Antimicrobial resistance of Escherichia coli and Enterococcus
faecalis in housed laying-hen flocks in Europe
S. VAN HOOREBEKE1*, F. VAN IMMERSEEL2, A. C. BERGE1, D. PERSOONS1,
J. SCHULZ3, J. HARTUNG3, M. HARISBERGER4, G. REGULA4, L. BARCO5,
A. RICCI5, J. DE VYLDER2, R. DUCATELLE2, F. HAESEBROUCK2
AND J. DEWULF1
1Veterinary Epidemiology Unit, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary
Medicine, Ghent University, Merelbeke, Belgium
2Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University,
3Institute for Animal Hygiene, Animal Welfare and Farm Animal Behaviour, University of Veterinary Medicine
Hanover, Foundation, Germany
4Veterinary Public Health Institute, Vetsuisse Faculty, University of Bern, Liebefeld, Switzerland
5Public Health and Risk Analysis Department, Istituto Zooprofilattico Sperimentale delle Venezie, Italy
(Accepted 3 November 2010; first published online 7 December 2010)
The aim of this study was to determine the potential association between housing type and
multiple drug resistance (MDR) in Escherichia coli and Enterococcus faecalis isolates
recovered from 283 laying-hen flocks. In each flock, a cloacal swab from four hens was
collected and produced 1102 E. coli and 792 E. faecalis isolates. Broth microdilution was used
to test susceptibility to antimicrobials. Country and housing type interacted differently with
the MDR levels of both species. In the E. coli model, housing in a raised-floor system was
associated with an increased risk of MDR compared to the conventional battery system
[odds ratio (OR) 2.12, 95% confidence interval (CI) 1.13–3.97)]. In the E. faecalis model the
MDR levels were lower in free-range systems than in conventional battery cages (OR 0.51,
95% CI 0.27–0.94). In Belgium, ceftiofur-resistant E. coli isolates were more numerous than
in the other countries.
Key words: Antimicrobial resistance in agricultural settings, Enterococcus, Escherichia coli,
The therapeutic, prophylactic and metaphylactic use
of antimicrobials is common practice in modern food-
animal husbandry [1–3]. Concerns have grown that
this widespread use of antimicrobialdrugs may lead to
an increase in antimicrobial resistance in numerous
bacteria potentially affecting public health [4, 5].
grammes are necessary to monitor the occurrence and
persistence of antimicrobial resistance in food animals
[3, 6, 7]. Indicator bacteria are generally used to
monitor antimicrobial resistance since they can be
bacteria acquire antimicrobial resistance faster than
* Author for correspondence: Dr S. Van Hoorebeke, Veterinary
Epidemiology Unit, Department of Reproduction, Obstetrics and
Herd Health, Faculty of Veterinary Medicine, Ghent University,
Salisburylaan 133, 9820 Merelbeke, Belgium.
Epidemiol. Infect. (2011), 139, 1610–1620.
f Cambridge University Press 2010
other commonly found bacteria [6, 8, 9]. Commensal
Escherichia coli and Enterococcus faecalis are inter-
nationally used as respective Gram-negative and
Gram-positive indicator bacteria for monitoring
antimicrobial resistance because of their common
presence in the animal intestinal tract [10–12]. Sur-
veillance of antimicrobial resistance is performed
in several countries , yet these surveillance pro-
grammes have generally been focused on cattle,
pig and broiler production. Programmes in laying
hens are still scarce. Therefore, there is a need to
monitor antimicrobial resistance development in lay-
ing hens .
According to the Council Directive 1999/74/EC,
from January 2012 onwards conventional battery
cages will be forbidden in the European Union (EU)
. Only enriched cages and non-cage housing
systems will be allowed. Non-cage housing systems
consist of an indoor area that may or may not be
combined with covered (‘wintergarden’) or un-
covered (‘free-range’) outdoor facilities [14, 15]. The
non-cage systems can be categorized into two groups:
single level systems with a ground floor area which is
fully or partially covered with litter and aviaries,
consisting of a ground floor area plus one or more
platforms [14, 15]. Free-range organic flocks have the
same structure as a free-range system but there are
some additional requirements concerning maximum
flock size, beak trimming and the origin of the feed.
