Emergence of a Debate: AGPs and Public Health

Abstract

A Heidelberg Appeal Nederland Report

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Emergence of a Debate:
AGPs and Public Health
A. Bezoen
W. van Haren
J.C. Hanekamp*
A
Heidelberg Appeal Nederland

Emergence of a Debate
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Emergence of a Debate: AGPs and Public Health
A. Bezoen
W. van Haren
J. C. Hanekamp,* PhD, CEO HAN
Supervision:
J.C. Hanekamp,* PhD, CEO HAN
Prof. Dr. A.W.C.A. Cornelissen
At the request of the FEFANA, the HAN Foundation has carried out an independent study
into the potential human health hazards related to the use of AGP’s in livestock feed. The
study is conducted under the auspices of the board of the HAN Foundation and an inde-
pendent scientific supervisory committee.
Scientific Committee:
- Prof. Dr. A.W.C.A. Cornelissen (Division of Parasitology, Utrecht University)
- Prof. Dr. W. Gaastra (Division of Bacteriology, Utrecht University)
- Prof. Dr. W.P.M. Hoekstra (Department of Molecular and Cellular Biology, Utrecht University)
- Prof. Dr. J. Verhoef (Clinical Microbiology Group, Utrecht University)
- Prof. Dr. A. Bast (Human Pharmacology and Toxicology; Maastricht University)
- Prof. Dr. R.H. Meloen (Molecular Recognition, Utrecht University)
© HAN, 1999
All rights reserved. No part of this publication may be reproduced and/or published by
print, photo–print, microfilm or any other means without the previous written consent of the
editor. Citing this report is authorised with explicit reference to this report.
In case this report is the result of a research program commissioned by a third party, the
rights and obligations of the contracting parties are subject to the relevant agreement con-
cluded between the contracting parties. This report remains the intellectual property of
HAN.
ISBN 90–76548–06–4
NUR 600
HAN
*hjaap@xs4all.nl +31(0)79 346 03 04/+31(0)79 346 06 43 (fax)
www.stichting–han.nl

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Table of Contents
EXECUTIVE SUMMARY 7
1THE ISSUE 11
1.1 Introduction 11
1.1.1 General 11
1.1.2 Overview 12
1.2 Objectives and methods 13
1.2.1 Objectives 13
1.2.2 Methods 13
2ASSESSING THE RISK 15
2.1 Introduction 15
2.2 The risk chain 16
2.3 Questions and answers 19
2.4 Reassessing the risk 21
3ANTIBIOTICS: USE AND RESISTANCE MECHANISMS 25
3.1 Summary 25
3.2 Antibiotics: categories 26
3.2.1 General 26
3.2.2 Categories of antibiotics 26
3.3 Antibiotic usage 28
3.3.1 General 28
3.3.2 Antibiotics used in animal husbandry 29
3.3.3 Regulations for the use of antimicrobial agents 31
3.3.4 Antibiotics in animal feed 32
3.3.5 Antibiotics in human health care 34
3.4 Cellular processes and antibiotics 36
3.4.1 Cell wall synthesis 36
3.4.2 Bacterial protein synthesis 3 6
3.5 Bacterial resistance and its transfer: basics 38
3.5.1 Location of resistance genes 38
3.5.2 Intrinsic and acquired resistance 39
3.6 Biochemical defence mechanisms against antibiotics 41
3.6.1 General 41
3.6.2 Bacterial cell wall defences 42
3.6.3 Bacterial protein synthesis defences 43

