

Vibrio cholerae cholera toxin (CT) Bacteriophage

Corynebacterium diphtheriae diphtheria toxin (DT) Bacteriophage

Clostridium botulinum botulinum toxin (BT) Bacteriophage

Escherichia coli heat labile enterotoxin (LT) plasmid

heat stable enterotoxin (ST) plasmid

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Clostridium tetani tetanus toxin plasmid

Bacillus anthracis anthrax toxin complex plasmid

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8.4.2 BACTERIAL ENDOTOXIN

As the name suggests, endotoxin is a component of the Gram negative bacterial cell wall that happens to
be toxic. Annually, large numbers of people develop overwhelming infections from Gram negative
bacteria, usually as a complication of other illnesses. Multiplication of bacteria in the blood
(septicaemia) kills many people in hospitals every day and the endotoxin is one of the toxic components
that triggers numerous host responses in a bid to contain the bacterial infection. Endotoxin is not very
toxic in itself but large amounts can be released in septicaemia.

Whereas the exotoxins discussed above are proteins actively secreted across the cytoplasmic membrane,
bacterial endotoxin is released into the environment passively, as part of the growth process via blebbing
of the outer membrane or when Gram negative bacteria are lysed through antibiotics or host defence
mechanisms. In septicaemia with Neiserria meningitidis, the release of fragments of cell wall into the
bloodstream will bind complement components and antibodies and protect (at least partially) the intact
organism.

The part of the Gram negative cell wall that is endotoxin is the lipopoly saccharide (LPS). LPS consists
of three components:

]i|>id-A - core polysaccharide- O-sidc chain

Biirird \Vi ihc^iuter

iti<inil}r:5nt".

The toxic component.

Highly rouse rved

r:liL^jt>i[:al]v acrcjHS Gram

ne:galiv<tl>acl^ria.

M [ n I r. r.'il i: I V £ :[ Jilsf: rv t:i] .
N on -toxic Ijv itsalt.

A |■]^]ly.'ia(:^J^;5PU■lL^ i-.hain
thai vark^s 1x:lwi^L^iL
specie*; (and n1 rains) u{

Haccs the aqueous
environment (extends
outward from <5 Liter
membrane) thcrelore
readily ^seen' Ijy
antiliodie:!!.

Compared with protein exotoxins, the glycolipid components of endotoxin confers a degree of resistance

to inactivation by heat (heat stable). Its chemistry also makes it considerably less immunogenic than the

protein exotoxins which helps account for the absence of protective immunity in most people, young or

old.

Endotoxin has a number of effects on the body. The most feared end result is probably shock, the

collapse of blood pressure with concomitant failure of the vascular central organs such as liver, kidneys

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and brain. Shock is probably caused by the triggering of multiple signalling events induced by LPS. A
few examples include the ability of LPS to induce fever (pyrogenicity) by triggering macrophages to
release pyrogenic interleukins. Red blood cells will coagulate in septicaemia following endotoxin
activating the clotting systems.

Bacterial LPS is a significant health problem not because of its toxicity - LPS is only weakly toxic - but
because of its ubiquity. Anywhere Gram negative bacteria multiply, LPS is released. This poses
problems to manufacturers of sterile products for injection such as saline solutions for intravenous drips,
blood transfusion products and all

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injectable preparations. Solutions for injection may be sterilised by filtration, but this does not remove
endotoxin. Pharmaceutical manufacturers put considerable effort into preventing (or at least minimising)
the growth of contaminating bacteria in the solutions before sterilisation. Detection of LPS itself is
challenging because of the problems of preparing water, plastics and glassware that are free of
detectable LPS (e.g. 250°C for 30 minutes for all glassware, please).

8.5 ENVIRONMENTAL SIGNALS REGULATING VIRULENCE IN

BACTERIA

The interactions between the host and the pathogen are not fixed and predetermined but a dynamic
interplay with the host and invading microbe attempting to thwart the other's plans. The host has
numerous defence mechanisms whereas bacteria will need to not just avoid ehmination but multiply. In
evolutionary terms the organism will seek to optimise the chances of reproduction in the infected host
such that further rephcation can occur in a subsequent host. These outcomes are often but not always
(see vertical transmission) favoured by producing vast numbers of organisms which increase the chances
of infecting other hosts.

An infection by an organism typically involves the six stages of attachment, invasion, multiplication,
spread, evasion of host defence and exit. It follows then that the organism will need different virulence
determinants to deal with each stage of the process. In order to conserve resources, bacteria will tend to
synthesise the appropriate virulence factors at the appropriate time. This reflects the tight control that
bacteria have over their gene expression and biochemical pathways; indeed, virulence factors are coded
for in genes and are synthesised by specific biochemical pathways. It makes economical sense then to
regulate the synthesis virulence determinants as and when they are needed rather than manufacture them
continually. Again, as with the control of gene expression and biochemical metabohc pathways, the
organism will respond to environmental signals and produce the virulence determinants accordingly.
Some of the triggers that have been recognised as environmental signals are:

• iron,

• temperature,

• osmolarity,

• gaseous conditions,

• stress.

It is easy to see how these triggers can be used by a bacterium to help identify where it is both before
and during an infection (see Box 8.2 ).

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8.5.1 HOW DO THESE ENVIRONMENTAL CUES ACT?

8.5.1.1 2-component signalling

Bacteria need to monitor their environment so as to make efficient use of the cellular energy. Nutrients
are often only transiently available and the competition with other organisms is fierce. The rapid growth
of the bacteria will favour those organisms that quickly adapt to the prevailing conditions. Bacteria
couple the environmental signals to intracellular events through signal transducers. A transducer simply
converts a signal from one form to another. Temperature recorders convert the temperature into a signal
(voltage) that can drive an ink pen on a paper recorder. Likewise, signal trans-

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■ BOX 8.2 A DAY IN THE LIFE OF VIBRIO CHOLERAE

To illustrate the importance of environmental signalling in the behaviour of a pathogenic organism,
we will construct a theoretical series of events that are likely to occur in the course of events leading
to a case of cholera Vibrio cholerae causes the severe secretory diarrhoea in humans known as
cholera. The organism is excreted in high numbers in the fluid faeces and transmitted via the faeco-
oral route. The organism thus depends on the faecal contamination of drinking water/food. Once
deposited in a stream (as a result of a patient contaminating the river upstream) the organism infects
a new host. The organism will immediately notice changes in temperature as it passes from the
ambient temperature of the stream to the 37°C of the intestine of the patient. The stomach will
provide an immediate pH challenge and the subsequent exposure to the proximal small intestine will
then send the pH towards neutrality along with exposure to bile salts. The osmolarity will also have
changed from that of the river water and, as the bacteria approach the epithelial cells of the intestine
(enterocytes), the oxygen tension will drop dramatically. It is clear that the organism has
encountered numerous stimuli which can be used to trigger the virulence factors such as synthesis of
adhesins for attachment, mucolytic enzymes and cholera-toxin production. Once shed in the watery
faeces, the changes that occur upon the organism finding itself in a new water source will cue the
organism to stop the production of the virulence factors.

ducers convert the environmental signals such as temperature or pH into chemical signalling pathways
that result in alterations in metabolism and gene expression.

Stimulus -^ signal transduction -^
response

This can be expanded to show the two key components that mediate the process:

Stimulus -^ sensor -^ response regulator -^
response

The sensor (component I) detects the environmental signals. This activation of the sensor causes a
protein kinase to become phosphorylated. The phosphorylated kinase then activates the response
regulator (component II), also by phosphorylation which mediates the response, for example
transcription of certain virulence factors. Whilst we have used virulence factors as the example, 2
component signalling is employed by bacteria to react to environmental stimuU such as changes in
ammonium and phosphate concentrations, gradients of attractant and repellant compounds (which
regulate chemotactic responses via flagellar activity).

8.6 ORIGINS OF VIRULENCE DETERMINANTS

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The proportion of bacteria that are pathogenic for humans and animals is small when compared to the
total number of cultured bacterial species. Bacteria that normally exist in non-human natural
environments, such as soil or water, will be adapted to survive in this econiche. They possess genes
appropriate to life in soil and water. If any of those bacteria possess the ability to adapt to replication in
humans, they will need a different set of genes appropriate to deal with the conditions in human tissues.
This predicts that pathogenic bacteria will possess two classes of genes: those necessary for life in the
environment (soil, water, etc.) and those necessary for life in a susceptible host. The

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Page 204

latter will be absent from organisms that are non-pathogenic for humans. What about bacteria that Hve as
normal harmless commensals (as part of the normal flora)? The same concept applies. What differs is
the definition of 'normal environment'. The normal flora reside on the host but do not invade tissues.
The genes necessary for invasion will be absent in the members of the normal flora.
The key difference between primary pathogens and non-pathogenic bacteria appear to be the possession
of the virulence determinants. With the exception of obligate intracellular pathogens such as Rickettsia,
it does not appear obvious that pathogens are markedly defective in their basic physiology compared
with commensals. In contrast, pathogens possess additional distinct characteristics that can cause disease
that are absent in the harmless species. These characteristics will often function as virulence factors.
The source of the virulence factors in most instances are not known, but the fact that many are carried on
mobile genetic elements helps explain the evolution of pathogenic bacteria. Table 8.3 lists a few
examples of genes that code for virulence factors that are located on plasmids and bacteriophages. As
stated above, certain virulence determinants are also found within transposable elements (transposons)
which will permit genetic mobihty such as the incorporation of the genes onto the bacterial
chromosome.

The accumulation of virulence determinants in mobile genetic elements has resulted in large regions of
genes that all code for virulence determinants, called pathogenicity islands. The characters coded for by
pathogenicity islands include production of haemolysins, fimbriae, ability to invade cells, and an abihty
to acquire iron. Pathogenicity islands have certain unique features:

• they are bracketed by DNA sequences characteristic of transposons, i.e. direct repeat sequences or
insertion sequences.

• pathogenicity islands delete with a higher frequency than native bacterial DNA.

• the DNA base composition (measured as G:C ratio) is distinct from native chromosome sequence.

• pathogenicity islands insertion sites match those for phage insertion during transduction.

As pathogenicity islands are not present in non-pathogenic bacteria, these characteristics support the
view that they have spread amongst bacterial populations through horizontal gene transfer mechanisms.
The collection of virulence determinants in one package helps the bacterium co-ordinate the expression
of these genes as and when necessary.

8.6.1 WHY THEN HAVEN'T ALL ORGANISMS BECOME PRIMARY

PATHOGENS?

Presumably, the successful incorporation of pathogenicity islands into the normal regulation of the
bacterial cell is not always achieved. The expression of functional virulence factors may not be
compatible in all bacteria, despite the presence of the appropriate genes within the island itself.
Furthermore, unless the additional genetic load proves essential for survival and multipHcation, the
pathogenicity islands will become just additional baggage such that the organism will lose out in

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competition with the wild type strains.

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8.7 HOST RESPONSE AS CAUSE OF DAMAGE/SYMPTOMS:
CHOLERA VERSUS DYSENTERY

Armed with all this information, can one predict the outcome of an infection? We shall close this chapter
by comparing the pathogenesis of two bacteria that cause gastrointestinal disease, Vibrio cholerae, the
aetiological agent of cholera and Shigella dysenteriae, the aetiological agent of bacillary dysentery. Both
organisms are strict pathogens of humans with no reservoir other than humans. The infectious dose of
Vibrio cholerae is high, thought to be at least 10^ organisms, whereas Shigella dysenteriae has a low
infectious dose of 100-1000 organisms. The pathology of cholera results virtually solely from the action
of protein exotoxins, in particular cholera toxin (CT), secreted by the organism once attached to the
small intestine. CT causes profound diarrhoea by activating ion secretion in the host epithelia with
almost no inflammatory component. Dysentery, however, is an infection of the colon and the diarrhoea
is a result of tissue destruction following the invasion of the shigellae into the epitheha. An
inflammatory response is provoked and the ulcers that form leak pus and blood along with the diarrhoea.
The shigellae possess plasmid-borne toxins that inhibit protein synthesis (shiga toxin) in the endothehum
of the intestinal blood vessels. In both organisms the toxins are coded for by mobile genetic elements
(phage and plasmids) yet the diseases are very different. In cholera the host immune response appears to
play almost no part in the disease, whereas dysentery is characterised by the tissue damage and
aggressive host responses (inflammation) in the bowel. This comparison tells us that host specificity,
infectious dose and site of infection do not predict the outcome of infection.

■ SUMMARY

Bacteria that are pathogenic to humans represent a relatively small fraction of the numbers
of organisms that Uve on humans as commensals and those that we encounter only
transiently in our environment. Those bacteria that possess the abihty to cause damage to
humans appear to do so through the use of virulence factors acquired as pathogenicity
islands. Primary pathogens cause disease by gaining access to niches in the host that are
usually unavailable to non-pathogens. Pathogencity is a quahtative expression of this
abihty. The extent to which this disease occurs is described quantitatively as 'virulence'.
Secondary pathogens are unable to breach the host defences. It is possible to define six key
steps in the pathogenic process and name some of the appropriate virulence factors:

(i) attachment to specific tissues via adhesins,

(ii) invasion of host tissues via invasins,

(iii) multiply through acquisition of nutrients via pabulins,

(iv) spread within host tissue if necessary,

(v) evade or resist host defences via evasins.

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(vi) exit.

Pathogenesis, however, is more accurately seen as the total outcome of the interactions
between the bacterium and the host. Pathogenesis is thus more complex than a list of six
steps. The genetic determinants for virulence factors are often located within distinct
regions of the bacterial genome called 'pathogenicity islands'. Genetic manipulation of
virulence factor genes provides a valuable step in determining the role of the microbial
products but is very dependent (i.e. vary) according to the model system in which the
experiments are performed.

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Page 206

RECOMMENDED READING

Alouf, J.E. and Freer, J.H. (eds) (1999) The Comprehensive Sourcebook of Bacterial Protein Toxins, 2nd
edition, Academic Press, London, UK.

Davies, D.H., Halablab, M.A., Clarke, J., Cox, F.E.G. and Young, T.W.K. (1999) Infection and

Immunity, Taylor & Francis, London, UK.

Finlay, B.B. and Falkow, S. (1989) Common themes in microbial pathogenicity. Microbiol. Rev. 53,

210-30.

Finlay, B.B. and Falkow, S. (1997) Common themes in microbial pathogenicity revisited. Microbiol.

Mol. Biol. Rev. 61, 136-69.

Mims, C.A., Nash, A. and Stephen, J. (2000) Mims' Pathogenesis of Infectious Disease, 5th edition.

Academic Press, London, UK.

Orndorff, P.E. Bacterial pathogenicity, in Balows, A., Tmper, H.G., Dworkin, M., Harder, W. and

Schleifer, K.-H. (1991) The Prokaryotes. A Handbook on the Biology of Bacteria, 2nd edition.

Springer, Berlin.

Salyers, A. A. and Whitt, D.D. (1994) Bacterial Pathogenesis: a Molecular Approach, ASM Press,

Washington, USA.

Schlaecter, M., Engelberg, N.C., Eisenstein, B.I. and Medoff, G. (eds) (1998) Mechanisms of

Microbial Disease, 3rd edition, Lippincott, Williams & Wilkins, Baltimore, USA,

REVIEW QUESTIONS

Question 8.1 What infectious diseases do not follow the six steps followed by a pathogen?

Question 8.2 If rapid replication rate is a key feature of bacterial pathogens, is Mycobacterium
tuberculosis a successful pathogen?

Question 8.3 Compare and contrast bacterial exotoxins and endotoxin.

Question 8.4 What are the problems with the original Koch's postulates?

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Page 207

VIRUSES AS PARASITES

At a conservative estimate, there are at least thirty different viruses from fifteen taxonomic families that
are common causes of human illness. This number does not take into consideration the serological types
that exist in many of these viruses. So the common cold we are taking as one virus, whereas there are
over eighty serological types of rhinovirus. As every human will be infected by viruses in their lifetime,
we must concede that viruses are efficient parasites of humans. This chapter outlines some of the
features of viruses that contribute to their success as human pathogens.

9.1 TYPES OF CLINICAL INFECTION

The four broad types of viral infections recognised in humans are hsted in Table 9.1 , and the relative

time course for each is shown in Figure 9.1 .

Because it is only those people with symptoms who come to our attention, it is easy to overlook the fact

that most viral infections are asymptomatic. For the people who demonstrate cUnical illness, the severity

will range between mild and severe. The proportion of susceptible people who develop illness from

those who are infected is called the attack rate ( Table 9.2) , although it is not a true 'rate' and gives no

indication of the total number of people infected.

The route of transmission does not fully account for the variation in attack rate between viruses,

although clearly direct inoculation by parenteral transmission is likely

Table 9.1 Types of viral infection

Duration

Extent

Examples

Acute

Local

rhinovirus: the common cold

Systemic

measles

Persistent

chronic hepatitis B
Epstein Barr virus

Latent

Herpes simplex: cold sore
Varicella zoster: shingles

Slow

Bovine spongieform
encephalopathy

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• Figure 9.1 Viral infections. The time course taken by different types of virus
infection. Note that the time scale varies between types of infection

Time (days)

Time (years)

Time (days)

Time (years)

Table 9.2 Attack rates for viruses'*

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Disease Virus Attack rate

Poliomyelitis poliovirus <\%

Infectious mononucleosis Epstein Barr virus \%

Rubella rubella virus 50%

Rabies rabies virus 95%

*The attack rate may also be called the Subclinical/Clinical ratio of those people infected. The values give the
percentage of those infected that have clinical features. Remember that the attack rate is not the same as
infectivity. A virus may be highly infectious but show no clinical features.

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Page 209

to be more efficient in establishing an infection than transmission via aerosols. UnUke bacterial
infections, many viral infections have a more damaging effect in older people compared to children. The
childhood viral infections such as chickenpox and mumps are famously unforgiving in adults, whereas
in children they cause rashes but otherwise usually fail to bother the child unduly. Why this should be is
largely unknown.

9.2 STRATEGIES FOR SUCCESSFUL VIRAL INFECTIONS

Successful? By this we mean reproductive success: able to rephcate and persist within a population. As
obhgate intracellular parasites, viruses have an absolute requirement for their host. If the virus causes
damage or even death of the host it must do so after it has had sufficient time to manufacture high
numbers of new viruses and release them into the environment. Note also that whilst many bacteria can
multiply in the environment without the requirement of animal tissue, viruses can only multiply in a
host. Periods spent in the environment are simply a holding event for viruses in which no multiphcation
can occur. Viral rephcation can be seen from a perspective in which the availabihty of the host
determines the strategy adopted for infecting humans. In dense populations rapid and short periods of
rephcation serve to transmit the virus such that new (and readily available) hosts becoming infected. In
sparsely populated areas, the virus wih need to produce virus over a longer period so that the few new
hosts that come into contact with the infected host can be infected. This distinction is reflected in two
broad groupings of viral infections of humans: acute and persistent. The differences are summarised in
Table 9.3 .

The acute infections rely on rapid multiplication of virus in order to manufacture and shed (transmit)
new virus before the host has had time to mount a neutrahsing immunity. Examples will be measles and
influenza viruses, both RNA viruses and ones that require sufficient numbers of susceptible hosts in
order to maintain themselves in the community. With the measles virus, humans appear to be the only
host. With influenza virus, however, a wide range of animals can be infected and the virus is therefore a
zoonosis, periodically sweeping into town as an epidemic having transferred from an animal reservoir.
Persistent infections in the host (not the environment) are those in which the virus is not ehminated but,
having established itself, will persist for the hfetime of the host. The virus is either replicating at a very
low level continually, such as chronic hepatitis virus infections, or is latent for most of the time but
periodically undergoes a short burst of full rephcation in which infectious virus is manufactured and
released, e.g. Herpes simplex in cold sores. Many persistent virus infections are acquired as

Table 9.3 Acute and persistent virus infections

Acute viral infections Persistent viral infections

Typically RNA virus with high mutation rate Typically DNA virus

No persistence following infection Persistence in host

Symptomatic infection Infections often asymptomatic

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Large numbers of virus produced in short period Virus numbers released episodically or

chronically over long period
Wide host range Limited host range

Horizontal transmission suited to high Vertical transmission, sustainable in low

population density density populations

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Page 210

children with little or no pathology and then persist for life. This lifetime of infection will optimise the
chances of transmission, preferably to other children. Persistent infections, by definition, require that the
virus is maintained within the host cell and thus seeks to avoid kilUng the cell (is not cytolytic).
Integration of the viral nucleic acid into the host chromosome is an appropriate means to help promote
long-term survival in the host and avoid contact with the immune system which will also seek to destroy
the infected cells. Whilst persistent virus infections are mostly DNA viruses such as Herpes and
papillomaviruses, retroviruses will insert themselves as proviral DNA into the host genome following
reverse transcription from RNA and many retrovirus infections are persistent.

