Hugo and Russell's Pharmaceutical Microbiology Seventh Edition PDF

Hugo and Russell’s Pharmaceutical Microbiology EDITED BY Stephen P Denyer B Pharm PhD FRPharmS Welsh School of Pharmacy Cardiff University Cardiff Norman A Hodges B Pharm PhD MRPharmS School of Pharmacy and Biomolecular Sciences Brighton University Lewes Road Brighton Sean P Gorman BSc PhD MPS School of Pharmacy Queen’s University Belfast Medical Biology Centre University Road Belfast SEVENTH EDITION Blackwell Science Hugo and Russell’s Pharmaceutical Microbiology Hugo and Russell’s Pharmaceutical Microbiology EDITED BY Stephen P Denyer B Pharm PhD FRPharmS Welsh School of Pharmacy Cardiff University Cardiff Norman A Hodges B Pharm PhD MRPharmS School of Pharmacy and Biomolecular Sciences Brighton University Lewes Road Brighton Sean P Gorman BSc PhD MPS School of Pharmacy Queen’s University Belfast Medical Biology Centre University Road Belfast SEVENTH EDITION Blackwell Science © 1977, 1980, 1983, 1987, 1992, 1998, a Blackwell Publishing company Blackwell Science, Inc., 350 Main Street, Blackwell Publishing Ltd, 9600 Garsington Blackwell Science Asia Pty Ltd, 550 Swanston The right of the Author to be identiﬁed accordance with the Copyright, Designs and All rights reserved. No part of this publication system, or transmitted, in any form or by photocopying, recording or otherwise, except and Patents Act 1988, without the prior First published 1977 Second edition 1980 Third edition 1983 Reprinted 1986 Fourth edition 1987 Reprinted 1989, 1991 Library of Congress Cataloging-in-Publication Hugo and Russell’s pharmaceutical microbiology Hodges, Sean P. Gorman. — 7th ed. p. cm. Rev. ed. of: Pharmaceutical microbiology Includes bibliographical references and index. ISBN 0-632-06467-6 1. Pharmaceutical microbiology. [DNLM: 1. Anti-Infective Agents. 2. H895 2004] I. Title: Pharmaceutical microbiology. Barry) III. Denyer, S. P. IV. Hodges, Pharmaceutical microbiology. QR46.5.P48 2004 615¢.1¢01579 — dc22 ISBN 0–632–06467–6 A catalogue record for this title is available Set in Sabon 9.5/12 pt by SNP Best-set Typesetter Printed and bound in the United Kingdom Commissioning Editor: Maria Khan Managing Editor: Rupal Malde Production Editor: Fiona Pattison Production Controller: Kate Charman For further information on Blackwell Publishing, http://www.blackwellpublishing.com Contents Contributors, vii Preface to Seventh Edition, ix Preface to First Edition, x Part 1: Biology of Microorganisms 1. Introduction to Pharmaceutical Microbiology, 3 Stephen Denyer, Norman Hodges and Sean Gorman 2. Fundamental Features of Microbiology, 9 Norman Hodges 3. Bacteria, 23 David Allison and Peter Gilbert 4. Fungi, 44 Kevin Kavanagh and Derek Sullivan 5. Viruses, 59 Jean-Yves Maillard and David Stickler 6. Protozoa, 82 Tim Paget 7. Principles of Microbial Pathogenicity and Epidemiology, 103 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 Antimicrobial Agents, 152 A Denver Russell Contents 23. The Manufacture and Quality Control of Immunological Products, 398 Michael Corbel 24. Pharmaceutical Biotechnology, 416 Miguel Cámara vi Contributors Dr David Allison School of Pharmacy and Pharmaceutical Sciences University of Manchester Oxford Road Manchester M13 9PL UK UK Dr Rosamund Baird Visiting Senior Lecturer School of Pharmacy and Pharmacology University of Bath Claverton Down Wales Bath BA2 7AY UK Dr Miguel Cámara Senior Lecturer in Molecular Microbiology Institute of Pharmaceutical Sciences Oxford Rd School of Pharmaceutical Sciences University of Nottingham UK Nottingham NG7 2RD UK Dr Michael Corbel National Institute for Biological Standards and Control Blanche Lane South Mimms Potters Bar Hertfordshire EN6 3QG UK Professor Stephen Denyer Welsh School of Pharmacy Wales Cardiff University Cardiff CF10 3XF UK Professor Roger Finch Professor of Infectious Diseases Clinical Sciences Building University of Nottingham The City Hospital Nottingham NG5 1PB UK UK Contributors Dr Anthony Smith Dr David Stickler Department of Pharmacy and Pharmacology University of Bath (5 West — 2.18) Claverton Down Main Building Bath BA2 7AY Museum Avenue UK PO Box 915 Cardiff CF10 3TL Professor JMB (Sandy) Smith Wales Head of Department of Microbiology Otago School of Medical Sciences Dr Derek Sullivan University of Otago Dunedin New Zealand Trinity College Ireland viii 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. Preface to the First Edition 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 x Part 1 Biology of Microorganisms Chapter 1 Introduction to pharmaceutical microbiology Stephen Denyer, Norman Hodges and Sean Gorman 1 Microorganisms and medicines 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 Chapter 1 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 4 Table 1.1 Microorganisms in pharmacy: Beneﬁts or uses The manufacture of: antibiotics steroids technology therapeutic enzymes Microbial genetics polysaccharides products of recombinant DNA technology Use in the production of vaccines Quality control of As assay organisms to determine antibiotic, vitamin and amino acid concentrations To detect mutagenic or carcinogenic activity 5 Chapter 1 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 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-acquired 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. 