Moreover, the application of antimicrobials in these
organic flocks is strictly restricted to therapeutic
usage. Presently it is not clear whether the different
housing and management systems for laying hens will
influence the occurrence of antimicrobial resistance.
Recent reports indicate that both in poultry and other
animal species the move from conventional indoor
production towards free-range and organic pro-
duction exerts a beneficial effect on the levels of anti-
bacteria [16–19]. On the other hand it has been shown
that this move to non-cage housing systems resulted
in an increased incidence of bacterial diseases [20, 21],
which could potentially lead to increased anti-
microbial usage. However, epidemiological data on
the prevalence of antimicrobial resistance in the
above-mentioned indicator bacteria in laying hens in
different housing systems is still limited.
The aim of this paper was to investigate the preva-
lence of antimicrobial resistance in E. coli and
E. faecalis isolates recovered from 283 laying flocks in
four European countries and to evaluate the potential
association between housing systems and observed
MATERIAL AND METHODS
Flocks were selected from a registry list of laying-hen
farms provided by the official identification and
registration authorities of the participating countries
(Belgium, Germany, Italy, Switzerland). The farm
size (>1000 laying hens) and the housing type were
the only two selection criteria. The distribution of
sampled farms aimed for was 20% conventional bat-
tery cage systems and 80% non-cage (alternative)
housing systems. Within the group of alternative
housing systems an equal number of aviaries, raised-
floor, free-range and organic farms was targeted. The
farmers were contacted by telephone and the purpose
of the study was explained. The planned date of de-
population was noted to make sure that the farm
could be sampled in the month prior to depopulation.
Sampling of the laying-hen farms
Four hens in the sampled flock were evenly selected
throughout the house and from each hen one cloacal
swab was taken by inserting a sterile cotton-tipped
swab about 5 cm into the cloaca. The swabs were di-
rectly placed in individual tubes containing Ames
A questionnaire was completed during an on-farm
interview at the same time that collection of samples
took place. The questionnaire consisted of 92 ques-
tions, with 31 open questions and 61 closed questions.
Prior to the study, the questionnaire was tested on
two Belgian laying-hen farms, one with conventional
battery cages and one with a free-range production
system in order to check whether the questions were
relevant to the aim of this study. The questions were
related to general farm and flock characteristics
such as flock size, breed, age of hens and biosecurity
measures. Special attention was paid to the housing
system of the sampled flock and the antimicrobial
treatments the hens had received during the current
production cycle. The same questionnaire was used in
all participating countries; a translation from English
to Dutch, German and Italian was performed during
a meeting with all participating countries. For each of
the participating countries one person was designated
to collect the samples and conduct the interview with
Antimicrobial resistance in laying hens 1611
the farmer. Questionnaires can be obtained from
corresponding author upon request.
Bacteriological examination of the samples
For the isolation of E. coli the swabs were plated onto
MacConkey plates (Oxoid1, France) and incubated
aerobically for 24 h at 37 xC. From each primary
plate, one colony was selected and plated again onto a
MacConkey agar plate. Suspected colonies were con-
firmed as E. coli by positive glucose/lactose fermen-
tation, gas production and absence of H2S production
on Kligler iron agar (Oxoid) and absence of aesculin
hydrolysis (bile aesculin agar; Oxoid). In Switzerland,
the following equivalent method was used: the cloacal
swabs were plated onto MacConkey agar (Oxoid) and
incubated for 24 h at 37 xC under aerobic conditions.
Strains which were lactose-positive were subcultured
on Brolacin agar (Merck1, Germany) and incubated
for 24 h at 37 xC under aerobic conditions. Confir-
mation of the strains as E. coli was performed using
RapiD 20 E (bioMe ´ rieux, France).