The differences in acute and persistent infections have consequences for antiviral treatments. Specific
antiviral chemotherapy will only have significant effects on persistent infections where there is
continued production of virus. In acute infections, the virus is produced in greatest numbers just prior to
the development of symptoms. By the time the patient has sought help, the virus is no longer in full
manufacture.

Other differences result from the properties of DNA and RNA viruses. You may recall from Chapter 3
that viruses contain either DNA or RNA and not both. RNA viruses have a reduced gene capacity
compared with DNA viruses. DNA viruses often possess sufficient genetic space (several hundred
genes) such that roughly 50 per cent code for proteins that can interfere with the host immunity (see
below, p. 219). RNA viruses do not have sufficient gene space and utiHse genetic variation in order to
generate new variants of virus.

9.3 STAGES IN HUMAN INFECTIONS

The key stages in the process of an infection are:

• entry into host,

• primary replication,

• spread within host,

• exit from host,

• evade host response.

9.3.1 ENTRY INTO HOST

The routes of transmission will depend on the site of the body that represent the exit site for the
infectious virus. The exit of virus from the body can occur via several vehicles: urine, faeces, semen,
saUva, blood, milk, skin, squamous epitheha. Hepatitis B virus is released into the bloodstream from the

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liver and the transfer of infected blood is the major vehicle for transmission. Examples include the
sharing of needles amongst drug abusers and the accidental transmission following blood transfusion.
With hepatitis B the transmission is parenteral and therefore the virus cannot be transmitted by sneezing
or touching. Note how the site of multiplication (the liver) need not be the infectious site (blood).
Conversely, rhinoviruses, the aetiological agents of the common cold, multiply in the upper respiratory
tract epithelia and are transmitted via the secretions from the runny nose.

There are several ways of listing routes of transmission of infectious agents (as given in Chapter 7 ). The
traditional list is as follows:

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• aerosol,

• faeco-oral,

• parenteral (including vertical transmission),

• sexually,

• direct contact.

The routes of horizontal transmission can be related to the sites in the human body that represent the
major sites of entry:

• skin,

• gastrointestinal tract,

• respiratory tract,

• genito-urinary tract.

This list can be sensibly reduced by grouping the last three into one to yield three key routes of entry for
a virus into a new human host:

• Entry through mucous membranes: respiratory, genito-urinary and gastrointestinal tracts and the
conjunctiva.

• Entry through skin: insect bites (e.g. yellow fever virus); animal bites (e.g. rabies virus); skin to
skin (e.g. papillomavirus (warts, veruccas), Orf virus).

• Parenteral transmission: vertical transmission, direct inoculation into blood (needle, insect or
animal bite) or blood transfer through trauma (including sexual transmission).

Vertical transmission has clear advantages in that the foetus has the same blood supply as the mother
and has no defences against virus attack other than those present in the mother. One of the drawbacks of
vertical transmission is the risk of damaging the developing child in utero, i.e. foetal death and abortion.
A less extreme outcome is teratogenesis (see Box 9.1 ). Vertical transmission also includes transmission
via the germ line (i.e. through infected sperm or ovaries).

The properties of the virus will also influence those routes that are likely to be successful for the virus.
Viruses that cause gastroenteritis will need to resist exposure to

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■ BOX 9.1 VIRAL TERATOGENESIS

Teratogenesis is the ability to cause defects in the foetus. The rubella virus is one of very few
viruses that are able to cause congenital malformations in the developing foetus. The vertical
transmission of the virus occurs simply through the unfortunate timing whereby the mother becomes
infected with the virus for the first time (i.e. is susceptible) in the first three months of pregnancy.
When the mother becomes viraemic the virus is able to traverse the defences of the placenta and
infect the growing child in utero. Up to 30 per cent of babies infected in the first trimester are bom
with malformations such as neuronal, eye and heart defects. Infections acquired in the last two
trimesters rarely result in any defects. Congenital rubella occurs because the virus infects cells in a
key stage of development. Severe infection of the foetus will be unsustainable and lead to stillbirth or
abortion. Children born with congenital rubella are likely to represent those that have been able to
repair and develop sufficiently, in spite of the viral damage.

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gastric acid, bile acids and intestinal proteases if they are to reach the small intestine in any number and
condition. As exempHfied by rotaviruses, viruses causing gastroenteritis are not enveloped (which would
be dissolved by bile acids) and tolerate acid pH and digestion by proteases. In fact, certain viruses are
'activated' by host proteases which act on surface proteins on the viral capsid. Numerous viruses that
infect the respiratory tract are enveloped (notably influenza), reflecting the absence of lipid-dissolving
secretions such as bile in the respiratory tract lining. For this reason the term 'gastric flu' is a misnomer.
Influenza viruses cannot infect the stomach.

9.3.2 PRIMARY REPLICATION

This depends on the site of entry: local infections will usually replicate at the site of entry. For example,
the rhinoviruses that cause the common cold will repHcate in the epithelia in the nose and upper
respiratory tract. Local infections may invade the local lymph nodes but otherwise are restricted to the
site of entry. Systemic infections tend to have two-stage infection. The primary replication site serves as
an amplification of the virus from the small initial infectious dose and will be asymptomatic. Once the
virus has replicated at the local lymph node or epithelium, large numbers of virus are then released into
the bloodstream where they can attack the secondary tissues with corresponding tissue damage.

9.3.3 SPREAD WITHIN HOST

The common cold will mostly restrict itself to the upper respiratory tract. Not all virus infections are
limited to locaHsed infections but, instead, spread throughout the body (systemic infection) and have
secondary rounds of replication in other organs, yet not every cell in the body will succumb to viral
infection. The pathology and symptoms help distinguish different infections (e.g. rubella virus often
causes joint inflammation, whereas hepatitis viruses target the Hver). The reason that viruses show such
tissue tropism (a restricted range of tissues that become infected) depends in part on the presence or
absence of appropriate receptor molecules on the host cell membranes and the relative permissiveness of
the cell type within that organ. This combination of factors means that the distribution of viral receptors
may be wider than any of the observed tissue tropism of the virus. Conversely, cell types other than
those in tissues displaying symptoms may be permissive for the virus but asymptomatic. Unless one
investigates all these other cells that may be infected, one can never be sure.
Many of the highly infectious virus infections of childhood (measles, chickenpox, rubella, mumps)
cause systemic infections reflecting rounds of viral replication in more than one organ of the body. The
virus enters at the appropriate site (skin, mucous membrane) and multipHes at the nearest available
tissue (e.g. epithelia). The virus spreads to the local lymph nodes from which it is released into the
bloodstream. This first viraemic phase carries the virus throughout the body and seeds the virus in
organs such as the liver, spleen and bone marrow. After a further round of multipUcation, a second

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viraemic phase will deposit the vims in organs throughout the body. The second viraemia may be the
release of large numbers of virus into the bloodstream or the movement of virus within the cells of the
immune system (EBV, measles, HIV). The actual organ that is targeted from the secondary viraemia is
called the 'target organ' and will probably manifest itself in the symptoms. Only at this stage, however,
does overt disease become apparent with the patient showing signs and symptoms typical of the
infection in question. The childhood rashes seen with measles and chickenpox will appear following the
second viraemia. The exact cause of the viral rashes varies. Some

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rashes represent virus-infected cells being destroyed by the immune response, whereas others represent
blood vessel damage due to virus.

Having presented a generalised pattern of systemic viral infections, it is important to point out that
certain virus infections do not employ viraemic phases to spread through the body. Rabies virus moves
along the nerve fibres from the site of the bite towards the central nervous system. A significant
advantage of this pathway is the relative inaccessibility of the nervous system to immune surveillance
and the specific protection from the circulating antibodies, which cannot penetrate cells.
The different pathways taken by viruses in the course of an infection will often be reflected in the
incubation period. Local infections have shorter incubation periods (up to a week) whereas the systemic
infections incubate for 10 days or more. The important feature of this process is the long incubation
period during which the patient becomes infectious but shows no clinical disease. (The time from
between becoming infected and becoming infectious is called the 'latent period' .) This is typical of
many viral infections, where the development of disease occurs after maximum numbers of virus are
being excreted. In this way transmission is favoured because the susceptible hosts are not running the
other way having seen the rash!

9.3.4 EXIT FROM HOST

What is the exit point from one host is likely to be the entry point for infection in the the next host. In
other words, the sites of viral replication will determine how and where the virus is transmitted to the
next host. Local infections will be shed from the local mucosa in secretions. The common cold is a
testament to the efficiency of the 'secretions'. Blood-borne infections are that much more difficult to
transmit horizontally but nevertheless are able to escape through blood via insects, blood transfusion,
and trauma during sexual contact.

A list of secretions that can contain viruses (saliva and nasal secretions, semen, milk, urine, vesicle
fluids and faeces) does not take into account the duration of excretion of the virus, nor the numbers of
virus, both of which can vary between virus depending on the strategy adopted. Is it better to shed large
numbers of virus for a short while or smaller numbers over a longer period?

9.3.5 EVADE HOST RESPONSE

This is dealt with in a later section (pp. 219-22).

9.4 INTERACTIONS BETWEEN VIRUSES AND EUKARYOTIC CELLS

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Having considered the broad strategy for viral replication in humans, what are the cellular events that
underlie the infection in the whole animal? The suggested reasons as to why some viruses are
pathogenic will be considered by examining the effects of viruses on single cells and the response of the
host to the virus.

Viral infections do not always result in manufacture of new virus. The terms productive or non-
productive infections reflect whether manufacture of new infectious virus is achieved or not. Cells that
do not permit the immediate manufacture of infectious virus are also described as non-permissive.
Note, however (see 'latent infections', below, p. 215), non-productive outcomes are not necessarily
without consequence. Table 9.4 summarises the outcomes of virus infections and their impact on the
cells. One key point about arranging the outcomes in such a manner is that cell death in virus-infected
cells provides no long-term survival in the host (literally, a 'dead

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Table 9.4 Virus-cell outcomes

Virus infection Cell outcome Mechanism

Productive Lethal Lysis

Non-lethal Persistence

Transformation
(Latency)
Non-productive Lethal Apoptosis

Non-lethal Transformation

(Latency)

end' !) whereas non-lethal infections permit the development of alternative stategies for replication and
spread. Latency is in parentheses because at some point complete virus is produced within the cell. This
means that the infected cell is productive. However, latent infections typically occur in cells that are
largely non-permissive.

Perhaps the ideal strategy for a virus is the productive, non-lytic, 'silent' infection akin to the carrier
state in certain bacterial infections. In this way the virus will be able to multiply and transmit itself
without the host developing adverse illness. Such virus infections of humans probably exist but the
problems of such a strategy are twofold: the host will develop immunity to the virus and the virus needs
to transmit itself to new hosts. The persistent and latent infections are close in terms of strategy to this
silent infection model, but they both have drawbacks. Persistent infections such as chronic hepatitis B
infection results in Hver damage. Latent infections such as Herpes simplex cold sores have to reactivate
occasionally in order to spread to new hosts. The development of the cold sore betrays the presence of
the virus to the host as well as risking treatment with antiviral drugs. For many viral infections, only a
minority of those people infected develop clinical disease. Alternatively, if as is the case with persistent
infections, the virus cannot remain silent indefinitely, then it will at least remain silent for as long as
possible in order to produce enough virus for transmission to the next host.

Under the heading of productive or non-productive we can discuss the outcomes of a virus infecting a
cell as listed on the right-hand side of Table 9.4

9.4.1 LYTIC INFECTION

Lysis of virus-infected host cells will occur either as a consequence of massive release of new virus
through the host plasma membrane or through the disruption of host metabolism and protein synthesis as
a result of the virus diverting the host machinery into virus manufacture.

9.4.2 PERSISTENT INFECTION

Here, virus-infected cells are not killed quickly but continue to produce intact virus. For viruses to

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persist it is important that they delay or prevent effective immunity developing against them. Hepatitis B
virus infections of the liver become chronic when an effective cell-mediated immunity fails to develop
and the virus is not eradicated. The host gains from this apparent failure of immunity in that liver failure
through clinical hepatitis is avoided, but the payback is the persistence of the virus and subsequent risk
of chronic hepatitis and liver cancer.

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9.4.3 LATENT INFECTION

Latent infections (dormant infections) result from vims incorporation into the host genome such that
complete virus is not manufactured. Typically they are DNA viruses. A subset of viral genes are
expressed so as to produce viral proteins necessary for regulation and maintenance of the viral genome
in the cell. Herpes simplex (HSV) and Varicella zoster (VZV) viruses both become latent by 'hiding' in
the sensory nerve ganglia and, when reactivated, cause cold sores and shingles respectively. Reactivated
virus, infected cells are damaged as a result (HSV-infected cells are lysed in vitro) whereas, in the latent
stage, the cells are intact. Reactivation from latency risks the host developing immunity, consequently
VZV can only occur once. HSV appears to avoid generating protective immunity. It is interesting to note
that the HSV is latent in nerve cell type. Nerves are postmitotic, i.e. non-dividing cell type, and
therefore the host genome will not replicate and expose the virus. Epstein Barr virus establishes latency
in B lymphocytes, cells which can undergo periodic repeated proliferation, thereby permitting
opportunities for the virus to multiply and infect new hosts.

9.4.4 ABORTIVE INFECTION

Viruses do not always gain control of the infected cell so that, in abortive infections, replication of the
virus is not completed and the viral genetic material is destroyed. The host cell may lack suitable
enzymes for the complete manufacture of new virion or virus produced is incomplete or defective in
some way. This restriction to viral replication has practical implications. Different cell lines are needed
for the successful replication of viruses in cell culture systems. Abortive infections may cause cytopathic
damage (CPE) because of the viral replication process, but the release of infectious virus is not achieved.

9.4.5 TRANSFORMING INFECTION

Occasionally, cells that have been infected with certain viruses exhibit a number of characteristics that
are not seen in 'normal' uninfected cells. These newly acquired features are related to the regulation of
the growth and multiplication of the cells. Essentially the virus causes the cell to extend its normal
replicative lifespan. The normal limited number of times the cell would replicate before senescing (i.e.
stop proliferating but still remain metabolically active) is extended in transformed cells. In other words,
the cells keep multiplying. As intracellular parasites, viruses can only benefit from the cell upregulating
biosynthetic machinery (and make more virus components) and prolong the number of times it divides
(thereby increasing the virus yield, for longer). The properties of transformed cells are given in Box 9.2 .
Viral transformation occurs following infection with viruses that are able to incorporate viral DNA into
the host cell chromosome. Once inserted into the host genome, the virus is called a provirus. This

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ability is limited to DNA viruses (the viral DNA incorporates directly)

■ BOX 9.2 CHARACTERISTICS OF TRANSFORMED CELLS

• Not inhibited by contact with other cells (absent contact inhibition) and hence will
grow as multiple layers.

• Much extended, if not indefinite, number of divisions (continuous cell line).

• Reduced requirement for serum in order to grow.

• Altered number of chromosomes (aneuploid).

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Table 9.5 Transforming viruses

Virus

Family

Viral nucleic acid

Papovavirus

DNA

Papovavirus

DNA

Herpes virus

DNA

Retrovirus

RNA

Retrovirus

RNA

SV40

Human papillomavirus'''

Epstein Barr virus*

Rous sarcoma virus

Human T-cell lympho trophic virus*

*Viruses that infect humans.

and retroviruses (the DNA is synthesised from the viral RNA using reverse transcriptase and
subsequently incorporated into the host genome). It is important to place these events in perspective.
Transformation is a rare event since only certain viruses can transform human cells ( Table 9.5) and the

frequency is low (e.g. approximately 1 in every 10^ cells infected with SV40 vims). Finally, cells that
are transformed are not necessarily neoplastic (tumour forming) because transformation is only one
stage of many in the process where a eukaryotic cell becomes neoplastic and it does not follow that a
transformed cell will become malignant.

The use of the term 'transformation' appHes to virus-infected eukaryotic cells and should be
distinguished from the transformation of bacterial cells by the uptake of naked DNA (as
described in Chapter 1) .

9.5 THE RESPONSE OF HOST CELLS TO VIRUS INFECTION

An immune response is necessary for the prevention of uncontrolled prohferation of virus. Viruses will
need to suppress the immune response sufficient to permit adequate rephcation and
transmission. Complete suppression is unnecessary and would be costly in terms of viral resources and
would allow the prohferation of other micro-organisms.

The balance that exists between mounting a vigorous immune response that is damaging to
the host tissues and not being able to contain the virus can be illustrated in experiments in
people suffering from the common cold. The general miserable symptoms were reduced by
patients who took anti-inflammatory pain killers but the group who took nothing had a shorter
period of illness at the expense of feeling worse.

It is said that viruses do not produce typical toxins as seen in toxigenic bacteria. This might reflect the
overall Hmits in our knowledge of viral genomes. Certainly it has become clear that viruses do code for
proteins that are potentially harmful. Several of the viruses that are linked with human cancers produce

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proteins that act to prevent the host cell from undergoing apoptosis (programmed cell suicide).
Apoptosis should be contrasted with necrosis. It is not possible to summarise the toxicity of viruses into
one dominant effect because of the diverse strategies employed by the viruses to successfully replicate
and be transmitted.

The mammalian cell has developed several mechanisms to try to limit the virus from overwhelming the
host. Whilst we all recognise the symptoms of a developing cold or other viral infection, the extent to
which this directly represents the toxicity of the viral infection or our reaction to an invader is not clear.
The host response to infection at the cellular level is an appropriate place to examine the fight back.
The human host responds to all infectious agents with cell-mediated and humoral responses. Against
intracellular pathogens, cell-mediated responses are generally more important since, although antibodies
can mop up free virus in body fluids such as the bloodstream, they cannot penetrate cells. For antiviral
immunity, one important group of polypeptides are the chemokines and cytokines. These small
molecular weight proteins regulate the interaction between immediate immune response (the innate
immunity) and the adaptive immune response that confers specific immunity. Chemokines and
cytokines have homeostatic and proinflammatory functions. The former concerns the maintenance of
haematopoietic system (lymphoid cell lineages from the bone marrow) and the latter is involved in the
recruitment of leukocytes from the circulation to the sites of infection. Chemokines/cytokines act on
cells through

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binding to appropriate cell surface receptors triggering the synthesis of antiviral proteins. Chemokine
receptors have proved to be important in viral infections through the discovery that the HIV binds to a
particular chemokine receptor on lymphocytes. The people with the rare congenital deficiency in this
receptor are resistant to infection with HIV. Cytokines are released from activated immune cells in
response to infection and they exert antiviral activity. There are increasing numbers of cytokines being
identified but the key antiviral cytokines are interferon-alpha, -beta, -gamma and tumour necrosis
factor. Cytokines bind to specific receptors on the appropriate host cells (typically macrophages and
lymphocytes) which activate signalling pathways that, in turn, activate various intracellular antiviral
events. Many of the mechanisms are as yet unknown but the inhibition of the replication of the virus is
the net effect. The antiviral mechanisms activated by cytokines can result in the lysis of the infected cell
or more subtle interference of viral replication without killing the infected host cell. Destroying infected
cells may be too damaging when dealing with cells that cannot regenerate, for example nerves or in
massive infections of a vital organ (e.g. extensive liver damage by hepatitis viruses) and a non-cytolytic,
non-cytopathic mechanism is required.

9.5.1 PRODUCTION OF INTERFERON

It was demonstrated in the 1950s that the culture medium bathing virus infected cell lines could inhibit
the multiplication of viruses in separate flasks. The active substances, termed interferons, are cytokines
(as discussed above) and are produced within hours of virus infection to limit the spread of virus in the
host whilst specific immune responses are developing. Most cell types can synthesise interferon but they
can be grouped into three broad types according to the cell type that manufactures them.

• IFN-H (alpha): leucocyte interferon,

• IFN-P (beta): fibroblast interferon,

• IFN- f^ (delta): immune interferon (activated T-lymphocytes and NK cells).