6 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 Chapter 1 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 8 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 1.2.2 Fungi 1.2.3 Protozoa 2 Naming of microorganisms 3 Microbial metabolism 4 Microbial cultivation 4.1 Culture media 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 Chapter 2 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 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 Table 2.1 Distinguishing features of prokaryotes Characteristic Eukaryotes Size Normally > 10 µm Location of chromosomes Within a cytoplasm Nuclear division Exhibit mitosis and meiosis Nucleolus Present Reproduction Asexual Chromosome number >1 Mitochondria and chloroplasts May be present Cell membrane composition Sterols present Cell wall composition Cell walls cellulose Ribosomes Cytoplasmic ribosomes are 80S Flagella Structurally complex Pili Absent Fimbriae Cilia Storage compounds Poly-b-hydroxybutyrate absent 10 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-negative 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 causing 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. of pharmaceutical and medical importance possess cell walls (and are therefore relatively 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 structurally more complex and varied in appear- ance. Fungi are considered to be non-photosynthe- Chapter 2 Fig. 2.1 (a) A growing culture of Bacillus (arrowed) prior to separation. (b) A growing (c) The mould Mucor plumbeus exhibiting (arrowed) are formed on specialized hyphae. ﬁlaments that superﬁcially resembles Micrococcus luteus inoculated to produce which the actively growing periphery of asexual spores have developed. 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 12 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 reactions. 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). Chapter 2 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 lism between the organism to be killed and the human host; without such a difference the ic would be very toxic 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 14 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 Chapter 2 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 109 cells ml-1 or more following overnight incubation in common liquid media. At concentrations below about 107 cells ml-1 culture 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 cells can give rise to colonies following 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 16 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 Table 2.2 Traditional and rapid methods Traditional methods Viable counts Total counts 1 Pour plate (counting colonies in agar) 2 Surface spread or surface drop counting chambers) (Miles Misra) methods (counting colonies on agar surface) 3 Membrane ﬁlter methods or cultures) (colonies growing on membranes on agar surface) 4 Most probable number (counts determinations based on the proportion of liquid cultures growing after receiving low inocula) Chapter 2 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 metabolic capabilities. Such diversity allows bacte- ria to grow in a multiplicity of environments rang- ing from hot sulphur springs (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- 24 Capsule Fimbriae Ribosomes Slime Mesosome Cytoplasm Membrane Plasmid Cell Wall Nucleoid Fig. 3.1 Diagram of a bacterial cell. Features above the dotted line are only found in some bacteria, whereas those below the line are common to all bacteria. Often bacteria remain together in speciﬁc arrangements after cell division. These arrange- ments are usually characteristic of different isms and can be used as part of a preliminary identiﬁcation. Examples of such cellular 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 Chapter 3 Surface Teichoic acid protein Peptidoglycan Cytoplasmic membrane 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 differences 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 polysaccharides called teichoic acids; these are either ribitol phosphate or glycerol phosphate molecules that are connected by phos- 26 Outer Fig. 3.4 Structure of the Gram-negative cell envelope. Fig. 3.5 Schematic representation of lipopolysaccharide (LPS). proteins are found at either 2004 by Blackwell Science Ltd Malden, Massachusetts 02148-5020, USA Road, Oxford OX4 2DQ, UK Street, Carlton, Victoria 3053, Australia as the Author of this Work has been asserted in Patents Act 1988. may be reproduced, stored in a retrieval any means, electronic, mechanical, as permitted by the UK Copyright, Designs permission of the publisher. Italian edition 1991 Fifth edition 1992 Reprinted 1993, 1994, 1995 Sixth edition 1998 Reprinted 1999, 2000, 2002, 2003 Seventh edition 2004 Data / edited by Stephen Denyer, Norman A. / edited by W.B. Hugo and A.D. Russell. Technology, Pharmaceutical. QV 250 II. Hugo, W. B. (William Norman A.V,. Gorman, S. P. VI. 2003024264 from the British Library Ltd., Hong Kong by Ashford Colour Press, Gosport visit our website: 11. Laboratory Evaluation of Antimicrobial Agents, 187 JMB Smith 12. Mechanisms of Action of Antibiotics and Synthetic Anti-infective Agents, 202 Peter Lambert 13. Bacterial Resistance to Antibiotics, 220 Anthony Smith 14. Clinical Uses of Antimicrobial Drugs, 233 Roger Finch Part 3: Microbiological Aspects of Pharmaceutical Processing 15. Ecology of Microorganisms as it Affects the Pharmaceutical Industry, 251 Elaine Underwood 16. Microbial Spoilage, Infection Risk and Contamination Control, 263 Rosamund Baird 17. Chemical