Antibiotic resistance of faecal enterococci in poultry, poultry farmers and poultry slaughterers

A. E. van den Bogaarda,*, R. Willemsb, N. Londona, J. Topb and E. E. Stobberingha

a Laboratory of Medical Microbiology, University of Maastricht, PO Box 616, 6200 MD Maastricht; b Research Laboratory for Infectious Diseases, National Institute of Public Health and the Environment, 3720 BA Bilthoven, The Netherlands


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The prevalence of resistance in enterococci to antibiotics, commonly used for therapy in poultry or as antimicrobial growth promoters (AMGPs), was determined in faecal samples of two chicken populations: broilers in which antibiotic and AMGP use is common and laying-hens with a low antibiotic usage. In addition faecal samples were examined from three human populations: broiler farmers, laying-hen farmers and poultry slaughterers. MICs of an extended panel of antibiotics for a randomly chosen gentamicin- or vancomycin-resistant enterococcal isolate from each faecal specimen were also determined. The prevalence of resistance for all antibiotics tested was higher in broilers than in laying-hens. Resistance in faecal enterococci of broiler farmers was for nearly all antibiotics higher than those observed in laying-hen farmers and poultry slaughterers. The overall resistance in broilers was correlated with the resistance in broiler farmers and in poultry slaughterers. No correlation between the results obtained in the laying-hens with any of the other populations was found. The 27 gentamicin-resistant isolates all showed high-level resistance to gentamicin and two of these isolates, both Enterococcus faecium, were resistant to all antibiotics tested, except vancomycin. The 73 vancomycin-resistant enterococci (VRE) isolated from the five populations belonged to four different species and in all isolates the vanA gene cluster was detected by blot hybridization. The pulsed-field gel electrophoresis (PFGE) patterns of these vancomycin-resistant enterococci were quite heterogeneous, but Enterococcus hirae isolates with the same or a closely related PFGE pattern were isolated at two farms from the broiler farmer and from broilers. Molecular characterization of vanA-containing transposons of these isolates showed that similar transposon types, predominantly found in poultry, were present. Moreover, similar vanA elements were not only found in isolates with the same PFGE pattern but also in other VRE isolated from both humans and chickens. The results of this study suggest transmission of resistance in enterococci from animals to man. For VRE this might be clonal transmission of animal strains, but transposon transfer seems to occur more commonly.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Acquired resistance against commonly used antibiotics has been observed ever since these agents were introduced in human and veterinary medicine. However, the rate of development of resistance appears to have accelerated in the past decade1 and today multiple resistant bacteria constitute a global problem.2,3 In the modern poultry industry antibiotics are used in high quantities not only for therapy and prevention of bacterial diseases, but also as antimicrobial growth promoters (AMGPs) in animal feeds.4 In 1990 in The Netherlands 80 000 kg of antibiotics (active substance) were used in humans and 300 000 kg on veterinary prescription in animals.5 This was in both populations equivalent to c. 100 mg of active substance/kg body weight/year. However, c. 26% of the veterinary used antibiotics were intended for poultry, mainly broilers, resulting in a yearly exposure of c. 430 mg of antibiotics/kg/year for poultry. This was considerably higher than the antibiotic usage in other food animal populations. In addition to these therapeutic antibiotics, food animals received more or less the same amount of antibiotics in their feeds as AMGP. These amounts have not changed much during subsequent years and similar figures have been published for 1997.6 This high antibiotic usage in poultry may compromise veterinary therapy but is also of public health concern. Antibiotic use selects not only for resistance in pathogenic bacteria, but also in the endogenous flora of exposed animals.

Enterococci belong to the endogenous flora of man and other animals and are intrinsically resistant to various antibiotics including cephalosporins, penicillinase-resistant penicillins, and clinically available levels of lincosamides and aminoglycosides. Enterococci are not important pathogens for animals; in humans, however, they have been implicated in infective endocarditis and urinary tract infections for nearly a century.7 Over the last 10 years, enterococci have emerged as major nosocomial pathogens. Approximately 12% of all nosocomial infections in the USA are caused by enterococci.8,9 The emergence of enterococci as nosocomial pathogens has very likely been caused by the increasing numbers of immunocompromised patients and their exposure to antibiotics against which enterococci are intrinsically resistant such as third- and fourth-generation cephalosporins.