Double-stranded RNA is the most potent stimulus for the production and release of interferon alpha and
beta which act to prevent or limit the surrounding cells from becoming infected. Interferons are
prophylactic rather than curative. The actions of interferons are somewhat varied in that they can
modulate the activity of immune cells as well as induce resistance to viral infection. Two important
antiviral processes induced by interferon in virus-infected cells are:

• the inhibition of viral-induced protein synthesis, and

• degradation of viral mRNA and rRNA.

The central player in cells stimulated by IFN is protein kinase R (PKR). The letter R is taken from the
viral dsi?NA that activates the transcription of this protein kinase. PKR produced by cells stimulated by
IFN inhibits viral driven protein synthesis by binding to the double- stranded RNA. PKR will also trigger
apoptosis, presumably as a last resort following uncontrolled viral replication.

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9.5.2 TRIGGERING APOPTOSIS

Controlled cell death (apoptosis) is a mechanism by which multicellular organisms remove unwanted
cells either during development (wonderfully illustrated by the loss

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of the tail in the developing tadpole) or respond to genomic abnormalities (pre-cancerous changes).
Apoptosis is also of great value as a defence mechanism against virus infection in cells. Apoptosis, in
contrast to necrosis, is a tightly controlled process that leaves no mess and causes no damage to
surrounding cells. As with host cell nucleases, viruses have acquired mechanisms with which to
counteract the triggering of apoptosis.

9.6 VIRUS-INDUCED TISSUE DAMAGE: VIRUS OR HOST?

To what extent does the rephcation of the virus in the tissue cause the disease? The varied outcomes
following a virus infecting a cell are given above (p. 213). Some of these help explain why virus
infections can cause cell damage. For example, lytic infections will result in the loss of function of the
infected cells. If sufficient numbers of cells are damaged in this way then the function of the organ may
be compromised. Rotaviruses cause diarrhoea in humans and animals. The infected cells of the vilh of
the ileum are shed resulting in the loss of absorptive surface area contributing to the diarrhoea. The
diarrhoea is a direct consequence of the viral damage in the enterocytes. Other examples of tissue
damage are indirect. In true chnical pohomyehtis the virus damages the nerve cells that serve the muscle
cells of the hmbs. The damage to the nerve cells by the virus causes the muscle to atrophy. Virus cannot
be found in the affected muscles themselves. Furthermore, pohomyehtis virus normahy only multiphes
within the enterocytes of the smah intestine and they show no morphological alterations. This serves to
remind us that the CPE observed in the cell lines used to grow viruses in the laboratory are not
necessarily reflected in the natural host cells.

The common symptoms of many viral infections, myalgia and headache, are usually indirect
consequences of the host immune response (often cytokines such as tumour necrosis factors)
rather than direct viral rephcation in the muscles and brain.

Different infection strategies are employed by different viruses. The extremes are an acute, hit-and-run
approach or a chronic persistent type of infection. Viruses choosing the former option wih have less
concern for the effects of rapid viral multiphcation on the host. Such viruses wih tend to suppress or
inactivate acute (innate) mechanisms of host defence so as to gain time for the rapid multiphcation of
new virus. If the host is fatally damaged by the infection, as long as sufficient virus has been
manufactured such that the infection is transmitted to new hosts, then the death of the host is of no
concern. Aggressive virus multiphcation wih probably result in protective immunity such that, if the
host recovers, he or she wih not be available for the virus to reinfect. Chronic infections wih need to
adopt those strategies that deal with the longer term problems of specific immunity, i.e. the development
of specific antibodies and cell-mediated cytotoxicity.
The reaction of the host to intracellular parasites, viruses in particular, will often result in tissue damage.

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Immunopathological damage will cause tissue pathology by at least two mechanisms.

■
9.6.1 IMMUNE CELL-MEDIATED CYTOTOXICITY

In tissues with rapid turnover, virus-infected cells that are lost may be replaced (e.g. intestinal and skin
epithelia). In non-replicating tissues like the heart and nerve tissue virus-infected cells cannot be lost so
readily without possible functional deficit. If, therefore, the immune response damages these cells, the
organ suffers from impaired function. Hepatitis viruses do not cause lytic infections of the hepatocyte
but instead evoke a lymphocytic cell inflammation. This cell-mediated host response is what damages
the liver tissue with resulting impairment of function.

■
9.6.2 IMMUNE COMPLEX REACTIONS

Immune complex reactions are typical of persistent infections. The antigen-antibody complexes can get
stuck ('deposit' themselves) in arteries to cause vasculitis (which

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manifests itself as a skin rash) or in the basement membrane of the glomeruli to cause kidney damage.
Both are seen with chronic hepatitis B infections.

9.7 HOW VIRUSES DEAL WITH HOST IMMUNE RESPONSES

The host immune response to viruses is both humoral and cell-mediated. Circulating antibody will
neutrahse extracellular virus in the body fluids (including mucous membranes) and the cell-mediated
arm will attack cells that express viral proteins. Previous infection or vaccination will be the two
relevant factors that dictate whether a person is able to swiftly neutrahse an infection or not. In hght of
these protective mechanisms, viruses have developed strategies to try to minimise their contact with the
immune response of the host. Viruses have adopted various strategies to out-manoeuvre the host, in
particular to evade or suppress the immune response that attempts to develop antiviral products.
It is useful to consider the broader strategies before looking at cellular mechanisms.

• Evasion of immune response. Viruses can rephcate in tissues that are relatively protected from
surveillance by immune cells (e.g. brain, dermis). Alternatively, they can avoid extracellular stages
in the course of infection. Fusion of infected cells, budding within cell vesicles and rephcation
within lymphocytes all avoid exposure to neutrahsing antibodies.

• Suppression of immune response. Some virus infections infect the immune cells that mount the
immune response in order to suppress them. Strategically this makes good sense; hence, many
viruses infect lymphocytes, e.g. B lymphocytes are infected by EBV and T lymphocytes are
infected by HIV. (It is interesting to note that intracellular bacteria tend to infect macrophages, e.g.
Mycobacterium tuberculosis.)

Vims infections of the neutrophils appear to be either unusual or of little value to the virus, possibly
because neutrophils do not hve long enough for adequate viral rephcation. Neutrophils hve for only 12-
18 hours, whereas lymphocytes and macrophages have long lifespans which make them worthwhile
targets.

9.7.1 CELLULAR MECHANISMS BY WHICH VIRUSES MODULATE THE

HOST IMMUNE RESPONSES

As we have seen, the first line response (innate immunity) of the host to viral infections is dominated by
the actions of the chemokines called interferons. Interferon will need to contain viral infection until the
specific immunity has developed. Many viruses have developed the abihty to block the actions of
interferon-based responses by producing proteins that interfere at various points in the action of the
different chemokines/cytokines. It is obvious that viruses need to thwart the early attempts of the

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immune system to kill them. In order to multiply and then exit the host before the specific immunity
develops, the virus must suppress the innate immunity for as long as possible. It has been pointed out
that total suppression of the immune response is not a feature of viral infections. Despite being costly in
terms of genome space for the virus, this strategy will be of benefit since it wants to multiply in the host
at the expense of other invading parasites. Selective suppression of the innate immunity still enables the
host to repel other pathogens.

The host response to virus infection will generate extracellular signals (cytokines) and these will bind to
cell receptors and trigger intracellular events that will aim to

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inhibit viral replication. It follows that viruses can inhibit both the extracellular and intracellular
signalUng events. The following Ust illustrates how viruses have attempted to modulate various points of
the immune response.

• Minimise recognition by the host. For viruses that cause systemic infections, the first entry of
virus into the bloodstream during a viraemic phase will attract the attention of Complement
components. Certain viruses produce molecular mimics (homologues) of Complement components
which will block the Complement cascade attacking the free virion. These homologues are termed
viroceptors. Mimicry is also used by enveloped viruses which include host proteins in the viral
envelope to mask their recognition as 'foreign'. Virus-infected cells also secrete proteins that
suppress their recognition by Complement, thereby protecting the manufacture of new virus.

• Inhibit production of interferon. Interferon production is an important early step in the defence
against virus infections. Viruses need to interfere with the action of interferon to estabhsh an
infection. One way to counter the action of interferon, is for viruses to secrete molecular mimics of
IFN receptors. In this way IFN binds to the competing mimic receptor rather than real receptor on
the virus-infected cell. As molecular mimics of receptors, they are called viroceptors. Other viral
proteins have been found to target the cellular signalUng pathways that occur following binding of
IFN to the cell receptor.

• Modulation of cytokine action. The mechanisms that viruses target when interfering with the
action of cytokines, most notably interferon, may be at any point in the communication network
employed by cytokines:

interfering with cytokine production by cells
inhibiting cytokine binding to cells and
interfering with the intracellular action of
cytokines

The action of viruses on cytokines, however, is not always inhibitory. Some viruses actually recruit
cytokines that promote cellular proUferation. In this way the net production of the virus will be
enhanced.

• Prevent apoptosis. Apoptosis, programmed cell suicide, occurs only if a cell receives a signal to
proceed. That signal may be triggered by the presence of a virus. In general, viruses try to prevent
the cell from carrying out this self-destruction and numerous examples exist of viruses producing
proteins that inhibit apoptosis. Apoptosis of eukaryotic cells can be triggered following activation
of proteins such as p53 and ICE-Uke proteases. Certain viruses produce proteins that inhibit the
functioning of these two triggers.

In order to produce viroceptors and other homologues, the virus must possess sufficient gene space. This
is only possible in the larger DNA viruses such as Herpes and pox viruses. RNA viruses do not have the
genome size to carry the necessary protein coding. To compensate, RNA viruses will more often choose
to outwit the host by exploiting the genetic plasticity inherent in repUcating RNA. One of the

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mechanisms for creating genetic variability and thereby generating time whilst the host develops specific
immunity is antigenic variation. The host has spent a week manufacturing immunity against the
original strain of virus but antigenic variation ( Figure 9.2) creates a new strain against which the host is
immunologically naive.

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• Figure 9.2 Antigenic drift and antigenic shift. Antigenic drift (left) shows
how the random mutations of a single base during replication of the viral RNA
results in a new amino acid within a surface protein (haemaglutinin or
neuraminidase in influenza A virus). The modified antigen is recognised
partially by antibodies generated against the original version. Antigenic shift
(right), however, represents a more substantial gene rearrangement. In this case
the protein is substantially altered through reassortment from a cell co-infected
by two distinct strains of virus such that there is no protection conferred from
antibodies against previous types of the antigen. Epidemics and pandemics
may follow antigenic shift as the population is effectively completely
susceptible again

Aniigcnic drift

Antigenic shift

9.7.1.1 Antigenic variation

Antigenic variation occurs both during repeated infections over the lifetime of the host (influenza
viruses) and during the course of a single infection of the host (HIV virus). As antigenic variation means
that some types will be more immunogenic than others, the strategy is one of some risk but selection will
promote those variants that are most suited to infect and transmit themselves through a population.

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Viruses exploit three mechanisms for creating genetic diversity:

• spontaneous mutation,

• recombination,

• reassortment.

Spontaneous mutation. In the course of replicating RNA mutations arise with a much higher frequency
than in DNA replication. The spontaneous mutation rate is close to one mutation every round of
replication of the genome, the highest rate in all living organisms when compared to rates of <0.01 for
DNA viruses and bacteria. The major cause of this hypermutability is the poor accuracy (low fidelity) of
the RNA-dependent RNA polymerase which has only a nominal ability to check and correct mistakes in
the base sequences. Whilst mutations may be good (increase fitness) or bad (decrease fitness) other
features of viral infections serve to increase the rate at which

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new viruses evolve. This includes viral replication itself, which yields large numbers of new virus in a
short period of time. Within a human host, selection will eliminate those mutants that are less well
suited, with the result that only a tiny fraction of the new viruses will be generated within the course of
an infection. The total number of viruses manufactured in a host will be a collection of closely related
genomes, dominated by the 'wild type' genome but including a range of mutants of rapidly diminishing
proportions. This distribution of non-identical but related viruses is called a quasispecies. The variation
in the genome that leads to minor sequence base changes are exempUfied as antigenic drift in influenza
viruses ( Figure 9.2) . The antigenic variation occurs in the viral haemagglutinin, a protein that projects
from the virion and mediates binding to red blood cells. Antigenic drift results in the reduced affinity of
host-neutrahsing antibodies.

Recombination. Viral recombination occurs when a cell is infected with two different RNA viruses and
the RNA produced from each virus undergoes strand switching. Exchanges of genome fragments under
these circumstances is not strictly controlled and the products may be deleterious mutants. Viral
recombination is able to introduce more genetic changes than single mutations, but for entire genes to
mix a virus needs genetic reassortment.

Reassortment. Influenza virus has a segmented genome and when two strains infect the same cell, the
reassortment of segments from the two genomes are packaged in new combinations to yield a new strain
of virus. Reassortment is responsible for the phenomenon of antigenic shift ( Figure 9.2) where both
haemagglutinin and neuraminidase proteins of influenza virus are antigenically distinct from the
originals. The loss of protection of previous neutraUsing antibodies partly explains why epidemic
influenza occurs after antigenic switching.

The importance of viral genetic variation Ues in the ability it gives RNA viruses to repeatedly infect the
same host, as exempUfied by annual rounds of new influenza virus strains. The impHcations of viral
quasispecies are witnessed in AIDS patients where microbiologists have had to introduce multiple
antiviral drug combinations for HIV treatment so as to minimise resistance developing from the
selection for resistant mutants by one drug. Similarly, there are impHcations for vaccine design if new
variants are readily generated, leaving the immunised population susceptible.

9.8 VIRUSES AND CANCER

It may surprise people to hear that 10-15 per cent of all cancers across the globe can be attributed to
viruses. This figure is hkely to increase as research uncovers more viral genes within the tumour cells.
One small benefit that has come about from the pandemic of AIDS has been the discovery that a new
herpes virus, human herpes virus-8 (HHV-8) has been identified to play a role in the development of
Kaposi's sarcoma. The importance of understanding the role of viruses in the aetiology of cancer cannot
be overemphasised. Studies of viruses have led to many of the critical discoveries in the workings of
eukaryote cell biology: gene mutations in bacteriophages, cellular oncogenes, tumour suppressor genes.

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bacterial restriction enzymes and viral reverse transcriptase

All of these have been uncovered through the study of viruses. The study of such fundamental
mechanisms of cell biology provide new targets for the design of novel anticancer and antiviral drugs.
Perhaps the most powerful implication from studying infectious links with cancer is the remarkable
finding that vaccination can be used to prevent cancer. Vaccination against hepatitis B has reduced
morbidity and mortality of liver cancer in Taiwan.

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Cancer is the uncontrolled proliferation of cells resulting from the accumulation of mutations in genes
that regulate the cell cycle. Normally, cell proliferation is tightly controlled such that cell division is
matched by cell loss in order to maintain the size of an organ (e.g. the liver). The cells are able to
respond in a balanced manner to the signals that promote or inhibit growth. In cancer the regulatory gene
products malfunction and the cell divides continuously. Carcinogenesis is a multistep process: a series of
events (up to nine in the case of colonic cancer) need to occur before the cancerous properties are fully
developed. Cancer is also multifactorial in origin: several different triggers are required to cause the
mutations, of which viral infection may be just one.

A number of viruses have been shown to directly cause cancer in animals. Rous in 1910 induced
sarcoma in chickens by injecting filterable extracts from diseased animals. The filterable agent turned
out to be a retrovirus and numerous retroviruses have been found subsequently to cause animal tumours.
Such a direct causal relationship has not been so easy to establish in humans. Some of the viruses that
have been linked with human cancer are as follows.

• Epstein Barr virus and Burkitt's lymphoma. The first association of a virus with a human
cancer was in 1964 with the direct observation of Epstein Barr virus particles by electron
microscopy in cell cultures from humans with Burkitt' s lymphoma. This cancer is geographically
limited to the malaria belt of Africa. Hybridisation with viral nucleic acid was also found in tumour
biopsies.

• Hepatitis B and hepatocellular carcinoma. Seroepidemiological studies in the 1970s linked the
virus with hepatitis. Hepatoma is most prevalent in parts of the world where HB V is most common.
Vaccination against childhood hepatoma has fallen significantly since introduction of hepatitis B
vaccine. A precedent was found in animals when HB V-like viruses were found in cases of hepatitis
and hepatoma in woodchucks!

• Human papillomavirus (HPV) and cervical carcinoma. There are over 80 antigenic types of
HPV which infect skin or mucous membranes. HPV show strong tissue tropism, hence the different
antigenic types are associated with particular lesions: mostly these are benign cellular proliferations
such as warts, macules and papillomas of skin and mucous membranes. HPV are DNA viruses
which cannot be cultured in vitro but, instead, require nucleic acid hybridisation for identification
in tissue. Cervical carcinoma is the most common in the developing world and because it is rare in
nuns and very rare in virgins compared with people of multiple sexual partners, the sexually
transmitted Herpes simplex virus was originally suspected. However, HPV came under suspicion in
1975 following weak hybridisation in cervical tissue using a probe from HPV of warts. In 1983,
HPV- 16 and HPV- 18 genome were isolated from cervical cancer biopsies. HPV virus genes are
expressed in most malignant tissues and in the HeLa cervical cell line. A series of trials are
underway in the UK to examine the effect of vaccination against HPV- 1 6 on cervical cancer.

9.8.1 PROOF OR EVIDENCE?

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A number of general observations can be made concerning the role of viruses in cancer. Cancer develops
decades after primary infection, giving ample opportunity for chemical and physical (radiation)
carcinogens to act. Many of the suspect viruses (EB V, HPV, hepatitis viruses) are ubiquitous across the
globe yet only a small percentage of infected people develop the associated cancer. Most people will
clear the viral genome or, failing that, coexist without symptoms or disease.

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9.8.2 CELLULAR MECHANISMS OF VIRAL CARCINOGENESIS

There are two sets of genes and their products that are involved in the mechanisms by wiiich viruses can
trigger immortalisation of cells:

• tumour suppressor genes,

• oncogenes.

9.9.2.1 Tumour suppressor genes

Most of the cells in the human body are not dividing but quiescent (metabolically active but not
dividing). They are growth-arrested and, in terms of the cell cycle, in GO/Gl phase. Throughout the cell
cycle there are checkpoints during which the damaged or mutated DNA is either repaired or the cell
undergoes apoptosis. Of the proteins involved in surveillance, the two most important are p53 and pRb
and, because they regulate the removal or repair of potentially malignant cells, they are termed tumour
suppressor genes. Viruses need to prevent cells from recognising the foreign viral DNA and
suppressing any proliferation (viral transformation). Likewise, apoptosis must be prevented or delayed
until viral replication has finished. DNA viruses will force the cell to undergo DNA synthesis by
producing early viral proteins that trigger DNA replication by sending the cell from GO/Gl into S phase.
This can be achieved by either inhibiting the proteins (p53 and pRb) that prevent progression into S
phase or stimulating the expression of growth-promoting genes.

9.9.2.2 Oncogenes

Genes that cause transformation of cells are called oncogenes. Strictly, oncogenes are mutated forms of
proto-oncogenes. Proto-oncogenes are normal cell genes that are involved in cell signalling events that
regulate growth and division. Proto-oncogenes are also referred to as c-oncs to distinguish them from
viral oncogenes (y-oncs). Viral oncogenes are modified from cellular oncogenes and present only in
RNA viruses. DNA viruses do not posses oncogenes. For RNA viruses to exert an oncogenic effect they
must have a stage in their replication cycle when they produce DNA and insert this as a provirus in the
host genome. Retroviruses are such viruses (although hepatitis B virus can also do this). Oncogenic
viruses that transform cells act by inserting a \-onc into the genome or by insertional activation (the viral
promotor promotes the transcription of a c-onc). The mechanisms are illustrated in Figure 9.3 .

9.9 CAUTIONARY POINTS

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Before one leaves with the impression that all viruses cause cancer, we should remind ourselves that the
multistep nature of carcinogenesis means that a virus that has one or two of the properties described is
still unlikely to induce malignant growth. For example, EBV is known to immortalise B-lymphocytes (in
vitro) from all people, not just people in the malaria belt, thus indicating that additional events are
required to lead to lymphoma. Indeed the multiple trigger hypothesis might explain geographic
restriction of certain tumours where malaria, burnt salt fish, herbal snuffs and other phenomena peculiar
to specific cultures act as cofactors. Some viruses can immortalise cell lines in vitro but have no known
associated human cancer (polyoma viruses). Conversely, some viruses (e.g. HBV linked with hepatoma)
do not immortalise cell lines.