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3.7 Selective pressure and resistance 45
3.7.1 The costs and benefits of resistance: a bacterial viewpoint 45
3.7.2 Reversal of resistance 45
4BACTERIAL ANTIBIOTIC RESISTANCE AND HUMAN HEALTH 47
4.1 Summary 47
4.2 Introduction 48
4.3 ‘Spreading the disease’ 49
4.4 Infectious bacteria 53
4.5 Resistance selection through antibiotics 56
4.5.1 Glycopeptides as human medicine and AGP 56
4.5.2 Virginiamycin used as AGP 57
4.5.3 AGPs also acting against Gram–negative bacteria 57
4.6 Research efforts and data compatibility 58
4.6.1 General 58
4.6.2 Research methods, data compatibility and reproducibility 58
4.6.3 Isolation of resistant strains and phenotypic characterisation 59
4.6.4 Determining resistance data: phenotypic characterisation 61
4.6.5 Genotypic characterisation 64
5PREVALENCE OF BACTERIA RESISTANT ANTIBIOTICS 69
5.1 Introduction 69
5.2 Resistance prevalence 69
5.2.1 Prevalence of vancomycin–resistant enterococci 69
5.2.2 Origin, transfer and spread of VRE 70
5.2.3 Meat as a possible source of resistant bacteria in humans? 7 2
5.2.4 Genetic identification of similarities and differences between
VRE in animals, meat, sewage and humans 74
5.2.5 Comparison of genes and intergenic sequences in Tn1546 75
5.3 Resistance to MLSB antibiotics 7 8
5.3.1 Prevalence 78
5.3.2 Human antibiotics and resistance prevalence 80
5.3.3 Genetic analysis of MLSB resistance 83
5.3.4 Prevalence of resistance against Zn–bacitracin 84
5.4 Conjugational transfer of genetic material 85
5.5 The animal–human link? 87
5.5.1 General 87
5.5.2 Cases providing evidence? 88
5.5.3 In conclusion 89
REFERENCES 91

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APPENDICES
APPENDIX IDEFINITIONS 111
APPENDIX II AGP DOSAGES 113
APPENDIX III HUMAN INTESTINAL FLORA 115
APPENDIX IV RESISTANCE GENES AGAINST STREPTOGRAMINS
LINCOSAMIDE AND MACROLIDES 117
APPENDIX V MIC VALUES 119
APPENDIX VI INFECTIOUS GRAM + BACTERIA 121
APPENDIX VII MAJOR NOSOCOMIAL INFECTIONS 123
APPENDIX VIII ZN–BACITRACIN RESISTANCE IN GRAM +
BACTERIA OVER THE LAST 40 YEARS 125

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Executive Summary
The HAN foundation
The HAN foundation (stichting Heidelberg Appeal Nederland) was established in 1993 in
the Netherlands and is registered in the Chamber of Commerce in Amsterdam. HAN is an
independent non-profit making alliance of scientists and science supporters whose aim is to
ensure that scientific debates are properly aired, and that decisions which are taken and ac-
tion that is proposed are founded on sound scientific principles. Members are accepted
from all walks of life and all branches of science. HAN has at present over 800 donors, in-
cluding almost 200 professors. HAN will be particularly concerned to address issues where
it appears that the public and their representatives, and those in the media are being given
misleading or one-sided information. Our primary role is to contribute to the scientific de-
bate itself. Our second role is to provide an independent voice to the media, the general
public and the educators, and by doing so, HAN aims to provide a balance on scientific
issues. One of the activities of the HAN Foundation is to conduct scientific research at the
request of third parties. Such research is performed by the HAN foundation only, supported
by an independent scientific supervisory committee. To ensure that the study is executed in
an independent fashion the HAN foundation has the right to publication regardless of the
outcome of the research. The content of this particular report is approved by the HAN
board of directors and the independent scientific supervisory committee only.
The issue
The question has been raised whether the use of antimicrobial growth promoters (AGPs) in
animals can result in resistance within human bacteria. Transfer of resistance to antibiotics
from livestock to humans is the point of concern here. The question is whether or not this
implies a threat to human health. FEFANA (Fédération Européenne des Fabricants
d’Adjuvants pour la Nutrition Animale; European federation of feed additive producers)
asked the HAN foundation to re-evaluate the risk associated with the use of antimicrobial
(antibiotic) growth promoting agents in livestock feed in relation to public health.
In a simplified manner, the risk issue concerning AGP use and human health can be de-
picted as follows, keeping in mind that any type of use (‘presence’) of antibiotics will result
in the rise of resistant bacteria, in the species in which it is being used:
Figure 1 Possible sources of human bacterial antibiotics resistance
Contribution to bacterial resistance in humans:
0 % 100 %
Human
antibiotic contribution
?
AGP contribution