Disinfectants, Antiseptics and Preservatives, 285 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 Sterility Assurance, 346 Stephen Denyer and Norman Hodges 21. Factory and Hospital Hygiene, 376 Robert Jones 22. Manufacture of Antibiotics, 387 Sally Varian v 25. Additional Applications of Microorganisms in the Pharmaceutical Sciences, 441 Denver Russell Index, 459 Professor James Ford Dr Robert Jones School of Pharmacy and Chemistry School of Pharmacy and Biomedical Sciences Liverpool John Moores University University of Portsmouth Byrom Street St Michael’s Building Liverpool L3 3AF White Swan Road Portsmouth PO1 2DT UK Dr James Furr Welsh School of Pharmacy Dr Kevin Kavanagh Cardiff University Head of Medical Mycology Unit King Edward VII Avenue Department of Biology Cardiff CF10 3XF National University of Ireland Maynooth Co. Kildare Professor Peter Gilbert Ireland School of Pharmacy and Pharmaceutical Sciences Dr Peter Lambert University of Manchester Aston Pharmacy School Aston University Manchester M13 9PL Aston Triangle Birmingham B4 7ET UK Professor Sean Gorman Professor of Pharmaceutical Microbiology Dr Jean-Yves Maillard School of Pharmacy School of Pharmacy and Biomolecular The Queen’s University of Belfast Sciences Belfast BT9 7BL University of Brighton Northern Ireland Lewes Rd Brighton BN2 4GJ Dr Mark Gumbleton UK Welsh School of Pharmacy Cardiff University Dr Tim Paget King Edward VII Avenue Department of Biological Sciences Cardiff CF10 3XF University of Hull Hull HU6 7RX UK Dr Norman Hodges Principal Lecturer in Pharmaceutical Professor A Denver Russell Microbiology Welsh School of Pharmacy School of Pharmacy and Biomolecular Cardiff University Sciences King Edward VII Avenue University of Brighton Cardiff CF10 3XF Lewes Road Wales Brighton BN2 4GJ Dr Eileen Scott School of Pharmacy The Queen’s University of Belfast Belfast BT9 7BL Northern Ireland vii Dr Elaine Underwood School of Biosciences SMA Nutrition Cardiff University Huntercomb Lane South Taplow Maidenhead Berks SL6 0PH UK Dr Sally Varian Consultant Microbiology Research Unit Ulverston School of Dental Science Cumbria LA12 8PT UK Dublin 2 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 and expertise. Finally, this addition is a tribute to the farsighted- ness of A.D. Russell and W.B. Hugo who took up the challenge in 1977 to produce a popular and con- cise read for pharmacy students required to study pharmaceutical microbiology. We are delighted that this current edition recognizes these origins by continuing the association with Hugo and Russell in its revised title. S.P. Denyer S.P. Gorman N.A. Hodges ix the following pages. The editors must bear respon- sibility for any omissions, a point which has most concerned us. Length and depth of treatment were determined by the dictate of our publishers. It is hoped that the book will provide a concise reading for pharmacy students (who, at the moment, lack a textbook in this subject) and help to highlight those parts of a general microbiological training which impinge on the pharmaceutical industry. In conclusion, the editors thank most sincerely the contributors to this book, both for complying with our strictures as to the length of their contribu- tion and for providing their material on time, and our publishers for their friendly courtesy and efﬁ- ciency during the production of this book. We also wish to thank Dr H.J. Smith for his advice on vari- ous chemical aspects, Dr M.I. Barnett for useful comments on reverse osmosis, and Mr A. Keall who helped with the table on sterilization methods. W.B. Hugo A.D. Russell 2 The scope and content of the book the most consistently successful and important in- dustries in many countries, not only in the tradi- tional strongholds of North America, Western Europe and Japan but, increasingly, in Eastern Eu- rope, the Indian subcontinent and the Far East. Worldwide sales of medicines and medical devices are estimated to have exceeded $US 401 billion (ap- proximately £250 billion) in 2002, and this ﬁgure is rising by 8% per annum. In the UK alone, the value of pharmaceutical exports is currently £10.03 bil- lion each year, a ﬁgure that translates to more than £150 000 for each employee in the industry. The growth of the pharmaceutical industry in re- cent decades has been paralleled by rising standards for product quality and more rigorous regulation of manufacturing procedures. In order to receive a manufacturing licence, a modern medicine must be shown to be effective, safe and of good quality. Most medicines consist of an active ingredient that is formulated with a variety of other materials (ex- cipients) that are necessary to ensure that the medi- cine is effective, and remains stable, palatable and safe during storage and use. While the efﬁcacy and safety aspects of the active ingredient are within the domain of the pharmacologist and toxicologist, respectively, many other disciplines contribute to the efﬁcacy, safety and quality of the manufactured product as a whole. Analytical chemists and phar- macists take lead responsibility for ensuring that the components of the medicine are present in the correct physical form and concentration, but quality is not judged solely on the physicochemical properties of the product: microorganisms also have the potential to inﬂuence efﬁcacy and safety. It is obvious that medicines contaminated with potentially pathogenic (disease-causing) micro- 3 contribute to the discipline of pharmaceutical microbiology. Commercial antibiotic production began with the manufacture of penicillin in the 1940s, and for many years antibiotics were the only signiﬁcant example of a medicinal product that was made using microorganisms. Following the adoption in the 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 in the manufacture of medicines has gathered great momentum. 