For the isolation of Enterococcus the swab was plated
on Slanetz & Bartley agar (Oxoid). After incubation
for 48 h at 42¡1 xC, one suspected E. faecalis colony
per sampled animal was purified and verified by using
Switzerland, E. faecalis was isolated from the cloacal
swabs on Enterococcosel agar (Becton, Dickinson
and Company, USA) and incubated for 48 h at 37 xC
under aerobic conditions. On Columbia 5% sheep
blood agar (Oxoid) strains were subcultured and
incubated for 24 h at 37 xC under anaerobic con-
ditions. Confirmation of the strains as E. faecalis was
performed using Rapid ID 32 STREP strips (bio
Me ´ rieux).
strips (bioMe ´ rieux).In
Antimicrobial susceptibility testing
For both indicator bacteria, susceptibility was tested
by means of broth microdilution using custom made
Sensititre1plates (Trek Diagnostics Systems Ltd,
UK). The antimicrobials tested and their ranges are
listed in Tables 1 and 2 for E. coli and E. faecalis,
respectively. The results were read visually after 24 h
incubation at 37 xC and the minimum inhibitory
concentration (MIC) was defined as the lowest
concentration of the antimicrobial that completely
inhibited visible growth. E. coli ATCC 25922 and
E. faecalis ATCC 29212 were used for quality con-
trol. The European Committee on Antimicrobial
Table 1. Minimum inhibitory concentration (MIC) distribution (%) for 1102 Escherichia coli isolates
(vertical black line indicates cut-off value)
Distribution (%) of MICs (µg/ml)
0·015 0·03 0·060·12 0·25 0·5124816 3264 128256512 10242048
89·60·5 1·3 5·60·0
81·1 0·80·80·2 17·0
1612 S. Van Hoorebeke and others
Susceptibility Testing (EUCAST) epidemiological
breakpoints for MIC determination were used. When
these EUCAST breakpoints were not available
(which was the case for apramycin, sulfamethoxazole
and kanamycin), the breakpoints mentioned in the
reports of the Danish Integrated Antimicrobial
Resistance Monitoring and Research Programme
were used .
Statistical data analysis
An isolate was defined as multiple drug-resistant
(MDR) if it exhibited resistance to two or more anti-
microbials. For both the E. coli and E. faecalis
models, MDR was used as an outcome variable in
statistical analysis. For E. coli, ceftiofur resistance
was also used as an outcome variable. Three separate
statistical models were developed to test these out-
come variables. The questionnaire responses were
transformed to binary or categorical variables.
Pearson’s x2test was used to determine potentially
significant differences between categorical explorative
variables (P<0.05), and those factors that were sig-
nificant in this univariate analysis were further tested
in the multivariate models. The predictive categorical
factors, consisting of country, housing type, presence
of other farm animals (e.g. pigs, cattle, sheep) on
farm, the presence of hens in the flock originating
fromdifferent rearingsites andantimicrobial
treatment of the flock were tested for inclusion in the
models. A stepwise forward selection process was
used for the variable selection in a population average
logistic regression model with a P value f0.2 for en-
try, and with a P value f0.10 for retention in the
model. This model did not control for clustering of
isolates within farms but was used for preliminary
testing of significant predictive factors. The factors
that were significant in this model were introduced
into a model using generalized estimating equations
(GEE) to control for clustering of samples within a
farm using an independent correlation matrix. Inter-
action effects were tested for variables retained in the
final GEE model. Odds ratios and 95% confidence
intervals were calculated for the parameters that
were retained in the GEE model and only factors with
a P value f0.05 were retained in the final GEE
model. The statistical software package SAS (SAS for
Windows, version 9.1, SAS Institute Inc., USA) was
used for data analysis.
Antimicrobial resistance patterns were described
using cluster analysis. Binary cluster analysis was
performed using the Jaccard matching coefficient and
the centroid method to obtain discrete clusters with
no intra-cluster variability. Due to the large number
of antimicrobial resistance clusters, the descriptive
and stratified analysis was limited to the 15 most
common resistance patterns describing more than
80% of the isolate dataset.
Table 2. Minimum inhibitory concentration (MIC) distribution (%) for 792 Enterococcus faecalis isolates
(vertical black line indicates cut-off value)
Distribution (%) of MICs (µg/ml)
Tigecycline 0·1 0·125·8 2·1 4·121·8 45·4
7·7 20·5 59·8
0·0150·030·06 0·12 0·250·51248 163264128 256512 1024 2048
Antimicrobial resistance in laying hens 1613
In total, 1102 E. coli isolates and 792 E. faecalis
isolates were collected from 283 laying-hen flocks
(69 Belgian, 85 German, 30 Italian and 99 Swiss
flocks). The participation rate was more than 90% in
Belgium, Italy and Switzerland and 70% in Germany.
