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Hugo and Russell’s

Pharmaceutical Microbiology

Queen’s University Belfast

Medical Biology Centre

Pharmaceutical Microbiology

Hugo and Russell’s

Pharmaceutical Microbiology

Queen’s University Belfast

Medical Biology Centre

a Blackwell Publishing company

Blackwell Science, Inc., 350 Main Street, Malden, Massachusetts 02148-5020, USA Blackwell Publishing Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK

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Library of Congress Cataloging-in-Publication Data

Hugo and Russell’s pharmaceutical microbiology / edited by Stephen Denyer, Norman A Hodges, Sean P Gorman — 7th ed.

Pharmaceutical microbiology.

QR46.5.P48 2004

615¢.1¢01579 — dc22

2003024264 ISBN 0–632–06467–6

A catalogue record for this title is available from the British Library

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Contributors, vii

Preface to Seventh Edition, ix

Preface to First Edition, x

Part 1: Biology of Microorganisms

Peter Gilbert and David Allison

Part 2: Antimicrobial Agents

8 Basic Aspects of the Structure and Functioning

of the Immune System, 117

Mark Gumbleton and James Furr

9 Vaccination and Immunization, 138

Peter Gilbert and David Allison

10 Types of Antibiotics and Synthetic

Sean Gorman and Eileen Scott

18 Non-Antibiotic Antibacterial Agents: Mode

of Action and Resistance, 306

Stephen Denyer and A Denver Russell

19 Sterile Pharmaceutical Products, 323

James Ford

20 Sterilization Procedures and SterilityAssurance, 346

Stephen Denyer and Norman Hodges

21 Factory and Hospital Hygiene, 376

Robert Jones

22 Manufacture of Antibiotics, 387

Sally Varian

Contents

23 The Manufacture and Quality Control of

Visiting Senior Lecturer

School of Pharmacy and Pharmacology

Senior Lecturer in Molecular Microbiology

Institute of Pharmaceutical Sciences

School of Pharmaceutical Sciences

Professor Stephen Denyer

Welsh School of Pharmacy

Cardiff University

Cardiff CF10 3XF

UK

Professor Roger Finch

Professor of Infectious Diseases

Clinical Sciences Building

Dr Kevin Kavanagh

Head of Medical Mycology Unit Department of Biology National University of Ireland Maynooth

Co Kildare Ireland

Dr Peter Lambert

Aston Pharmacy School Aston University Aston Triangle Birmingham B4 7ET UK

Dr Jean-Yves Maillard

School of Pharmacy and Biomolecular Sciences

University of Brighton Lewes Rd

Brighton BN2 4GJ UK

Dr Tim Paget

Department of Biological Sciences University of Hull

Hull HU6 7RX UK

Professor A Denver Russell

Welsh School of Pharmacy Cardiff University King Edward VII Avenue Cardiff CF10 3XF Wales

Dr Eileen Scott

School of Pharmacy The Queen’s University of Belfast Belfast BT9 7BL

Northern Ireland

Professor James Ford

School of Pharmacy and Chemistry Liverpool John Moores University Byrom Street

Liverpool L3 3AF UK

Dr James Furr

Welsh School of Pharmacy Cardiff University King Edward VII Avenue Cardiff CF10 3XF Wales

Professor Peter Gilbert

School of Pharmacy and Pharmaceutical Sciences

University of Manchester Oxford Rd

Manchester M13 9PL UK

Professor Sean Gorman

Professor of Pharmaceutical Microbiology School of Pharmacy

The Queen’s University of Belfast Belfast BT9 7BL

Northern Ireland

Dr Mark Gumbleton

Welsh School of Pharmacy Cardiff University King Edward VII Avenue Cardiff CF10 3XF Wales

Dr Anthony Smith

Department of Pharmacy and Pharmacology

University of Bath (5 West — 2.18)

Claverton Down

Bath BA2 7AY

UK

Professor JMB (Sandy) Smith

Head of Department of Microbiology

Otago School of Medical Sciences

Maidenhead Berks SL6 0PH UK

Dr Sally Varian

Consultant Ulverston Cumbria LA12 8PT UK

Dr David Stickler

School of Biosciences Cardiff University Main Building Museum Avenue

PO Box 915 Cardiff CF10 3TL Wales

Dr Derek Sullivan

Microbiology Research Unit School of Dental Science Trinity College Dublin 2 Ireland

Preface to the Seventh edition

We were much honoured to be recommended by

Professor A.D Russell to act as editors for the 7th

edition of Pharmaceutical Microbiology All three

of us have used this textbook in its various editions

throughout our careers as teachers and researchers,

and we recognize the important role it fulfils

As might be anticipated when a new editorial

team is in place, a substantial number of changes

have been made Well over half the chapters have

new authors or co-authors We also use Chapter 1

to give a rationale for the scope and content of the

book, emphasizing the interrelated character of the

discipline of pharmaceutical microbiology In

addi-tion, by combining and reorganizing chapters, by

introducing new material and through a revised

page format we have tried to provide readers with a

distinctive 7th edition

We must thank our contributors for their willing collaboration in this enterprise, especially Professor Russell for his continuing contri-butions, and our publishers for their support andexpertise

Finally, this addition is a tribute to the ness of A.D Russell and W.B Hugo who took upthe challenge in 1977 to produce a popular and con-cise read for pharmacy students required to studypharmaceutical microbiology We are delightedthat this current edition recognizes these origins bycontinuing the association with Hugo and Russell

farsighted-in its revised title

S.P DenyerS.P GormanN.A Hodges

When we were first approached by the publishers to

write a textbook on pharmaceutical microbiology

to appear in the spring of 1977, it was felt that such

a task could not be accomplished satisfactorily in

the time available

However, by a process of combined editorship

and by invitation to experts to contribute to the

various chapters this task has been accomplished

thanks to the cooperation of our collaborators

Pharmaceutical microbiology may be defined as

that part of microbiology which has a special

bear-ing on pharmacy in all its aspects This will range

from the manufacture and quality control of

phar-maceutical products to an understanding of the

mode of action of antibiotics The full extent of

microbiology on the pharmaceutical area may be

judged from the chapter contents

As this book is aimed at undergraduate

pharmacy students (as well as microbiologists

en-tering the pharmaceutical industry) we were under

constraint to limit the length of the book to retain it

in a defined price range The result is to be found in

the following pages The editors must bear sibility for any omissions, a point which has mostconcerned us Length and depth of treatment weredetermined by the dictate of our publishers It ishoped that the book will provide a concise readingfor pharmacy students (who, at the moment, lack atextbook in this subject) and help to highlight thoseparts of a general microbiological training whichimpinge on the pharmaceutical industry

respon-In conclusion, the editors thank most sincerelythe contributors to this book, both for complyingwith our strictures as to the length of their contribu-tion and for providing their material on time, andour publishers for their friendly courtesy and effi-ciency during the production of this book We alsowish to thank Dr H.J Smith for his advice on vari-ous chemical aspects, Dr M.I Barnett for usefulcomments on reverse osmosis, and Mr A Keall who helped with the table on sterilization methods

W.B HugoA.D Russell

Preface to the First Edition

Biology of Microorganisms

1 Microorganisms and medicines

Despite continuing poverty in many parts of the

world and the devastating effects of HIV and AIDS

infection on the African continent and elsewhere,

the health of the world’s population is progressively

improving This is reflected in the increase in life

expectancy that has been recorded for the great

majority of the countries reporting statistics to the

World Health Organization over the last 40 years

In Central America, for example, the life expectancy

has increased from 55 years in 1960 to 71 years in

2000, and the increase in North (but not

sub-Saharan) Africa is even greater, from 47 to 68 years

Much of this improvement is due to better nutrition

and sanitation, but improved health care and the

greater availability of effective medicines with

which to treat common diseases are also major

contributing factors Substantial inroads have been

made in the prevention and treatment of cancer,

cardiovascular disease and other major causes of

death in Western society, and of infections and

diar-rhoeal disease that remain the big killers in

develop-ing countries Several infectious diseases have been

eradicated completely, and others from substantial

parts of the world The global eradication of

small-pox in 1977 is well documented, but 2002 saw three

of the world’s continents declared free of polio, and

the prospects are good for the total elimination of

polio, measles and Chagas disease

The development of the many vaccines and other

medicines that have been so crucial to the

improve-ment in world heath has been the result of the large

investment in research by the major international

pharmaceutical companies This has led to the

manufacture of pharmaceuticals becoming one of

the most consistently successful and important dustries in many countries, not only in the tradi-tional strongholds of North America, WesternEurope and Japan but, increasingly, in Eastern Eu-rope, the Indian subcontinent and the Far East.Worldwide sales of medicines and medical devicesare estimated to have exceeded $US 401 billion (ap-proximately £250 billion) in 2002, and this figure isrising by 8% per annum In the UK alone, the value

in-of pharmaceutical exports is currently £10.03 lion each year, a figure that translates to more than

bil-£150 000 for each employee in the industry

The growth of the pharmaceutical industry in cent decades has been paralleled by rising standardsfor product quality and more rigorous regulation ofmanufacturing procedures In order to receive amanufacturing licence, a modern medicine must beshown to be effective, safe and of good quality.Most medicines consist of an active ingredient that

re-is formulated with a variety of other materials cipients) that are necessary to ensure that the medi-cine is effective, and remains stable, palatable andsafe during storage and use While the efficacy andsafety aspects of the active ingredient are within thedomain of the pharmacologist and toxicologist, respectively, many other disciplines contribute tothe efficacy, safety and quality of the manufacturedproduct as a whole Analytical chemists and phar-macists take lead responsibility for ensuring thatthe components of the medicine are present in the correct physical form and concentration, butquality is not judged solely on the physicochemicalproperties of the product: microorganisms alsohave the potential to influence efficacy and safety

(ex-It is obvious that medicines contaminated withpotentially pathogenic (disease-causing) micro-