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 the principal 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 become increasingly important, not only in the pharmacy curriculum but also in those of other disciplines em- ployed in the pharmaceutical industry. Table 1.1 summarizes these beneﬁts and uses of microorgan- isms in pharmaceutical manufacturing, together with the more widely recognized hazards and problems that they present. Looking ahead to the early decades of the 21st century, it is clear that an understanding of the physiology and genetics of microorganisms will also become more important, not just in the pro- duction of new therapeutic agents but in the under- standing of infections and other diseases. Several of the traditional diseases that were major causes of death before the antibiotic era, e.g. tuberculosis and diphtheria, 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 like Creutzfeldt–Jakob disease, West Nile virus and severe acute respiratory syndrome (SARS) that have only been recognized in recent years. Not only has the development of resistance to established antibiotics become a challenge, so too has the ability of microorganisms to take advantage of changing practices and procedures in medicine and surgery. Microorganisms are found almost everywhere in our surroundings and they possess beneﬁts and problems Related study topics Harmful effects Study topic Good manufacturing practice May contaminate non- Non- sterile medicines: Industrial ‘fermentation’ sterile and sterile medicines Enumeration of microorganisms in the with a risk of infection manufacturing environment (environmental monitoring) and in raw materials and manufactured products Identiﬁcation and detection of speciﬁc organisms Sterile medicines: Sterilization methods immunological products Sterilization monitoring and validation procedures Sterility testing Assay methods Assessment and calculation of sterility assurance Aseptic manufacture Ames mutagenicity test May contaminate non- Enumeration, identiﬁcation and detection as above, plus sterile and sterile medicines with Characteristics, selection and testing of a risk of product deterioration antimicrobial preservatives Cause infectious and other diseases Immunology and infectious diseases Characteristics, selection and use of vaccines and antibiotics Use of biocides in infection and contamination control Control of antibiotic resistance Cause pyrogenic reactions (fever) Bacterial structure when introduced into the body Pyrogen and endotoxin testing even in the absence of infection Provide a reservoir of antibiotic Microbial genetics resistance genes Introduction to pharmaceutical microbiology Clearly, a knowledge of the mechanisms whereby microorganisms are able to resist antibiotics, colonize medical devices and cause or predispose humans to other disease states is essential in the development not only of new antibiotics, but of other medicines and health-care practices that miminize the risks of these adverse situations developing. 2 The scope and content of the book to Criteria and standards for the microbiological quality of medicines depend upon the route of administration of the medicine in question. The vast majority of medicines that are given by mouth or placed on the skin are non-sterile, i.e. they may contain some microorganisms (within limits on type and concentration), whereas all injections and ophthalmic products must be sterile, i.e. they contain no living organisms. Products for other anatomical 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 the concentration of organisms that may be present and requiring 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 manu- facturing environment, raw materials and ﬁnished product 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 of microbial growth and spoilage during storage and use by the patient (Chapters 16 and 17). For a sterile product the criterion of quality is simple; there should be no detectable microorgan- isms whatsoever. The product should, therefore, be able to pass a test for sterility, and a knowledge of the procedures and interpretation of results of such tests is an important aspect of pharmaceutical microbiology (Chapter 20). Injections are also sub- ject to a test for pyrogens; these are substances that cause a rise in body temperature when introduced Introduction to pharmaceutical microbiology have properties that make them unique. It is far more 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 the need to demonstrate that a new antibiotic (or any new medicine) affords a real advantage over estab- lished drugs. Because of this range and diversity of products, pharmacists are now far more commonly called upon to advise on the relative merits of the antibiotics available to treat particular categories of infection than was the case hitherto (Chapters 10, 12 and 14). A prerequisite to provide this informa- tion is a knowledge not only of the drug in question, but the infectious disease it is being used to treat and the 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 