The aim of this study was to analyse the influence of exposure to antibiotics used in The Netherlands as AMGPs or for veterinary therapy in poultry on the resistance of faecal enterococci recovered from poultry, poultry farmers and poultry slaughterers. In addition, the antibiotic susceptibility to several antibiotics for enterococci cultured on antibiotic-free agar plates was determined. The poultry population consisted of two groups with a different usage of antibiotics: broilers, young chickens raised within 8 weeks for slaughter, and laying-hens producing eggs for human consumption. Broilers are fed continuously on AMGP agents and these drugs are not used in laying-hens. Moreover, broilers have a relatively high use of antibiotics on veterinary prescription, while laying-hens are exposed, at least during their productive life, to a relatively low amount of antimicrobial agents.

In addition, the prevalence and degree of resistance against the same antibiotics was assessed in faecal enterococci of three populations of humans with a different risk of exposure to faecal bacteria from chickens: broiler and laying-hen farmers, who have a daily close contact with chickens with a high and low exposure, respectively, to antibiotics, and workers in a poultry processing plant, who handled broilers or broiler products on a daily basis. Finally, possible sharing of vancomycin-resistant enterococci (VRE) between chickens and humans was assessed by genotyping of VRE by pulsed-field gel electrophoresis (PFGE). The similarity of vancomycin resistance elements found in chicken and human isolates was evaluated by comparing Tn1546 derivatives found in chicken and human isolates of VRE.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Collection of the faecal samples

From September to December 1997 [c. 6 months after the suspension of the use of avoparcin (a glycopeptide antibiotic, like vancomycin) as an AMGP by the European Commission] c. 250 farmers in the south of The Netherlands, keeping either broilers or laying-hens were asked by letter to submit one fresh faecal sample from themselves and a mixed sample consisting of fresh faecal droppings of three different chickens from the oldest flock at their farms. In addition 100 poultry slaughterers working at the poultry-processing plant, where the broilers of the participating broiler farmers were slaughtered, were asked to provide one faecal specimen from themselves. All participants were requested to send the samples on the day of collection to the bacteriological laboratory together with a completed questionnaire about recent hospital stay, antibiotic usage by themselves, family members or their animals during the 3 months preceding sample collection and whether they kept food and/or pet animals. The samples (collected in small plastic vials without transport medium) were sent to the laboratory by parcel post. On the day of arrival at the laboratory within 24 h after collection the samples were diluted (10-1) in 0.9% NaCl (w/v) with 20% (v/v) glycerol and stored frozen at -20°C until assayed (3 months maximum).

Isolation of (resistant) enterococci

After thawing the samples 40 µL of 10-1 and 10-3 dilutions in 0.9% NaCl (w/v) were inoculated on to KF–Streptococcus agar plates (Oxoid CM701, Basingstoke, UK) with and without antibiotics using a spiral plater (Salm en Kip BV, Utrecht, The Netherlands) as described previously.10,11 The antibiotics were selected because they, or related antibiotics, that are known to show cross-resistance with the tested antibiotics, had been regularly used in poultry either on veterinary prescription or as AMGPs. The antibiotic concentrations incorporated in the agar are shown in Table 1Go and are the same as used in previous studies10–12 to make results comparable. If after 48 h of incubation no VRE were detected on the vancomycin-containing agar plate, 0.5 mL of the 10-1 dilution was incubated overnight in nutrient broth containing 10 mg/L vancomycin and 0.4 g/L sodium azide and the next morning 0.5 mL of this broth was plated out on a vancomycin (10 mg/L)-containing agar plate.


View this table:
[in this window]
[in a new window]
 
Table 1. Prevalence of antibiotic-resistant enterococci and prevalence of high degree of resistance in faecal samples from broilers, laying-hens, broiler farmers, laying-hen farmers and poultry slaughterers
 
Enterococci appeared as typical red or pink colonies on KF–Streptococcus agar. After 48 h incubation at 42°C only the typical pink colonies were counted. The minimum detection level, as assayed with spiked faeces samples, was c. 300 cfu/g faeces.

The prevalence of antibiotic resistance (%) in a population was calculated as the number of samples showing growth of enterococci on the antibiotic-containing plates, divided by the total number of samples tested x 100.