Chapter 1

Introduction to pharmaceutical microbiology

Stephen Denyer, Norman Hodges and Sean Gorman

organisms are a safety hazard, so medicines

administered by vulnerable routes (e.g injections)

or to vulnerable areas of the body (e.g the eyes)

are manufactured as sterile products What is less

predictable is that microorganisms can, in addition

to initiating infections, cause product spoilage by

chemically decomposing the active ingredient or

the excipients This may lead to the product being

under-strength, physically or chemically unstable

or possibly contaminated with toxic materials

Thus, it is clear that pharmaceutical microbiology

must encompass the subjects of sterilization and

preservation against microbial spoilage, and a

pharmacist with responsibility for the safe, hygienic

manufacture and use of medicines must know

where microorganisms arise in the environment,

i.e the sources of microbial contamination, and the

factors that predispose to, or prevent, product

spoilage In these respects, the pharmaceutical

microbiologist has a lot in common with food and

cosmetics microbiologists, and there is substantial

scope for transfer of knowledge between these

disciplines

Disinfection and the properties of chemicals

(bio-cides) used as antiseptics, disinfectants and

preserv-atives are subjects of which pharmacists and other

persons responsible for the manufacture of

medi-cines should have a knowledge, both from the

per-spective of biocide use in product formulation and

manufacture, and because antiseptics and

disinfec-tants are pharmaceutical products in their own

right However, they are not the only antimicrobial

substances that are relevant to medicine; antibiotics

are of major importance and represent a product

category that regularly features among the top five

most frequently prescribed The term ‘antibiotic’ is

used in several different ways: originally an

anti-biotic was defined as a naturally occurring substance

that was produced by one microorganism that

inhibited the growth of, or killed, other

micro-organisms, i.e an antibiotic was a natural product,

a microbial metabolite More recently the term has

come to encompass certain synthetic agents that are

usually used systemically (throughout the body) to

treat infection A knowledge of the manufacture,

quality control and, in the light of current concerns

about resistance of microorganisms, the use of

antibiotics, are other areas of knowledge that

contribute to the discipline of pharmaceutical microbiology

Commercial antibiotic production began withthe manufacture of penicillin in the 1940s, and formany years antibiotics were the only significant example of a medicinal product that was madeusing microorganisms Following the adoption inthe 1950s of microorganisms to facilitate the manu-facture of steroids and the development of recombi-nant DNA technology in the last three decades

of the 20th century, the use of microorganisms inthe manufacture of medicines has gathered greatmomentum It led to more than 100 biotechnology-derived products on the market by the new millennium and another 300 or more in clinical trials While it is true to say that traditionally theprincipal pharmaceutical interest in microorgan-isms is that of controlling them, exploiting micro-bial metabolism in the manufacture of medicines is

a burgeoning area of knowledge that will becomeincreasingly important, not only in the pharmacycurriculum but also in those of other disciplines em-ployed in the pharmaceutical industry Table 1.1summarizes these benefits and uses of microorgan-isms in pharmaceutical manufacturing, togetherwith the more widely recognized hazards and problems that they present

Looking ahead to the early decades of the 21stcentury, it is clear that an understanding of thephysiology and genetics of microorganisms willalso become more important, not just in the pro-duction of new therapeutic agents but in the under-standing of infections and other diseases Several ofthe traditional diseases that were major causes ofdeath before the antibiotic era, e.g tuberculosis anddiphtheria, are now re-emerging in resistant form —even in developed countries — adding to the problems posed by infections in which antibiotic re-sistance has long been a problem, and those likeCreutzfeldt–Jakob disease, West Nile virus and severe acute respiratory syndrome (SARS) thathave only been recognized in recent years

Not only has the development of resistance to established antibiotics become a challenge, so toohas the ability of microorganisms to take advantage

of changing practices and procedures in medicineand surgery Microorganisms are found almosteverywhere in our surroundings and they possess

Immunology and infectious diseases Characteristics, selection and use of vaccines and antibiotics Use of biocides in infection and contamination contr

the potential to reproduce extremely rapidly; it is

quite possible for cell division to occur every 20

minutes under favourable conditions These

characteristics mean that they can adapt readily to

a changing environment and colonize new

niches One feature of modern surgery is the

ever-increasing use of plastic, ceramic and metal devices

that are introduced into the body for a wide variety

of purposes, including the commonly encountered

urinary or venous catheters and the less common

intra-ocular lenses, heart valves, pacemakers and

hip prostheses Many bacteria have the potential to

produce substances or structures that help them to

attach to these devices, even while combating the

immune system of the body Thus, colonization

often necessitates removal and replacement of the

device in question — often leading to great

discom-fort for the patient and substantial monetary cost to

the health-care service It has recently been

estimat-ed that, on average, a hospital-acquirestimat-ed infection

results in an extra 14 days in hospital, a 10%

in-crease in the chance of dying and more than £3000

additional expenditure on health care The

devel-opment of strategies for eliminating, or at least

restricting, the severity or consequences of these

device-related infections is a challenge for

pharma-cists and microbiologists within the industry, and

for many other health-care professionals

In addition to an improved understanding of

the mechanisms of antibiotic resistance, of the links

between antibiotic resistance and misuse, and of the

factors influencing the initiation of infections in the

body, our insights into the role of microorganisms

in other disease states have broadened significantly

in recent years Until about 1980 it was probably

true to say that there was little or no recognition of

the possibility that microorganisms might have a

role to play in human diseases other than clear-cut

infections In recent years, however, our perception

of the scope of microorganisms as agents of disease

has been changed by the discovery that

Helicobac-ter pylori is intimately involved in the

develop-ment of gastric or duodenal ulcers and stomach

cancer; by the findings that viruses can cause

cancers of the liver, blood and cervix; and by the

suspected involvement of microorganisms in

di-verse conditions like parkinsonism and Alzheimer’s

disease

Clearly, a knowledge of the mechanisms wherebymicroorganisms are able to resist antibiotics, colonize medical devices and cause or predisposehumans to other disease states is essential in the development not only of new antibiotics, but ofother medicines and health-care practices that miminize the risks of these adverse situations developing

2 The scope and content of the book

Criteria and standards for the microbiologicalquality of medicines depend upon the route of administration of the medicine in question Thevast majority of medicines that are given by mouth

or placed on the skin are non-sterile, i.e they maycontain some microorganisms (within limits ontype and concentration), whereas all injections andophthalmic products must be sterile, i.e they contain no living organisms Products for otheranatomical sites (e.g nose, ear, vagina and bladder)are often sterile but not invariably so (Chapter 19).The microbiological quality of non-sterile medi-cines is controlled by specifications defining theconcentration of organisms that may be present andrequiring the absence of specific, potentially haz-ardous organisms Thus the ability to identify the organisms present, to detect those that are prohibited from particular product categories and

to enumerate microbial contaminants in the facturing environment, raw materials and finishedproduct are clearly skills that a pharmaceutical microbiologist should possess (Chapters 2–6) So,too, is a familiarity with the characteristics of antimicrobial preservatives that may be a compo-nent of the medicine required to minimize the risk ofmicrobial growth and spoilage during storage anduse by the patient (Chapters 16 and 17)

manu-For a sterile product the criterion of quality issimple; there should be no detectable microorgan-isms whatsoever The product should, therefore, beable to pass a test for sterility, and a knowledge ofthe procedures and interpretation of results of suchtests is an important aspect of pharmaceutical microbiology (Chapter 20) Injections are also sub-ject to a test for pyrogens; these are substances thatcause a rise in body temperature when introduced

into the body Strictly speaking, any substance

which causes fever following injection is a pyrogen,

but in reality the vast majority are of bacterial

origin, and it is for this reason that the detection,

assay and removal of bacterial pyrogens

(endotox-ins) are considered within the realm of microbiology

(Chapter 19)

Sterile medicines may be manufactured by two

different strategies The most straightforward and

preferred option is to make the product, pack it in

its final container and sterilize it by heat, radiation

or other means (terminal sterilization, Chapter 20)

The alternative is to manufacture the product from

sterile ingredients under conditions that do not

permit the entry of contaminating organisms

(asep-tic manufacture, Chapters 15 and 21); this latter

option is usually selected when the ingredients or

physical form of the product render it heat- or

radiation-sensitive Those responsible for the

manufacture of sterile products must be familiar

with the sterilization or aseptic manufacturing

pro-cedures available for different product types, and

those who have cause to open, use or dispense

ster-ile products (in a hospital pharmacy, for example)

should be aware of the aseptic handling procedures

to be adopted in order to minimize the risk of

prod-uct contamination

The spoilage of medicines as a result of microbial

contamination, although obviously undesirable,

has as its main consequence financial loss rather

than ill health on the part of the patient The other

major problem posed by microbial contamination

of medicines, that of the risk of initiating infection,

although uncommon, is far more important in

terms of risk to the patient and possible loss of

life (Chapters 7 and 16) Infections arising by this

means also have financial implications, of course,

not only in additional treatment costs but in terms

of product recalls, possible litigation and damage to

the reputation of the manufacturer

The range of antimicrobial drugs used to

prevent and treat microbial infections is large; for

example, a contemporary textbook of

antimicro-bial chemotherapy lists no fewer than 43 different

cephalosporin antibiotics that were already on the

market or the subject of clinical trials at the time of

publication Not only are there many antibiotic

products, but increasingly, these products really

have properties that make them unique It is farmore difficult now than it was, say, 20 years ago, for

a manufacturer to obtain a licence for a ‘copycat’product, as licensing authorities now emphasize theneed to demonstrate that a new antibiotic (or anynew medicine) affords a real advantage over estab-lished drugs Because of this range and diversity ofproducts, pharmacists are now far more commonlycalled upon to advise on the relative merits of theantibiotics available to treat particular categories ofinfection than was the case hitherto (Chapters 10,