Whilst immunosuppression by viruses (e.g. HIV) results in increased rates of cancer incidence (Kaposi's
sarcoma), HIV does not cause cancer directly. This suggests that immune systems hold these infections
in check but profound immunosuppression

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• Figure 9.3 Oncogenesis from integrated virus genes. The effects of proviral
genome on gene transcription can occur in at least three ways. (A) The viral
promotors cause read through on virus genes including a w-onc. (B) A virus
without an oncogene may still activate c-oncogenes because of the read
through driven by viral promotors. (C) A viral gene product enhances
transcription of a cellular gene at a distant site

(A)

V-OtiC

V
Provinis

(B>

II 1

*i-iitK

(C)

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Cellular ui;nt

cannot. Finally, viruses may act indirectly by increasing replication of cells. Faster-replicating cells are
more prone to mutagenic events induced by cofactors, hence HB V may promote replication of liver cells
such that other mutagenic agents (e.g. ingested fungal aflatoxins) have a greater effect. Incidentally, a
similar sequence of events is postulated to occur in the development of gastric lymphoma following
Helicobacter pylori infections.

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■ SUMMARY

Viruses are very successful parasites. Viruses tend to cause asymptomatic infections in
humans with only a small fraction of the total population exposed developing symptoms. In
patients who become ill, they are usually most infectious just prior to showing symptoms so
that the infection is not displayed to the next host and transmission is not compromised.
Important differences are seen in DNA and RNA viruses, most notably in acute and
persistent infections. Viral infections evoke immune cells to release cytokines in order to
curtail the infection and mobilise the immune system. Viruses have numerous mechanisms
to modulate the immune response and remain within the host (latency). Such adaptations
have resulted in viruses being significant factors in the development of carcinogenesis.

RECOMMENDED READING

Alcami, A. and Koszinowski, U.H. (2000) Viral mechanisms of immune evasion. Mol. Med. Today 6,
365-72.

Collier, J. and Oxford, J. (2000) Human Virology, 2nd edition, Oxford University Press, Oxford, UK.

Domingo, E. and Holland, J.J. (1997) RNA virus mutations and fitness for survival. Ann. Rev.

Microbiol. 51, 151-78.

Everett, H. and McFadden, G. (1999) Apoptosis: an innate immune response to virus infection.

Trends Microbiol. 7, 160-5.

Kalvakolanu, D.V. (1999) Virus interception of cytokine-regulated pathways. Trends Microbiol. 7,

166-71.

Porterfield, J.S. (1992) Pathogenesis of viral infections; in McGee, J.O.D., Isaacson, P.O. and

Wright, N.A. (eds) Oxford Textbook of Pathology, Oxford University Press, Oxford, UK.

Villarreal, L.P., Defilippis, V.R. and GottUeb, K.R. (2000) Acute and persistent viral Ufe strategies

and their relationship to emerging diseases. Virology 272, 1-6.

REVIEW QUESTIONS

Question 9.1 Does the target site of viral replication have any significance on the severity and
duration of the infection?

Question 9.2 List the reasons to explain why viruses cannot be stated as sole causes of cancer.

Question 9.3 What is a viral quasispecies?

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Question 9.4 Why is the incubation period between 7 and 14 days for many childhood viral
infections?

Question 9.5 How do RNA and DNA viruses differ in their strategies of human infections?

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10
FUNGI AS PARASITES

The impact of fungi on humans is potentially considerable when one considers the possible targets. As
parasites fungi primarily infect plants, and the h-ish famine of the 1850s in which the potato crop was
ruined by Phytopthera infestans represents an alarming example of the damaging effects fungal infection
has on foodstuffs. Two million people died during the five-year period in which the crops were lost.
Less dramatically, fungi also cause spoilage of pharmaceutical and cosmetic products, as well as
attacking the timbers of our homes.

Only a small proportion of fungi (<0.1 per cent) exist as parasites of humans and animals, fungi mostly
acting as saprophytes (organisms that absorb their organic nutrients from dead substrates). By definition,
then, saprophytic organisms are not parasitic because their nutritional sources are not alive; instead the
organisms are acting as decomposers and recyclers. As most fungal infections are acquired from an
environmental source, fungi are not obligate human parasites but, instead, human mycoses can be
considered accidental infections that are not designed to facilitate the spread of the organism to new
hosts. Hence, human to human spread is very rare for most fungal infections with the exception of the
dermatophytes (see below, p. 229).
Clinically, human fungal infections can be grouped into:

(i) systemic disease (infections of internal organs of the body), and

(ii) localised or superficial infections (infections confined to the skin or mucous membranes that do
not invade into deeper tissues or organs - see Table 10.1) . This classification is anthropomorphic
in that it considers the diseases as they affect man. In order to see what are the important features
of the fungi themselves, it is appropriate to group the organisms into diseases by primary
pathogenic fungi and secondary pathogenic fungi. The tiny fraction of fungi that can cause
human disease, despite the daily exposure to large numbers of airborne fungal spores, can be
taken to mean that most fungi cannot overcome human defence mechanisms.

10.1 PRIMARY PATHOGENIC FUNGI

The fungi that are primary pathogens are those that can infect and cause disease in healthy people;
'healthy' in that there are no recognisable predisposing features which

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Mycosis

Table 10.1 Examples of the two major types of human mycoses

Aetiolosical asent(s)

Page 228

Systemic mycoses

Histoplasmosis
Systemic candidosis
Cryptococcosis
Aspergillosis
Localised infections
Dermatophytoses

Candidiasis
Pityriasis versicolor

Histoplasma capsulatum
Candida albicans
Cryptococcus neoformans
Aspergillus spp.

Epidermophyton spp., Trichosporum spp.,
Microsporum spp.
Candida albicans
Pityrosporum spp.

facilitate infection. The diseases that are seen may be systemic infections or superficial infections. This
immediately demonstrates how the severity of the infection is not necessarily an indication of the
pathogenic potential of the organism.

10.1.1 SYSTEMIC INFECTIONS

Fungi that are able to cause systemic illness in healthy people are rare and confined to specific
geographic locations across the world. The fungus Histoplasma capsulatum causes histoplasmosis
which is endemic to certain areas in the southern states of America. Another infection is
coccidiomycosis, caused by Coccidioides immitis. Like histoplasmosis, the fungus is found between the
southern states of America, Mexico and the northern-most countries of South America. These two
infections are caused by inhalation of the fungus, which exhibits dimorphism, i.e. can exist as a yeast or
a mould. The organisms are acquired by inhalation of the conidia from soil, and develop in the lungs as
yeasts. The abihty to switch between forms is seen following the change in temperature so that
Histoplasma capsulatum is a mould when grown at 25°C but grows as yeast at body temperature. This
property is then more accurately described as thermal dimorphism. The switch from being a mould in
the soil where it adopts a saprophytic existence to a yeast when at 37°C presumably favours the parasitic
existence in a warm-blooded animal. Dimorphism is a particular feature of the endemic fungi that cause
systemic infections but it is not restricted to pathogenic fungi ( Table 10.2) .

Table 10.2 Fungal morphology as saprophytes and pathogens

Mycosis

Fungus

Sap rophytic fo rm

Parasitic form(s)

Coccidiomycosis
Histoplasmosis

Coccidioides immitis

Histoplasma
capsulatum

branching hyphae
branching hyphae

spherules (sacs of

endospores)

yeast

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Dermatophytosis Dermatophytes branching hyphae b ranching hyphae

Cryptococcosis Cryptococcus yeast encapsulate yeast

neoformans
Candidiasis Candida albicans yeast yeast, hyphae,

pseudohyphae

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10.1.2 SUPERFICIAL INFECTIONS: THE DERMATOPHYTOSES

The infections caused by the group of fungi collectively termed the dermatophytes are best known by
the infection ringworm. Nothing to do with worms, ringworm is a fungal infection of the skin which
shows up as a circular area of redness in which the fungal mycelia are radially extending outwards
through the superficial skin squames. The sites of infection are reflected in the medical terms tinea
barbae (beard), tinea capitis (head), tinea corporis (body), tinea cruris (groin), tinea pedis (foot) and tinea
unguinum (nail). The different sites of infection on the body reflect the ability of dermatophytes to
invade keratinised tissues (skin, hair and nails) but no further, since these fungi have the ability to obtain
nitrogen from the keratin present only in the (dead) tissues in the body.

The dermatophytes are a group of approximately 40 species that are classified by their anamorphic
(asexual) stages. They fall into three genera: Trichophyton, Microsporum and Epidermophyton within
the Fungi Imperfecta (Deuteromycota). The ability to reproduce sexually is very rarely found in these
organisms, and this is thought to represent the intermediate transfer from environmental organisms to a
strictly parasitic existence on humans and animals. The organisms can be described according to their
ecological location:

• anthropophilic dermatophytes: exclusively infect humans,

• zoophiUc dermatophytes: animal parasites that accidentally infect humans,

• geophiUc dermatophytes: soil inhabitants that can infect humans and animals.

As zoophilic dermatophyte infections are transmissible from animals to humans, anthropophilic
infections are transmissible from person to person, a property unusual in human mycoses ( Figure
10.1) . In addition, the organisms vary in their geographical

• Figure 10.1 Lifecycles of dematophytes. Humans are accidental hosts for
zoophilic and geophilic dermatophytes but the only recognised host for
anthropophilic infections

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IlLtini^ns

An(hropophil]c

Humans
Ajiimals

Zuupliilic

GccjphiLic

Animals
SoiJ

lliiniaii!:

Ilitnian!),

Soil

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distribution with some widespread (globally) and others restricted to specific areas of the world.
As the dermatophytes infect only keratinised tissues, they do not invade Uving tissues. In keeping with
the descriptions of radial growth in Chapter 4 , the lesions radiate as a ring with the advancing hyphae
migrating into new tissues, and the pathology shows varying degrees of cell-mediated inflammation with
an increased rate of division and keratinisation of the skin layers. This increased cell turnover will
provide an increased supply of nutrients for the fungus. The infections with the most degree of
inflammation are the zoophiUc and geophilic species, unhke the anthopophilic infections, which are
often asymptomatic. Such findings support the idea that the most host-adapted species (the
anthropophilic species) are better suited to maintaining itself within a human population.

10.2 SECONDARY FUNGAL PATHOGENS

The three fungi that we will consider are all able to cause systemic infections, but it appears that
predisposing factors in the host are required for secondary pathogenic fungi.

It is because of the advances in medicine that opportunistic infections have become a greater problem,
both in incidence and chnical management of the patient. The increase in the number of successful
organ transplants, for example, means that such patients are at risk of developing infections due to
opportunist organisms, of which fungi have been particularly problematic. We will consider three fungi
that have become regular culprits in causing infections in patients with underlying disease: Cryptococcus
neoformans, Aspergillus spp. and Candida albicans. In all three organisms, the increased opportunities
for invading the patient have arisen from the following factors:

• increased numbers of patients with immunosuppression: naturally occurring such as AIDS or
through more successful medical immune manipulation during transplantation.

• continued use of broad spectrum antibiotics.

• increased use of intravenous lines.

• increased rates of survival in premature babies.

10.2.1 CRYPTOCOCCUS NEOFORMANS

Cryptococcus neoformans is a yeast that has become a frequent invader of patients with impaired cell-
mediated immunity, whereas chnical disease is rare in healthy people. In such patients the yeast often
invades the central nervous system and is particularly frequent in causing meningitis, although
pneumonia is not uncommon. The organism can be recovered from soil throughout the world, thus
differing from the primary pathogens described above which have a restricted geographical location.
Cryptococci will favour nitrogen-rich soil, typically achieved through bird droppings. There are
approximately twenty species of cryptococci but only C. neoformans causes disease in humans.

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Unusual amongst fungal pathogens is the production of a polysaccharide capsule by C. neoformans. All
cryptococci can produce polysaccharide capsules but, when growing in soil, the capsule is usually not
evident. However, when nitrogen or moisture levels drop, capsule synthesis is stimulated. In this way the
capsule can provide a reservoir of water during an oncoming period of drought and protect the yeast
from dehydration. C. neoformans will grow at 37°C but most species of cryptococcus do not tolerate this

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temperature, preferring ambient temperatures. Birds typically have body temperatures over 40°C and so,
whilst cryptococci might be carried in the avian gut, they do not cause illness in birds.
Humans are exposed to the organism via the respiratory tract. The dehydrated yeast cell will be of
sufficiently small diameter to act as an aerosol such that the yeasts may enter the respiratory tract,
whereas yeast cells with abundant capsule present will be too large to be effective aerosols. The increase
in carbon dioxide and fall in nitrogen concentrations that the yeast encounters once inhaled stimulate the
synthesis of the capsule. These changes are good examples of the environmental signalling discussed in
Chapter 8 . Cryptococcus neoformans will multiply within phagocytes, resistant to the antimicrobial
attack of the phagolysosome. As with bacterial capsules, the crytococcal capsule has a number of
functions: the polysaccharide capsule is a polymer of mannan, a typical component of fungal cell walls,
which is poorly immunogenic. The mannan appears to inhibit effective phagocytosis by the host
neutrophils unless specific antibodies are produced. The capsule is also a weak activator of the host
immune response if not intrinsically immunosuppressive. Shed capsular material will also act to bind
host antibody and complement components, thereby protecting the organisms itself.
Additional virulence factors have been suggested and the production of a phenoloxidase enzyme may
contribute to the neurotropism of the yeast. The phenoloxidase enzyme catalyses the conversion of
neurotransmitters such as dopamine, which are particularly abundant in brain tissue, into melanin.
Melanin acts as a scavenger for reactive oxygen species such as superoxide ion. This may protect the
yeast from such damage due to reactive oxygen species by leucocytes.

To keep perspective, it should be remembered that C. neoformans is rarely seen as a cause of disease in
healthy people despite the fact that many people are likely to encounter the organism. Only patients with
impaired or defective immune systems are likely to become ill with this organism. Interestingly, patients
with deficiencies in their cell-mediated immunity develop infections with this organism. These patients
are typically people with late stage AIDS and patients with immunosuppressive treatment for organ
transplant or autoimmune disease like Hodgkin's disease. The role of the humoral arm of the immune
system is less clear. Significant antibody titres in serum do not appear to provide protection in these
patients, which supports the critical role of the cell-mediated arm.

10.2.2 ASPERGILLUS INFECTIONS

Aspergilli are moulds and, consistent with a free-living fungus, they are ubiquitous in the environment
where they can be cultured from soil, compost and grain. They are also easily cultured from foods and
plants in indoor environments. The abundance and small size of the spores produced (2-3 |im) means
that most people will encounter them as aerosols, able to penetrate the lower respiratory tract.
Fortunately, healthy people can deal with the spores, i.e. destroy or eliminate them, and hence do not
become ill, but the groups of patients who develop systemic illness with opportunistic fungi are
sufficiently immunocompromised that the spores are able to establish themselves within the host and
germinate into hyphae. Typically, Aspergillus infections occur in patients with prolonged and severe
neutropenia (very low numbers of neutrophils in the blood) and often colonise pre-existing lesions such

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as old tubercular lesions or lung cancer sites. Of the numerous species within the genus, Aspergillus
fumigatus is the most frequent species isolated from patients with disease. Quite what distinguishes this
species from the rest, in terms of virulence capacity, is not known.

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A. fumigatus grows across a broad temperature range (from 12-55°C), a feature which is an obvious
prerequisite for a free-hving organism to multiply in the human host. The spores are thought to be more
resistant to the attack of neutrophils than the hyphae, and this might correspond with the need for severe
neutropenia in order for the organism to estabUsh in the lungs. The spores are also adhesive to fibrin and
laminin components of lung tissues. AspergilH, Uke all fungi, produce a number of proteolytic enzymes
such as proteases, phospholipases and elastases. As elastin is a major constituent of lung tissue, it is
suggested that such enzymes will faciUtate penetration of lung tissue by the hyphae. The ease with
which AspergilU can be cultured from the environment means that vulnerable patients in hospital wards
are continually exposed to Aspergillus spores, not least because building works are often ongoing in
hospital grounds. It has been recommended that special air filtration or positive pressure environments
are needed for bone marrow transplant patients but the expense is clearly substantial and the possible
protective benefits have not been shown.

10.2.3 CANDIDA ALBICANS

There are greater than 100 species of Candida but only C. albicans is regularly isolated from humans,
either as a member of the normal flora or from ill patients. Infections by C. albicans have increased in
incidence over the last 20 years as more and more medical advances provide opportunities for the
organism to colonise ill patients such that C. albicans is now the most common fungal infection in
humans. The reasons must he partly with the fact that C. albicans is readily isolated as a member of the
normal flora of humans (mouth, gut and genitourinary tract) and any reduction in colonisation resistance
by antibiotics will permit a swift increase in numbers of Candida. Candida albicans causes localised
infections such as vaginal thrush which occur in otherwise healthy people, but is also an important cause
of systemic disease in hospitaUsed patients. The risk of colonisation by an endogenous organism is
clearly greater than with exogenously-acquired organisms. Correspondingly, infections by C. albicans
are invariably the same strain as that present in the normal flora and the patient is Ukely to be colonised
prior to developing cUnical infection.

In systemic candidosis the site of invasion varies. Where the site of infection seeds directly into the
bloodstream (for example an infected intravenous cannula), the natural defences of the skin are
bypassed. Alternatively, invasion from the mucous membranes is likely as these are the normal sites of
residence for the organism. Once in the bloodstream, the organisms will be distributed throughout the
body (haematogenous spread) and multiple organs may become infected.

The virulence factors that have been proposed for C. albicans include the abihty of the organism to
adhere to numerous substrates including the skin, epithelia and IV Hne plastic, the production of
proteinase enzymes and the property of dimorphism. As mentioned above, dimorphism is the ability to
switch from hyphal forms to yeast forms and back again depending on environmental signals, notably
temperature. When C. albicans is exposed to serum at 37°C it produces hyphal forms which are
particularly adherent to human tissues. Mutants unable to exhibit the hyphal forms are less virulent in
animal models than the wild type. Dimorphism is seen in other primary fungal pathogens such as

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Histoplasma capsulatum where the hyphal form in the soil switches to the yeast form in human tissue. It
is interesting to note that in C. albicans both hyphal and yeast forms can be observed in infected human
tissues. The hyphal forms appear early on in the course of an infection and release of the proteolyic
enzymes from the hyphal tips can be seen as valuable in the organism penetrating the tissues. The
penetra-

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tion of tissues by hyphae is termed thigmotropism. Once simple nutritional substances are available the
hyphae revert to the yeast form. The signalling systems for Candida are not fully elucidated but the
principle of the organism adapting to its environment is unmistakable.

The virulence of C. albicans is compUcated due to the genetic variabiUty of the organism. The yeast is
diploid but considered strictly asexual. Not being tied down to sexual reproduction via meiois, C.
albicans can vary the level of expression of the virulence genes from the two chromosomes very readily.
There are implications for the research worker attempting to identify the virulence determinants in
C. albicans in that, during the course of an experiment, the yeast may alter the expression of the
targeted gene under investigation if both alleles are not ablated.

Having considered three different fungi you will notice that unifying themes are not very evident. The
primary pathogens, the dermatophytes, do not cause systemic disease whereas the secondary pathogen,
Candida, does cause invasive, systemic disease. Thus, the severity of the disease is not an indication that
the causative agent is a primary pathogen. The exogenous infections, cryptococcocosis and aspergillosis,
are both acquired via the respiratory tract but both neither are infectious between humans.

10.3 PATHOGENIC MECHANISMS

From the above examples what are the key virulence factors of fungi that enable human infections?
Numerous fungi infect plants and cause considerable destruction to industrial farming and horticulture,
and plant mycologists have considerable expertise on the pathogenic mechanisms of fungal infections.
With human disease, insufficient research has, to date, been responsible for a hmited understanding of
fungal pathogenesis. Hence, the extent to which pathogenic fungi follow the six characteristic steps
described for bacterial pathogens is largely speculative.

adhere,

invade,

multiply,

spread,

avoid host defence mechanisms,

exit the host.