The degree of antibiotic resistance (%) of each faecal sample tested was determined from the number of enterococcal colonies on the antibiotic-containing plate divided by the total number of enterococcal colonies on the antibiotic-free plate x 100. Two degrees of antibiotic resistance can be distinguished: low degree of resistance, i.e. when <50% of the enterococci present in a faecal sample are resistant and high degree, when 50% or more (thus the majority) are resistant to that particular agent.11,13 The prevalence (%) of high degree resistance is the number of samples with a high degree of resistance to a particular antibiotic divided by the total number of samples tested x 100.

Because Enterococcus faecalis is considered to be intrinsically resistant to dalfopristin–quinupristin, a sample was only considered to contain enterococci resistant to this drug if at least one Enterococcus faecium was isolated and identified from the dalfopristin–quinupristin-containing agar plate. For this reason the prevalence (%) of high degree of resistance could only be calculated for the total enterococcal population in the specimen.

Identification and antibiotic susceptibility testing

If growth was observed on a vancomycin- or gentamicin-containing agar plate, one colony from these plates was selected at random for identification and determination of the MIC of an extended panel of antibiotics with a microbroth dilution method in IsoSensitest Broth (Oxoid CM473) using an inoculum size of 5 x 105 cfu/mL. E. faecalis ATCC 29212 was used as reference strain. Identification to species level was performed according to the criteria of Devriese et al.14,15

PFGE analysis of VRE

If VRE were recovered from a chicken and a farmer from the same farm these isolates were genotyped by PFGE, the resistance genes identified and the vanA transposons analysed.

PFGE was performed after DNA digestion with Sma I as described previously.16 To assess the similarity of the different patterns the criteria of Tenover et al.17 were used: if an isolate differed from a main type by only three or less bands, it was considered as a subtype. Comparison of the PFGE fingerprints was made using computer assisted analysis (BioNumerics, Applied Maths, Kortrijk, Belgium). Comparisons of patterns were made using the unweighted pair group method using arithmetic averages (UPGMA) clustering method by using the Dice coefficient.

Detection of vanA, vanB and vanC genes

The vanA, vanB and vanC genes were detected by hybridization with specific probes as described previously.10

Molecular characterization of Tn1546 derivatives

Characterization of the vanA-containing transposons was performed by means of restriction fragment length polymorphism (RFLP) analysis and DNA sequencing of Tn1546-specific PCR products as described previously.10,18 Tn1546 derivatives were classified by type in concordance with the nomenclature used previously.10,18 All VRE isolates were analysed for the presence of the point mutations at positions 1226, 4847, 7658, 8234 and 9692, for left and right end deletions and for the exact integration site and orientation of IS1216V downstream of vanX in the type B and E transposons.

Statistical analysis

A one-way analysis of variance was used to estimate overall differences between the group means. Group means were compared pairwise using t-tests controlled for overall error rate (Bonferroni test) where P < 0.05 was regarded as statistically significant. Correlations between populations were calculated using the Pearson's product moment correlation coefficient test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Study population

From the 100 poultry slaughterers asked to participate in the study 46 responded and 31% from the poultry farmers: 51 of 150 broiler farmers and 26 of 100 laying-hen farmers. One broiler farmer had no animals on the farm at the moment of sample collection so only a human faecal sample was supplied. From 46 faecal samples from slaughterers 41 (89%) yielded enterococci. All 50 broiler samples, all 25 samples of laying-hen farmers and 24 (96%) of the samples of the laying-hens, and 49 (96%) of 51 stools of broiler farmers also yielded enterococci. Sixty-five sam-ples produced growth of enterococci on the vancomycincontaining agar plates and an additional 19 samples yielded enterococci after enrichment (Table 1Go).

