Table Of ContentSTUDIES in the AGRICULTURAL and FOOD SCIENCES
A series of high-level monographs which review recent research in various
areas of agriculture and food science
Consultant Editors:
D. J. A. Cole University of Nottingham
W. Haresign University of Nottingham
W. Henrichsmeyer Director, Institut für Agrarpolitik,
University of Bonn
J.P. Hudson formerly Director, Long Ashton Research Station,
University of Bristol
G. Kimber Professor of Agronomy, University of Missouri-
Columbia
J.L. Krider Professor of Animal Sciences, Purdue University
D.E. Tribe Director, Australian Universities' International
Development Program, Canberra
V. R. Young Professor of Nutritional Biochemistry,
Massachusetts Institute of Technology
Already published:
Recent Advances in Animal The Calf— Fourth Edition
Nutrition—1978 J. H.B.Roy
Edited by W. Haresign and D.
Energy Metabolism
Lewis
Edited by Lawrence E. Mount
Recent Advances in Animal Growth in Animals
Nutrition—1979 Edited by T.L.J. Lawrence
Edited by W. Haresign and D.
Mineral Nutrition of Fruit Trees
Lewis
Edited by D. Atkinson, R.O.
Recent Advances in Animal Sharpies, J.E. Jackson and W.M.
Nutrition—1980 Waller
Edited by W. Haresign
Sheep Breeding—Second edition
Recent Advances in Animal Edited by G.J. Tomes, D.E.
Nutrition—1981 Robertson and R. J. Lightfoot
Edited by W. Haresign Revised by W. Haresign
Recent Advances in Animal Mineral Nutrition of Animals
Nutrition—1982 V.l. Georgievskii, B.N. Annenkov
Edited by W. Haresign and V.T. Samokhin
Recent Advances in Animal Protein Contribution of Feedstuff's for
Nutrition—1983 Ruminants
Edited by W. Haresign Edited by E.L. Miller and I.H. Pike
in association with A.J.M. van Es
Plant Breeding for Pest and Disease
Resistance Advances in Agricultural Microbiology
G.E. Russell Edited by N.S. Subba Rao
Antimicrobials and Agriculture
The Proceedings of the 4th International
Symposium on Antibiotics in Agriculture:
Benefits and Malefits
Malcolm Woodbine MSc, PhD, FRSC, FIBiol
Professor of Agricultural Microbiology, University of Nottingham
BUTTERWORTHS
London Boston Durban Singapore Sydney Toronto Wellington
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First published 1984
© The several contributors named in the list of contents, 1984
British Library Cataloguing in Publication Data
International Symposium on Antibiotics in Agriculture:
Benefits and Malefits (1983)
Antimicrobials and agriculture. (Studies in the
agricultural and food sciences)
1. Anti-infective agents in agriculture
I. Title II. Woodbine, Malcolm III. Series
668'.6 SF98.A4/
ISBN 0-408-11155-0
Library of Congress Cataloging in Publication Data
International Symposium on Antibiotics in Agriculture: Bene
fits and Malefits (4th : 1983 : University of Nottingham)
Antimicrobials and agriculture.
Bibliography: p.
Includes indexes.
1. Antibiotics in veterinary medicine Congresses.
2. Antibiotics in agriculture Congresses. 3. Anti-infective
agents Congresses. I. Woodbine, Malcolm. II. Title.
SF918.A5158 1983 636.089'5329 84-5002
ISBN 0-408-11155-0
Filmset by Butler and Tanner Ltd, London and Frome
Printed and bound in Great Britain by Anchor Brendon Ltd, Tiptree, Essex
PREFACE
The Easter Schools in Agricultural Science began in 1953 with the one on
Micropedology, organized by Professor Gordon Hallsworth. This success
encouraged the following Easter Schools and led to two on Antibiotics in
Agriculture. The first of these—originally envisaged for 1956 or 1957—was
anticipated by the First International Conference on Antibiotics in Agricul
ture, which was held in Washington in 1955 and published in 1956 (as
publication 297 of the National Academy of Science-National Research
Council). The Americans decided not to sponsor another such conference
and so the Second International Conference on Antibiotics in Agriculture
was held at Sutton Bonington in 1962 (as the 9th Easter School) and published
by Butterworths in 1962. The success attached to this effort and the continuing
one of Easter Schools led to the prospect of a Third International Conference
and 25th Easter School in Agricultural Science on Antibiotics in Agriculture.