One or more of the steps can be found in most pathogens, but the complete set are difficult to clearly
identify in many pathogenic fungi (to date anyway). For those fungi that do possess parasitic modes of
existence, a number of adaptations have accompanied the shift from saprophytic to parasitic existence.

10.3.1 ADAPTATION TO INCREASED TEMPERATURES (THERMAL

TOLERANCE)

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The temperature difference between the soil and warm-blooded animals is significant in light of the
serious penalties incurred on enzyme function as the temperature increases. Those fungi that cause
infections of the skin (dermatophytes) are restricted to temperatures cooler than 37°C, and may illustrate
the problems of adapting to wider temperatures. For those fungi that do tolerate body temperatures, the
shift in temperature is used as a signal for gene expression (a feature observed in pathogenic bacteria).

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10.3.2 ANATOMICAL STRUCTURES

The ability of certain fungi to use specialised structures to penetrate the host tissues is widespread
amongst plant pathogens and also occurs in human fungal pathogens. The ability to switch between
hyphal and yeast forms (dimorphism) is commonly observed when infecting animal hosts in the course
of an infection. Candida develop hyphae (pseudohyphae) when invading human tissue. In contrast to
bacterial pathogens, the use of capsules as virulence factors is almost unknown in fungal pathogens with
the exception of Cryptococcus neoformans.

10.3.3 SECRETION OF NUMEROUS HYDROLYTIC ENZYMES

The heterotrophic nature of fungal nutrition means that they secrete numerous degradative enzymes in
large quantities, many of which may attack host tissues. Consequently, it is difficult to assess
experimentally the contribution of individual enzymes as the other enzymes may compensate for any
loss. Furthermore, the property is characteristic of most fungi, yet very few are pathogenic to humans.

10.3.4 EVADE IMMUNE RESPONSES

Fungal pathogens that persist in human hosts do so by various mechanisms:

1 . infection of immuno-priviliged sites (skin, central nervous system),

2 . fungal spores are relatively resistant to phagocytosis compared with hyphae,

3 . release of immuno-modulatory substances or molecular mimics.

This chapter has outlined four fungal infections to illustrate the variety in conditions that underlie these
events. They highlight the need to consider both the state of the host and the fungus in conjunction;
viewing either in isolation is insufficient. With fungal infections of humans that are accidental to the
normal lifecycle of the organism, the virulence factors are unlikely to be specific to that organism but
instead common to most fungi. The reason disease has developed is because of an accidental encounter.
Accidental human mycoses have little opportunity for transmission to new hosts in sharp contrast to
well-adapted pathogens, the dermatophytes and Candida albicans. Studies on the pathogenesis of fungi
have lagged behind those of bacteria and viruses, although the rising incidence of mycoses will stimulate
much needed funds for new

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■ BOX 10.1 TOXIC PRODUCTS

Whilst we are primarily concerned with how microbes infect humans, it is important to remember
that certain human diseases result from fungal growth in products that we consume. Fungal
poisoning of foodstuffs can be separated into:

• mycetism, the ingestion of poisonous mushrooms, and

• mycotoxicosis, the ingestion of food products containing toxins (mycotoxins) secreted
by fungi that have contaminated the raw material or product. The mycotoxicoses
include ergotism, in which cereal grain is infected with Claviceps purpurea (and was
once a common illness known as 'St Anthony's fire') and ingestion of aflatoxin.
Aflatoxins are potent carcinogens produced by Aspergillus spp., linked with the
development of liver cancer. Peanuts are often found to contain high concentrations of
these toxins as a result of fungal contamination during storage.

Finally, we should perhaps include alcoholism as a widespread type of mycotoxicosis !

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studies. However, the methodology will be more complicated than with bacteria as genetic complexities
of most fungi will complicate the 'virulence gene' deletion studies.

■ SUMMARY

As saprophytes, fungi are mostly accidental pathogens of humans. Primary fungal
pathogens (certain dimorphic fungi and the dermatophytes) are able to infect humans
without recognisable predisposing factors in the host. Primary pathogens cause systemic or
superficial (skin) infections. The dimorphic fungi responsible for systemic infections are
acquired via inhalation of spores and are able to replicate in the niche (human tissue). The
superficial dermatophyte infections are restricted to keratinised tissues. Secondary fungal
pathogens replicate in humans only through predisposing factors such as
immunosuppression or traumatic implantation of the organisms into the tissues but, as a
consequence, are more likely to cause systemic disease. The virulence factors recognised
include dimorphism, thermal tolerance and immunomodulatory abilities, but increasing
numbers of immunosuppressed hosts through medical improvements or disease such as
AIDS means that fungal infections are increasing in number. This increase indicates the
importance of host condition in the balance between fungal and human factors in whether
infections are established.

RECOMMENDED READING

Cutler, J.E. (1991) Putative virulence factors of Candida albicans. Annu. Rev. Microbiol. 45, 187-218.
Denning, D.W. (1991) Epidemiology and pathogenesis of systemic fungal infections in the
immunocompromised host. /. Antimicrob. Chemother. 28, Suppl. B, 1-16.
Fridkin, S.K. and Jarvis, W.R. (1996) Epidemiology of nosocomial fungal infections. Clin.
Microbiol. Rev. 9, 499-511.

Hogan, L.H., Klein, B.S. and Levitz, S.M. (1996) Virulence factors of medically important fungi.
Clin. Microbiol. Rev. 9, 469-88.

Kobayashi, G.S. and Medoff, G. (1998) Introduction to the fungi and mycoses, in Schlaecter, M.,
Engelberg, N.C., Eisenstein, B.I. and Medoff, G. (eds) Mechanisms of Microbial Disease, 3rd
edition, Lippincott, Williams & Wilkins, Baltimore, USA.

Richardson, M.D. (1992) Fungal infections, in McGee, J.O.D., Isaacson, P.G. and Wright, N.A. (eds)
Oxford Textbook of Pathology, Oxford University Press, Oxford, UK.
Weitzman, I. and Summerbell, R.C. (1995) The dermatophytes. Clin. Microbiol. Rev. 8, 240-59.

REVIEW QUESTIONS

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Question 10.1 Which are the two most at risk population groups for fungal infections?
Question 10.2 To what extent is Candida albicans representative of fungi?
Question 10.3 Many dermatophytes are anamorphic. Explain the significance of this sentence.
Question 10.4 What is dimorphism?

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11
CONTROL OF MICROBIAL INFECTIONS

This chapter considers some of the options available to help to control the impact of infectious diseases
on populations. The study of diseases within populations is called epidemiology. For microbiologists,
infectious disease epidemiology is an increasingly important area of study as infectious diseases do not
appear to be diminishing in number. The increased mobihty of people across the globe has meant that
infectious diseases can be acquired in one part of the world and within hours transported to another. The
movement of micro-organisms will be the same. To keep an eye on the changes in distribution and
numbers of infectious diseases, national centres are required. Within the USA this is based at the Center
for Disease Control (CDC), Atlanta and in the UK, the Central PubHc Health Laboratory, Colindale,
London. This overview of national trends is part of the surveillance of infectious disease. The reason
that certain infectious diseases must be notified to the pubHc health department is because of the value of
national surveillance in identifying and controlUng epidemics. Box. 11.1 gives the important terms that
are used.

11.1 EPIDEMIOLOGY OF INFECTIOUS DISEASE

What are the features of micro-organisms that spread as infectious diseases through a community?
Classic infectious diseases such as measles can be modelled assuming that

■ BOX 11.1 EPIDEMIOLOGICAL TERMS
Incidence: number of new cases of infection occurring within a population over a defined period of
time ('per unit population per unit time').

Prevalence: the total number of cases in a defined population either at any one point in time (point
prevalence) or over a defined period (period prevalence). Prevalence gives an indication of how
widespread the infection is within a population, whereas incidence, by recording only the new cases,
tells you how rapidly the outbreak is growing.

Outbreak: the incidence of an infection increases within a population above that normally expected.
Only two cases represent an outbreak when considered against a background of an infection that is
normally an uncommon occurrence. An outbreak is thus defined relative to the normal incidence. A
sporadic case is a case of illness (infection) not considered part of an outbreak.

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the infection is transmitted randomly between infectious and susceptible people. The rate of
transmission will be proportional to the product of the number of susceptibles, multipHed by the number
of infectious people and is based on the mass action principle ( Figure 11.1) . If S is the number of
susceptibles and / is the number of infectious people, then the rate of new infections (AO will be the
product of the rate of mixing and the concentration of the two components (susceptibles and infected):

Mathematically, the proportional rate of increase means that there must be a constant (P) that moderates
the product. That constant represents the probability of effective transmission. For example, the
efficiency of transmission of hepatitis B virus being transmitted by sneezing is extremely low in
comparison to measles virus, which has a high value of P (closer to one). The transmission coefficient
(P) will vary between micro-organisms and their route of transmission. The other two components / and
S are more direct: the more infectious people (/) or susceptibles (S) there are, the greater the rate of new
infections occurring (AO.

• Figure 11.1 The law of mass action. Originally used to describe the motion
of two gases in a closed container, the law of mass action can be used to help
model ideas about the mixing of an infectious agent such as measles between
susceptible and infected people. (A) shows the two populations, susceptible (S,
open circles) and infected people (I, shaded circles) moving around at a fixed
rate (arrows). The rate of new infections (N) will be the product of S and I and
the transmission coefficient (P, see text). The rate of new infections can be
increased by either (B) increasing the number infected, or susceptible, as well
as (C) increasing p, the efficiency of transfer of the organism (indicated by
increased length of the arrows)

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(A)

^■0

9o^

(B)

(C)

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The number of people directly infected by someone who is infected by a particular micro-organism in an
entirely susceptible population is termed R^, the basic reproductive rate. Assuming that the infected

and susceptibles can mix randomly (homogenous mixing) we can use the mass action principle to
define R^ as the product of the susceptibles (S), the probability of effective transmission (P) and the

period of time each case is infectious (D).

The equation again shows that increasing any component will increase R^, e.g. increase the duration of

infectiousness and the number of people who become infected per case increases. The conditions
defined here give a numerical value to the incidence of new cases in a population consisting only of
susceptible and infected people, who mix randomly. These conditions are somewhat artificial because
the proportion of people who are immune is not considered, but nevertheless, such values help in
analysing the behaviour of different micro-organisms. Highly infectious diseases Uke measles have high
values of R^ (around 20) whereas poorly infectious diseases (famously leprosy) have R^ values much

closer to 1. Being highly infectious, organisms with high R^ infect people at a younger age than

organisms with higher R^. In other words, R^ and average age at infection are inversely related.

For the organism, three key limits are predicted:

fi,^ < 1; the infectjon declines and disaiipears from the comiTiuiiitv,

\ = I ; the Ltifectinji persists, Le, rematiis endejiiic,

R,^ > ] ; I he in Eh?:^:! I oil wiil iiirreafle in innicletirei And spread aaan epirlemir.

When an infectious disease with a high R^ such as measles invades a completely susceptible population

(an isolated island community) around 20 new cases arise from every infectious person and a short-lived
epidemic will occur. Fortunately it will burn itself out as the number of susceptibles is quickly
exhausted. The epidemic will occur more rapidly than with an organism of lower R^ since, once

infected, people will become immune (or perhaps die), the number of susceptibles falls and the virus
finds fewer and fewer people to infect. This phenomenon is critical in terms of the outbreak. When R^ <

1, less than one secondary case is produced for each infected individual and the epidemic fades. Thus the
actual or effective reproductive rate (R) falls during the course of an outbreak because the number of
susceptibles that become immune is not replenished. The effective reproductive rate is therefore variable
and related to the basic reproductive rate thus:

R = R^x

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where x is the proportion of the population susceptible, e.g. HRq is 20 and 50 per cent of the population
are immune, then /^=20Xfl,'i= LO.

Knowing that 'V. ~ 1 represents a threshold point below which the epidemic will die out, we have
obtained a theoretical minimum number of susceptibles required to maintain the organism within the
community. From our equation, this threshold - the numerical density threshold {Dj) - can be

predicted by setting /?(, to 1 .

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Page 239
Therefore:

Dj will be high for organisms with high R^ because they spread so effectively through the population

and therefore need high densities of susceptibles.

The outcome of our proposed measles epidemic can therefore be predicted to some extent. The key

parameters in an epidemic are:

• the size of the population, and,

• the rate of new susceptibles appearing.

The epidemic will fade and disappear as R falls to less than unity, but if the population density is high
enough (>Dj) the organism may persist and reappear later as a second epidemic. This pattern may repeat

and give epidemics every couple of years, following the oscillation of R around unity ( Figure 11.2 ). The

length of time between epidemics will depend on the arrival of new susceptibles (such as births or

migrations).

Should all organisms seek a high R^l Not necessarily. Microbes with high R^ run the risk of quickly

eliminating all potential hosts as they sweep through th e community. They may be viewed as too
effective, but in large, dense cities this strategy is workable. More patient strategies are needed if the
population is sparse. Production of robust and

• Figure 11.2 Effect of introducing vaccination on epidemics. The effect of the
introduction of a vaccination campaign with coverage levels below that needed
to eradicate the disease. (A) shows the inter-epidemic period lengthens but the
disease does not disappear altogether because the number of susceptibles has
not been reduced sufficiently to eradicate the disease completely. (B) shows
how the age range changes upon introduction of a vaccination campaign. Note
the increase in age of infection in the vaccinated population (dotted line)
compared with the unvaccinated population (entire line)

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\kcci nation begins

(A)

Numbers

Ptrcciil

suiiceplibic

Time (days)

LQD

bic

V-~

— ^i, 1 — ^::::;=:a„,^ 1

1 vuEir

25 ytan

Age

50 yi^ass

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Page 240

long-living infective forms is one option. Bacterial endospores are a good example. Viruses are less well
equipped to cope with long periods outside a host and some have adopted vertical transmission as the
ultimate adaptation to ensuring the transmission of the organism when low-density populations exist (e.
g. chronic Hepatitis B infection).

11.1.1 THE VALIDITY OF Rq

One of the values of R^ lies in what its composition tells us. The characteristics that comprise R^ must be

essential and important for the organism, therefore reduce any of these values and the efficiency of
spread will be impaired. If any of the parameters are reduced to zero, in theory the epidemic will be
halted.

If I I then the number of susceptibles (S), the efficiency of transmission (P) and infectious period (D)

will all be suitable targets for control of the epidemic. The easiest target will often be to reduce the

number of susceptibles through vaccination.

As a marker of the ability of an organism to spread, Rq gives an indication of the propensity of an

organism to invade a population (cause an epidemic). Organisms of high R^ will spread very efficiently

and infect the very young. R^ can be used to predict the levels of vaccination needed to eradicate

infectious disease. The effective reproductive rate R must be less than one for the disease to disappear.

Earlier, we defined I I, where x is the fraction of the population susceptible. If a completely susceptible

population is represented as unity, then the actual fraction of the population immunised can be

represented as ( P), hence:
Substituting R with 1 yields:

This can be rearranged to find the percentage (p) that need to be vaccinated as follows:

p = 1 - I /ft,,

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p will be the boundary condition, i.e. it sets the minimum proportion of the population that needs to be
vaccinated in order for the effective reproduction rate to fall to one. It follows that vaccination rates

must not be less than that set by ^ ~ ' ^"■■.

To combat measles it is necessary to achieve very high vaccination rates (around 95 per cent uptake) in

children as young as possible. It is clearly not that helpful to vaccinate children at an age that is older

than the average age of infection.

Does the incidence of infection drop with increasing vaccination rates? Mathematical modelling

indicates that the relationship is not linear, as shown in Figure 11.3 . Only once R has been reduced to

values approaching one do noticeable reductions in infection result. Conversely, however, the value of/?

required to eliminate an infectious disease does not have to be 100 per cent coverage. The protection of

the few unvaccinated by the immunity of the bulk of the population is called 'herd immunity' . The

protection lies in the absolute numbers of susceptibles falling below the critical threshold value set by

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• Figure 11.3 The non- linear relationship predicted between the proportion of
the population vaccinated and the reproductive rate of an infectious disease.
The slope of the graph shows how vaccination will reduce the incidence of the
disease with greatest effect when the effective reproductive rate approaches 1.
Note also that the uptake of vaccination needs to be greater than 95 per cent to
see an infectious disease with a high Rq (such as measles, dashed line)

ehminated but less with a disease such as poliomyelitis with a lower R^ (dotted

line)

JOOn

Proi>ortioii
vaccinated
(% hnmniic)

What measures can be implemented to reduce P and D ? For highly infectious diseases such as measles
the idea of mass action as the basis for describing the random mixing of infectious and susceptible
people appears to be a good model for describing what actually happens. Hence, R^ proves useful in

comparing the infectiousness of different organisms. With measles, diphtheria and rubella, each confer
lifelong immunity. This characteristic helps in monitoring a population for immunity as the protective
antibodies, all developed against a single dominant antigenic type, can readily be measured in the serum
of people. The variation in antigenic types seen with influenza vims make predictions of the virus almost
impossible, as well as the manufacture of correct vaccine that will be protective every year. R^ can be

estimated from serological surveys of the population.

The age at which antibodies appear can be used to calculate R^ (the younger the age at which the

infection occurs equates to a higher R^ and vice versa). Again, such methods are problematic for

influenza virus because different strains of influenza vims may circulate through the population

periodically, making the serological testing complicated.

Not all infectious diseases spread horizontally like measles. Hepatitis B vims is acquired parenterally or

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vertically. Such transmission is not random; the chances of all individuals in the population encountering
each other are not equal, thus mass-action models do not apply. An additional difficulty is that the
efficiency of transmitting the infection may also vary between different groups of people. Sexually
transmitted diseases are an example of a non-homogenous mixing of the population (celibate people will
not become infected) as well as examples of differences in the efficiency of transmission (transmission
of HIV and Hepatitis B virus is less efficient in heterosexual partners than in homosexuals).

11.2 VACCINATION

What measures exist to prevent and reduce the incidence of infection? Arguably the provision of clean
drinking water, nutrition, sanitation and general economic health

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Page 242

have a profound effect, but to these vaccination must be added. Only vaccination has resulted in an

infectious disease being eradicated from the human population, with the global vaccination against

smallpox declared successful by the World Health Organisation in 1979.

The mechanisms by which vaccination works in humans is essentially immunology and will not be

covered here. The area of interest to us is what vaccination tells us about infectious diseases of humans.

The success of vaccination depends foremost on our understanding the physiology and ecology of the

organism in question.

Whilst vaccination protects the individual, there are benefits to the whole community when vaccination

uptake rates are high. The few remaining individuals that have not been vaccinated are protected by the

herd immunity principle. As discussed previously, Dj represents the minimal number of susceptibles

that are needed for a particular organism. Herd immunity arises if the number of susceptibles fall below
that value.

Herd immunity in humans only applies to infectious diseases that are strict human pathogens. Any
infection that is acquired from the environment or from animals will be unaffected by the reduction in
susceptibles (Dj) because it has a natural reservoir distinct and separate from humans (i.e. the

environment or animals). For example, the reservoir for tetanus bacilli is the soil and rabies virus is a
zoonosis. Neither are caught from other humans. Thus, vaccination of other people provides no
protection to the individual who has caught tetanus from a rose bush thorn or rabies from an infected
dog. Note how influenza virus epidemics cloud the distinction. Influenza originates from an avian
reservoir (notably ducks) and once transferred to humans, spreads as epidemic influenza from person to
person. Strictly, this qualifies influenza as a zoonosis. The vaccination coverage is selective, i.e. targeted
to the most susceptible, such as elderly people with chest disease. In this circumstance vaccination rates
are unlikely to ever impact on the Dj, hence effective reproductive rate (R) is unaffected and herd

immunity is unlikely to occur.

Ideally, a vaccine should be stable enough to withstand being transported across the country without
loss of potency. One of the reasons for the success of the smallpox vaccine was its stability at high
temperatures whilst being delivered to the populations close to the Equator. Also, a vaccine should be
totally safe and effective, cheap and easy to administer (preferably oral) and provide lifelong immunity.
The reahty is that some vaccines have many of these features but none possess them all. Despite this
vaccines have reduced the incidence of infectious disease, reduced the incidence of lifelong sequelae and
disability and reduced the mortality rate.

DDD, the value of vaccines:

• Reduce Disease,

• Reduce Disability,

• Reduce Death.

The types of vaccine that are produced commercially can be separated according to whether the vaccine

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uses intact organisms, either live or killed, or components/ products of organisms. What follows are
selected examples and important features of the different types of vaccine currently used.