12 and 14) A prerequisite to provide this tion is a knowledge not only of the drug in question,but the infectious disease it is being used to treat andthe factors that might influence the success of antibiotic therapy in that situation (Chapter 7).While there was a belief among some commenta-tors a generation ago that infectious disease was

informa-a problem thinforma-at winforma-as well on the winforma-ay to perminforma-anentresolution owing to the development of effectivevaccines and antibiotics, such complacency hasnow completely disappeared Although cardiovas-cular and malignant diseases are more frequentcauses of death in many developed countries, infec-tious diseases remain of paramount importance inmany others, so much so that the five leading infec-tions — respiratory, HIV/AIDS, diarrhoeal disease,tuberculosis and malaria, accounted for 11.5 mil-lion deaths in 1999 The confidence that antibioticswould be produced to deal with the vast majority ofinfections has been replaced by a recognition thatthe development of resistance to them is likely tosubstantially restrict their value in the control ofcertain infections (Chapter 13) Resistance to an-tibiotics has increased in virtually all categories ofpathogenic microorganisms and is now so preva-lent that there are some infections and some organ-isms for which, it is feared, there will soon be noeffective antibiotics It has been estimated that theannual cost of treating hospital-acquired infec-tions may be as high as $4 billion in the USA alone The scale and costs of the problem are suchthat increasing attention is being paid to infectioncontrol procedures that are designed to minimizethe risk of infection being transmitted from one patient to another within a hospital The properties

of disinfectants and antiseptics, the measurement oftheir antimicrobial activity and the factors influenc-

ing their selection for use in hospital infection

control strategies or contamination control in the

manufacturing setting are topics with which both

pharmacists and industrial microbiologists should

be familiar (Chapters 11 and 18)

It has long been recognized that microorganisms

are valuable, if not essential, in the maintenance of

our ecosystems Their role and benefits in the

carbon and nitrogen cycles in terms of recycling

dead plant and animal material and in the fixation

of atmospheric nitrogen are well understood The

uses of microorganisms in the food, dairy and

brew-ing industries are also well established, but until the

late 20th century advances in genetics, immunology

and biotechnology, their benefits and uses in the

pharmaceutical industry were far more modest For

many years the production of antibiotics (Chapter

22) and microbial enzyme-mediated production of

steroids were the only significant pharmaceutical

examples of the exploitation of metabolism of

microorganisms The value of these applications,

both in monetary and health-care terms has been

immense Antibiotics currently have an estimated

world market value of $25 billion and by this

crite-rion they are surpassed as products of

biotechnolo-gy only by cheese and alcoholic beverages, but the

benefits they afford in terms of improved health and

life expectancy are incalculable The discovery of

the anti-inflammatory effects of corticosteroids had

a profound impact on the treatment of rheumatoid

arthritis in the 1950s, but it was the use of enzymes

possessed by common fungi that made cortisone

widely available to rheumatism sufferers The

syn-thesis of cortisone by traditional chemical methods

involved 31 steps, gave a yield of less than 0.2% of

the starting material and resulted in a product

cost-ing, even in 1950s terms, $200 per gram Exploiting

microbial enzymes reduced the synthesis to 11 stepsand the cost rapidly fell to $6 per gram

Apart from these major applications, however,the uses of microorganisms in the manufacture ofmedicines prior to 1980 were very limited Enzymeswere developed for use in cancer chemotherapy (asparaginase) and to digest blood clots (streptoki-nase), and polysaccharides also found therapeuticalapplications (e.g dextran — used as a plasma ex-pander) These were of relatively minor importance,however, compared with the products that followedthe advances in recombinant DNA technology in the1970s This technology permitted human genes to

be inserted into microorganisms, which were thusable to manufacture the gene products far more efficiently than traditional methods of extractionfrom animal or human tissues Insulin, in 1982, wasthe first therapeutic product of DNA technology to

be licensed for human use, and it has been followed

by human growth hormone, interferon, blood clotting factors and many other products DNAtechnology has also permitted the development ofvaccines which, like that for the prevention of he-patitis B, use genetically engineered surface antigensrather than whole natural virus particles, so thesevaccines are more effective and safer than those produced by traditional means (Chapters 9 and 23).All these developments, together with miscella-neous applications in the detection of mutagenicand carcinogenic activity in drugs and chemicalsand in the assay of antibiotics, vitamins and aminoacids (Chapter 25), have ensured that the role of microorganisms in the manufacture of medicines isnow well recognized, and that a basic knowledge

of immunology (Chapter 8), gene cloning and other biotechnology disciplines (Chapter 24) is anintegral part of pharmaceutical microbiology

1 Introduction

Microorganisms differ enormously in terms of their

shape, size and appearance and in their genetic and

metabolic characteristics All these properties are

used in classifying microorganisms into the major

groups with which many people are familiar, e.g

bacteria, fungi, protozoa and viruses, and into the

less well known categories like chlamydia,

rick-ettsia and mycoplasmas The major groups are the

subject of individual chapters immediately

follow-ing this, so the purpose here is not to describe any of

them in great detail but to summarize their features

so that the reader may better understand the

dis-tinctions between them A further aim of this

chap-ter is to avoid undue repetition of information in the

early part of the book by considering such aspects

of microbiology as cultivation, enumeration and

genetics that are common to some, or all, of the

various types of microorganism

1.1 Viruses, viroids and prions

Viruses do not have a cellular structure They are

particles composed of nucleic acid surrounded by

protein; some possess a lipid envelope and

associat-ed glycoproteins, but recognizable chromosomes,

cytoplasm and cell membranes are invariably

absent Viruses are incapable of independent cation as they do not contain the enzymes necessary

repli-to copy their own nucleic acids; as a consequence,all viruses are intracellular parasites and are repro-duced using the metabolic capabilities of the hostcell A great deal of variation is observed in shape(helical, linear or spherical), size (20–400 nm) andnucleic acid composition (single- or double-stranded, linear or circular RNA or DNA), but al-most all viruses are smaller than bacteria and theycannot be seen with a normal light microscope; in-stead they may be viewed using an electron micro-scope which affords much greater magnification.Viroids (virusoids) are even simpler than viruses,being infectious particles comprising single-strand-

ed RNA without any associated protein Those thathave been described are plant pathogens, and, sofar, there are no known human pathogens in thiscategory Prions are unique as infectious agents inthat they contain no nucleic acid A prion is an atyp-ical form of a mammalian protein that can interactwith a normal protein molecule and cause it to undergo a conformational change so that it, in turn,becomes a prion and ceases its normal function Prions are the agents responsible for transmissiblespongiform encephalopathies, e.g Creutzfeldt–Jakob disease (CJD) and bovine spongiform en-cephalopathy (BSE) They are the simplest and most

Chapter 2

Fundamental features of microbiology

Norman Hodges

1 Introduction

1.1 Viruses, viroids and prions

1.2 Prokaryotes and eukaryotes

1.2.1 Bacteria and archaea

5 Enumeration of microorganisms

6 Microbial genetics 6.1 Bacteria 6.2 Eukaryotes 6.3 Genetic variation and gene expression

7 Pharmaceutical importance of the major categories of microorganisms

recently recognized agents of infectious disease,

and are important in a pharmaceutical context

owing to their extreme resistance to conventional

sterilizing agents like steam, gamma radiation and

disinfectants (Chapter 18)

1.2 Prokaryotes and eukaryotes

The most fundamental distinction between the

various microorganisms having a cellular structure

(i.e all except those described in section 1.1 above)

is their classification into two groups — the

prokaryotes and eukaryotes — based primarily on

their cellular structure and mode of reproduction

Expressed in the simplest possible terms,

prokary-otes are the bacteria and archaea (see section 1.2.1),

and eukaryotes are all other cellular

microorgan-isms, e.g fungi, protozoa and algae The crucial

dif-ference between these two types of cell is the

possession by the eukaryotes of a true cell nucleus in

which the chromosomes are separated from the

cytoplasm by a nuclear membrane The

prokary-otes have no true nucleus; they normally possess

just a single chromosome that is not separated from

the other cell contents by a membrane Other major

distinguishing features of the two groups are that

prokaryotes are normally haploid (possess only one

copy of the set of genes in the cell) and reproduceasexually; eukaroyotes, by contrast, are usuallydiploid (possess two copies of their genes) and nor-mally have the potential to reproduce sexually Thecapacity for sexual reproduction confers the majoradvantage of creating new combinations of genes,which increases the scope for selection and evolu-tionary development The restriction to an asexualmode of reproduction means that the organism inquestion is heavily reliant on mutation as a means ofcreating genetic variety and new strains with advan-tageous characteristics, although many bacteria areable to receive new genes from other strains orspecies (see section 6.1 and Chapter 3) Table 2.1lists some distinguishing features of the prokary-otes and eukaryotes

1.2.1 Bacteria and archaea

Bacteria are essentially unicellular, although some species arise as sheathed chains of cells Theypossess the properties listed under prokaryotes inTable 2.1, but, like viruses and other categories

of microorganisms, exhibit great diversity of form,habitat, metabolism, pathogenicity and other char-acteristics The bacteria of interest in pharmacy and medicine belong to the group known as the

Table 2.1 Distinguishing features of prokaryotes and eukaryotes

Location of chromosomes Within a true nucleus separated from the In the cytoplasm, usually attached to the cell

cytoplasm by a nuclear membrane membrane Nuclear division Exhibit mitosis and meiosis Mitosis and meiosis are absent

Reproduction Asexual or sexual reproduction Normally asexual reproduction

Cell wall composition Cell walls (when present) usually contain Walls usually contain peptidoglycan

cellulose or chitin but not peptidoglycan Ribosomes Cytoplasmic ribosomes are 80S Ribosomes are smaller, usually 70S

Storage compounds Poly-b-hydroxybutyrate absent Poly-b-hydroxybutyrate often present

eubacteria The other subdivision of prokaryotes,

the archaea, have little or no pharmaceutical

impor-tance and largely comprise organisms capable of

living in extreme environments (e.g high

tempera-tures, extreme salinity or pH) or organisms

exhibit-ing specialized modes of metabolism (e.g by

deriving energy from sulphur or iron oxidation or

the production of methane)

The eubacteria are typically rod-shaped

(bacil-lus), spherical (cocci), curved or spiral cells of

approximately 0.5–5.0 mm (longest dimension) and

are divided into two groups designated

Gram-posi-tive and Gram-negaGram-posi-tive according to their reaction

to a staining procedure developed in 1884 by

Chris-tian Gram (see Chapter 3) Although all the

patho-genic species are included within this category there

are very many other eubacteria that are harmless or

positively beneficial Some of the bacteria that

con-taminate or cause spoilage of pharmaceutical

mate-rials are saprophytes, i.e they obtain their energy

by decomposition of animal and vegetable

material, while many could also be described as

parasites (benefiting from growth on or in other

liv-ing organisms without causliv-ing detrimental effects)

or pathogens (parasites damaging the host)