a problem that was well on the way to permanent resolution owing to the development of effective vaccines and antibiotics, such complacency has now completely disappeared. Although cardiovas- cular and malignant diseases are more frequent causes of death in many developed countries, infec- tious diseases remain of paramount importance in many 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 antibiotics would be produced to deal with the vast majority of infections has been replaced by a recognition that the development of resistance to them is likely to substantially restrict their value in the control of certain infections (Chapter 13). Resistance to an- tibiotics has increased in virtually all categories of pathogenic 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 no effective antibiotics. It has been estimated that the annual 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 such that increasing attention is being paid to infection control procedures that are designed to minimize the risk of infection being transmitted from one patient to another within a hospital. The properties of disinfectants and antiseptics, the measurement of their antimicrobial activity and the factors inﬂuenc- 7 microbial enzymes reduced the synthesis to 11 steps and the cost rapidly fell to $6 per gram. Apart from these major applications, however, the uses of microorganisms in the manufacture of medicines prior to 1980 were very limited. Enzymes were developed for use in cancer chemotherapy (asparaginase) and to digest blood clots (streptoki- nase), and polysaccharides also found therapeutical applications (e.g. dextran — used as a plasma ex- pander). These were of relatively minor importance, however, compared with the products that followed the advances in recombinant DNA technology in the 1970s. This technology permitted human genes to be inserted into microorganisms, which were thus able to manufacture the gene products far more efﬁciently than traditional methods of extraction from animal or human tissues. Insulin, in 1982, was the 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. DNA technology has also permitted the development of vaccines which, like that for the prevention of he- patitis B, use genetically engineered surface antigens rather than whole natural virus particles, so these vaccines 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 mutagenic and carcinogenic activity in drugs and chemicals and in the assay of antibiotics, vitamins and amino acids (Chapter 25), have ensured that the role of microorganisms in the manufacture of medicines is now well recognized, and that a basic knowledge of immunology (Chapter 8), gene cloning and other biotechnology disciplines (Chapter 24) is an integral part of pharmaceutical microbiology. 4.2 Cultivation methods 4.3 Planktonic and sessile growth 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 absent. Viruses are incapable of independent repli- cation as they do not contain the enzymes necessary to copy their own nucleic acids; as a consequence, all viruses are intracellular parasites and are repro- duced using the metabolic capabilities of the host cell. A great deal of variation is observed in shape (helical, linear or spherical), size (20–400 nm) and nucleic acid composition (single- or double- stranded, linear or circular RNA or DNA), but al- most all viruses are smaller than bacteria and they cannot 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 that have been described are plant pathogens, and, so far, there are no known human pathogens in this category. Prions are unique as infectious agents in that they contain no nucleic acid. A prion is an atyp- ical form of a mammalian protein that can interact with 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 transmissible spongiform encephalopathies, e.g. Creutzfeldt– Jakob disease (CJD) and bovine spongiform en- cephalopathy (BSE). They are the simplest and most 9 copy of the set of genes in the cell) and reproduce asexually; eukaroyotes, by contrast, are usually diploid (possess two copies of their genes) and nor- mally have the potential to reproduce sexually. The capacity for sexual reproduction confers the major advantage of creating new combinations of genes, which increases the scope for selection and evolu- tionary development. The restriction to an asexual mode of reproduction means that the organism in question is heavily reliant on mutation as a means of creating genetic variety and new strains with advan- tageous characteristics, although many bacteria are able to receive new genes from other strains or species (see section 6.1 and Chapter 3). Table 2.1 lists some distinguishing features of the prokary- otes and eukaryotes. crucial dif- 1.2.1 Bacteria and archaea Bacteria are essentially unicellular, although some species arise as sheathed chains of cells. They possess the properties listed under prokaryotes in Table 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 and eukaryotes Prokaryotes Typically 1–5 µm true nucleus separated from the In the cytoplasm, usually attached to the cell by a nuclear membrane membrane Mitosis and meiosis are absent Absent or sexual reproduction Normally asexual reproduction 1 Absent Sterols absent (when present) usually contain Walls usually contain peptidoglycan or chitin but not peptidoglycan Ribosomes are smaller, usually 70S Structurally simple Present Present Poly-b-hydroxybutyrate often present