11.2.1 HETEROLOGOUS VACCINE

The use of cow pox virus as a vaccine for smallpox by Edward Jenner is an example of a live,
heterologous vaccine. The term 'heterologous' is used to describe how a micro-organism (cow pox
caused by Vaccinia virus) that causes infections in other hosts (cows) is able to induce protective
immunity to the human form of the disease (smallpox caused by Variola virus) if used as a vaccine.

11.2.2 LIVE, ATTENUATED VACCINE

The BCG is a live strain of Mycobacterium tuberculosis that has been cultivated on culture medium
containing bile, thereby reducing the virulence of the organism compared

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Page 243
with the wild type. The Sabin poho vaccine is another example of a Uve, attenuated vaccine.

■

11.2.3 KILLED ORGANISMS

Influenza and rabies vaccines are killed organisms, achieved by treating the virus with P-propiolactone,
a chemical not dissimilar to formaldehyde, which does not destroy the protective antigens on the virus.
The difference between influenza and rabies vaccine is that rabies is a monotypic virus (has one
antigenic type), unlike influenza virus which undergoes antigenic modulation and requires a new
vaccine for each type.

■

11.2.4 SUBUNIT VACCINES

Inactivated intact bacteria have been largely replaced (where possible) by the use of the appropriate
component that provides protective immunity. This reduces the risk of any contamination of vaccine by
organisms that are not killed. The protective antigens on an organism are likely to be exposed on the
surface. Several vaccines use the appropriate structures in place of intact organisms, e.g. the
polysaccharide capsules from Haemophilus influenzae. Neisseria meningitidis and Streptococcus
pneumoniae.

■
11.2.5 TOXOIDS

Toxoids are the secreted exotoxins from toxigenic pathogens such as Clostridium tetani (tetanus toxin)
inactivated by formaldehyde. The formaldehyde treatment inactivates the toxin but does not destroy the
antigenic determinants that evoke the immune response. In this case the antigens that protect against the
disease are coded on the toxin rather than a surface structure of the organism. Such vaccines prevent
clinical illness developing but not necessarily infection.

■
11.2.6 PASSIVE VACCINES

Passive vaccines differ fundamentally from the other types of vaccine. Passive vaccines are simply
injections of pre-formed immunoglobulin raised against the organism in question. Hepatitis A virus is
usually acquired from infected seafood, particularly mussels and other filter feeders. The virus is passed
into the sea water from faeces of infected patients but are concentrated in the mussels growing in the
contaminated water. People travelling to areas of particular high risk of Hepatitis A infection are given
anti-hepatitis A immunoglobulin as immunoprophylaxis. Such a procedure provides protection for the

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individual only, driven by the particular circumstances rather than national campaigns. The protection is
finite, lasting only the duration of the immunoglobulin in the bloodstream (approximately 3 months).

11.2.7 WHAT DO THESE VACCINES HOPE TO ACHIEVE?

With smallpox vaccine, global eradication was achieved following a WHO campaign that chased all
cases of the disease across the world. Usually vaccination is used to limit the impact (morbidity and
mortality) of an infectious disease rather than eliminate it. It is only realistic to attempt eradication of an
infectious disease that has no inanimate or animal reservoir, i.e. a strict human pathogen. Even many
strict human infectious diseases may be impossible to eradicate because of antigenic variation in the
organism, or short-lived protection against the disease. For infectious diseases that cause childhood
epidemics, such as measles, vaccination seeks to protect the community as well as the individual.
Vaccination is also used for individuals at high risk of acquiring specific infections, usually related to
their work. For example, vaccination against anthrax for

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people who work with animal skins/hides/carcasses or vaccination against hepatitis B virus for
healthcare workers. Such specific vaccination has no expectation of reducing the incidence of the
disease in the population at large, nor of providing herd immunity. Instead, these people are vaccinated
because they are at greater risk of encountering the organism.

Vaccination against certain infectious agents may be futile. It is suggested that chickenpox (Varicella
virus) is usually sufficiently mild when caught as a child and vaccination simply will be an unnecessary
financial cost and the associated vaccine-related side effects will increase.

11.2.8 THE PROBLEMS OF VACCINATION

There is no such thing as a perfectly safe medical procedure. Ironically, vaccination suffers from poor
public perception as a result of its effectiveness in eliminating the infectious disease. By ehminating
whooping cough and all the associated crippHng illness that Bordetella pertussis creates, the only events
that attract attention are adverse reactions in a small proportion of children vaccinated. Vaccine-induced
disease will always accumulate in absolute numbers against a background of falHng or absent numbers
of cases.

If the vaccination uptake rate has not reached the threshold level for /? = 1 then the organisms will still
circulate (i.e. remain endemic) in the population. In these circumstances vaccination can be envisaged to
increase the average age at which people become infected because it is taking longer for the organism to
encounter the susceptible fraction of the population ( Figure 11.2b) . With rubella virus (German measles)
infections in older children may coincide with pregnancy. Pregnant women that contract rubella virus in
the first three months of pregnancy are at risk of the virus also infecting the developing foetus. Such an
infection causes permanent damage such as impaired vision, deafness, cardiac defects and other
abnormalities (congenital rubella syndrome, CRS). Thus, vaccination rates below that required for
eradication will inadvertently place women at increased risk of CRS. A less catastrophic consequence is
seen with mumps virus infections in men. With an increased age at infection, the illness has more
serious effects such as orchitis (inflammation of the testicles, with possible temporary sterility) and even
meningitis.

Maintaining the necessary high vaccination rates against infectious diseases such as measles requires
continued public health momentum. Complacency will occur if the disease is not seen for several years
and people will forget or relegate the childhood disease to history. Compulsory vaccination in order to
attend school has been one approach to counter this problem.

Vaccines need to cover the window of susceptibility in newborn children. The maternal antibodies
provide immunity for the first 3 months but the levels of circulating immunoglobulins in the newborn
child will disappear over time. The period that follows (3-12 months of age) is when the child is most
vulnerable to acquiring highly infectious diseases such as measles and Haemophilus influenzae capsule
type b. Unfortunately, vaccines tend to be less efficient in children of this age range (for a variety of
reasons such as poor immunogenicity of the vaccine target and residual maternal antibodies). This leaves
the child vulnerable. The concept of timing of vaccination obviously means that vaccines must be given

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before the average age of infection.

Microbes that undergo antigenic variation will be under selective pressure such that the antigenic variant
that is not in the vaccine will be selected for. The continual surveillance of influenza virus for new
antigenic types is the only option for creating an appropriate vaccine until a generic cross-protecting
antigen can be found.

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Table 11.1 If vaccination against smallpox worked, why not measles?

Smallpox Measles Poliomyelitis

Reservoir:

Acute infection (no persistence)
Number of serotypes
Infectious during prodrome
Subclinical cases

Average age of infection

Percentage vaccination uptake for elimination (p)

Humans

Yes

12-20

15yrs

2-5yrs

lOyrs

80%

>90%

>80%

Table 11.1 indicates why smallpox was such a good target for eradication. A stable, one-course vaccine
could be given well before the average age of infection. The smallpox vims has a low R^ and,

accordingly, a high average age at first exposure to the vims. The low R^ results in fewer people needing

to be vaccinated to eliminate the organism. The virus has no animal reservoir, is antigenically stable and
a short infectious period results in a readily identifiable illness (the horrid lesions covering the entire
face of smallpox victims is unmistakable). Thus, the disease is easy to trace, a factor that helped during
the global vaccination campaign. Measles will be more difficult to eradicate but has many of the
important features that helped eliminate smallpox. Likewise with poliomyelitis.

11.3 MICROBIAL CONTROL (OTHER THAN VACCINATION)

The old adage that prevention is better than cure is undeniable because it will be impossible to ehminate
pathogenic micro-organisms from our environment (despite the apparent success in ehminating
smallpox virus). Rather than attempt to kill organisms in their natural habitat, which will include soil
and animal sources, the control of infectious diseases aims to reduce contact between the organism and
potential hosts. Three targets can be proposed:

• reducing the source of the infection,

• reducing the opportunities for transmission,

• increasing the resistance of the host.

The measures that a country takes (for it must be a national programme if there is to be any real chance
of being effective) are usually the following:

• general public health: provision of clean drinking water and disposal of sewerage,

• control of animals that might cause zoonoses,

• vaccination.

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• immuno- or chemo-prophylaxis,

• vector control.

11.3.1 GENERAL PUBLIC HEALTH

In the nineteenth century, considerable improvements in public health resulted from the introduction of
clean water supplies and public sanitation systems. These, and other improvements that followed
(nutrition, housing, education, childcare improvements)

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drastically cut morbidity and mortality rates in city and rural populations across Europe. Even before the
introduction of antibiotics, the infectious disease burden had started to fall in England, a testament to the
importance of these measures. With increasing biomedical sophistication it is easy to lose sight of the
value of the essential pubHc health measures in favour of costly new innovations. Faeco-oral
transmission of Vibrio cholerae is prevented when clean water suppUes are maintained. Vaccination
should not seek to replace or obviate the need for water suppUes free of sewerage. The high childhood
mortaUty rates in countries with inadequate water suppUes are due to infectious diseases and illustrate
the impact of faiUng in these basic needs. Although entirely preventable by public sanitation, high
childhood mortaUty tends to lead to high population growth and poverty which compound the problems.
A word of warning, however, about cleaning up water suppUes: poUomyeUtis (see Box 11.2)

11.3.2 CONTROL OF ZOONOSES

Zoonotic sources of infections will be impossible to eradicate without kilUng all the animal reservoirs.
Alternative strategies are the only hope and, with over 200 organisms that can cause zoonoses in
humans, control rather than eradication is of great importance. The transmission of many zoonoses from
animals to humans occurs via insect vectors, for example louse-borne infections by Rickettsia
spp. (typhus), tickborne infections by Borrelia spp. (e.g. Lyme's disease), yellow fever virus is
transmitted by mosquitoes of the gtmx^Aedes, and plague {Yersinia pestis) transmitted by fleas. Perhaps
the most famous zoonosis, rabies is caught directly from the bite of infected animals, typically dogs.
With deforestation and increased expansion of cities (the slum regions, that is!) humans are encountering
arthropod vector-borne diseases more frequently. The displaced vectors are driven from forests into new
geographical locations and the micro-organisms spread into new vectors.

Less shocking than exotic infections such as Ebola virus outbreaks, food acts as the source and vehicle
for transmission of infections such as salmonellosis and campylobacteriosis and thus may be zoonoses.
Food products are regulated at several levels but simple hygienic precautions will prevent many
infections. Correct storage temperatures will prevent multipUcation of microbes and adequate cooking
temperatures will

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■ BOX 11.2 POLIOMYELITIS

Whilst the improvements in deahng with urban sewage disposal ehminated faeco-oral diseases such
as typhoid and cholera from England, the reduction in exposure to other faeco-orally-spread
microbes had an unexpected consequence: an increase in paralytic poliomyehtis. Poliomyelitis virus
is an enterovirus; it is excreted from the intestine and transmitted faeco-orally. In developing
countries, the virus is widespread and can be readily isolated from sewage. Like other human virus
infections the organism is mostly asymptomatic (95 per cent of infections are without symptoms) but
the incidence of symptomatic illness, notably paralytic poliomyehtis, increases the older the age of
the patient on first exposure. If the virus is circulating widely in the community then the age of
exposure is very young. Improvements in sanitation will result in a reduction in prevalence of the
virus and the average age of infection increases and, consequently, the incidence of paralytic
pohomyelitis increases disproportionately. Thus in contrast to many infectious diseases,
improvements in sanitation result in an increase of clinical disease, although the overall incidence of
the infection is reduced overall.

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kill organisms. Pasteurisation of milk is an outstanding example of the efficacy of disinfection
eliminating a disease; sterilisation of milk at extreme temperature levels is not necessary to kill
Mycobacterium bovis. The control of food-borne disease will depend on removal of the pathogens in the
original foodstuff (e.g. salmonellae in chickens and their feed) as well as maintaining correct handling
and cooking methods. Each of the various steps in the food chain (source, manufacture, distribution and
destination) should offer control points at which microbial contamination can be checked.

11.3.3 CONTROL OF VECTORS

The control of vector-borne disease has significantly greater problems than in directly acquired zoonoses
such as food poisoning. With the increases in the distribution and number of cases of Dengue fever, an
arthropod-borne illness caused by a flavivirus, we have problems similar to those in controlling malaria,
notably controlling the mosquitoes that carry the virus. The mosquitoes that carry dengue virus, Aedes
spp., have spread back into the central Americas having once been eradicated. This means that Dengue
is now the most common arbovirus infection in humans responsible for millions of cases annually. It is
clear that eradication is likely to be impossible for any zoonosis because it will be unlikely that the
reservoir(s) or vectors can be eradicated, especially if the organism is able to reside in a variety of host
and vector species. With dengue virus, the virus is maintained in monkeys and transferred by the
mosquito within forests (hence termed sylvatic cycle). Disturbingly, the virus has widened its vector
range by using the Aedes species that prefer urban environments {Aedes aegypti) such that dengue is
maintained within human populations (the urban cycle). Note then that urban dengue is not a zoonosis).
You should note that with malaria in humans, the species responsible (protozoa of the genus
Plasmodium) are restricted to, and transmitted only between, humans. Malaria is therefore not a
zoonosis. Theoretically at least it is possible to eradicate malaria if you eradicate the vector. With
zoonoses, eradication of the natural host is likely to be impossible. Under such circumstances one can
only hope for controlling the infection rather than eradicating the infection. The term 'eradication' is not
used to describe the treatment of an individual patient with antimicrobial drugs, but should be reserved
when discussing the elimination of a disease from a geographical zone (or, famously, smallpox from the
world!).

To stand any chance of controlling vector-borne infections, an understanding of the biology of the vector
itself is essential. As microbiologists, one may slip into thinking that expertise in the virus or bacterium
is sufficient, but the behaviour of the vector is critical in devising strategies of control. For example, the
sixty species of Anopheles mosquito that can transmit malaria to humans differ in their biting times and
preference for breeding sites. The key features include knowledge of their breeding sites, biting
behaviour, resting sites and host preferences (animal or human).

Female mosquitoes are blood sucking because of the nutritional demands of producing eggs.
Males can live off nectar.

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Wholescale spraying with insecticides such as DDT has been effective in the past, but the problem of the
mosquito developing resistance to the insecticide has been steadily increasing. At first sight DDT
appears to be attractive: stable (it remains active on the walls of sprayed rooms for days) and selectively
toxic; weight for weight the toxicity is greatest against insects than against higher animals.
Unfortunately, DDT has a half life of greater then 100 years and accumulates to toxic levels in the food
chain. The environmental side effects of DDT have led to the more controlled use of DDT and other less
potent chemical insecticides exemplified best in the development of impregnated bed nets. Of the
various biological controls tested the use of a larvacidal

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toxin has had the most success. Bacillus thuringiensis produces a pore-forming toxin that kills insect
larvae and, being a member of the genus Bacillus, produces endospores which makes it feasible to spray
large areas with a suspension of the organism in a suitably robust form.

Prompt diagnosis and treatment of individual cases of vector-borne diseases will also serve to
reduce the opportunities for transmission from infected hosts.

The control of vectors is a national effort with the assistance of the World Health Organisation to co-
ordinate such programmes. The financial resources and political commitment need to be maintained if
any prolonged effects are to be realised. The return of mosquitoes to areas that have been previously
cleared has been witnessed in many areas of the globe. Most vector control programmes aim to reduce
the population size of the vector. Eradication being that much more difficult to sustain, it will usually
stand better chances of success on islands rather than mainland areas. Table 11.2 compares control and
eradication strategies.

11.3.4 IMMUNO- AND CHEMOPROPHYLAXIS

Immunoprophylaxis is the administration of immune serum to people who are considered at acute risk
of developing diseases against which there is a protective anti-serum, for example, tetanus, botulism and
diphtheria. Such protection is effective but of limited scope in that it protects the few people at risk for a
limited period of time but has no effect on the control of the disease in the community. There is little, if
any, distinction between the terms 'immunoprophylaxis' and 'passive vaccination'.
Chemoprophylaxis is the use of antibiotics in two circumstances:

1 . to protect contacts of patients with infectious diseases from developing clinical disease, and

2 . to provide temporary protection during the course of a medical intervention or surgical operation.

Again, chemoprophylaxis provides cover for the immediate contacts and cannot be employed to provide
prolonged, widespread protection for communities. Antibiotics have not eliminated infectious disease or
infections from the world, nor in fact have they significantly reduced their incidence. It may be argued
that the prophylactic use of antibiotics has prevented infections in specific circumstances such as
outbreaks of meningococcal meningitis in schools. In these circumstances close contacts (e.g. school
children in the same school) are given antibiotics to prevent the disease developing. Immunisation will
be inappropriate (assuming there is a suitable vaccine) because the time needed to develop immunity (2-
4 weeks) will leave the contacts unprotected in the short term. Meningococcal meningitis can occur in
clusters of people in a pattern

Table 11.2 Comparison of control and eradication programmes for arthropod-borne diseases

Control Eradication

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Objective Reduce the incidence of the disease Stop transmission and eliminate human

infection
Duration Indefinite Limited period of time

Area of Focused to key areas (urban environments, intense All areas where transmission occurs

operation transmission)

Long-term risks Indefinite, recurring costs Imported cases

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consistent with a common source outbreak. As vaccines are not available for all the serogroups of
Neisseria meningitidis (antibodies against the polysaccharide capsules are protective) antibiotics are
given to prevent the disease developing.

Another area where antibiotics have prevented infection is the prophylactic cover for surgical
operations. For protection during surgery, the antibiotics need to be broad spectrum and given so that the
peak levels are obtained during the operation. Whilst this has not abolished post-operative infections, the
incidence of post-operative infections would undoubtedly be higher if such prophylaxis was omitted.
Table 11.3 summarises some of the factors that are likely to have an impact in reducing the infectious
disease burden on a country. As stated earlier, the epidemics that scourged England were falling in both
number and mortality before the advent of vaccines and antibiotics.

11.4 ANTIBIOTIC RESISTANCE (BACTERIA)

The most pressing problem facing medicine is the growing number of micro-organisms that are now
resistant to a wide range of antibiotics. The term antibiotic resistance describes the condition where
bacteria have developed or acquired means to overcome the inhibitory effect of one or more
antibiotics. A chilHng example is multidrug-resistant Mycobacterium tuberculosis. Without the
development of new antibiotics, thoracotomy (removal of the affected lobes of the lung) appears to be
the only treatment option.

The mechanisms by which bacteria develops resistance to antibiotics are either due to mutation or
acquisition of resistance genes from other organisms.

11.4.1 MUTATION

CHnically, mutation leading to resistance has not been a major problem compared with the acquisition of
resistance genes. In general, the effect of mutation will be to modify the target protein such that the
binding affinity of the antibiotic is reduced. The protein will tolerate a certain loss of efficiency due to
mutations, but constraints will limit the number and frequency of viable mutations in the active site. The
affinity of binding will fall, but not such that the protein function is lost completely. If the binding site
for the antibiotic is distinct from the active site of the protein, then there will be more scope for mutation
to occur without significant loss of function. For example, the binding of an antibiotic on a ribosome
might sterically hinder the correct binding of the tRNA and lead to misreading. If the antibiotic
competed with the tRNA for the same binding site, then mutation of the tRNA binding site will limit the
extent of mutation.

Mutational events reduce the potency of the antibiotic for the target site and this is observed by small
increases in the concentration of antibiotic needed to inhibit the

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Table 11.3 Likely causes for control of certain infectious diseases

Reduced host Reduced transmisssion Improved Improved treatment

susceptibility diagnosis

Improved nutrition immunisation antibiotics rapid diagnostic

tests
Improved housing improved public health: water, food,

sanitation, antibiotics

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growth of the organism (minimum inhibitory concentration, MIC); for example, the MIC of Staph,
aureus to penicillin may increase from 0.01 |Jg/ml to 0.05 |ig/mL. By comparison, acquisition of
resistance genes usually results in large increases in the MIC (e.g. from 1 |ag/ml to >64 |ag/mL).
An example of mutational resistance of clinical importance is the resistance of Mycobacterium
tuberculosis to the aminoglycoside streptomycin. During the treatment of patients with tuberculosis, the
proportion of bacteria that are resistant to the effects of streptomycin increases steadily with time due to
mutations in the ribosomal binding site for streptomycin. The frequency of mutation of Mycobacterium
tuberculosis is approximately 1 in 10^, meaning that one in every 10 million cells undergoes a
mutational event that affects streptomycin activity. The mutation rate is fixed and therefore occurs
irrespective of the presence of streptomycin. Thus, in patients taking streptomycin, the selective pressure
results in the resistant mutants continuing to grow (unlike the susceptible cells), displacing the falling
numbers of streptomycin susceptible cells until eventually all the bacteria will be resistant to
streptomycin. If the antibiotic is withdrawn then the selective pressure vanishes and the susceptible cells
will regain dominance because of the cost of streptomycin resistance in terms of reduced efficiency of
ribosomal function. The key feature here is the constant rate of mutation; all that changes is the selective
pressure on the organism by exposing the organisms to streptomycin.