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The risk assessment thus revolves around the question to what extent, if at all, the use of
AGPs in animal rearing contributes to bacterial antibiotic resistance already present in hu-
mans.
The data
A prerequisite in this hazard scheme is the transfer of animal bacterial antibiotic resistance
from animals to humans. A risk assessment thus in part requires data concerning this resis-
tance transfer. Unfortunately, these data are in essence non-existent. Van den Bogaard et al.
(1997b) claimed that a turkey and a farmer had the same strain of vancomycin-resistant E.
faecium. Until now this letter is the only one that describes indistinguishable strains in ani-
mals and humans suggesting a possible transfer of bacteria. However, it was not proved that
this strain really colonised the human gut. Furthermore, since other reports describing
similar cases are not available, reproducibility is absent. Generalisation from this particular
observation is scientifically unsound and without foundation as transfer mechanisms of
DNA are manifold taking into account the different bacteria species and genera and the
several resistant traits of interest.
Resistance transfer -although crucial- is, however, only part of the total risk assessment
process. The acquiring of resistance by micro-organisms under selective antibiotics pressure
is far from uniform and in many cases not fully resolved. Furthermore, the epidemiological
consequences of resistance transfer from animals to humans, once established in a repro-
ducible manner, need to be taken into account. Epidemiological data to this date do not
show that use of AGPs in animal rearing compromised the use of related antibiotics in hu-
man medicine. Therefore, past experiences do not reveal that AGPs are a major source of
resistance within human bacteria even after 30 years of use. Moreover, there are no indica-
tions that human infectious diseases are on the increase as a result of the use of AGPs. Risk
analysis also requires the positive (health) effects to be taken into account such as improved
animal welfare and the reduction of the shedding of pathogenic zoonotic micro-organisms.
It is clear that reproducible and documented data concerning antibiotic resistance transfer
from animals to humans is lacking. This makes a formal risk assessment of this issue not
possible. By definition risk assessment can not be based only on the possibility (the hazard
identification) that antibiotic resistance could in theory be transferred from animals to hu-
mans. A quantitative scientific basis is needed for that. Risk analysis guarantees that sound
scientific data are applied in weighing both the positive- against the negative health effects.
In conclusion
- The human health risk concerning the use of AGPs cannot be properly assessed for lack of
data.
- The contribution to human bacterial antibiotic resistance from animal bacterial resistance can-
not be fully assessed for lack of data.
- Sofar, AGP use did not compromise the human therapeutic use of related antibiotics.
- Sofar, epidemiological data do not show an increase of infectious diseases as a result of the
use of AGPs.
- Thorough documented
in vivo
cases showing the spread of antimicrobial resistant Gram-
positive bacteria from livestock to humans are in essence non-existent.

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- Resistance transfer from animals to humans is only part of the entire risk chain. The major parts
of this chain of events comprise of a micro-biological/ genetic part, an animal-human transfer
part and an epidemiological part.
- Assessing the human health risk in relation to AGPs involves making a full scientific inventory.
Beneficial aspects such as animal welfare in relation to the use of AGPs and the influence of
AGPs on the spread of pathogenic zoonotic organisms also need to be taken into considera-
tion.
- A comprehensive multidisciplinary research effort is needed to properly assess all aspects of
the use of AGPs in animal husbandry.