Rick-ettsia and chlamydia are types of bacteria that are

obligate intracellular parasites, i.e they are

inca-pable of growing outside a host cell and so cannot

easily be cultivated in the laboratory Most bacteria

of pharmaceutical and medical importance possess

cell walls (and are therefore relatively resistant to

osmotic stress), grow well at temperatures between

ambient and human body temperature, and exhibit

wide variations in their requirement for, or

toler-ance of, oxygen Strict aerobes require atmospheric

oxygen, but for strict anaerobes oxygen is toxic

Many other bacteria would be described as

faculta-tive anaerobes (normally growing best in air but can

grow without it) or micro-aerophils (preferring

oxygen concentrations lower than those in normal

air)

1.2.2 Fungi

Fungi are eukaryotes and therefore differ from

bacteria in the ways described in Table 2.1 and are

structurally more complex and varied in

appear-ance Fungi are considered to be

non-photosynthe-sizing plants, and the term fungus covers both

yeasts and moulds, although the distinction tween these two groups is not always clear Yeastsare normally unicellular organisms that are largerthan bacteria (typically 5–10 mm) and divide either

be-by a process of binary fission (see section 4.2 andFig 2.1a) or budding (whereby a daughter cell aris-

es as a swelling or protrusion from the parent thateventually separates to lead an independent exis-

tence, Fig 2.1b) Mould is an imprecise term used to

describe fungi that do not form fruiting bodies ible to the naked eye, thus excluding toadstools andmushrooms Most moulds consist of a tangled mass(mycelium) of filaments or threads (hyphae) whichvary between 1 and > 50 mm wide (Fig 2.1c); theymay be differentiated for specialized functions, e.g absorption of nutrients or reproduction Somefungi may exhibit a unicellular (yeast-like) ormycelial (mould-like) appearance depending uponcultivation conditions Although fungi are eukary-otes that should, in theory, be capable of sexual reproduction, there are some species in which thishas never been observed Most fungi are sapro-phytes with relatively few having pathogenic poten-tial, but their ability to form spores that are resistant

vis-to drying makes them important as contaminants

of pharmaceutical raw materials, particularly materials of vegetable origin

1.2.3 Protozoa

Protozoa are eukaryotic, predominantly cellular microorganisms that are regarded as ani-mals rather than plants, although the distinction between protozoa and fungi is not always clear andthere are some organisms whose taxonomic status

uni-is uncertain Many protozoa are free-living motileorganisms that occur in water and soil, althoughsome are parasites of plants and animals, includinghumans, e.g the organisms responsible for malariaand amoebic dysentery Protozoa are not normallyfound as contaminants of raw materials or manu-factured medicines and the relatively few that are ofpharmaceutical interest owe that status primarily

to their potential to cause disease

2 Naming of microorganisms

Microorganisms, just like other organisms, are

normally known by two names: that of the genus

(plural = genera) and that of the species The former

is normally written with an upper case initial letterand the latter with a lower case initial letter, e.g

Staphylococcus aureus or Escherichia coli These

may be abbreviated by shortening the name of the genus provided that the shortened form is

Fig 2.1 (a) A growing culture of Bacillus megaterium in which cells about to divide by binary fission display constrictions (arrowed) prior to separation (b) A growing culture of the yeast Saccharomyces cerevisiae displaying budding (arrowed) (c) The mould Mucor plumbeus exhibiting the typical appearance of a mycelium in which masses of asexual zygospores (arrowed) are formed on specialized hyphae (d) The bacterium Streptomyces rimosus displaying the branched network of

filaments that superficially resembles a mould mycelium (e) The typical appearance of an overnight agar culture of

Micrococcus luteus inoculated to produce isolated colonies (arrowed) (f) A single colony of the mould Aspergillus niger in

which the actively growing periphery of the colony (arrowed) contrasts with the mature central region where pigmented asexual spores have developed.

unambiguous, e.g Staph aureus, E coli Both the

full and the shortened names are printed in italics to

designate their status as proper names (in old

books, theses or manuscripts they might be in

roman type but underlined) The species within a

genus are sometimes referred to by a collective

name, e.g staphylococci or pseudomonads, and

neither these names, nor names describing groups

of organisms from different genera, e.g coliforms,

are italicized or spelt with an upper case initial

letter

3 Microbial metabolism

As in most other aspects of their physiology,

microorganisms exhibit marked differences in their

metabolism While some species can obtain carbon

from carbon dioxide and energy from sunlight or

the oxidation of inorganic materials like sulphides,

the vast majority of organisms of interest in

pharmacy and medicine are described as

chemo-heterotrophs — they obtain carbon, nitrogen and

energy by breaking down organic compounds The

chemical reactions by which energy is liberated by

digestion of food materials are termed catabolic

reactions, while those that use the liberated energy

to make complex cellular polymers, proteins,

car-bohydrates and nucleic acids, are called anabolic

reactions

Food materials are oxidized in order to break

them down and release energy from them The term

oxidation is defined as the removal or loss of

elec-trons, but oxidation does not invariably involve

oxygen, as a wide variety of other molecules can

accept electrons and thus act as oxidizing agents As

the oxidizing molecule accepts the electrons, the

other molecule in the reaction that provides them is

simultaneously reduced Consequently, oxidation

and reduction are invariably linked and such

reac-tions are often termed redox reacreac-tions The term

redox potential is also used, and this indicates

whether oxidizing or reducing conditions prevail

in a particular situation, e.g in a body fluid or a

culture medium Anaerobic organisms prefer low

redox potentials (typically zero to -200 mV or less)

while aerobes thrive in high redox potential

envi-ronments (e.g zero to +200 mV or more)

There are marked similarities in the metabolicpathways used by pathogenic bacteria and by mam-mals Many bacteria use the same process of glycol-ysis that is used by humans to begin the breakdown

of glucose and the release of energy from it ysis describes the conversion of glucose, through aseries of reactions, to pyruvic acid, and it is aprocess for which oxygen is not required, althoughglycolysis is undertaken by both aerobic and anaerobic organisms The process releases only arelatively small amount of the energy stored in asugar molecule, and aerobic microorganisms, incommon with mammals, release much more of theenergy by aerobic respiration Oxygen is the molecule at the end of the sequence of respiratoryreactions that finally accepts the electrons and al-lows the whole process to proceed, but it is worthnoting that many organisms can also undertake

Glycol-anaerobic respiration, which uses other final

electron acceptors, e.g nitrate or fumarate

As an alternative to respiration many organisms use fermentation as a means of releasingmore energy from sugar; fermentation is, by defini-tion, a process in which the final electron acceptor is

micro-an orgmicro-anic molecule The term is widely understood

to mean the production by yeast of ethanol and bon dioxide from sugar, but in fact many organismsapart from yeasts can undertake fermentation andthe process is not restricted to common sugar (sucrose) as a starting material or to ethanol andcarbon dioxide as metabolic products Many pathogenic bacteria are capable of fermenting sev-eral different sugars and other organic materials togive a range of metabolic products that includesacids (e.g lactic, acetic and propionic), alcohols(e.g ethanol, propanol, butanediol) and other com-mercially important materials like the solvents ace-tone and butanol Fermentation is, like glycolysis,

car-an car-anaerobic process, although the term is monly used in the pharmaceutical and biotechnolo-

com-gy industries to describe the manufacture of a widerange of substances by microorganisms where thebiochemical process is neither fermentative noreven anaerobic, e.g many textbooks refer to anti-biotic fermentation, but the production vessels areusually vigorously aerated and far from anaerobic.Microorganisms are far more versatile than mam-mals with respect to the materials that they can use

as foods and the means by which those foods are

broken down Some pathogenic organisms can

grow on dilute solutions of mineral salts and sugar

(or other simple molecules like glycerol, lactic or

pyruvic acids), while others can obtain energy from

rarely encountered carbohydrates or by the

diges-tion of proteins or other non-carbohydrate foods

In addition to accepting a wide variety of food

ma-terials, many microorganisms can use alternative

metabolic pathways to break the food down

depending on the environmental conditions, e.g

facultative anaerobes can switch from respiration

to fermentation if oxygen supplies are depleted It is

partly this ability to switch to different metabolic

pathways that explains why none of the major

an-tibiotics work by interfering with the chemical

reac-tions microorganisms use to metabolize their food

It is a fundamental principle of antibiotic action

that the drug must exploit a difference in

metabo-lism between the organism to be killed and the

human host; without such a difference the

antibiot-ic would be very toxantibiot-ic to the patient too However,

not only do bacteria use metabolic pathways for

food digestion that are similar to our own, many of

them would have the ability to switch to an

alterna-tive energy-producing pathway if an antibiotic was

developed that interfered with a reaction that is

unique to bacteria

The metabolic products that arise during the

pe-riod when a microbial culture is actually growing

are termed primary metabolites, while those that

are produced after cell multiplication has slowed or

stopped, i.e in the ‘stationary phase’ (see Chapter

3), are termed secondary metabolites Ethanol is a

primary metabolite of major commercial

impor-tance although it is only produced in large

quanti-ties by some species of yeast More common than

ethanol as primary metabolites are organic acids, so

it is a common observation that the pH of a culture

progressively falls during growth, and many

organ-isms further metabolize the acids so the pH often

rises after cell growth has ceased The metabolites

that are found during secondary metabolism

are diverse, and many of them have commercial or

therapeutic importance They include antibiotics,

enzymes (e.g amylases that digest starch and

proteolytic enzymes used in biological washing

powders), toxins (responsible for many of the

symptoms of infection but some also of therapeutic

value, e.g botox — the toxin of Clostridium botulinum) and carbohydrates (e.g dextran used as

a plasma expander and for molecular separations

by gel filtration)

4 Microbial cultivation

The vast majority of microorganisms of interest inpharmacy and medicine can be cultivated in the lab-oratory and most of them require relatively simpletechniques and facilities Some organisms are para-sites and so can only be grown inside the cells of ahost species — which often necessitates mammaliancell culture facilities — and there are a few (e.g theorganism responsible for leprosy) that have neverbeen cultivated outside the living animal