Mutational resistance that causes problems for the treatment of a bacterial infection is largely restricted
to Mycobacterium tuberculosis. Why?

The organism has a long generation time (2-4 hours) compared with 'fast' growing organisms such as
Esch. coli with a generation time of 20-30 minutes and Escherichia coli has multiple copies of the
rRNA gene whereas Mycobacterium tuberculosis has only one. Any mutation in one of the appropriate
genes in Escherichia coli will be recessive to the unaffected gene copies whereas mutations in the single
copy of the gene in Mycobacterium tuberculosis will be expressed.

11.4.2 ACQUISITION OF RESISTANCE GENES (HORIZONTAL GENE

TRANSFER)

Resistance genes are thought to exist in populations of bacteria that will be exposed to antibiotics in their
natural environment. Bacteria in the soil will encounter antibiotics produced by fungi and Streptomyces
spp. The strong selection pressures exerted by antibiotic use in agriculture (animal feeds, crop spraying,
etc.), hospitals and the home (in countries where antibiotics can be bought freely at the pharmacy
without prescription) has resulted in bacterial pathogens acquiring resistance genes from other
organisms. The horizontal gene exchange of transmissible plasmids containing resistance genes means
that effective antibiotic resistance is acquired in one step rather than waiting for mutational events to
develop. The resistance genes have become widespread throughout different pathogens across the globe.
Their mobility is conferred by becoming incorporated in transposons that, in turn, accumulate as gene
cassettes in mobile plasmids called multidrug resistance plasmids (often abbreviated to R plasmids).
They resemble the pattern observed with pathogenicity islands, clusters of genes all coding for related

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functions.

Mutational events may be the only option for organisms that do not readily exchange genes horizontally.
Again, Mycobacterium tuberculosis is a good example. The cell wall structure of Mycobacterium
tuberculosis has the extra mycolic acid/arabinogalactan layers that may prevent transformation,
conjugation and transduction.

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11.4.3 RESISTANCE TO ANTIBIOTICS: CELLULAR MECHANISMS

11.4.3.1 Enzymatic inactivation of the antibiotic

A good example is the production of ^-lactamase, an enzyme that hydrolyses the P-lactam nucleus of P-
lactam antibiotics (penicillins). These enzymes may be found on the chromosome or on resistance
plasmids and are expressed constitutively or as an inducible enzyme, only when P-lactams are present.
The strategy adopted is exempHfied in the Gram positive organism Staphylococcus aureus which
produces inducible P-lactamase to be released extracellularly. In contrast the Gram negative Escherichia
coli constitutively produces much smaller amounts of ^-lactamase but sites it in the periplasmic space.
In this way Escherichia coli chooses to only degrade P-lactam antibiotic that permeates through the
porins in the outer membrane. Inactivating enzymes are also found that attack aminoglycosides and
chloramphenicol.

11.4.3.2 Active efflux of antibiotic

The bacterium expends energy to actively extrude antibiotic from within the bacterial cell. The efflux
pumps responsible may be able to pump more than one type of antibiotic (multidrug resistance efflux
pumps).

11.4.3.3 Reduced uptake

An alternative to removing antibiotic once it has reached the cytosol is to mutate the mechanisms that
are responsible for the uptake of the antibiotic in the first place. Such a strategy relies on active, i.e.
energy expending, mechanisms of uptake. Passive diffusion will not apply.

11.4.3.4 Modification of drug target

Mutations will result in alterations of the binding sites in antibiotic target proteins. Penicillin binding
proteins, for example, can reduce their binding affinity for -lactam antibiotics. Alterations in the amino
acid sequences of the ribosomal proteins can also result in reduced binding of the antibiotics that act on
protein synthesis.

11.4.3.5 Overproduction of target

With antibiotics such as the sulphonamides and trimethoprim, competitive inhibitors of enzymes
involved in the biosynthesis of folic acid, it is logical to see how bacteria have developed resistance to
these drugs by simply overproducing enzymes. In the case of trimethoprim, the organism produces

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excess dihydrofolate reductase in order to overcome the competition from the drug.

Some of these mechanisms are produced by mutation, others are simply acquired through horizontal

gene transfer. The origins of the resistance mechanisms lie in other organisms, presumably those that

produce antibiotics themselves. When an organism is under prolonged pressure through exposure to

antibiotics, in time, the appropriate resistance mechanism will eventually make itself available.

It is important to realise that bacteria can be 'resistant' to the inhibitory effects of antibiotics through

mechanisms that are neither mutational events nor resulting from acquisition of exogenous resistance

genes.

• Antibiotics have a particular spectrum of activity. Those organisms that are unaffected by a
particular antibiotic may lack the target site or the mechanisms by which the antibiotic enters the
bacterium.

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Even though several antibiotics are bactericidal (e.g. P-lactams), they can only exert this effect on
actively growing bacteria; the organisms in stationary phase are not killed. This is a general
phenomenon and applies not just to antibiotics but to disinfectants and other antibacterial
conditions (starvation, drought). Endospores represent the extreme example of this strategy:
completely inert structures that dominate in unfavourable conditions that are effectively unaffected
by antibiotics.

Dense collections of bacteria as biofilms offer protection partially through physical restriction and
binding of the antibiotic so as to reduce the active concentration that can penetrate the biofilm.

11.5 ANTIVIRAL DRUG RESISTANCE

When an antibacterial or antiviral agent acts at a single target site then resistance can develop through
mutations at that site. The high mutation rates in RNA viruses will mean that such problems will occur
more readily than with DNA-based bacteria and this is witnessed in HIV therapies. Again, as with
antibiotic pohcies, certain manoeuvres can be employed to minimise resistance developing. These
include the use of combinations of antiviral compounds, switching between two unrelated agents
(although sequential use of antivirals has been criticised for simply promoting resistance rather than
preventing it) and the use of high concentrations to prevent any viral replication ('knock out')- This,
theoretically, prevents any viable mutants being released.

Resistance to antiviral agents will result from mutations in the viral genome. The modified proteins may
act on cell events that occur upstream of the active compound (e.g. mutations in viral thymidine kinase
will reduce its affinity of binding to the nucleoside) or mutations may occur in the target protein itself (e.
g. retroviral reverse transcriptase). Whilst mutation is a frequent event in RNA viruses, and resistance
develops frequently during the course of an infection, the spontaneous mutation events in a DNA virus
like Herpes simplex will be of much lower frequency. Note that latent Herpes simplex virus does not
express TK or DNA polymerase, hence the virus remains unaffected by the drug.
The laboratory testing for resistance to antiviral agents has a number of problems. Direct testing of the
inhibitory concentrations of antiviral agent (phenotypic testing) gives a concentration that is active, at
least under laboratory conditions. Phenotypic testing requires that the organism has been cultured and
this is not possible for those viruses that have not yet been successfully cultured in vitro. Molecular
mechanisms avoid this problem. Genotype testing can, in theory, be carried out on cUnical samples as
well as cultured virus. Polymerase chain detection for resistant gene sequences are increasingly available
and are less technically demanding than phenotypic tests. The interpretation of both types of testing is
somewhat arbitrary and, Uke antibiotic susceptibihty testing in bacteria, will only indicate possible
outcomes or provide frames of reference.

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■

11.6 SURVEILLANCE

In the latter half of the 1980s, the incidence of tuberculosis had increased to 'epidemic' proportions in a
developed city, New York, in the most prosperous country in the world. How could such a well-
recognised infectious disease against which an effective vaccine and a range of antibiotics are available
spread unnoticed in such a city? The reasons illustrate how good public health infrastructure can be
undermined if unsupported. Famously associated with poverty, tuberculosis had steadily increased in a
place where HIV was endemic amongst people living in overcrowded, poor housing. The treatment of
tuberculosis uses three antibiotics over 6 months and patients with pulmonary disease who are the
infectious sources quickly become non-infectious following

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antibiotic treatment. If patients fail to complete the treatment, they will relapse into infectious cases and
antibiotic resistance may result. Of the measures taken, DOT (direct observation of therapy) was
crucial to the management of the epidemic. DOT targets the source by effecting a reduction of the
proportion of infectious cases. One of the reasons for the return of tuberculosis was the reduced funding
to public health departments responsible for monitoring (surveillance) and treating the disease. Partly
this was a consequence of years of success in reducing the incidence of tuberculosis such that
complacency had set in. Not surprisingly the cost of bringing the epidemic under control was more than
the savings made in the first place.

The value of quarantine measures to screen imported animals for infectious diseases needs
consideration. The ancient practice of retaining ships in port for 40 days ('quarantina')
applied to all trade, not just to animals, and was driven mostly by a need to prevent the spread
of plague. With the spatial isolation of an island, quarantine can prevent the introduction of a
disease. With increasing international travel by air, the principle has been undermined. The
building of a tunnel under the English channel to connect the United Kingdom with the rest of
Europe means the isolation is breached again.

11.7 FUTURE EPIDEMICS

Epidemics have been a regular feature of human existence throughout history, so it is easy to state with
confidence that they will continue to be so. The reasons for such a gloomy outlook are not difficult to
find (see Box 11.3) . As populations have increased in size, particularly in cities, so has the incidence of
infectious disease. The proportion of people infected by certain infectious diseases (tuberculosis is again
an example) may fall, but the absolute numbers of people infected will still increase. The present
population forecasts indicate increasing numbers of large (greater than 10 million people) cities, mostly
in ports and concentrated in developing countries. Other than providing a critical population threshold to
sustain diseases Hke measles, sexually transmitted diseases and the common cold, large cities need
global commerce to support them, as well as attracting migration, which both increase the inward and
outward flux of people, goods and microbes. The pubHc health infrastructure needs to be appropriately
sized and funded to prevent the resurgence of old infections that, although once under control, will
readily return without continued control. The expansion of the areas of known infectious disease is a
result of expanding urban slums into the country such that the associated risks of deforestation and
monoculture of crops all result in disturbing zoonotic cycles. It is also depressing to note the regular
appearance of epidemics that accompany war, a habit that, far from diminishing, seems to be on the
increase and risks employing biological weapons. Although poverty is famously Unked with poor health,
notably tuberculosis, wealthy nations are not excluded from risk as advances in medicine keep more
people aUve with increasing opportunities for infections.

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International surveillance will become increasingly important as the world shrinks through globalisation
in order that infectious diseases can be tracked. The bioterrorist threat, exemplified by the crippling cost
of sending a few samples of anthrax spores

■ BOX 11.3 EXAMPLES OF CHANGING PATTERNS IN INFECTIOUS
DISEASE

• Previously unrecognised diseases: Lyme disease (Borrelia burgdoferi), Helicobacter
pylori gastritis.

• Increasing zone of disease: increasing malaria and Dengue due to the spread of
mosquito vectors into new areas.

• Resurgence of 'old' infections: epidemic wave of diphtheria in Europe affecting the
old communist bloc.

• Apparent synergy in tuberculosis with HIV in AIDS patients.

• Increasing incidence of drug-resistant tuberculosis.

• Food-borne infections increasing with globalisation of food products.

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through the post, is a further call to adequate microbiological infrastructure and expertise. The factors
that are important in protecting the pubUc from biological weapons are similar to those that are
considered in all microbiology laboratories. The comparisons are given in Box 11.4 .
The need for microbiologists is obvious but should that idea need reinforcement then it is worth noting
that the cost of eradication of poliomyelitis in the USA has paid for itself every 26 days since 1977. The
financial losses to a country incurred through infectious disease every year may yet make governments
take notice.

Having described the human activities that promote infectious disease, what are the features of the
organisms that will continue to create problems? RNA viruses are the organisms with the highest
mutation rates. It can be expected that such organisms will continue to cause epidemics, just as HIV has
been the most frightening of recent times because of the genetic variation that occurs through inaccurate
transcription and genetic reassortment. HIV has demonstrated the potential impact that an RNA virus
can have if it is able to cross the species barrier (from apes to humans). The risk of mutational events
resulting in greater virulence in bacteria and fungi is less than for RNA viruses, but the increasing
recognition that toxins and pathogenicity islands are coded for on mobile genetic elements means that
bacteria will continue to present threats. The increase in antibiotic resistance genes through horizontal
gene transfer over the last 50 years reminds us that we have not yet overcome the existing microbial
threats.

■ BOX 11.4 HAZARD GROUPS

In order to protect the staff in microbiology laboratories as well as the public and the environment,

micro-organisms have been categorised into four Hazard Groups. The groups, in increasing order of

hazard, are as follows:

Group 1 : Unlikely to cause human disease.

Group 2: Can cause disease and may be a hazard to employees.

Unlikely to spread to the community.

Effective prophylaxis or treatment available.
Group 3: Can cause human disease.

Risk of spread to the community.

Effective prophylaxis or treatment available.
Group 4: Can cause severe human disease.

Likely risk of spread into the community.

No effective prophylaxis and treatment.
The micro-organisms considered the most effective biological weapons are chosen because
they:

• are easily disseminated into the public or are highly infectious (readily transmitted
from person to person).

• cause high mortahty.

• require special action for public health control.

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The organisms mostly fall into Hazard Group 4. Viruses such as Variola major virus
(smallpox) and Ebola and Marburg viruses are not easily treated with antiviral agents but
there are effective vaccines available (although not routinely used). Bacillus anthracis and
Yersinia pestis (the aetiological agent of plague) can be treated with antibiotics.

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Page 255

11.7.1 RABBIT OR THE TORTOISE?

Headline infections such as AIDS and cholera, in which epidemics sweep through populations with
dramatic consequences, will eclipse the fact that the most frequent cause of death due to an infectious
micro-organism is tuberculosis. With chronic but steady progression, the disease is quietly spreading
through developing countries such that almost 2 billion people are thought to be infected, 2 million of
whom die every year. Mycobacterium tuberculosis is a strict human pathogen and has no other reservoir.
It is interesting to compare how M. tuberculosis has obtained this position of dominance with organisms
like Vibrio cholerae or HIV. The organism has a cell wall structure that is distinct from the archetypal
Gram positive and Gram negative bacteria; instead it is dominated by a thick layer of complex
glycolipids. The organism has one of the slowest growth rates of all pathogens. It has no known protein
exotoxin or type III secretion systems or recognised pathogenicity islands, and horizontal gene transfer
appears to be rare. The pathology instead results mostly from the host responses. Whereas most bacterial
pathogens are driven from carbohydrate metabolism, M. tuberculosis utilises lipids as principle
metabolic fuels and then uses lipid products to build the cell wall. Lipid-based compounds are
manufactured to act as immunomodulatory molecules that confer the ability to persist for life in the host,
residing within macrophages. Spread via aerosols from infectious cases, the organism adopts a common
mode of transmission. What distinguishes M. tuberculosis from another aerosol-transmitted infectious
disease like measles is the duration of infection (lifelong). By adopting a 30 year latency period within
the host, the infectious cases are virtually guaranteed to encounter a susceptible host at some stage.
Although the difficulties in genetically manipulating mycobacteria and having no suitable animal
models of latency have delayed research on putative virulence factors, an understanding of disease
epidemiology suggests that the long-term approach to parasitism is an important feature. Whatever the
mechanisms, the genus Mycobacterium appears to be well suited to the parasitic existence both in
humans and other animals; Mycobacterium bovis is the major pathogen of animals.

■ SUMMARY

Epidemiological analyses have become increasingly important in the control of infectious

diseases. The concept of the reproductive rate has proven to be a useful starting point for

understanding the epidemiology of infectious diseases and permits estimates of the

percentage vaccination uptake that is required to eliminate an infectious disease. Similarly,

the concept of the persistence threshold number can predict the persistence or periodic

epidemic waves of an infectious agent.

An increasing number of vaccines, both in range of targets and vaccine type, have been

developed and still they provide the most effective means of controlling infectious diseases

in populations. The use of antibiotics is becoming increasingly threatened through the

development of resistance mechanisms by numerous pathogenic micro-organisms.

The patterns of human behaviours are powerful influences in promoting opportunities for

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new infectious disease. As human behaviour continues to change local and global ecology,
new infections and epidemics will continue to appear. Thus the role of public health
measures remain as important today as they were in the early nineteenth century, but
international surveillance is likely to be the next crucial strategy in attempting to minimise
the developing microbial threats to human health.

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Page 256

RECOMMENDED READING

Ciba Foundation Symposium, Number 207 (1997) Antibiotic Resistance: Origins, Evolution, Selection
and Spread, Wiley, Chichester, UK.

Davies, J. (1994) Inactivation of antibiotics and the dissemination of resistance genes. Science 264,

375-82.

Nikaido, H. (1994) Prevention of drug access to bacterial targets: permeability barriers and drug

efflux. Science 264, 382-8.

Noah, N. and O'Mahony, M. (eds) (1998) Communicable Disease Epidemiology and Control, John

Wiley & Sons Ltd, Chichester, UK.

Nokes, D.J. and Anderson, R.M. (1988) The use of mathematical models in the epidemiological

study of infectious diseases and in the design of mass immunisation programmes. Epidemiol. Infect.

101, 1-20.

Spratt, B.G. (1994) Resistance to antibiotics mediated by target alterations. Science 264, 388-93.

REVIEW QUESTIONS

Question 11.1 What are the mechanisms by which bacteria resist the action of antibiotics?

Question 11.2 Define the reproductive rate {R^. How does it differ from the net reproductive rate
(/?)?

Question 11.3 What are Arbovirus infections and why are they difficult to control?

Question 11.4 What is passive vaccination?

Question 11.5 Compare and contrast control and eradication programmes.

Question 11.6 List factors that may be thought responsible for the development of 'new' epidemics
or infectious diseases.