The mean ± s.d. log10 cfu of enterococci per gram of faeces in positive faecal samples was 5.4 ± 1.4 for humans and 7.7 ± 0.4 in chicken faecal samples. In the 3 months preceding the sample collection two poultry slaughterers and one laying-hen farmer had been hospitalized. None of the laying-hen farmers had taken antibiotics, but in four cases one member of their family had; four broiler farmers (8%) and two family members had used antibiotics; and four slaughterers (9%) and two members of their respective families had. In all three groups c. 55% kept pet animals, mainly dogs and cats. Thirteen of the laying-hen farmers (52%), 18 of the broiler farmers (35%) and six poultry slaughterers (13%) also kept other food animals, mainly pigs. There were no farmers in the study who kept both laying-hens and broilers. None of the poultry slaughterers kept poultry. There were no significant differences observed within the same group between people keeping pigs and those who did not (data not shown). From the laying-hen flocks two (8%) and 23 (46%) of the 50 broiler flocks had received antibiotics on veterinary prescription whilst on the farm: one laying-hen flock had been treated with amoxicillin and the other with oxytetracycline. From the broiler flocks 14 had been treated with oxytetracycline, three with co-trimoxazole, two with either amoxicillin or a combination of lincomycin and spectinomycin and one with flumequine. One broiler flock had been treated twice: first with tylosin and subsequently with enrofloxacin. No information was obtained about AMGP use.

Prevalence and degree of resistance

The prevalence of resistance and the prevalence of high degree resistance for the tested antibiotics of the populations studied are presented in Table 1Go.

The highest percentage of samples with a high degree of resistance was observed in the faecal samples from broilers. The prevalence of resistance was significantly higher in broilers for quinupristin–dalfopristin and vancomycin than in laying-hens (P < 0.05). For oxytetracycline, erythromycin and gentamicin the prevalence of resistance was of the same order in both chicken populations, but the prevalence of high degree was significantly higher in broilers than in laying-hens (P < 0.05). In the human samples, amoxicillin resistance was only observed in faecal samples of broiler farmers and high-level gentamicin resistance in a few samples of broiler farmers and poultry slaughterers. Tetracycline resistance was high in all three human populations. The prevalence of resistance for vancomycin and quinupristin–dalfopristin was higher in broiler farmers than in laying-hen farmers and poultry slaughterers, but this was only significant for vancomycin (P < 0.05).

In general, the prevalence of resistance against all antibiotics correlated well between broilers and broiler farmers (correlation coefficient: 0.95) and between broilers and poultry slaughterers (correlation coefficient: 0.96, P < 0.01), while the prevalence of antibiotic resistance of laying-hens showed no significant correlation with any of the three human populations studied.

Resistance of isolates

All 27 isolates that were isolated from the gentamicincontaining agar plates had MIC > 500 mg/L. This high-level resistance (HLR) to gentamicin, was observed in seven isolates from broilers that were identified as E. faecium. Three of these were also resistant to amoxicillin and two were additionally resistant to all antibiotics tested: oxytetracycline, erythromycin, quinupristin/dalfopristin and trimethoprim, but not to vancomycin. The other 20 gentamicin HLR isolates were identified as E. faecalis except for three isolates from broiler farmers, which were identified as E. faecium.

The MICs of glycopeptides and the identification of isolates from the vancomycin-containing plates are shown in Tables 2 and 3GoGo, respectively. Only 73 isolates were tested as one broiler isolate was lost during storage. In all 73 isolates but two (one broiler and one broiler farmer isolate, which were identified as E. durans and E. faecium respectively) the vanA gene cluster was detected by blot hybridization. The MIC for these vanA-containing isolates of vancomycin was 8 mg/L (Table 2Go). No vanB or vanC genes were found. Most vanA-containing isolates were identified as E. faecium (n = 41), but five isolates were E. faecalis, 16 E. hirae and nine E. durans.


View this table:
[in this window]
[in a new window]
 
Table 2. MIC values for enterococcal isolates (n = 73) from vancomycin-containing (10 mg/L) selective agar plates
 

View this table:
[in this window]
[in a new window]
 
Table 3. Identification of vancomycin-resistant enterococci (n = 73) from broilers, laying-hens, broiler farmers, laying-hen farmers and poultry slaughterers
 
Pulse-field gel electrophoresis (PFGE)

At 10 farms VRE were isolated from the farmer, and from his poultry. These 10 paired VRE isolates from the farmer and his chickens were analysed by PFGE (Figures 1, 2GoGo and Table 4Go). In general the PFGE patterns obtained were highly variable. Only at two farms (nos 14 and 31) were the isolates from the farmer and from the broilers of the same farm either identical (100%) or highly similar (93%). In addition, the VRE isolated from broiler farmer 40, showed a high degree of similarity (85%) to the VRE isolates from broiler farmer 14 and his broilers. These five isolates were identified as E. hirae.