Fundamental features of microbiology sizing plants, and the term fungus covers both yeasts and moulds, although the distinction be- tween these two groups is not always clear. Yeasts are normally unicellular organisms that are larger than bacteria (typically 5–10 mm) and divide either by a process of binary ﬁssion (see section 4.2 and Fig. 2.1a) or budding (whereby a daughter cell aris- es as a swelling or protrusion from the parent that eventually 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 vis- ible to the naked eye, thus excluding toadstools and mushrooms. Most moulds consist of a tangled mass (mycelium) of ﬁlaments or threads (hyphae) which vary between 1 and > 50 mm wide (Fig. 2.1c); they may be differentiated for specialized functions, e.g. absorption of nutrients or reproduction. Some fungi may exhibit a unicellular (yeast-like) or mycelial (mould-like) appearance depending upon cultivation conditions. Although fungi are eukary- otes that should, in theory, be capable of sexual reproduction, there are some species in which this has never been observed. Most fungi are sapro- phytes with relatively few having pathogenic poten- tial, but their ability to form spores that are resistant to drying makes them important as contaminants of pharmaceutical raw materials, particularly materials of vegetable origin. Most bacteria 1.2.3 Protozoa resistant to Protozoa are eukaryotic, predominantly uni- cellular microorganisms that are regarded as ani- mals rather than plants, although the distinction between protozoa and fungi is not always clear and there are some organisms whose taxonomic status is uncertain. Many protozoa are free-living motile organisms that occur in water and soil, although some are parasites of plants and animals, including humans, e.g. the organisms responsible for malaria and amoebic dysentery. Protozoa are not normally found as contaminants of raw materials or manu- factured medicines and the relatively few that are of pharmaceutical interest owe that status primarily to their potential to cause disease. 2.1 and are 11 megaterium in which cells about to divide by binary ﬁssion display constrictions culture of the yeast Saccharomyces cerevisiae displaying budding (arrowed). the typical appearance of a mycelium in which masses of asexual zygospores (d) The bacterium Streptomyces rimosus displaying the branched network of a mould mycelium. (e) The typical appearance of an overnight agar culture of isolated colonies (arrowed). (f) A single colony of the mould Aspergillus niger in the colony (arrowed) contrasts with the mature central region where pigmented is normally written with an upper case initial letter and 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 Fundamental features of microbiology There are marked similarities in the metabolic pathways 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. Glycol- ysis describes the conversion of glucose, through a series of reactions, to pyruvic acid, and it is a process for which oxygen is not required, although glycolysis is undertaken by both aerobic and anaerobic organisms. The process releases only a relatively small amount of the energy stored in a sugar molecule, and aerobic microorganisms, in common with mammals, release much more of the energy by aerobic respiration. Oxygen is the molecule at the end of the sequence of respiratory reactions that ﬁnally accepts the electrons and al- lows the whole process to proceed, but it is worth noting that many organisms can also undertake anaerobic respiration, which uses other ﬁnal electron acceptors, e.g. nitrate or fumarate. As an alternative to respiration many micro- organisms use fermentation as a means of releasing more energy from sugar; fermentation is, by deﬁni- tion, a process in which the ﬁnal electron acceptor is an organic molecule. The term is widely understood to mean the production by yeast of ethanol and car- bon dioxide from sugar, but in fact many organisms apart from yeasts can undertake fermentation and the process is not restricted to common sugar (sucrose) as a starting material or to ethanol and carbon dioxide as metabolic products. Many pathogenic bacteria are capable of fermenting sev- eral different sugars and other organic materials to give a range of metabolic products that includes acids (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, an anaerobic process, although the term is com- monly used in the pharmaceutical and biotechnolo- gy industries to describe the manufacture of a wide range of substances by microorganisms where the biochemical process is neither fermentative nor even anaerobic, e.g. many textbooks refer to anti- biotic fermentation, but the production vessels are usually vigorously aerated and far from anaerobic. Microorganisms are far more versatile than mam- mals with respect to the materials that they can use 13 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 in pharmacy and medicine can be cultivated in the lab- oratory and most of them require relatively simple techniques and facilities. Some organisms are para- sites and so can only be grown inside the cells of a host species — which often necessitates mammalian cell culture facilities — and there are a few (e.g. the organism responsible for leprosy) that have never been cultivated outside the living animal. in metabo- 4.1 Culture media antibiot- A signiﬁcant number of common microorganisms are capable of synthesizing all the materials they need for growth (e.g. amino acids, nucleotides and vitamins) from simple carbon and nitrogen sources and mineral salts. Such organisms can grow on truly synthetic (chemically deﬁned) media, but many organisms do not have this capability and need a medium that already contains these bio- chemicals. Such media are far more commonly used than synthetic ones, and several terms have been used to describe them, e.g. routine laboratory media, general purpose media and complex media. They are complex in the sense that their precise chemical composition is unknown and is likely to vary slightly from batch to batch. In general, they are aqueous solutions of animal or plant extracts that contain hydrolysed proteins, B-group vitamins and carbohydrates. Readily available and relatively inexpensive sources of protein include meat extracts (from those parts of animal carcasses that are not used for human or domestic animal consumption), milk and soya. The protein is hydrolysed to varying degrees to give peptones (by deﬁnition not coagulable by heat or ammonium sulphate) or amino acids. Trypsin or other proteolytic enzymes are preferred to acids as a means of hydrolysis because acids Fundamental features of microbiology suppress bacterial contaminants, and bile to sup- press organisms from anatomical sites other than the gastrointestinal tract. Many such additives are used in media for organism identiﬁcation purposes, and these are considered further in subsequent chapters. The term enrichment sometimes causes confusion in this context. It is occasionally used in the sense of making a medium nutritionally richer to achieve more rapid or profuse growth. Alterna- tively, and more commonly, an enrichment medium is one designed to permit a particular type of organ- ism to grow while restricting others, so the one that grows increases in relative numbers and is ‘enriched’ in a mixed culture. Solid media designed for the growth of anaerobic organisms usually contain non-toxic reducing agents, e.g. sodium thioglycollate or sulphur-con- taining amino acids; these compounds create redox potentials of -200 mV or less and so diminish or eliminate the inhibitory effects of oxygen or oxidiz- ing molecules on anaerobic growth. The inclusion of such compounds is less important in liquid media where a sufﬁciently low redox potential may be achieved simply by boiling; this expels dissolved oxygen, which in unstirred liquids, only slowly re- saturates the upper few millimetres of liquid. Redox indicators like methylene blue or resazurin may be incorporated in anaerobic media to conﬁrm that a sufﬁciently 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 the concentrations used. Some fungal media that are in- tended for use with specimens that may also contain bacteria may be supplemented with antibacterial antibiotics, 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) that separates the cell into two more-or-less equal com- partments each containing a copy of the genetic ma- terial. Septum formation is often followed by constriction such that the connection between the two cell compartments is progressively reduced (see 15 properties may be an aid in identiﬁcation proce- dures (see Chapter 3). Anaerobic organisms may be grown on Petri dishes provided that they are incubated in an anaer- obic jar. Such jars are usually made of rigid plastic with airtight lids, and Petri dishes are placed in them together with a low temperature catalyst. The cata- lyst, consisting of palladium-coated pellets or wire, causes the oxygen inside the jar to be combined with hydrogen 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 the oxygen is removed, an anaerobic atmosphere is achieved and this is monitored by an oxidation- reduction (redox) indicator; resazurin is frequently used, as a solution soaking a fabric strip. Yeast colonies often look similar to those of bac- teria, although they may be larger and more fre- quently coloured. The appearance of moulds growing on solid microbiological media is similar to their appearance when growing on common foods. The mould colony consists of a mycelium that may be loosely or densely entangled depending on the species, often with the central area (the oldest, most mature region of the colony) showing pigmentation 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. bacterial 4.3 Planktonic and sessile growth overnight in- Bacteria growing in liquid culture in the laboratory usually exist as individual cells or small aggregates of cells suspended in the culture medium; the term planktonic is used to describe such freely suspended cells. 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 com- monly grow attached to a surface which, for many species, may be solid, e.g. soil particles, stone, metal or glass, or for pathogens an epithelial surface in the body, 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 all the testing procedures that have been designed to Fundamental features of microbiology 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, in many 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 regu- latory agencies to mean a viable count that records all the different species or types of microorganism that might be present in a sample. Table 2.2 lists the more common counting meth- ods available. The ﬁrst three traditional methods of viable counting all operate on the basis that a living cell (or a small aggregate or ‘clump’ of cells) will give rise to a visible colony when