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1 The Issue
1 . 1 Introduction
1. 1. 1 General
Resistance of bacteria against antibiotics, meaning that antibiotics do not have a bactericidal
or bacteriostatic effect due to the rise or inherent capability to withstand the antibiotics in
question, used in human medical treatment can be a serious public health risk. It is known
that the use of antibiotics can lead to the origination/emergence of antibiotics resistant bac-
teria (Levy, 1997). Examples of bacteria that have become resistant to human antibiotics
are:
- Methicillin–resistant
Staphylococcus aureus
(MRSA)
- Penicillin–resistant pneumococci
- Vancomycin–resistant enterococci (VRE)
It seems that there is hitherto no well–founded consistent scientific basis for the suggestion
that these resistances –in part– originate from the several decades of animal feed additives
use (the so–called Antimicrobial Growth Promoters (AGPs)), with a possible exception of
the VREs. There is, however, extensive evidence and clinical experience that links these
bacterial resistances with the human use of medicinal antibiotics both in hospitals and the
local community. In spite of all this, bacterial resistance originating from animal use o f
antibiotics has become a subject of extensive political and scientific debate within the Euro-
pean Community.
Antibiotics, when added to the feed, decrease the time and the amount of feed needed to
reach slaughter weight (Nefato, 1997). It has been shown that the use of antibiotics for this
goal selects for resistant bacteria in animals (Hummel et al., 1986; Bager et al., 1997; Klare
et al., 1995; Van den Bogaard et al., 1996, 1997b; Aarestrup et al., 1997, 1998). Some of
the growth promoters used in feed are structurally related to antibiotics used in human
medicine. Their mode of action on bacterial cells can then be identical (or highly compara-
ble). Resistant bacteria found in animals might in this way be resistant to antibiotics used in
human medicine. This is called cross–resistance. The concern now is that resistance, as
found in animals, might spread to humans. This spread might add to the already widespread
existence of bacterial resistance within humans resulting from human use of antibiotics. The
reasoning behind this is simple and straightforward, albeit tentative:
Scheme 1.1.1.1 Risk scheme concerning AGPs and human health
Bacteria in the animal gut and faeces contain resistant bacteria, caused by the use of anti-
biotics as growth promoters in livestock feed, which might be transferred to humans in one
way or the other. Those resistant bacteria might themselves be a human health threat or
they might transfer their resistance to other bacteria capable of colonising the human gut.
Virulent resistant strains might cause illness not easily treated by known antibiotics.
In other words the human gut might be colonised by resistant bacteria previously present in
animals. The second possibility is the transfer of resistance determinants from bacteria pre-
viously present in animals to human bacteria commonly present in the gut or to human
pathogens. If resistance in the animal is due to the use of antibiotics in the feed, mixing
antimicrobials with feed could in theory contribute to the emergence of serious infections in
man.