A detailed description of the number of isolates per
housing type and per country is presented in Table 3.
The median flock size of the sampled flocks was 7612
laying hens (range 1000–84000 hens). The flock size in
conventional battery cage houses was significantly
higher than the flock size in non-cage systems.
Twenty-four of the 283 sampled flocks were treated
with antimicrobials during the current production
cycle according to the declaration of the farmers.
The number of treated flocks differed significantly
between countries (P<0.05) but not between housing
types. In Italy none of the sampled flocks were
treated with antimicrobials, for Belgium this was 3/69
sampled flocks, for Switzerland 5/99 flocks and for
Germany 16/85 sampled flocks. Colistin was the most
(five flocks), neomycin (two flocks) and enrofloxacin
(one flock). There was a significant difference between
countries in the number of laying-hen farms where
other production animals were also kept (P<0.05). In
Italy, only 16.7% of the sampled laying-hen farms
managed other animal production on the same site,
whereas in Germany, Belgium and Switzerland this
was 38.5%, 43.8% and 75.5%, respectively.
The distribution of the MICs (in %) of each anti-
microbial is described in Table 1 for E. coli and in
Table 2 for E. faecalis.
MDR in E. coli
The majority of the isolates (55.0%) were susceptible
to all 18 antimicrobials, 16.9% were resistant to one
antimicrobial and the remaining 28.1% were multi-
resistant. The 15 most common resistance phenotypes
of antimicrobial resistance clusters are described in
Table 4. The housing of hens in raised-floor systems,
compared to conventional battery cages (P=0.02)
and country (P=0.03) turned out to be risk factors
for higher levels of MDR in the final GEE logistic
regression model (Table 5). Other factors such as
other animal production on the farm, the presence
of hens originating from different rearing farms in
the sampled flock and antimicrobial treatment of the
flock were not retained in the final model. When
looking at factors affecting ceftiofur resistance in
Table 3. Number of isolates per country and per housing type for Escherichia coli and Enterococcus faecalis
No. of flocks
No. of isolates
No. of flocks
No. of isolates
No. of flocks
No. of isolates
No. of flocks
No. of isolates
Success rate of isolation for E. coli=97.3%; for E. faecalis=70.0%.
1614S. Van Hoorebeke and others
Table 4. The 15 most common antimicrobial resistance clusters in Escherichia coli isolated from European laying hens
isolates% AR freq#
AMC AMP APR CEFCEP CHLCIP COLFLO GEN NALNEO SPT STR SULTETTRI XNL
AMC, Amoxicillin/clavulanic acid; AMP, ampicillin; APR, apramycin; CEF, cefpodoxime; CEP, cefalothin; CHL, chloramphenicol; CIP, ciprofloxacin; COL, colistin;
FLO, florfenicol; GEN, gentamicin; NAL, nalidixic acid; NEO, neomycin; SPT, spectinomycin; STR, streptomycin; SUL, sulfamethoxzole; TET, tetracycline; TRI,
trimethoprim; XNL, ceftiofur.
* AR cluster=antimicrobial resistance cluster describing the pattern of resistance of the isolates.
# AR freq=antimicrobial resistance frequency: the number of antimicrobials to which the E. coli were classified as resistant (R).
$ The number and percentage of isolates described in this table represent 81.8% of the 1102 total isolates included in this study.
Antimicrobial resistance in laying hens
E. coli, Belgium was more likely to have ceftiofur-
resistant isolates than the other three countries.
Ceftiofur resistance varied from 0.0% (Switzerland),
>2.5% and 4.5% (Italy and Germany, respectively),
to 12.1% in Belgium. No significant potential associ-
ation between the other tested risk factors and
ceftiofur resistance was observed.
MDR in E. faecalis
The majority of E. faecalis isolates were multi-
resistant (51.1%), 34.5% were resistant to one
antimicrobial and only 14.4% of all isolates were
pan-susceptible. The 15 most common resistance
phenotypes of antimicrobial resistance clusters are
described in Table 6. The results of the GEE logistic
regression model, showing factors associated with
MDR in E. faecalis are presented in Table 7. The
isolates from laying hens housed in free-range systems
were more likely to have lower levels of MDR
(P=0.03) compared to conventional battery cage
systems. Isolates from Belgian hens had lower levels
of resistance than hens in Germany and Italy. Similar
to the observations in E. coli, other factors such as
other animal production on the farm, antimicrobial
treatment of the flock and the presence of hens
originating from different rearing plants in the flock
did not significantly interact with the levels of MDR.