View larger version (23K):
[in this window]
[in a new window]
 
Figure 1. PFGE patterns and dendrogram of 10 paired vancomycin-resistant enterococci isolated from faecal samples of farmers and poultry from the same farm after total DNA digestion with Sma I.

 


View larger version (20K):
[in this window]
[in a new window]
 
Figure 2. Genetic map of Tn1546 and four Tn1546 derivatives. The thick horizontal lines represent Tn1546 and Tn1546 types E8–112. The position of genes and open reading frames (orf) and direction of transcription is depicted by open arrows. Dotted boxes represent IS elements. The position of the first nucleotide upstream and downstream from IS insertion sites are depicted. Filled arrows indicate the transcriptional orientation of inserted IS elements. Deletions are indicated by dotted lines.

 

View this table:
[in this window]
[in a new window]
 
Table 4. Characteristics of paired GRE isolated from samples of farmers and chickens from the same farm
 
Tn1546 types among different VRE isolates

Eight different vanA transposon types were found among the 18 VRE isolated from poultry and farmers (Figure 1Go). Tn types A1, A2, B2, E7 and E11 have been described previously.10,18 In short, type A1 is identical to Tn1546.19 Type A2 is characterized by a G -> T point mutation at position 8234 and an IS1216V–IS3 insertion at the left end of the transposon (Figure 2Go). Type B2 contains an IS1216V insertion in the vanXvanY intergenic region. Type E7 contains also the IS1216V insertion in the same region as in type B2 as well as a large left end deletion encompassing the orf1 gene and a part of the orf2 gene, while type E11 contains an additional deletion at the right end of the transposon including the vanZ gene. Three new transposon types were found: types E14, E17 and E19. Types E14 and E17 are closely related to type E11. The only difference resides in the size of the small deletion flanking the IS1216V insertion site downstream of vanX. Type E19 is comparable to type E7, with differences in the size of the left end deletion and in the size of the small deletions adjacent to the IS1216V insertion site in the vanXvanY intergenic region. Comparative analysis of the Tn1546- like elements of the paired VRE strains revealed that in addition to the two sets of paired strains with comparable PFGE patterns, 14 and 31, the paired strains of broiler and broiler farmer 3 also contained identical transposon types. In the other cases different transposon types were present in chicken and farmer isolates. In conclusion, in three of the eight paired strains that were analysed an identical vanA transposon was present in both the animal and farmer isolate.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The overall prevalence of antibiotic resistance between broilers and broiler farmers and poultry slaughterers indicates that contact with broilers is a risk factor for colonization of humans with resistant bacteria. In laying-hens and laying-hen farmers this was only the case for tetracycline and erythromycin resistance, which correlated with the increased risk for humans from animals with a high degree of resistance. The prevalence of 6% amoxicillin-resistant faecal enterococci in broiler flocks and the absence in laying-hens, was most likely a result of the higher overall antibiotic usage in broilers than in laying-hens, which favours the selection of intrinsically less amoxicillinsusceptible E. faecium in the intestinal flora. The presence of amoxicillin-resistant enterococci only in the faecal flora of broilers and broiler farmers and not in any other of the studied populations nor in healthy suburban residents in The Netherlands12 is suggestive of a broiler to farmer transfer of resistant enterococci.

The significantly higher fluoroquinolone resistance observed in broilers compared with the other populations is most likely due to the more common use of enrofloxacin and flumequine, a less potent fluoroquinolone than enrofloxacin, in broilers than in laying-hens. The fluoroquinolone-resistant enterococci isolated from laying-hens might be due to the use of quinolones during the rearing period. The prevalence of resistance in broiler farmers, however, was not different from the other populations studied. As clonal transmission of fluoroquinoloneresistant E. coli strains from poultry to humans has been described,20 this might suggest that colonization of humans by poultry enterococci occurs less readily than colonization by poultry E. coli. As resistance to fluoroquinolones is caused by chromosomal mutations and is not transferable, this could explain the observed lack of correlation in the prevalence of ciprofloxacin-resistant enterococci between broilers and broiler farmers and suggests that for other antibiotics the exchange of resistance genes between poultry enterococci and endogenous human enterococci is a more important phenomenon for resistance transfer than colonization of the human intestinal tract by poultry enterococci alone.