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It should be noted that most AGPs are active against Gram–positive bacteria and not against
Gram–negative bacteria.1 Antibiotics that are active against Gram–negative bacteria are usu-
ally not active against Gram–positive bacteria and vice versa. Examples of Gram–negative
bacteria are Escherichia coli and Salmonella typhimurium. An example of a Gram–positive
bacterium is Staphylococcus aureus. The antibiotics discussed in this report are active
against the Gram–positive bacteria group. The bacteria considered in this report belong to
the Gram–positive group. The antibiotics resistance transfer issue is thus limited to the
Gram–positive bacteria group when discussing the relevant AGPs.
1. 1 .2 Overview
Vancomycin (an antibiotic) resistant enterococci (VRE) were first detected in hospital pa-
tients in Europe in the late 1980s. Since then these bacteria have been isolated frequently in
all parts of the world (Bates, 1997). VRE can be a problem for immuno–compromised pa-
tients, who have a severe disease or have been surgically operated. Also people who are
wounded by an accident or carry medical devices like catheters have shown to suffer infec-
tions caused by VRE (Weinstein, 1998; Bogle and Bogle, 1997). In hospitals, the majority
of VRE are isolated from patients in intensive care units and other specialised wards (Bates,
1997).
Later it appeared that not only patients with clear symptoms of infection carried VRE, but
also other patients in the hospital and people on admission to the hospital (Jordens et al.,
1994; Gordts et al., 1995; Klare et al., 1995). This indicated that the problem was not solely
a hospital matter.
It was found that within community people VRE was also quite widespread. These bacteria
were also detected in sewage, waste water, animals and meat. Where these VRE originated is
not always clear.
It is necessary to elucidate how VRE emerge and to find the source of VRE in community
and hospitalised people. Do these bacteria arise in humans or are bacteria or resistance
genes transferred from other sources to humans adding to the resistance of human bacteria?
Glycopeptide antibiotics, like avoparcin, vancomycin and teicoplanin, can cause emer-
gence/selection of resistant bacteria. This has been show in humans who received vancomy-
cin or teicoplanin (Van der Auwera et al., 1997), as well as in animals which received avop-
arcin as growth promoter (Bager et al., 1997; Klare et al., 1995, Van den Bogaard et al.,
1996). As glycopeptide antibiotics are rarely used to treat patients in Europe, the use of
avoparcin as growth promoter in feed was suggested as source for resistant bacteria present
in humans (due to cross–resistance).
Avoparcin has been used in Europe in animal feed until 1997. At the moment up to ten
other antimicrobials are allowed as growth promoter in animal feed. So avoparcin is not the
only feed additive that may have an effect on the prevalence of resistant bacteria in humans.
1 The plasma membrane of Gram-positive bacteria is surrounded by a thick cell wall, typically 250 Å
wide, composed of peptidoglycan and teichoic acid. Gram-negative bacteria on the other hand have a more
complex membrane system. Their plasma membrane is surrounded by a 30 Å wide peptidoglycan wall,
which in turn is covered by an 80 Å outer membrane comprising of protein, lipid and lipopolysaccharide.
Because of the different layered cell-wall structure of the Gram-negative bacteria in comparison to the
Gram-positive bacteria, antibiotics against Gram-positive bacteria are mostly inactive against Gram-
negative bacteria.

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In the USA avoparcin is not used as a growth promoter in animal feed. When comparing
European and USA data about the prevalence and relatedness of VRE a better insight in the
epidemiology (emergence and spread) of VRE might be obtained.
1 .2 Objectives and methods
1. 2.1 Objectives
The main objective of this report is to reassess the risk to human health caused by antimi-
crobial growth promoters (AGP) used as feed additives. To be able to do this, several
sub–questions have to be answered.
- Does the use of antimicrobial growth promoters (antibiotics) lead to the spread of AGP resis-
tance beyond the sphere of livestock production? There is strong evidence for the presence
and emergence of bacteria in animals resistant to antibiotics present in the feed. This is not a
point of controversy at the moment. It is useful to know which antibiotics are used to promote
animal growth. Then it will be made clear which of them possibly form a threat to human health.
The prevalence of resistance to these antibiotics (and their structural analogues used in human
medicine) will be listed.
- Are there documented cases that show the spread of antimicrobial resistant bacteria from live-
stock to humans? Resistance to avoparcin–vancomycin (used in feed and to treat humans re-
spectively) is quite wide spread among pigs and poultry (less in cows). Articles that describe
the spread of VRE or other resistant bacteria from livestock to humans, if present, will be
evaluated.
- What is the risk of the use of antibiotics in feed to human beings and how does this relate to
other risk factors? Risk factors for humans concerning antibiotic resistance will be discussed.
The use of antibiotics in feed as a risk factor will be evaluated.
- Can these data be generalised to all AGPs? Not only avoparcin and its relation with resistance
to vancomycin will be studied, also other antibiotics used in feed and showing
cross–resistance with antibiotics used in human health care will be included.
1. 2 .2 Methods
Literature of the last decade containing data about resistant bacteria in animals and humans
will be analysed. First, factors leading to the emergence of resistant bacteria will be studied.