introduced into or onto the surface of a suitable medium and incubat- ed. Thus, the procedure for pour plating usually in- 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 into empty 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 surface spread technique the sample (usually 0.1–0.25 ml) is spread over the surface of agar which has of enumerating cells Rapid methods (Indirect viable counts) 1 Direct microscopic counting 1 Epiﬂuorescence (uses dyes that give (using Helber or haemocytometer characteristic ﬂuorescence only in living cells) often coupled to image analysis 2 Turbidity methods (measures 2 Adenosine triphosphate (ATP) methods turbidity (opacity) in suspensions (measures ATP production in living cells using bioluminescence) 3 Dry weight determinations 3 Impedance (measures changes in 4 Nitrogen, protein or nucleic acid resistance, capacitance or impedance in growing cultures) 4 Manometric methods (measure oxygen consumption or CO2 production by growing cultures) 17 Some of the relative merits of these procedures are described in Table 2.3. Most probable number (MPN) counts may be used when the anticipated count is relatively low, i.e. from 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 transport rates. This advantage allows bacteria far more rapid growth rates than eukaryotic cells. While the classiﬁcation of bacteria is immensely complex, nowadays relying very much on 16S ribo- somal DNA sequencing data, a more simplistic approach is to divide them into major groups on purely morphological grounds. The majority of bacteria are unicellular and possess simple shapes, e.g. round (cocci), cylindrical (rod) or ovoid. Some rods are curved (vibrios), while longer rigid curved organisms with multiple spirals are known as spirochaetes. Rarer morphological forms include the actinomycetes which are rigid bacteria resem- bling fungi that may grow as lengthy branched ﬁla- ments; the mycoplasmas which lack a conventional peptidoglycan (murein) cell wall and are highly pleomorphic organisms of indeﬁnite shape; and some miscellaneous bacteria comprising stalked, sheathed, budded and slime-producing forms often associated with aquatic and soil environments. Bacteria [NAM NAG NAM NAG] Pili L-ala L-ala interbridge D-glu D-glu Meso-DAP Meso-DAP D-ala Flagella D-ala D-ala DAP D-glu represented L-ala NAG NAM NAG Fig. 3.2 Structure of Escherichia coli peptidoglycan. host. Examples of such targets will be noted in the following sections. organ- 2.2.1 Cell wall arrange- The bacterial cell wall is an extremely important structure, being essential for the maintenance of the shape and integrity of the bacterial cell. It is also chemically unlike any structure present in eukary- otic cells and is therefore an obvious target for antibiotics that can attack and kill bacteria without harm to the host (Chapter 12). The primary function of the cell wall is to provide a strong, rigid structural component that can with- stand the osmotic pressures caused by high chemi- cal concentrations of inorganic ions in the cell. Most bacterial cell walls have in common a unique structural component called peptidoglycan (also called murein or glycopeptide); exceptions include the mycoplasmas, extreme halophiles and the archaea. Peptidoglycan is a large macromolecule containing glycan (polysaccharide) chains that are cross-linked by short peptide bridges. The glycan chain acts as a backbone to peptidoglycan, and is composed of alternating residues of N-acetyl muramic acid (NAM) and N-acetyl glucosamine (NAG). To each molecule of NAM is attached a tetrapeptide consisting of the amino acids l- alanine, d-alanine, d-glutamic acid and either ly- sine or diaminopimelic acid (DAP). This glycan tetrapeptide repeat unit is cross-linked to adjacent glycan chains, either through a direct peptide link- age or a peptide interbridge (Fig. 3.2). The types and numbers of cross-linking amino acids vary from or- ganism to organism. Other unusual features of the 25 Lipoteichoic acid Fig. 3.3 Structure of the Gram-positive cell wall. phodiester bridges. Because they are negatively charged, teichoic acids are partially responsible for the negative charge of the cell surface as a whole. Their function may be to effect passage of metal cations through the cell wall. In some Gram-posi- tive bacteria glycerol–teichoic acids are bound to membrane 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 generally found 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 is found outside the peptidoglycan layer. This outer membrane is asymmetrical, composed of proteins, lipoproteins, phospholipids and a component unique to Gram-negative bacteria, lipopolysaccha- ride (LPS). Essentially, the outer membrane is attached to the peptidoglycan by a lipoprotein, one end of which is covalently attached to peptidogly- can and the other end is embedded in the outer membrane. The outer membrane is not a phospho- lipid bilayer although it does contain phospholipids in the inner leaf, and its outer layer is composed of LPS, a polysaccharide–lipid molecule. Proteins are also found in the outer membrane, some of which form trimers and traverse the whole membrane and in so doing form water-ﬁlled channels or porins through which small molecules can pass. Other Bacteria Lipopolysaccharide Receptor protein Porin Lipoprotein membrane Periplasmic protein Periplasm Peptidoglycan Lipid A KDO Core O-antigen

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