The levels of MDR in E. faecalis were lower in free-
range laying hens than in the conventional battery
cage system, whereas increased levels of MDR were
seen in E. coli in raised-floor hens. This is in contrast
to the studies of Schwaiger et al. [17, 18] who found
significantly lower levels of antimicrobial resistance in
E. coli and faecal enterococci in free-range organic
laying hens compared to laying hens housed in con-
ventional battery cages. There are several possible
explanations for the ambiguous association between
the non-cage housing types and the level of MDR.
A first important factor is exposure to antimicrobials.
In the current study, apart from the free-range or-
ganic flocks, the reported antimicrobial use in laying
hens in the non-cage systems was not significantly
lower than in conventional battery cages. The higher
incidence of bacterial diseases in laying hens housed
in non-cage systems as described in several studies
[20, 21] may have resulted in a more frequent use of
antimicrobials. If this were the case, it raises the
question whether, besides the advantages at the an-
imal-welfare level, there will be any adverse conse-
quences for public health on the level of spread and
persistence of antimicrobial resistance in laying hens
in the future. Second, in non-cage systems the chance
of oro-faecal transmission of bacteria is much higher
than in conventional battery cages, both between hens
and between the animals and the environment. This
could also be of importance since, apart from anti-
microbial usage, other factors such as localization and
size of the microbial population , and immunity
and contact intensity of the host  play a role in
antimicrobial resistance development. Finally, the
fact that many of the sampled farms with non-cage
Table 5. Results of the GEE logistic regression analysis for the identification
of risk factors for the presence of multiple drug resistance in Escherichia
coli from European laying hens
OR 95% CIP value
Type of housing
Conventional battery (ref.)
OR, Odds ratio; CI, confidence interval.
1616 S. Van Hoorebeke and others
production systems made the change to the new pro-
duction systems only a few years before the onset of
the study and that these new production systems are
often located in the same house where previously the
battery cages were present, might also explain the
observed results. It has been described that the de-
crease in antimicrobial resistance following changes in
the production system or in antimicrobial usage
Table 6. The 15 most common antimicrobial resistance clusters in Enterococcus faecalis isolated from
European laying hens
freq# AMP AVI CHL CIP DAP ERY FLO GEN KAN LIN SAL STR TET TIG VAN
AMP, ampicillin; AVI, avilamycin; CHL, chloramphenicol; CIP, ciprofloxacin; DAP, daptomycin; ERY, erythromycin;
FLO, florfenicol; GEN, gentamicin; KAN, kanamycin; LIN, linezolid; SAL, salinomycin; STR, streptomycin; TET,
tetracycline; TIG, tigecyclin; VAN, vancomycin.
* AR cluster=antimicrobial resistance cluster describing the pattern of resistance of the isolates.
# AR freq=antimicrobial resistance frequency: the number of antimicrobials to which the E. faecalis were classified as
$ The number and percentage of isolates described in this table represent 81.2% of the 803 total isolates included in this
Table 7. Results of the GEE logistic regression analysis for the
identification of risk factors for the presence of multiple drug resistance in
Enterococcus faecalis from European laying hens
OR95% CIP value
Type of housing
Conventional battery (ref.)
OR, Odds ratio; CI, confidence interval.
Antimicrobial resistance in laying hens1617
policy in food-producing animals is very slow and
that resistance against certain antimicrobials can
still be detected long after direct selection pressure by
antimicrobial usage has ended [25, 26].
The difference in effect of the housing system on
MDR in E. coli and E. faecalis might be the result of
different biological characteristics of both bacterial
species. E. coli is typically an inhabitant of the intes-
tinal tract and is to a considerable extent present in the
hens’ faeces. The animals have frequent oro-faecal
contact in non-cage indoor production systems, re-
sulting in an intense exchange of E. coli between hens.
This high-contact intensity could cause the higher
levels of MDR in laying hens in raised-floor systems.