4.1 Culture media

A significant number of common microorganismsare capable of synthesizing all the materials theyneed for growth (e.g amino acids, nucleotides andvitamins) from simple carbon and nitrogen sourcesand mineral salts Such organisms can grow ontruly synthetic (chemically defined) media, butmany organisms do not have this capability andneed a medium that already contains these bio-chemicals Such media are far more commonly usedthan synthetic ones, and several terms have beenused to describe them, e.g routine laboratorymedia, general purpose media and complex media.They are complex in the sense that their precisechemical composition is unknown and is likely tovary slightly from batch to batch In general, theyare aqueous solutions of animal or plant extractsthat contain hydrolysed proteins, B-group vitaminsand carbohydrates

Readily available and relatively inexpensivesources of protein include meat extracts (from thoseparts of animal carcasses that are not used forhuman or domestic animal consumption), milk andsoya The protein is hydrolysed to varying degrees

to give peptones (by definition not coagulable byheat or ammonium sulphate) or amino acids.Trypsin or other proteolytic enzymes are preferred

to acids as a means of hydrolysis because acids

cause more amino acid destruction; the term

‘tryp-tic’ denotes the use of the enzyme Many

micro-organisms require B-group vitamins (but not the

other water- or fat-soluble vitamins required by

mammals) and this requirement is satisfied by yeast

extract Carbohydrates are used in the form of

starch or sugars, but glucose (dextrose) is the only

sugar regularly employed as a nutrient

Micro-organisms differ in terms of their ability to ferment

various sugars and their fermentation patterns

may be used as an aid in identification Thus, other

sugars included in culture media are normally

present for these diagnostic purposes rather than as

carbon and energy sources Sodium chloride may be

incorporated in culture media to adjust osmotic

pressure, and occasionally buffers are added to

neutralize acids that result from sugar metabolism

Routine culture media may be enriched by the

addition of materials like milk, blood or serum, and

organisms that need such supplements in order to

grow are described as ‘exacting’ in their nutritional

requirements

Culture media may be either liquid or solid; the

latter term describes liquid media that have been

gelled by the addition of agar, which is a

carbohy-drate extracted from certain seaweeds Agar at a

concentration of about 1–1.5% w/v will provide a

firm gel that cannot be liquefied by the enzymes

nor-mally produced during bacterial growth (which is

one reason it is used in preference to gelatin) Agar is

unusual in that the melting and setting

tempera-tures for its gels are quite dissimilar Fluid agar

solutions set at approximately 40°C, but do not

reliquefy on heating until the temperature is in

excess of 90°C Thus agar forms a firm gel at 37°C

which is the normal incubation temperature for

many pathogenic organisms (whereas gelatin does

not) and when used as a liquid at 45°C is at a

sufficiently low temperature to avoid killing

microorganisms — this property is important in

pour plate counting methods (see section 5)

In contrast to medium ingredients designed to

support microbial growth, there are many

materi-als commonly added to selective or diagnostic

media whose function is to restrict the growth of

certain types of microorganism while permitting or

enhancing the growth of others Examples include

antibacterial antibiotics added to fungal media to

suppress bacterial contaminants, and bile to press organisms from anatomical sites other thanthe gastrointestinal tract Many such additives areused in media for organism identification purposes,and these are considered further in subsequentchapters The term enrichment sometimes causesconfusion in this context It is occasionally used inthe sense of making a medium nutritionally richer

sup-to achieve more rapid or profuse growth tively, and more commonly, an enrichment medium

Alterna-is one designed to permit a particular type of ism to grow while restricting others, so the one thatgrows increases in relative numbers and is

organ-‘enriched’ in a mixed culture

Solid media designed for the growth of anaerobicorganisms usually contain non-toxic reducingagents, e.g sodium thioglycollate or sulphur-con-taining amino acids; these compounds create redoxpotentials of -200 mV or less and so diminish oreliminate the inhibitory effects of oxygen or oxidiz-ing molecules on anaerobic growth The inclusion

of such compounds is less important in liquid mediawhere a sufficiently low redox potential may beachieved simply by boiling; this expels dissolvedoxygen, which in unstirred liquids, only slowly re-saturates the upper few millimetres of liquid Redoxindicators like methylene blue or resazurin may beincorporated in anaerobic media to confirm that asufficiently low redox potential has been achieved.Media for yeasts and moulds often have a lower pH (5.5–6.0) than bacterial culture media(7.0–7.4) Lactic acid may be used to impart a low

pH because it is not, itself, inhibitory to fungi at theconcentrations used Some fungal media that are in-tended for use with specimens that may also containbacteria may be supplemented with antibacterialantibiotics, e.g chloramphenicol or tetracyclines

4.2 Cultivation methods

Most bacteria and some yeasts divide by a process

of binary fission whereby the cell enlarges or elongates, then forms a cross-wall (septum) thatseparates the cell into two more-or-less equal com-partments each containing a copy of the genetic ma-terial Septum formation is often followed byconstriction such that the connection between thetwo cell compartments is progressively reduced (see

Fig 2.1a) until finally it is broken and the daughter

cells separate In bacteria this pattern of division

may take place every 25–30 minutes under optimal

conditions of laboratory cultivation, although

growth at infection sites in the body is normally

much slower owing to the effects of the immune

sys-tem and scarcity of essential nutrients, particularly

iron Growth continues until one or more nutrients

is exhausted, or toxic metabolites (often organic

acids) accumulate and inhibit enzyme systems

Starting from a single cell many bacteria can

achieve concentrations of the order of 109cells ml-1

or more following overnight incubation in common

liquid media At concentrations below about 107

cells ml-1culture media are clear, but the liquid

becomes progressively more cloudy (turbid) as the

concentration increases above this value; turbidity

is, therefore, an indirect means of monitoring

cul-ture growth Some bacteria produce chains of cells,

and some elongated cells (filaments) that may

ex-hibit branching to produce a tangled mass

resem-bling a mould mycelium (Fig 2.1d) Many yeasts

divide by budding (see section 1.2.3 and Fig 2.1b)

but they, too, would normally grow in liquid media

to produce a turbid culture Moulds, however, grow

by extension and branching of hyphae to produce a

mycelium (Fig 2.1c) or, in agitated liquid cultures,

pellet growth may arise

When growing on solid media in Petri dishes

(often referred to as ‘plates’) individual bacterial

cells can give rise to colonies following overnight

in-cubation under optimal conditions A colony is

sim-ply a collection of cells arising by multiplication of a

single original cell or a small cluster of them (called

a colony-forming unit or CFU) The term ‘colony’

does not, strictly speaking, imply any particular

number of cells, but it is usually taken to mean a

number sufficiently large to be visible by eye Thus,

macroscopic bacterial colonies usually comprise

hundreds of thousands, millions or tens of millions

of cells in an area on a Petri dish that is typically

1–10 mm in diameter (Fig 2.1e) Colony size is

lim-ited by nutrient availability and/or waste product

accumulation in just the same way as cell

concen-tration in liquid media Colonies vary between

bac-terial species, and their shapes, sizes, opacities,

surface markings and pigmentation may all be

characteristic of the species in question, so these

properties may be an aid in identification dures (see Chapter 3)

proce-Anaerobic organisms may be grown on Petridishes provided that they are incubated in an anaer-obic jar Such jars are usually made of rigid plasticwith airtight lids, and Petri dishes are placed in themtogether with a low temperature catalyst The cata-lyst, consisting of palladium-coated pellets or wire,causes the oxygen inside the jar to be combined withhydrogen that is generated by the addition of water

to sodium borohydride; this is usually contained

in a foil sachet that is also placed in the jar As theoxygen is removed, an anaerobic atmosphere isachieved and this is monitored by an oxidation-reduction (redox) indicator; resazurin is frequentlyused, as a solution soaking a fabric strip

Yeast colonies often look similar to those of teria, although they may be larger and more fre-quently coloured The appearance of mouldsgrowing on solid microbiological media is similar

bac-to their appearance when growing on commonfoods The mould colony consists of a myceliumthat may be loosely or densely entangled depending

on the species, often with the central area (the oldest, most mature region of the colony) showingpigmentation associated with spore production (Fig.2.1f) The periphery of the colony is that part which

is actively growing and it is usually non-pigmented

4.3 Planktonic and sessile growth

Bacteria growing in liquid culture in the laboratoryusually exist as individual cells or small aggregates

of cells suspended in the culture medium; the termplanktonic is used to describe such freely suspendedcells In recent years, however, it has become recog-nized that planktonic growth is not the normal situ-ation for bacteria growing in their natural habitats

In fact, bacteria in their natural state far more monly grow attached to a surface which, for manyspecies, may be solid, e.g soil particles, stone, metal

com-or glass, com-or fcom-or pathogens an epithelial surface in thebody, e.g lung or intestinal mucosa Bacteria attached to a substrate in this way are described

as sessile, and are said to exhibit the biofilm or microcolony mode of growth

Planktonic cells are routinely used for almost allthe testing procedures that have been designed to

assess the activity of antimicrobial chemicals and

processes, but the recognition that planktonic

growth is not the natural state for many organisms

prompted investigations of the relative

susceptibili-ties of planktonic- and biofilm-grown cells to

antibiotics, disinfectants and decontamination or

sterilization procedures In many cases it has been

found that planktonic and sessile bacteria exhibit

markedly different susceptibilities to these lethal

agents, and this has prompted a reappraisal of the

appropriateness of some of the procedures used (see

Chapters 11 and 13)

5 Enumeration of microorganisms

In a pharmaceutical context there are several

situa-tions where it is necessary to measure the number of

microbial cells in a culture, sample or specimen:

• when measuring the levels of microbial

contami-nation in a raw material or manufactured medicine

• when evaluating the effects of an antimicrobial

chemical or decontamination process

• when using microorganisms in the manufacture

of therapeutic agents

• when assessing the nutrient capability of a

growth medium

In some cases it is necessary to know the total

number of microbial cells present, i.e both living

and dead, e.g in vaccine manufacture dead and

living cells may both produce an immune response,and in pyrogen testing both dead and living cells in-duce fever when injected into the body However, inmany cases it is the number or concentration of

living cells that is required The terminology in

mi-crobial counting sometimes causes confusion A

total count is a counting procedure enumerating both living and dead cells, whereas a viable count,

which is far more common, records the living cells

alone However, the term total viable count (TVC)

is used in most pharmacopoeias and by many latory agencies to mean a viable count that recordsall the different species or types of microorganismthat might be present in a sample

regu-Table 2.2 lists the more common counting ods available The first three traditional methods ofviable counting all operate on the basis that a livingcell (or a small aggregate or ‘clump’ of cells) willgive rise to a visible colony when introduced into oronto the surface of a suitable medium and incubat-

meth-ed Thus, the procedure for pour plating usually volves the addition of a small volume (typically 1.0 ml) of sample (or a suitable dilution thereof)into molten agar at 45°C which is then poured intoempty sterile Petri dishes After incubation the resultant colonies are counted and the total is multi-plied by the dilution factor (if any) to give the con-centration in the original sample In a surfacespread technique the sample (usually 0.1–0.25 ml)

in-is spread over the surface of agar which has

Table 2.2 Traditional and rapid methods of enumerating cells

Traditional methods

1 Pour plate (counting colonies 1 Direct microscopic counting 1 Epifluorescence (uses dyes that give

2 Surface spread or surface drop counting chambers) cells) often coupled to image analysis (Miles Misra) methods (counting 2 Turbidity methods (measures 2 Adenosine triphosphate (ATP) methods

colonies on agar surface) turbidity (opacity) in suspensions (measures ATP production in living cells

(colonies growing on membranes on 3 Dry weight determinations 3 Impedance (measures changes in

agar surface) 4 Nitrogen, protein or nucleic acid resistance, capacitance or impedance in

4 Most probable number (counts determinations growing cultures)

previously been dried to permit absorption of the

added liquid The Miles Misra (surface drop

method) is similar in principle, but several

individ-ual drops of culture are allowed to spread over

dis-crete areas of about 1 cm diameter on the agar

surface These procedures are suitable for samples

that are expected to contain concentrations in

ex-cess of approximately 100 CFU ml-1so that the

number of colonies arising on the plate is

sufficient-ly large to be statisticalsufficient-ly reliable If there are no

clear indications of the order of magnitude of the

concentration in the sample, it is necessary to plate

out the sample at each of two, three or more

(deci-mal, i.e 10-fold) dilutions so as to obtain Petri

dish-es with conveniently countable numbers of colonidish-es

(usually taken to be 30–300 colonies)

If 30 is accepted as the lowest reliable number to

count and a pour plate method uses a 1.0-ml

sam-ple, it follows that the procedures described above

are unsuitable for any sample that is expected to

contain <30 CFU ml-1, e.g water samples where the

count may be 1 CFU ml-1or less Here, membrane

filter methods are used in which a large, known

vol-ume of sample is passed through the membrane

which is placed, without inversion, on the agar

sur-face Nutrients then diffuse up through the

mem-brane and allow the retained cells to grow into

colonies on it just as they would on the agar itself

Some of the relative merits of these procedures aredescribed in Table 2.3

Most probable number (MPN) counts may beused when the anticipated count is relatively low,i.e from <1 up to 100 microorganisms per ml Theprocedure involves inoculating multiple tubes ofculture medium (usually three or five) with threedifferent volumes of sample, e.g three tubes eachinoculated with 0.1 ml, three with 0.01 ml and threewith 0.001 ml If the concentration in the sample is

in the range indicated above, there should be a portion of the tubes receiving inocula in which nomicroorganisms are present; these will remain ster-ile after incubation, while others that received inocula actually containing one or more CFU showsigns of growth The proportions of positive tubesare recorded for each sample volume and the resultsare compared with standard tables showing theMPN of organisms per ml (or per 100 ml) of origi-nal sample The procedure is more commonly used

pro-in the water, food and dairy pro-industries than pro-in thepharmaceutical industry, nevertheless it is a validtechnique described in pharmacopoeias and appro-priate for pharmaceutical materials, particularlywater

Turbidity measurements are the most commonmeans of estimating the total numbers of bacteriapresent in a sample Measuring the turbidity using a

Table 2.3 The relative merits of the common viable counting procedures

Pour plate Requires no pre-drying of the agar surface Very small colonies of strict aerobes at the base of the agar

Will detect lower concentrations than surface may be missed spread/surface drop methods Colonies of different species within the agar appear similar

— so it is difficult to detect contaminants Surface spread Surface spread often gives larger colonies than Agar surface requires pre- drying to absorb sample

and surface drop pour plates — thus they are easier to count Possibility of confluent growth, particularly with moulds, methods Easier to identify contaminants by appearance masking individual colonies

of the colonies Membrane If necessary, will detect lower concentrations Viscous samples will not go through the membrane and

Antimicrobial chemicals in the sample can be restricting filtration capacity physically removed from the cells

spectrophotometer or colorimeter and reading the

concentration from a calibration plot is a simple

means of standardizing cell suspensions for use as

inocula in antibiotic assays or other tests of

anti-microbial chemicals Fungi cannot readily be

handled in this way because the suspension may not

be uniform or may sediment in a spectrophotometer

cuvette Consequently, dry weight determinations

on known volumes of culture are an alternative

means of estimating fungal biomass Direct

micro-scopic counting may be an appropriate method for

bacteria, yeasts and fungal spores but not for

moulds, and indirect measures of biomass like

as-says of insoluble nitrogen, protein or nucleic acids

are possible for all cell types, but rarely used outside

the research laboratory

Most of the traditional methods of viable

count-ing suffer from the same limitations:

• relatively labour intensive

• not easy to automate

• slow, because they require an incubation period

for colonies to develop or liquid cultures to become

turbid

• may require relatively large volumes of culture

media, many Petri dishes and a lot of incubator

space

For these reasons much interest and investigative

effort has been invested in recent years in the use of

so-called ‘rapid’ methods of detecting and counting

microorganisms (see also Chapter 3) These

methods enumerate viable organisms — usually

bacteria and yeasts rather than moulds — in a

mat-ter of hours and eliminate the 24–48-hour (or

longer) incubation periods that are typical of

tradi-tional procedures The rapid methods employ

various means of indirect detection of living cells,

but the following operating principles are the

most common:

• Epifluorescent techniques use fluorescent dyes

that either exhibit different colours in living and

dead cells (e.g acridine orange) or appear

colour-less outside the cell but become fluorescent when

absorbed and subjected to cellular metabolism (e.g

fluorescein diacetate)

• Living cells generate adenosine triphosphate

(ATP) that can readily be detected by enzyme

assays, e.g luciferin emits light when exposed

to firefly luciferase in the presence of ATP; light

emission can be measured and related to bacterialconcentration

• The resistance, capacitance or impedance of aculture medium changes as a result of bacterial oryeast growth and metabolism, and these electricalproperties vary in proportion to cell concentration

• Manometric techniques are appropriate formonitoring the growth of organisms that consume

or produce significant quantities of gas during theirmetabolism, e.g yeasts or moulds producing carbon dioxide as a result of fermentation

These methods are fast, readily automated andeliminate the need for numerous Petri dishes and incubators On the other hand they require expen-sive equipment, have limitations in terms of detec-tion limits and may be less readily adapted tocertain types of sample than traditional methods.Furthermore, there are problems in some cases withreconciling the counts obtained by rapid methodsand by traditional means The newer techniquesmay detect organisms that are metabolizing but notcapable of reproducing to give visible colonies, somay give values many times higher than traditionalmethods; this has contributed to the caution withwhich regulatory authorities have accepted the datagenerated by rapid methods Nevertheless, they arebecoming more widely accepted and are likely tobecome an integral part of enumeration procedures

in pharmaceutical microbiology in the foreseeablefuture

6 Microbial genetics

The nature of the genetic material possessed by amicrobial cell and the manner in which that geneticmaterial may be transferred to other cells dependslargely upon whether the organism is a prokaryote

or a eukaryote (see section 1.2)

circumstances may also be contained upon

plas-mids; these are usually similar in structure to

chro-mosomes but much smaller and replicate

independently (Chapters 3 and 13) The total

com-plement of genes possessed by a cell, i.e those in the

chromosome, plasmid(s) and any received from

other sources, e.g bacteriophages (bacterial

viruses), is referred to as the genome of the cell

Typically bacterial chromosomes are 1 mm or

more in length and contain about 1000–3000

genes As many bacterial cells are approximately

1 mm long, it is clear that the chromosome has to be

tightly coiled in order to fit in the available volume

Although all the genes are contained on a single

chromosome (rather than being distributed over

two or more), it is possible for a cell to contain

several copies of that chromosome at any one time.

Usually there are multiple copies during periods of

rapid cell division, but some species seem to have

many copies all the time The mechanisms by which

bacterial genes may be transferred from one

organ-ism to another are described in Chapter 3

Plasmids usually resemble chromosomes except

that they are approximately 0.1–1.0% of the size of

a bacterial chromosome, and there are a few that

are linear rather than circular Plasmid genes are not

essential for the normal functioning of the cell but

may code for a property that affords a survival

advantage in certain environmental conditions;

bacteria possessing the plasmid in question would

therefore be selected when such conditions exist

Properties which can be coded by plasmids include

the ability to utilize unusual sugars or food sources,

toxin production, production of pili that facilitate

the attachment of a cell to a substrate (e.g intestinal

epithelium) and antibiotic resistance A cell may

contain multiple copies of any one plasmid and may

contain two or more different plasmids However,

some plasmid combinations cannot co-exist inside

the same cell and are said to be incompatible; this

phenomenon enables plasmids to be classified into

incompatibility groups

Plasmids replicate independently of the

chromo-some within the cell, so that both daughter cells

contain a copy of the plasmid after binary fission

Plasmids may also be passed from one cell to

another by various means (Chapter 3) Some

plas-mids exhibit a marked degree of host specificity and

may only be transmitted between different strains

of the same species, although others, particularlythose commonly found in Gram-negative intestinalbacteria, may cross between different species with-

in a genus or between different genera Conjugative(self-transmissible) plasmids code for genes that facilitate their own transmission from one cell toanother by the production of pili These sex pili ini-tially establish contact between the two cells andthen retract, drawing the donor and recipient cellstogether until membrane fusion occurs