The high prevalence of HLR to gentamicin in the enterococcal faecal flora of both broilers and laying-hens in this study was unexpected. Thal et al. found no high-level gentamicin-resistant enterococci in faecal samples of poultry despite their presence in wild birds21–23 and environmental samples.24 In Denmark only 1% of the E. faecalis isolated from healthy broilers at slaughter were resistant to gentamicin and no gentamicin-resistant E. faecium were isolated.25 In this study in both species HLR to gentamicin has been observed. Gentamicin is not registered for use in poultry in The Netherlands, but the observed resistance might have been due to regular off label use in young chickens. High-level gentamicin resistance in enterococci has been reported in several studies from human infections,26–28 but in faecal enterococci from healthy Dutch suburban residents in 1997 no high-level gentamicin-resistant enterococci were detected.12 The prevalence of 10% in broiler farmers is most likely derived from their animals as gentamicin is practically only used in hospitals in The Netherlands and none of the farmers or family members had been in hospital in the 3 months before the study.

The prevalence of erythromycin resistance was in all populations high, but the high degree of resistance was significantly higher (P < 0.05) in broilers than in the three human populations, indicating transfer from broilers to humans. Erythromycin is fully cross-resistant with tylosin, a commonly used antibiotic for poultry as AMGP, and on veterinary prescription. Several studies have shown that in chickens raised on tylosin-containing feed not only the percentage tylosin-resistant enterococci in the faecal flora of exposed animals increased over time but also the relative numbers of E. faecium to the total numbers of enterococci present in the flora.29–32 Quinupristin–dalfopristin is like virginiamycin a mixture of two pristinamycins that act synergistically. In the EU virginiamycin has only been used sparsely for human therapy of staphylococcal infections and only in a few member states, but has been used extensively as AMGP for many years. Both compounds are cross-resistant. At the time of the study quinupristin– dalfopristin was not registered for any use in the EU. The high prevalence of quinupristin–dalfopristin resistance observed in broilers is therefore most likely caused by the common use of virginiamycin in broiler feeds as AMGP.

VRE were most common in the faecal samples from broilers. In broiler farmers a significantly higher prevalence and degree of VRE was observed than in the other two human populations in this study and in Dutch suburban residents.5 Because avoparcin does not select for vanB resistance the most common resistance gene cluster in animal isolates is of the vanA type, which confers resistance to both vancomycin and teicoplanin. In this study, however, >50% of the VRE were phenotypically susceptible to teicoplanin. This was due to the fact that the most prevalent transposon variant was a Tn1546 derivative, which contained a large left end deletion including the orf1 gene, an IS1216V insertion in the vanXvanY intergenic region and a deletion of the vanZ gene, which in the normal transposon confers teicoplanin resistance. These features found in the homologous types E11, E14 and E17 were present in half of the transposon types analysed. Interestingly, type E11 and the homologous types E9 and E10 were also predominantly present (9/13) in isolates recovered from turkeys and in almost half of the isolates (4/9) recovered from turkey farmers in The Netherlands.10 All Tn1546 variants found in the isolates from broilers and laying-hens contained at position 8234 the ‘G’, which is consistent with the results reported previously.10,18,33 This variant is also found in humans although the majority of human isolates and all isolates recovered from pigs contain a ‘T’ at this position.10,18,33 The finding that human isolates analysed in this study contain vanA transposon types predominantly found in poultry and not the most prevalent human transposon type is again an indication that poultry farmers have acquired vanA resistance genes from their animals. Since the isolates recovered from poultry and poultry farmers were in most cases genetically different, it is most likely that vancomycin resistance was transmitted from poultry enterococci to enterococci of the intestinal flora of the farmers by horizontal transfer of Tn1546 variants. That this was only found in three cases does not mean that it is a rarely occurring event since only one isolate was tested from each sample and, therefore, the method used is likely to have a very low sensitivity. Other studies have also pointed to a similarity between vanA-containing elements in animals and humans.10,18,34–38 It was interesting to note that all E. hirae isolates, irrespective of whether they originated from animal or human sources, contained the highly homologous vanA transposon types E11, E14 and E17. This could suggest a level of enterococcal species specificity of vanA transposon types and is consistent with the results of Butaye et al.39 who found that 50% of VRE from boilers and nearly all VRE from laying-hens were E. hirae.