Since enterococci are widely distributed in the soil
and the environment , access to a pasture may
exert some kind of diluting effect on the intestinal
enterococcal population, leading to lower levels of
MDR in free-range laying hens. However, although
these differences between the housing types are stat-
istically significant, it is not yet clear whether they
have some biological relevance and therefore further
study is necessary to confirm and clarify these find-
Although differences in methodology of sampling
and analysis between different studies have to be
taken into account, the results of our study illustrate
that, in general, the levels of antimicrobial resistance
in indicator bacteria in laying hens are relatively
low compared to broilers, pigs and – to a lesser
extent – cattle [12, 27, 28]. This is in accord with pre-
vious studies in laying hens [17, 18, 29]. The overall
limited antimicrobial usage in egg-producing laying
hens, as shown in the results, could play a role in this
observation since it is generally accepted that the use
of antimicrobials is one of the major risk factors for
the development and spread of antimicrobial resist-
ance [30, 31]. A reason for the lower levels of resist-
ance seen in the layers compared to broilers may be
that the layers in this study were sampled on average
at age 74 weeks, compared to broilers that are usually
aged 6 weeks at time of slaughter. It has been de-
scribed for several animal species that antimicrobial
resistance levels decrease with increasing age [32, 33].
From this point of view, it would be very interesting
to perform longitudinal studies to observe the inter-
action between antimicrobial resistance and the age of
the hens and to monitor the use of antimicrobials
during the rearing of the pullets.
The fact that the samples were analysed in different
laboratories in different countries may have slightly
influenced the results, despite the equivalent method-
ology that was used in all four participating countries.
However, the observed differences in MDR levels be-
tween countries, probably also result from regional
differences in animal husbandry and antimicrobial
usage and the fact that the distribution of the housing
system of the sampled farms was not the same in
each country. A marked finding in this respect is the
ceftiofur resistance in E. coli in Belgium. Whereas in
Germany, Italy and Switzerland only very low levels
of ceftiofur resistance were found, in Belgium this re-
sistance was 12.1% in E. coli. Consequentially the
odds to have resistance against ceftiofur were higher
in Belgium compared to the other countries, although
only the difference with Switzerland was significant.
This study result coincides with recent findings of high
levels of ceftiofur resistance in broilers in Belgium
. It has recently been stated by several authors that
one of the reasons for the increasing levels of ceftiofur
resistance may be the worldwide and systematic use of
ceftiofur in breeding eggs and 1-day-old chicks in the
hatcheries [34, 35]. In Belgium the use of ceftiofur in
poultry has not been licensed for a decade  but
might be continued off-label .
Another hypothesis for the increased ceftiofur re-
sistance might be the production on the same farm of
other animal species, in which the use of ceftiofur is
still permitted. This could result in horizontal trans-
mission of resistance genes between bacterial popu-
lations of different animal species [37, 38]. However,
the number of mixed-production farms in Belgium did
not significantly differ from the situation in Germany
and was even less than in Switzerland. Possibly the
high density of farms in the northern part of Belgium,
where 90% of all animal production is situated,
may enhance contact between the bacterial popula-
tions of different ecological systems, for example
surface water, leading to an efficient horizontal spread
of antimicrobial resistance. For humans, it has been
demonstrated that a higher population density en-
hances the development and spread of antimicrobial
resistance [39,40]. Further
studies are needed to elucidate this possibility.
The results of this study suggest that the levels of
antimicrobial resistance in indicator bacteria such as
E. coli and E. faecalis in laying-hen flocks are rela-
tively low. The differences observed between both
indicator bacteria with respect to the potential
1618 S. Van Hoorebeke and others
association between the housing system and MDR
suggest that it is important to not focus on a sole
bacterial species when attempting to assess risk
factors for antimicrobial resistance. It is crucial to
conscientiously monitor the prevalence and evolution
in time of antimicrobial resistance in laying hens,
both during the rearing period and the production
cycle, in order to be able to detect early changes
in antimicrobial resistance and to minimize the
spread of resistant bacteria to humans. Therefore it
studies in the field and under experimental conditions
on a broader spectrum of indicator bacteria are per-
This research was funded by the EU FP6, under the
contract 035547 (Safehouse project). The authors
thank all farmers for permission to sample their farm
and all laboratory technicians at the different insti-
tutions for their skilful help during the bacteriological
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