6.2 Eukaryotes

Eukaryotic microorganisms (yeasts, moulds, algaeand protozoa) possess a nucleus that normally con-tains one or more pairs of linear chromosomes, inwhich the ds DNA is complexed with protein Thecells may divide asexually and the nucleus under-goes mitosis — a sequence of events by which thenucleus and the chromosomes within it are replicat-

ed to give copies identical to the originals Most eukaryotes also have the potential for sexual reproduction during which the nucleus undergoesmeiosis, i.e a more specialized form of nuclear andchromosome division creating new gene combina-tions, so the offspring differ from the parents Despite this potential, there are some eukaryoticcells, particularly fungi, in which a sexual stage inthe life cycle has never been observed Many eukary-otic microorganisms possess plasmids, and somefungal plasmids are based on RNA instead of DNA

6.3 Genetic variation and gene expression

Microorganisms may adapt rapidly to new ronments and devise strategies to avoid or negatestressful or potentially harmful circumstances.Their ability to survive adverse conditions may result from the organism using genes it already possesses, or by the acquisition of new genetic information The term ‘genotype’ describes the ge-netic composition of an organism, i.e it refers to thegenes that the organism possesses, regardless ofwhether they are expressed or not It is not uncom-mon for a microbial cell to possess a particular genebut not to express it, i.e not to manufacture the protein or enzyme that is the product of that gene,

envi-unless or until the product is actually required; this

is simply a mechanism to avoid wasting energy For

example, many bacteria possess the genes that code

for b-lactamases; these enzymes hydrolyse and

inactivate b-lactam antibiotics (e.g penicillins) In

many organisms b-lactamases are only produced in

response to the presence of the antibiotic This form

of non-genetic adaptation is termed phenotypic

adaptation, and there are many situations in which

bacteria adopt a phenotypic change to counter

envi-ronmental stress But microorganisms may also use

an alternative strategy of genetic adaptation, by

which they acquire new genes either by mutation

or conjugation (Chapter 3); subsequently, a process

of selection ensures that the mutant organisms that

are better suited to the new environment become

numerically dominant

In bacteria, mutation is an important mechanism

by which resistance to antibiotics and other

anti-microbial chemicals is achieved, although the receipt

of entirely new genes directly from other bacteria

is also clinically very important Spontaneous

mutation rates (rates not influenced by mutagenic

chemicals or ionizing radiation) vary substantially

depending on the gene and the organism in

ques-tion, but rates of 10-5–10-7are typical These values

mean that, on average, a mutant arises once in every

100 thousand to every 10 million cell divisions Although these figures might suggest that mutation

is a relatively rare event, the speed with which microorganisms can multiply means, for example,that mutants exhibiting increased antibiotic resis-tance can arise quite quickly during the course oftherapy

7 Pharmaceutical importance of the major categories of microorganisms

Table 2.4 indicates the ways in which the differenttypes of microorganism are considered relevant inpharmacy The importance of viruses derives exclu-sively from their pathogenic potential Because oftheir lack of intrinsic metabolism viruses are notsusceptible to antibiotics, and the number of effec-tive synthetic antiviral drugs is limited Partly forthese reasons, viral infections are among the mostserious and difficult to cure, and of all the categories

of microorganism, only viruses appear in (the mostserious) Hazard Category 4 as classified by the Advisory Committee on Dangerous Pathogens Because they are not free-living, viruses are

Table 2.4 Pharmaceutical importance of the major categories of microorganisms

Pharmaceutical relevance

incapable of growing on manufactured medicines

or raw materials, so they do not cause product

spoilage, and they have no synthetic capabilities

that can be exploited in medicines manufacture

Viruses are relatively easy to destroy by heat,

radia-tion or toxic chemicals, so they do not represent a

problem from this perspective In this, they contrast

with prions; while some authorities would question

the categorization of these infectious agents as

microorganisms, they are included here because of

their undoubted ability to cause, as yet incurable,

fatal disease, and their extreme resistance to lethal

agents Pharmacists and health-care personnel in

general should be aware of the ability of prions to

easily withstand sterilizing conditions that would

be satisfactory for the destruction of all other

categories of infectious agent

There are examples of bacteria that are

impor-tant in each of the different ways indicated by the

column headings of Table 2.4 Many of the

med-ically and pharmaceutmed-ically important bacteria are

pathogens, and some of these pathogens are of

long-standing notoriety as a result of their ability to

resist the activity of antibiotics and biocides

(disin-fectants, antiseptics and preservatives) In addition

to these long-established resistant organisms, other

bacteria have given more recent cause for concern

including methicillin-resistant Staphylococcus

aureus, vancomycin-resistant enterococci and

mul-tiply resistant Mycobacterium tuberculosis

(Chapter 13) While penicillin and cephalosporin

antibiotics are produced by fungal species, the

ma-jority of the other categories of clinically important

antibiotics are produced by species of bacteria,

notably streptomycetes In addition, a variety of

bacteria are exploited commercially in the

manu-facture of other medicines including steroids,

en-zymes and carbohydrates The ability of bacteria to

grow on diverse substrates ensures that their

poten-tial as agents of spoilage in manufactured medicines

and raw materials is well recognized, and the ability

of many species to survive drying means that they

survive well in dust and so become important as

contaminants of manufactured medicines The

ability to survive not only in dry conditions but in

other adverse environments (heat, radiation, toxic

chemicals) is well exemplified by bacterial spores,

and their pre-eminence at or near the top of the

‘league table’ of resistance to lethal agents has sulted in spores acting as the indicator organismsthat have to be eliminated in most sterilizationprocesses

re-Like bacteria, fungi are able to form spores thatsurvive drying, so they too arise commonly as cont-aminants of manufactured medicines However, thedegree of resistance presented by the spores is usual-

ly less than that exhibited by bacteria, and fungi donot represent a sterilization problem Fungi do notgenerally create a significant infection hazard either; relatively few fungal species are consideredmajor pathogens for animals that possess a fullyfunctional immune system There are, however,several fungi which, while representing little threat

to immunocompetent individuals, are neverthelesscapable of initiating an infection in persons withimpaired immune function; the term opportunistpathogens is used to describe microorganisms (of all types) possessing this characteristic In thiscontext it is worth noting that the immunocom-promised represent an increasingly large group ofpatients, and this is not just because of HIV andAIDS Several other conditions or drug treatmentsimpair immune function, e.g congenital immuno-deficiency, cancer (particularly leukaemia), radio-therapy and chemotherapy, the use of systemiccorticosteroids and immunosuppressive drugs(often following tissue or organ transplants), severeburns and malnutrition

Protozoa are of significance largely owing to thepathogenic potential of a few species Because pro-tozoa do not possess cell walls they do not survivedrying well (unless in the form of cysts), so they arenot a problem in the manufacturing environment —and even the encysted forms do not display resis-tance to sterilizing processes to match that ofbacterial spores It should be noted that protozoalinfections are not currently a major problem tohuman health in temperate climates, although theyare more troublesome in veterinary medicine and inthe tropics There are concerns that the geographi-cal ranges of protozoal infections like malaria mayextend substantially if current fears about globalwarming translate into reality

1 Introduction

The smallest free-living microorganisms are the

prokaryotes, comprising bacteria and archaea (see

Chapter 2) Prokaryote is a term used to define cells

that lack a true nuclear membrane; they contrast

with eukaryotic cells (e.g plants, animals and

fungi) that possess a nuclear membrane and

inter-nal compartmentalization Indeed, a major feature

of eukaryotic cells, absent from prokaryotic cells, is

the presence in the cytoplasm of

membrane-enclosed organelles These and other criteria

differ-entiating eukaryotes and prokaryotes are shown

in Table 2.1

Bacteria and archaea share many traits and it wasnot until the early 1980s that differences first be-came evident from analyses of gene sequences Onemajor difference is the composition of cell walls Amore striking contrast is in the structure of the lipidsthat make up their cytoplasmic membranes Differ-ences also exist in their respective patterns of metabolism Most archaea are anaerobes, and areoften found inhabiting extreme environments It ispossible that their unusual membrane structure

6 Bacterial reproduction and growth kinetics

6.1 Multiplication and division cycle

6.3.1 Transformation 6.3.2 Transduction 6.3.3 Conjugation

7 Environmental factors that influence growth and survival 7.1 Physicochemical factors that affect growth and survival of bacteria

7.1.1 Temperature 7.1.2 pH 7.1.3 Water activity/solutes 7.1.4 Availability of oxygen 7.2 Nutrition and growth

8 Detection, identification and characterization of organisms of pharmaceutical and medical significance 8.1 Culture techniques

8.1.1 Enumeration 8.1.1.1 Enumeration media 8.1.1.2 Rapid enumeration techniques 8.1.2 Enrichment culture

8.1.3 Selective media 8.1.4 Identification media (diagnostic) 8.2 Microscopy

8.3 Biochemical testing and rapid identification 8.4 Molecular approaches to identification 8.5 Pharmaceutically and medically relevant microorganisms

9 Further reading

gives archaeal cells greater stability under extreme

conditions Of notable interest is the observation

that no disease-causing archaea have yet been

iden-tified The vast majority of prokaryotes of medical

and pharmaceutical significance are bacteria

Bacteria represent a large and diverse group of

microorganisms that can exist as single cells or as

cell clusters Moreover, they are generally able to

carry out their life processes of growth, energy

gen-eration and reproduction independently of other

cells In these respects they are very different to the

cells of animals and plants, which are unable to live

alone in nature and can exist only as part of a

multi-cellular organism They are capable of growing in a

range of different environments and can not only

cause contamination and spoilage of many

phar-maceutical products but also a range of different

diseases For this reason only bacteria will be

re-ferred to throughout the remainder of this chapter

1.1 Bacterial diversity and ubiquity

Bacterial diversity can be seen in terms of variation

in cell size and shape (morphology), adaptation to

environmental extremes, survival strategies and

metabolic capabilities Such diversity allows

bacte-ria to grow in a multiplicity of environments

rang-ing from hot sulphur sprrang-ings (65°C) to deep freezers

(–20°C), from high (pH 1) to low (pH 13) acidity

and high (0.7 M) to low osmolarity (water) In

addition, they can grow in both nutritionally rich

(compost) and nutritionally poor (distilled water)

situations Hence, although each organism is

uniquely suited to its own particular environmental

niche and rarely grows out of it, the presence of

bac-teria may be considered ubiquitous Indeed, there is

no natural environment that is free from bacteria

This ubiquity is often demonstrated by terms used

to describe organisms that grow and/or survive in

particular environments An example of such

descriptive terminology is shown in Table 3.1

2 Bacterial ultrastructure

2.1 Cell size and shape

Bacteria are the smallest free-living organisms, their

size being measured in micrometres (microns)