This was based on the view that the updating of earlier topics could be
regarded as either a good thing per se or as losing some appeal by virtue of
repetition.
The 1976 programme was designed to meet these views by updating the
1956 and 1962 volumes and to introduce a new aspect. Accordingly, contri
butions (and topics) were invited to give 'special reference to synergism'. This
volume was published in 1977 by Butterworths. Those attending the 25th
Easter School considered that another should be held under the title of
'Antibiotics in Agriculture: Benefits and Malefits', a suggestion put forward
by Sir James Howie.
In the event, the 1983 meeting was held as the Fourth International Sym
posium on Antibiotics in Agriculture—with the considerable support listed
in the acknowledgements—and is published, by Butterworths, under the
agreed title 'Antimicrobials and Agriculture'.
Sir James Howie chaired a short final session of the symposium, on the
topic Where to Now? It was then agreed (1) that the 'spine' of Soil-Plant-
Animal-Feed-Food-Public Health had provided a sound framework to dis
cuss Antibiotics and Antibiosis in Agriculture, (2) that, as far as the future
could be seen, it would be an excellent idea to have a 5th Symposium, possibly
about 1988, (3) that the present editor, his possible successor, or Professor
Alan Linton, be responsible, (4) that of the various suggestions on a theme,
v
vi Preface
the one suggested by Professor Alasdair Steele-Bodger received general ap
proval: 'Antibiotics and Agriculture: Remits and Responsibilities'.
Malcolm Woodbine
ACKNOWLEDGEMENTS
As on the previous occasions, any symposium or conference needs both
general and financial help for it to be organized at all. A very deep 'thank
you', therefore, for the generous financial support from the following organ
izations:
Aplin and Barrett Ltd
Beecham Pharmaceuticals Research Division
Cyanamid International
Dow Chemical Co. Ltd
Eli Lilly Research Ltd
Foundation for Microbiology
Glaxo Research
Hoechst (U.K.) Ltd
ICI Phamaceuticals
Olin Chemicals
Pfizer Ltd
SmithKline Animal Health Products
Squibb Europe Inc.
Upjohn Ltd
To my colleagues Dr Alan Seaman, Mr David Fowler, Mr John Rosillo,
Mrs Caroline Walker, and to my students: post-graduates Hisham Bashir,
Munim Amin; undergraduates Joanne Morley, Georgina Simpson, Sarah
Tullett, Bill Wadsworth; to our domestic, library and administrative col
leagues, especially to Ms Jeanette O'Neill for her admirable secretarial sup
port; to Ms Doreen Borrows who acted as Symposium Secretary; and to my
wife Hilda both for her general support and help with the references and
indexing with which my daughter Barbara was also associated. Many thanks
also to the staff at Butterworths for their help in the preparation of the
volume.
vu
1
PROLOGUE
MARK H. RICHMOND
Professor of Molecular Microbiology and
Vice-Chancellor, The University, Manchester, UK
In many ways, this symposium on the benefits and disadvantages of anti
biotics in agriculture is well timed. The antibiotic era—if one discounts the
sulphonamides as antibiotics—has now lasted for about 40 years and the
benefits and disadvantages of these agents are fairly clear for all to see. On
the one hand there are undoubted advantages: agents now exist which do
indeed have enormously valuable properties and many infectious conditions
are largely controlled—both in man and domestic animals—by their use.
Moreover, treatment is much more effective and rapid, with clear advantages
in morbidity and the incidence of unwanted side-effects.
Against this background there are some fairly notable disadvantages. Re
sistant populations of bacteria have emerged in many cases and these have
sometimes caused severe clinical problems. Perhaps few of these are actually
life-threatening, but often they delay recovery and lead to unwanted compli
cations.