In conclusion the results of this study provide evidence for dissemination of resistant enterococci from animals to man and, probably more importantly the exchange of resistance genes between poultry and human enterococci. As antibiotic resistance is not restricted to E. faecium, but also occurs regularly in other enterococcal species, monitoring of resistance should not be restricted to E. faecium or E. faecium and E. faecalis isolates alone.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study has been made possible by grant No. 28-2075-1 of the Dutch Prevention Fund.


    Notes
 
* Corresponding author. Tel: +31-43-3881015/6; Fax: +31-43-3884161; E-mail: A.vandenBogaard{at}CPV.Unimaas.NL Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Smith, D. W. (1999). Decreased antimicrobial resistance after changes in antibiotic use. Pharmacotherapy 19, S129–32.

2 . Obrien, T. F. (1997). The global epidemic nature of antimicrobial resistance and the need to monitor and manage it locally. Clinical Infectious Diseases 24, S2–8.[ISI][Medline]

3 . Shanahan, P. M. A., Thomson, C. J. & Amyes, S. G. B. (1994). The global impact of antibiotic-resistant bacteria: their sources and reservoirs. Review of Medical Bacteriology 5, 174–82.

4 . van den Bogaard, A. E. & Stobberingh, E. E. (1999). Antibiotic usage in animals – Impact on bacterial resistance and public health. Drugs 58, 589–607.[ISI][Medline]

5 . van den Bogaard, A. E. (1997). Antimicrobial resistance— relation to human and animal exposure to antibiotics. Journal of Antimicrobial Chemotherapy 40, 453–4.[Free Full Text]

6 . van den Bogaard, A. E. (2000). Veterinary use of antibiotics in the Netherlands—facts and figures. Tijdschrift Diergeneeskunde 125, 527–30.

7 . Murray, B. E. (1990). The life and times of the Enterococcus. Clinical Microbiology Reviews 3, 46–65.[ISI][Medline]

8 . Edmond, M. B., Wallace, S. E., McClish, D. K., Pfaller, M. A., Jones, R. N. & Wenzel, R. P. (1999). Nosocomial bloodstream infections in United States hospitals: A three-year analysis. Clinical Infectious Diseases 29, 239–44.[ISI][Medline]

9 . Emori, T. G. & Gaynes, R. P. (1993). An overview of nosocomial infections, including the role of the microbiology laboratory. Clinical Microbiology Reviews 6, 428–42.[Abstract]

10 . Stobberingh, E., van den Bogaard, A., London, N., Driessen, C., Top, J. & Willems, R. (1999). Enterococci with glycopeptide resistance in turkeys, turkey farmers, turkey slaughterers, and (sub)urban residents in the South of the Netherlands: evidence for transmission of vancomycin resistance from animals to humans? Antimicrobial Agents and Chemotherapy 43, 2215–21.[Abstract/Free Full Text]

11 . van den Bogaard, A. E., London, N. & Stobberingh, E. E. (2000). Antimicrobial resistance in pig faecal samples from The Netherlands (five abattoirs) and Sweden. Journal of Antimicrobial Chemotherapy 45, 663–71.[Abstract/Free Full Text]

12 . van den Bogaard, A. E., Mertens, P., London, N. H. & Stobberingh, E. E. (1997). High prevalence of colonization with vancomycin- and pristinamycin-resistant enterococci in healthy humans and pigs in The Netherlands: is the addition of antibiotics to animal feeds to blame? Journal of Antimicrobial Chemotherapy 40, 454–6.[Free Full Text]

13 . Levy, S. B., Marshall, B., Schluederberg, S., Rowse, D. & Davis, J. (1988). High frequency of antimicrobial resistance in human fecal flora. Antimicrobial Agents and Chemotherapy 32, 1801–6.[ISI][Medline]

14 . Devriese, L. A., Hommez, J., Wijfels, R. & Haesebrouck, F. (1991). Composition of the enterococcal and streptococcal intestinal flora of poultry. Journal of Applied Bacteriology 71, 46–50.[ISI][Medline]

15 . Devriese, L. A., van der Kerckhove, A., Klipper-Balz, R. & Schleifer, K. H. (1987). Characterization and identification of Enterococcus species isolated from the intestines of animals. International Journal of Systematic Bacteriology 37, 257–9.