Be-cause of this small size a microscope affording aconsiderable degree of magnification (¥400–1000)

is necessary to observe them Bacteria vary in sizefrom a cell as small as 0.1–0.2 mm in diameter tothose that are >5 mm in diameter Bacteria this large,

such as Thiomargarita namibiensis, are extremely

rare Instead, the majority of bacteria are 1–5 mmlong and 1–2 mm in diameter By comparison, eukaryotic cells may be 2 mm to > 200 mm in diame-ter The small size of bacteria has a number of impli-cations with regard to their biological properties,most notably increased and more efficient transportrates This advantage allows bacteria far morerapid growth rates than eukaryotic cells

While the classification of bacteria is immenselycomplex, nowadays relying very much on 16S ribo-somal DNA sequencing data, a more simplistic approach is to divide them into major groups onpurely morphological grounds The majority ofbacteria are unicellular and possess simple shapes,e.g round (cocci), cylindrical (rod) or ovoid Somerods are curved (vibrios), while longer rigid curvedorganisms with multiple spirals are known asspirochaetes Rarer morphological forms includethe actinomycetes which are rigid bacteria resem-bling fungi that may grow as lengthy branched fila-ments; the mycoplasmas which lack a conventionalpeptidoglycan (murein) cell wall and are highlypleomorphic organisms of indefinite shape; andsome miscellaneous bacteria comprising stalked,sheathed, budded and slime-producing forms oftenassociated with aquatic and soil environments

Table 3.1 Descriptive terms used to describe bacteria

Psychrophile Growth range -40°C to +20°C

Thermophile Growth range +40°C to +85°C Thermoduric Endure high temperatures

Obligate anaerobe Air (oxygen) poisoned Autotroph Utilizes inorganic material Heterotroph Requires organic material

Often bacteria remain together in specific

arrangements after cell division These

arrange-ments are usually characteristic of different

organ-isms and can be used as part of a preliminary

identification Examples of such cellular

arrange-ments include chains of rods or cocci, paired cells

(diplococci), tetrads and clusters

2.2 Cellular components

Compared with eukaryotic cells, bacteria possess a

fairly simple base cell structure, comprising cell

wall, cytoplasmic membrane, nucleoid, ribosomes

and occasionally inclusion granules (Fig 3.1)

Nev-ertheless it is important for several reasons to have a

good knowledge of these structures and their

func-tions First, the study of bacteria provides an

ex-cellent route for probing the nature of biological

processes, many of which are shared by

multicellu-lar organisms Secondly, at an applied level, normal

bacterial processes can be customized to benefit

society on a mass scale Here, an obvious example is

the large-scale industrial production

(fermenta-tion) of antibiotics Thirdly, from a pharmaceutical

and health-care perspective, it is important to be

able to know how to kill bacterial contaminants

and disease-causing organisms To treat infections

antimicrobial agents are used to inhibit the growth

of bacteria, a process known as antimicrobial

chemotherapy The essence of antimicrobial

chemotherapy is selective toxicity (Chapters 10, 12

and 14), which is achieved by exploiting differences

between the structure and metabolism of bacteria

and host cells Selective toxicity is, therefore, most

efficient when a similar target does not exist in the

host Examples of such targets will be noted in thefollowing sections

2.2.1 Cell wall

The bacterial cell wall is an extremely importantstructure, being essential for the maintenance of theshape and integrity of the bacterial cell It is alsochemically unlike any structure present in eukary-otic cells and is therefore an obvious target for antibiotics that can attack and kill bacteria withoutharm to the host (Chapter 12)

The primary function of the cell wall is to provide

a strong, rigid structural component that can stand the osmotic pressures caused by high chemi-cal concentrations of inorganic ions in the cell.Most bacterial cell walls have in common a uniquestructural component called peptidoglycan (alsocalled murein or glycopeptide); exceptions includethe mycoplasmas, extreme halophiles and the archaea Peptidoglycan is a large macromoleculecontaining glycan (polysaccharide) chains that arecross-linked by short peptide bridges The glycanchain acts as a backbone to peptidoglycan, and is

with-composed of alternating residues of N-acetyl muramic acid (NAM) and N-acetyl glucosamine

(NAG) To each molecule of NAM is attached atetrapeptide consisting of the amino acids l-alanine, d-alanine, d-glutamic acid and either ly-sine or diaminopimelic acid (DAP) This glycantetrapeptide repeat unit is cross-linked to adjacentglycan chains, either through a direct peptide link-age or a peptide interbridge (Fig 3.2) The types andnumbers of cross-linking amino acids vary from or-ganism to organism Other unusual features of the

Cytoplasm

Ribosomes

Nucleoid Plasmid

Fig 3.1 Diagram of a bacterial cell Features represented

above the dotted line are only found in some bacteria,

whereas those below the line are common to all bacteria.

L-ala

D-ala

D-glu Meso-DAP

Fig 3.2 Structure of Escherichia coli peptidoglycan.

cell wall that provide potential antimicrobial

tar-gets are DAP and the presence of two amino acids

that have the d-configuration

Bacteria can be divided into two large groups,

Gram-positive and Gram-negative, on the basis of a

differential staining technique called the Gram

stain Essentially, the Gram stain consists of

treat-ing a film of bacteria dried on a microscope slide

with a solution of crystal violet, followed by a

solu-tion of iodine; these are then washed with an

alco-hol solution In Gram-negative organisms the cells

lose the crystal violet–iodine complex and are

ren-dered colourless, whereas Gram-positive cells

retain the dye Regardless, both cell types are

counter-stained with a different coloured dye, e.g

carbol fuchsin, which is red Hence, under the light

microscope Gram-negative cells appear red while

Gram-positive cells are purple These marked

dif-ferences in response reflect difdif-ferences in cell wall

structure The Gram-positive cell wall consists

pri-marily of a single type of molecule whereas the

Gram-negative cell wall is a multilayered structure

and quite complex

The cell walls of Gram-positive bacteria are quite

thick (20–80 nm) and consist of between 60% and

80% peptidoglycan, which is extensively

cross-linked in three dimensions to form a thick

poly-meric mesh (Fig 3.3) Gram-positive walls

frequent-ly contain acidic pofrequent-lysaccharides called teichoic

acids; these are either ribitol phosphate or glycerol

phosphate molecules that are connected by

phos-phodiester bridges Because they are negativelycharged, teichoic acids are partially responsible forthe negative charge of the cell surface as a whole.Their function may be to effect passage of metalcations through the cell wall In some Gram-posi-tive bacteria glycerol–teichoic acids are bound tomembrane lipids and are termed lipoteichoic acids.During an infection, lipoteichoic acid molecules re-leased by killed bacteria trigger an inflammatory re-sponse Cell wall proteins, if present, are generallyfound on the outer surface of the peptidoglycan.The wall, or more correctly, envelope of Gram-negative cells is a far more complicated structure(Fig 3.4) Although they contain less peptidoglycan(10–20% of wall), a second membrane structure isfound outside the peptidoglycan layer This outermembrane is asymmetrical, composed of proteins,lipoproteins, phospholipids and a componentunique to Gram-negative bacteria, lipopolysaccha-ride (LPS) Essentially, the outer membrane is attached to the peptidoglycan by a lipoprotein, oneend of which is covalently attached to peptidogly-can and the other end is embedded in the outermembrane The outer membrane is not a phospho-lipid bilayer although it does contain phospholipids

in the inner leaf, and its outer layer is composed ofLPS, a polysaccharide–lipid molecule Proteins arealso found in the outer membrane, some of whichform trimers and traverse the whole membrane and

in so doing form water-filled channels or porinsthrough which small molecules can pass Other

Lipoteichoic acid Teichoic acid

Surface protein

Peptidoglycan

Cytoplasmic

cell wall.

proteins are found at either the inner or outer face

of the membrane

The LPS (Fig 3.5) is an important molecule

be-cause it determines the antigenicity of the

Gram-negative cell and it is extremely toxic to animal cells

The molecule consists of three regions, namely lipid

A, core polysaccharide and O-specific

polysaccha-ride The lipid A portion is composed of a

disaccha-ride of glucosamine phosphate bound to fatty acids

and forms the outer leaflet of the membrane It is the

lipid A component that is responsible for the toxic

and pyrogenic properties of Gram-negative

bacteria Lipid A is linked to the core

polysaccha-ride by the unique molecule ketodeoxyoctonate

(KDO), and at the other end of the core is the

O-polysaccharide (O-antigen), which usually

contains six-carbon sugars as well as one or more

unusual deoxy sugars such as abequose

Although the outer membrane is relatively

per-meable to small molecules, it is not perper-meable to

en-zymes or large molecules Indeed, one of the major

functions of the outer membrane may be to keep

certain enzymes that are present outside the plasmic membrane from diffusing away from thecell Moreover, the outer membrane is not readilypenetrated by hydrophobic compounds and is,therefore, resistant to dissolution by detergents.The region between the outer surface of the cyto-plasmic membrane and the inner surface of theouter membrane is called the periplasm This occu-pies a distance of about 12–15 nm, is gel-like in con-sistency and, in addition to the peptidoglycan,contains sugars and an abundance of proteins in-cluding hydrolytic enzymes and transport proteins.Table 3.2 summarizes the major differences in wall composition between Gram-positive andGram-negative cells

cyto-2.2.2 Cytoplasmic membrane

Biochemically, the cytoplasmic membrane is a fragile, phospholipid bilayer with proteins distrib-uted randomly throughout These are involved

in the various transport and enzyme functions

Lipoprotein Porin

Outer membrane

Periplasm

Lipopolysaccharide Receptor protein

Periplasmic protein

Peptidoglycan

Fig 3.4 Structure of the Gram-negative

cell envelope.

Fig 3.5 Schematic representation of

lipopolysaccharide (LPS).