Although it is clear that antibiotics have their advantages and disadvan
tages, it is often rather difficult to decide where the balance lies for any one
agent. If one goes to meetings on the newer /Mactam antibiotics, for example,
there is much talk of resistant organisms and /Mactamase production. Despite
this, however, very large amounts of benzyl penicillin and of phenoxymethyl
penicillin are still sold and are effective 40 years or so after their first use.
Contrariwise, most of the new /Mactams are primarily for hospital prescrip
tion and used to treat a minority of infections.
In fact, when one considers the occasions when the well-established 'old'
penicillins do not work, one commonly finds the infections in the very young,
in the very old, or in the compromised. Thus, there is a hint here that the
problems lie not so much with the organisms and their possible resistance,
but rather with the patient and his or her relative inability to marshal the
necessary defences to clear the infection.
In fact, when one reviews the position—and this is particularly clear for
the /Mactams—one finds a situation where one has a range of generally rather
effective 'old' compounds each with its typical therapeutic role and then a
large number of newer introductions, all very similar in properties, and all
seeking a niche in the market. The fact that the advantages of a particular
compound are only marginal makes it ever more difficult for the physician to
3
4 Prologue
decide which agent to use; in the face of a battery of advice, he tends to throw
in his hand and remain wedded to a well-tried, but often marginally less good,
alternative.
The fact that it is an impairment of the defences of the host which seems to
be of the greatest importance, now that we have a large range of effective
antibiotics, points the way to the future as far as research and development of
new anti-infectives is concerned. What seems to be needed is the agent to
augment the action of the 'old' antibiotics by potentiating the defence mech
anisms compromised in the host. Perhaps this end can be achieved by an
enhancement of the immune response, or by the potentiation of phagocytosis
or by some other alteration of the normal protective measures of the host.
If the future of antibiotic therapy is indeed destined to be part of a combined
therapy—part antibiotic and part modulator of the defence mechanisms—it
is clear that much work needs to be done; and it is in this connexion that one
has reached a critical point. The sort of research to generate the new com
pounds will be of a type very different from that which evolved the antibiotics
we now know. In general, synthetic chemistry will be less involved, and
modern biology—immunology, cell biology, development—will become par
amount.
In fact, this need to alter the point of research attack so radically will make
for major difficulties in the pharmaceutical companies. One can readily see at
least three. First, the type of research worker needed will change and the skills
which seemed of paramount importance in the past will be needed less.
Secondly, the direction of the research effort will give rise to difficulties. In
many companies the senior research directors have reached their positions
from a background of organic chemistry—a testimonial to the routes to
successful products in the past. Such people—often excellent and gifted
researchers—find the imperatives of biological research hard to grasp; and it
is often difficult for a biologist to urge a successful case for a new research
project in such an environment. Perhaps it is not accidental that several
recently appointed research directors of major companies are biologists intro
duced from academia rather than promoted from within the company.
Almost certainly the greatest difficulty at this present critical juncture,
however, is to know precisely where to concentrate one's research effort. If
one has the objective of supporting conventional antibiotics with agents
which enhance the normal defences of the host, where does one start? Too
little is known to be able to focus one's research; yet the resources available-
even to industry—are simply incapable of covering all the necessary areas.
It is against this background that one feels that university research may
soon become, once again, of paramount importance to the pharmaceutical
companies. In the recent past, when the semisynthetic modification of existing
nuclei was of supreme importance, most of the necessary effort and expertise
were available within the research departments of the companies. Now there
is a real need for a much greater insight into the fundamental biology of the
infectious process if the development of new agents is going to be achieved,
and this is likely to arise from work in academic departments. Indeed the
transition, which is the central point for discussion in this brief prologue,
gives major chances for the universities of the world. Their efforts will be of
crucial importance if we are to move successfully into the next phase of the
antibiotic era.