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INDEX

absorbance 59, 64

absorption zone 105

acetyl coenzyme A 68

N-acetyl glucosamine (NAG) 10, ii, 106

N-acetyl muramic acid (NAM) 10, J_i

acid fast bacilli ]3, H

acyclovir 141 , 252

adenosine triphosphate (ATP) 66-1 \

adhesin 2i, 193

Aedes aegypti 247

aflatoxin 234

agar 63, 65

AIDS, see human immunodeficiency virus

alanine jj_

alcohol(s) 130, ill

alkylating agents 139

amino acid (s) 166-7

D-10

L-10
p-aminobenzoic acid see para-amino benzoic acid
aminoglycosides 137
amphotericin B 144
anaerobic bacteria 52
anamorph 106, 112
anamorphic yeasts 106
animals, infections from, see zoonoses
Anopheles spp. 247
anthropophilic fungi 229
anti-toxin, see toxoid

antibacterial agents, see antimicrobial agents
antibacterial peptides 136
antibiotic(s) UO, 123, 132, 250

action of 134-40

bactericidal 120, 133

bacteriostatic 120 , 133

effect on normal flora 181

resistance 34, IM, 249-52

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antibody 100

fluorescent 100

titre 188
antifungal agents 143-5
antigenic drift 221 , 222
antigenic shift 221 , 222
antigenic variation 196 , 220, 241
antigen(s)

H- 12, 20, 157

O- 12, 20, 157
antiseptic 129

antiviral agents 139-43 , 252
apical growth 107 , 110
apoptosis 216, 217, 220, 224
arbovirus 174 , 246
arthropod-borne virus, see arbovirus
Ascomycota 116
ascospores 116
ascus 116

asexual reproduction 28, 29, 115 , 149
aspergillosis 231-2
Aspergillus sp. 228, 231-2
asynchronous growth 44
ATP see adenosine triphosphate
ATP synthase 70
attachment, bacterial 193
attack rate, viral 207, 208
attenuation 242-3
autoclave 126-7
autolysis 136
azoles 143-5
AZT 142

|3-lactam(s) 135
B-lymphocytes 224
Bacillus 22, 248

anthracis 197

stearothermophilus 128
bacteria, cocci 4-7

curved 5

cytoplasmic membrane 15-16

evolution 3

genetic plasticity 25-35

inclusions 18-19

mesophilic 50, 51

motility of 22-4

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psychrophilic 50, 5i

size of 4-7

spiral 5

survivor curve 122 , 124

thermophilic 50, 51

typing methods 156-9
bactericidal antibiotics 120 , 133
bacteriocins 180
bacteriophage 16, 27, 32, 79, 83, M, 200

structure of 84

temperate 32

typing 158
bacteriostatic antibiotics 120, 133
balanced growth 37

basic reproductive rate, see reproductive rate
Basidiomycota 116
basidiospore 117
batch culture 46-8
BCG vaccine 242
bile duct 176
binary fission

bacterial 37
of yeast 105-6
binomial distribution 61
binomial nomenclature 154
biofilm 21, 23, 25, 252
biological control of vectors 247-8
biological species concept 149
Bordetella pertussis 170 , 244
botulinum toxin 200
Buchnera sp. 166-7
budding 95, 105
Burkitt's lymphoma 223
butanediol 72
butyric acid 72

cancer 222

Candida albicans V\_, 228, 232

capacity test 132

caproic acid 72

capsid 80

capsomer 80

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capsule 20, 30, 1%, 230, 243
carbon sources 67-8
caries, dental 188
carrier(s) 176 , 189
catabolism, see metabolism
catalase 53, 54
cell, membrane 8

structure 7, 8

wall 7, 8, 103, KM, 106-7
cell-mediated immunity 231
cellulose 10, Ui
cephalosporins 135
cervical cancer 223
chemostat 63, 64
chemotaxis 24
chemotroph 68
chick embryo 97
chickenpox virus 244
chitin 10, 106, ill
Chlamydia 165, 166
chloramphenicol 137 , 138
chlorine 130
chlorophyll 115
cholera 203, 205, 255
cholera toxin 198, 199
cholesterol 144
chorioallantoic membrane 97
chromosomal DNA, see deoxyribonucleic acid
chromosome

bacterial 16, 25

length 16

supercoiling 16
cistron 26
classification 146

Adansonian 155

fungal 115

phenetic 152

special 152
Claviceps purpurea 234
clonal, populations 28, 29, 35, HI, 149
Clostridium spp. 22

botulinum 168 , 169

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dijficile 18i, 198
cluster analysis 155
coccidiodomycosis 128
coenocytic 104
colitis, diversion 181
colonisation 168 , 191
colonisation resistance 181
colony

rough 12

smooth 12
commensalism 163
competition 164, 179
complement 196 , 220
conidiophore 109 , 116
conidium (-ia) 116
conjugation 27, 29, 31
conjunctiva 211
continuous culture 63-5
Corynebacterium diphtheriae 77, 196
cowpox virus 242

Cryptococcus neoformans 151 , 228, 230-1
crystal violet 8
culture media 63
cytochrome systems 69-71
cytokines 216, 219, 220
cytopathic effect 98, 99
cytoplasmic membrane 9, 143
cytosine permease 143
cytoskeleton 107
cytotoxicity, of viruses 98-100 , 216, 218

D-value 123, 125

DDT (diphenyltrichloroethane) 247

decimal reduction time (D) 123, 125

dendrogram 155

dengue fever 247

deoxyribonucleic acid

AT:GC ratio 18

chromosomal 15-18

circular V]_

linear V]_

mutation 28

organisation 15-18 , 26

polymerase 90, Mi, 252

supercoiling 16, 139
dermatophyte fungi 228-30

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dermatophytoses 228-30
Deuteromycota 116 , 117
diarrhoea 198
diauxic growth 49
differential media 65
diffusion 5

dihydrofolate reductase 140
dikaryon 112
dilution rate 63-5
dimorphism 228, 232, 234

thermal 228
diphtheria 171

toxin 198
diploid iii, 112
disinfectants 129-32

methods of testing 132
disinfection 129-32
diversion colitis 181
DNA, see deoxyribonucleic acid
DNA gyrase 16, 17, 139
doubling time 37, 46
dyes 65
dysentery 205

Ebola virus 184, 185, 246

eclipse phase 87, 90

econiche 179

efficiency of infection 98

eggs 97

electron microscope 4, 100

electron, transport chain 66, 69, 70

electron transport chain phosphorylation 69

Embden-Meyerhof pathway 69, 72

end product

inhibition 73

repression 73, 75
endospore 3, 2i, 119, 126, 248
endosymbiont 166
endotoxin 13^, 201
enrichment culture 66
Enterobacteriaceae 151
enterotoxin 176 , 200
Enterovirus 151
Entner Doudoroff pathway 69
enzyme linked immunosorbant assay (ELISA) 101
enzymes

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autolytic 136

constitutive 251

extracellular HI

hydrolytic Ui, 234

inducible 35, 72, 73, 125

repression 35, 72, 73
epidemics 253

epidemiology of infectious disease 236-40 , 253-4
Epidermophyton 117 , 170
epithelium 180-1 , 194
Epstein Barr virus 223 , 224
eradication programmes 248
ergosterol 144, 145
ergotism 234

Escherichia coli 18, 25, 121, 1151, 156, 166, 169, 250, 251
ethidium bromide 139
ethylene oxide 129
evolution 146

of virulence 184-5
exotoxin(s) 198-200
exponential decline 120 , 121
exponential growth 37-47

facultative anaerobes 52-4

faecal-oral transmission 173-6

fatty acids 180

feedback inhibition 73-5

fermentation 69, 71, 111

filamentous fungi 103 , 104

filtration 128

fimbriae 8, 20

fission 105

flagella 8, 20, 23

flagellin 20

fleas 175

flies 175

flow cytometry 60

5 -flucytosine 143

foetus 174

folate synthesis 139

food poisoning 175 , 176 , 246

formaldehyde 129

fruiting body 103

fumarate 71

fungi

asexual reproduction 107 , 112 , 115

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cell wall 106-7

classification 115-17

genetics 111-15

spores 116-17
Fungi Imperfecta, 111 , 117
fungus, see fungi

gametes 112
gene

exchange 29-35

loss 14, 166-7

mutation 28, 29

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promoter 26

transfer 29-35 , 249
generation time 46
genome 25, 166
genotype 25, 148
gentamicin 137
genus, genera 150
geophilic fungi 229
germ tube 106
germination of spores 22
glucan 10, 106
D-glutamic acid 11, 197
glycogen 19
glycolysis 69
gnotobiotic animals 180
Gram stain 8
growth

bacterial 37-78

continuous culture 63

curve 46, 47

doubling time 37, 46

exponential 37

fungal 108-10

linear 38

logarithmic 37-46

measurement of 56-63

optimum temperature for 50-1

radial 109
rate 4, 42, 46, 195

specific growth rate 42, 44, 46, 64
growth phases 46, 47
gut 179-80

haemadsorption 99, 100

haemagglutinin 99, 221, 222

haemolysin 77, 200

haemolysis 77

hair, fungal infections of 172 , 229

hand washing 182

haploid 16, 25, HI, 112

hazard groups 254

heat.

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sterilisation 126-8

thermal death time 123
helical symmetry 81
hepatitis viruses 214, 218, 223, 241, 243
herd immunity 240, 242
Herpes simplex virus 170 , 171 , 252
heterothallic 112
histones 16

Histoplasma capsulatum 228
histoplasmosis 228

HIV, see human immunodeficiency virus
holomorph 106
homothallic 112

horizontal gene transfer 29, 250 , 254
horizontal transmission 167 , 174
hospital-acquired infection, see nosocomial

infections
host-parasite relationship 164-9
human carriers 176

human immunodeficiency virus (HIV), 142, 222, 224, 252, 254, 255
hydrogen electrode 55
hydrogen peroxide 53
hyphum (ae) 103, 231, 232

iatrogenic infection 183

icosahedron 81, 82

identification of micro-organisms 152^

immune complexes 218

immune electron microscopy 100

immune evasion tactics, viral 196 , 219

immunisation 240-5

immunity 238, 240

immunocompromised host 231

imunofluorescence 100

immunoglobulin

IgA 197

IgAse 197

IgG 100

IgM 100
immunopathology 218
immunoprophylaxis 243 , 245, 248
inactivation constant 122 , 123
incidence of infection 236
inclusion body 19, 98
incompatibility 112
induction, inducer 35

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infection 168, 192
types no, 182
infectious disease(s) 168
infectious dose 189 , 191
infectivity 191
influenza virus 221 , 243
innate immunity 216
insect vectors 174, 175, 247, 248
integration of DNA 28, 29-35
interferon 217, 220
intracellular micro-organisms 166-7
in-use test 132
invade, bacteria 194
ionising radiation 128
iron binding proteins 77
iron limitation 77, 194

k 121-3

karyogamy 112-14
keratin Ui, 229, 230
killing curve 120 , 121
Koch's postulates 172, 187

for genes 191
K0H16

lac operon 35, 74, 75
P-lactamase 251
lactic acid 72

fermentation 72
lactobacilli 18i
lactose 72, 74
lag phase 46, 47, 87
latency 215

latent heat of vapourisation 126
latent infection 170, 207, 213-15
LD5oi90

leprosy 196

leucotoxin 200

life cycle of arboviruses 247

of fungi 112-14
light microscope 4, 79
Linnaeus 149, 154
lipid A 12, 201

lipopolysaccharide (LPS) K), 12, IM, 201, 202
lipoprotein K)

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lipoteichoic acid 9, 12
lithotroph 68
logarithmic growth 38
lymph nodes 212
lymphocyte 219
lysis of cells 214
lysogeny 32
lysozyme JJ_, 180

MacConkey agar 65
macrolides 139
malachite green 14
malaria 247
mannan 231
mannoproteins 106
mating type 106, 112
mean generation time 37, 45
measles 237-45
media, culture

differential 65

enrichment 65

selective 65
meiosis 112
melanin 231
mesophile 50
metabolism 4, 34, 66

regulation of 72-6
metachromatic granules 19
methylene blue 20
microaerophile 52
micro-organisms

classification of 146-56

typing of 156-9
microscope

electron 4, 79, 100

light 4, 79

resolving power 100
milk 60

minimum inhibitory concentration (MIC) 250
mitochondria 167
molecular clock 147
MoUicutes 14
Monod growth equation 50
monolayer of cells 95-6
morphological species concept 149
mosaic, genes 35

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mosquito 246, 247

most probable number (MPN) 60-3

motility 22-3

tumbling 23, 24
mould 103

mucosal surfaces 211
MuUer's ratchet 167
multidrug resistance plasmids 250
multiple antibiotic resistance 183
mumps virus 170, 171 , 244
murein 9

mutation 28, 29, 22i, 249, 25i, 252
mutualism 164
mycelium (a) 103

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Page 260

Mycobacterium spp. \3_

bovis 247
leprae 13, 188

tuberculosis 13, 129, 137, Hi, 187, 196, 249, 250, 255
mycolic acid 13
Mycoplasma spp. 14

pneumoniae 14
mycotoxicosis 234
mycotoxin 234
myxomatosis 185

NAD/NADH, see nicotinamide adenine dinucleotide

N-acetyl glucosamine (NAG) 10, \\, 106

N-acetyl muramic acid (NAM) 10, J_i

N-acyl-homoserine lactones 25

nails, fungal infections of 229

necrosis 216

Neisseria gonorrhoea 170

Neisseria meningitidis 170

nervous system 213, 215

net reproductive rate, see reproductive rate

neuraminidase 221 , 222

neurotoxin 200

neutralism 163

neutropenia 231

nicotinamide adenine diucleotide (NAD) 66, 67, 70, 7]_

nitrogen 230-1

nomenclature 148, 153-4

normal flora 178, 204

nosocomial infections 182

nucleic acid,

alkylating agents 139

crosslinkers 139

hybridisation of 158

inhibitors of synthesis 135

intercalators 139
nucleoside analogues 141
numerical taxonomy 155
nutritional requirements of organisms 67, 68
nystatin 144

obligate aerobe 52^
obligate anaerobe 52-4

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oncogenes 224, 225
oncogenic viruses 222-4
Oomycota 116
operon 27, 35, 72, 74
organelle, intracellular 167
osmoregulation 13
osmotic lysis \3

pressure ]3
outbreak 236

outer membrane proteins 196
oxidation 55

oxidative phosphorylation, see electron chain phosphorylation
oxygen 5J_, 54

toxicity 53, 54

panmictic 35
papilloma virus 223
para-aminobenzoic acid 139 , 140
parasexual cycle 115
parasite(s)

ecto- 167

endo- 167

intracellular 165

strict human 1 69-70
parasitism 164, 168
parenteral transmission 211
passive diffusion 5-7
pasteurisation 247
pathogen 168

primary 168, 204, 227

secondary 168, 188, 227, 230
pathogenicity 189
pathogenicity island 34, 200 , 204
pathway

cyclic 139

feedback inhibition of 75

repression 75
penicillin 135 , 250

binding proteins 136 , 251
pentose phosphate pathway 69
peptidoglycan 8, 9, 135
peritrichous 2J_

persistent infections, viral 207-10 , 214
pH (hydrogen ion concentration) 56, 65
phage, see bacteriophage
phagocytosis 20

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phagolysosome 231

phases of growth, see growth

phenotype 25, 148

phospho-enol pyruvate 68

phospholipase 200

phosphorylation 203

phototroph 68

phylogeny 146

Phytopthera infestans 227

Picomaviridae 151

pill, pilus 8, 23

plague 246

plaques, viral 97, 158

plasmids 16, 18, 27, HI, 200, 250

conjugative 3J_, 32

copy number 27
plasmogamy 112

pneumococci, see Streptococcus pneumoniae
poisonous fungi 234
Poisson distribution 61
poliomyelitis virus 151, 170, 218, 245, 246
polycistronic RNA 18, 72, 137, 142
polyenes 143
polyhedron 81, 82

polymerase chain reaction (PCR) KH, 158, 159, 252
polymixin 136
polyphasic species 150
polyphosphate granules 20
polysome 137
polythetic species 150
population genetics 29-35
pore-forming toxins 198-200
porins 13^, 136 , 251
potassium hydroxide, see KOH
poxvirus 79, 220
pregancy 244
prevalence 236
promoter, see gene
propagation of virus 95-7
prophage 32
prophylaxis 217 , 248-9
proteases K)

protein synthesis inhibitors 135 , 137-9
proton(s) 66

pseudomembranous colitis 181
Pseudomonas aeruginosa 171

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psychrophile 50
public health 245
purines 29

pyogenic infection 172
pyrogenic infections 172
pyrogenicity 201
pyruvate 7]_, 72

quarantine 253

quasispecies 222

quaternary ammonium compounds 130

quinolones 139

quorum sensing 25-6

rabbit myxomatosis 185

rabies virus 98, 169, 213, 243, 240

rashes 219

reactive oxygen species (ROS) 53, 231

reassortment 222

receptors 212

recombination 222

redox potential 54^ 55

reducing agents 56

reducing equivalents 55

repression 35

reproductive rate 238, 242

reservoirs of infection 129 , 169 , 247

resistant bacteria, see antibiotics

respiration

aerobic 7J_

anaerobic 7J_

retrovirus 93, 210

reverse transcriptase, 93, 142

inhibitors 142
Reynold's number 23
ribonucleic acid 18, 139

messenger 18

polymerase 74, 91, 92, 221

ribosomal 147 , 150
ribosome 18, 19, 137-9
ribozymes 143
Rickettsia 165 , 166

prowazekii 9
ringworm 229
Ro, see reproductive rate
rough colony morphology 12

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Rous sarcoma virus 223
route of transmission 173
routes of infection 129 , 172 , 189
rubella virus JJO, 211, 244

S-layers 20

Sabin vaccine 243

salmonella food poisoning 175-6 , 246-7

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Salmonella spp. 154 , 169 , 247

typhi 169, 170, 171, 176, 189, 195
salt 65

sand fly virus 175
scanning electron microscope 100
Schizosaccharomyces 105
secondary metabolites 110 , 133
secretion systems in bacteria,, 15^ 194
selection pressure 250
selective media 65
selective toxicity 134
senescence zone 105
sepsis 215
septicaemia 201
serial dilution 58
serotype 12, 157-8
serum resistance 196
sessile bacteria 23
sewerage 245
sex pilus 2]_, 3J_
sexual reproduction 149

sexually transmitted infections 173, 174, 177, 188, 241
shedding of micro-organisms 198 , 218
Shigella dysenteriae 205
shingles, see varicella zoster virus
siderophore 77, 194
size, microbial 4
skin 172, 211, 229
skin flora 179
slow infection, viral 207
smallpox 242, 245
smooth colony morphology 12
soil 228, 230, 231, 233, 245
source of infection 129 , 169
species 149

specific growth rate 42, 44, 46, 64
specific immunity 216
spheroplasts 13^, 136
Spitzenkorper KM, 107
sporangiospores 116
sporangium (ia) 116
spore production 107, 109, UO, 116, 117

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spores 107, 177, 231

spread of organisms within host 195 , 196 , 212

Staphylococcus spp. 5

alpha toxin 198, 199

aureus 6, 65, IM, 121, IM, 251
steam 126, 127

effect of air on steam temperature 127

and pressure relationship 126
sterilisation 124-9
sterility assurance limit 171
storage granules 8, 19
storage zone, fungal 105
Streptococcus spp. 5

pneumoniae 50, 133

pyogenes 171
streptomycin 135 , 250
substrate level phosphorylation 69
sucrose 13^

sulphonamides 139, 140, 251
superoxide dismutase 53
surface area /volume ratio 5-7
sylvatic cycle 247
symbiosis HI , 164 , 165
synchronous growth 42
syncytia 99

synergy, antibiotic 139
syntrophy 76, 163
syphilis 171
systematics 146

taxa 155

TCA cycle, see tricarboxylic acid cycle
teichoic acid 9, HI
teleomorph 106, 112, HO
temperate phage 32
teratogenic infections 211
tetanus 200
toxin 200
tetracycline 137 , 138
thermal tolerance, fungal 233
thermophile 50
thigmotropism 233
thrush 232

thymidine kinase 141 , 252
ticks 175
tinea 229

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tissue culture 96

tissue tropism 212

topoisomerases 16, 17

total count 57

total viable count 57

toxins 33, 168, 198-200

toxoid 243

transduction 29, 32, 33

transferrin 77

transformation 29, 30, 215

translocation 194

transmission electron microscope 100

transmission of micro-organisms, 172 , 173 , 210, 211 , 236-41

efficiency 176-7

germline 174
horizontal 177 , 211

perinatal 174

postnatal 174
prenatal 174

vertical 177, 21i
transposon(s) 28, 31, 204
Treponema pallidum 170 , 171 , 188
tricarboxylic acid cycle 59, 69
Trichophyton spp. 117 , 170
trimethoprim 139, 140, 251
tuberculosis 252, 253
tumour necrosis factor 217
tumour suppressor genes 224
tumour viruses 223-5
turbidometry 59, 60
turbidostat 63, 64
turgor pressure 8, 1^
two component signalling 202
typhoid Mary 176
typhus 9
typing, bacterial 157-8

ultraviolet light 128
urban life cycle 247
urinary tract infection 169
urinogenital tract 173
use dilution test 132

vaccination 239-45
vaccine types 242-3
vaccinia virus 242

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vaginal tract 181

Varicella zoster virus 171 , 205 , 215

vectors 179, 246, 248

vegetative cell 22

ventilation 232

vertical transmission 29, 167 , 174

viable count 57

Vibrio cholerae 16, 76, 170, 203, 205

viraemia 212

viroceptors 220

viroid 79

virulence 177, 189-92

regulation of 202^
virus

classification 86, 114 , 148

envelope 80, 88, 89, 95

genome 84, 89-93

latent 215

nucleic acid 84, 85
viscosity 22, 23
vitamin(s) 180
volutin granules 19

warts 223
water 60

activity 56

drinking 245
whooping cough 244
World Health Organisation 242, 243, 248

yeast 105, 116, 179, 232
yellow fever virus 115 , 246
Yersinia pestis 246

Ziehl Neelsen stain 14

zoonosis (es) 169, 175, 242, 245, 246

zoophilic fungi 229

Zygomycota 104, 116

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