2
BETA-LACTAMASE INHIBITORS: BIOCHEMICAL
PROPERTIES AND BACTERIOLOGICAL APPLICATION
M. COLE
Beecham Pharmaceuticals Research Division, Epsom, UK
The interest in inhibitors of the /Mactamase enzymes (penicillinases and
cephalosporinases, etc.) arises from their importance as resistance determi
nants for /Mactam antibiotics. There are several mechanisms by which bac
teria can be resistant to the lethal effects of penicillins and cephalosporins,
but probably the most important involves destruction of the essential ß-
lactam ring of these molecules by /Mactamases in the bacterial cell wall. These
enzymes are surprisingly widely occurring and many different types exist,
several of the most important being plasmid mediated.
Much research effort has been and is still directed to finding members of
the /Mactam class of antibacterials which have stability to /Mactamases.
However, it has not proved easy to achieve stability to a wide range of these
enzymes while retaining previously existing desirable features. This situation
has encouraged the search for /Mactamase inhibitors which might enhance
the activity of labile compounds against resistant strains. For such inhibitors
to be of therapeutic use they must, of course, be able to protect the labile
compound from those /Mactamases which most readily attack it.
The /Mactamase inhibitors which were first found, for example methicillin
and cloxacillin, did not meet all of the above criteria. Much more promising
results have been obtained with recent compounds such as salts or derivatives
of clavulanic acid and penicillanic acid sulphone.
This chapter reviews the various /Mactamase inhibitors and discusses our
observations on some of the /Mactamases of veterinary isolates of bacteria,
their inhibition by potassium clavulanate and its ability to potentiate the
activity of amoxycillin against these bacteria.
Mechanism of penicillin resistance
The three main mechanisms by which bacteria may be resistant to the action
of penicillins are illustrated in Figure 2.1. First, the target site enzyme (trans-
peptidase) may have structural features which make it less readily inhibited
by penicillin than is the case in sensitive cells. This may be the situation in
methicillin-resistant staphylococci (Hayes et al, 1981). Secondly, the outer
layers of the cell wall may be of such a composition as to prevent or seriously
5
6 Beta-lactamase inhibitors: biochemical properties
Penicillin molecules (P)
Sections through bacterial cell walls
\MÎ
Outer cell wall
Peptidoglycan
Cytoplasmic ^ -
] WJ
membrane T~^
INSENSITIVE PERMEABILITY PENICILLIN
TARGET ENZYME BARRIER DESTRUCTION
Target enzyme is of a type Components of cell wall Penicillin is destroyed by
which is not readily ^^ prevent diffusion of ß-lactamase enzyme ^^
inhibited by penicillin ^Λ penicillin to target outside and inside the cell
enzyme ■■ wall rendering the
penicillin concentration too
low to inhibit the target
enzyme WM
Figure 2.1 Mechanisms of resistance to penicillins
retard the penetration of penicillin molecules to the target site enzyme. This
mechanism probably explains why benzylpenicillin has poor activity against
Gram-negative bacteria in spite of its good affinity for the penicillin-binding
proteins (Curtis et al., 1979).
The third mechanism involves destruction of penicillin molecules by bac
terial enzymes called /Mactamases. These enzymes are usually located in a
region of the cell wall just outside the cytoplasmic membrane. They are thus
in an ideal position to prevent the build-up of an inhibitory concentration of
penicillin at the target site enzyme. In some bacteria, for example certain
strains of Staphylococcus aureus, the /Mactamase is also liberated from the
cell (exocellular enzyme).
Enzymes which destroy penicillins and cephalosporins—the
/Mactamases
The /Mactamases catalyse the hydrolysis of the /Mactam ring of penicillins
and cephalosporins to give products devoid of antibacterial activity. Some
times the names 'penicillinase' and 'cephalosporinase' are used for these
enzymes, depending on the substrate, but this can be confusing as many of
the enzymes having penicillinase activity can also attack cephalosporins and
vice versa. Amoxycillin is unstable to several different types of /Mactamases,
the product of the reaction being an inactive penicilloic acid (see Figure 2.2).
The presence of a /Mactamase in a bacterial culture can be demonstrated
by disrupting the cells with ultrasonics, mixing the resulting crude enzyme
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