16 . van den Braak, N., van Belkum, A., van Keulen, M., Vliegenthart, J., Verbrugh, H. A. & Endtz, H. P. (1998). Molecular characterization of vancomycin-resistant enterococci from hospitalized patients and poultry products in The Netherlands. Journal of Clinical Microbiology 36, 1927–32.[Abstract/Free Full Text]

17 . Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing, D. H. et al. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 2233–9.[Free Full Text]

18 . Willems, R. J. L., Top, J., van den Braak, N., van Belkum, A., Mevius, D. J., Hendriks, G. et al. (1999). Molecular diversity and evolutionary relationships of Tn1546-like elements in enterococci from humans and animals. Antimicrobial Agents and Chemotherapy 43, 483–91.[Abstract/Free Full Text]

19 . Arthur, M. & Courvalin, P. (1993). Genetics and mechanisms of glycopeptide resistance in enterococci. Antimicrobial Agents and Chemotherapy 37, 1563–71.[ISI][Medline]

20 . van den Bogaard, A., London, N., Driessen, C. & Stobberingh, E. (2001). Antibiotic resistance of faecal Escherichia coli in poultry, poultry farmers and poultry slaughterers. Journal of Antimicrobial Chemotherapy 47, 763–71.[Abstract/Free Full Text]

21 . Thal, L. A., Welton, L. A., Perri, M. B., Donabedian, S., McMahon, J. W., Chow, J. W. et al. (1996). Antimicrobial resistance in enterococci isolated from turkeys fed virginiamycin. Program and Abstracts of the Thirty-sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, LA, September 15–18, 1996. C-120. American Society for Microbiology, Washington, DC.

22 . Thal, L. A. & Zervos, M. J. (1999). Occurrence and epidemiology of resistance to virginiamycin and streptogramins. Journal of Antimicrobial Chemotherapy 43, 171–6.[Free Full Text]

23 . Thal, L. A., Chow, J. W., Mahayni, R., Bonilla, H., Perri, M. B., Donabedian, S. A. et al. (1995). Characterization of antimicrobial resistance in enterococci of animal origin. Antimicrobial Agents and Chemotherapy 39, 2112–5.[Abstract]

24 . Rice, E. W., Messer, J. W., Johnson, C. H. & Reasoner, D. J. (1995). Occurrence of high-level aminoglycoside resistance in environmental isolates of enterococci. Applied and Environmental Microbiology 61, 374–6.[Abstract]

25 . Aarestrup, F. M., Bager, F., Jensen, N. E., Madsen, M., Meyling, A. & Wegener, H. C. (1998). Resistance to antimicrobial agents used for animal therapy in pathogenic-bacteria, zoonotic-bacteria and indicator bacteria isolated from different food animals in Denmark – a base-line study for the Danish integrated antimicrobial resistance monitoring program (Danmap). Acta Pathologica, Microbiologica et Immunologica Scandinavica 106, 745–70.

26 . Schouten, M. A., Voss, A. & Hoogkamp Korstanje, J. A. A. (1999). Antimicrobial susceptibility patterns of enterococci causing infections in Europe. Antimicrobial Agents and Chemotherapy 43, 2542–6.[Abstract/Free Full Text]

27 . Grayson, M. L., Eliopoulos, G. M., Wennersten, C. B., Ruoff, K. L., De Girolami, P. C., Ferraro, M. J. et al. (1991). Increasing resistance to beta-lactam antibiotics among clinical isolates of Enterococcus faecium: a 22-year review at one institution. Antimicrobial Agents and Chemotherapy35, 2180–4.[ISI][Medline]

28 . Patterson, J. E. & Zervos, M. J. (1990). High-level gentamicin resistance in Enterococcus: microbiology, genetic basis, and epidemiology. Reviews of Infectious Disease 12, 644–52.[ISI][Medline]

29 . Hinton, M. (1986). The ecology of Escherichia coli in animals including man with particular reference to drug resistance. Veterinary Record 119, 420–6.[ISI][Medline]

30 . Kaukas, A., Hinton, M. & Linton, A. H. (1988). The effect of growth-promoting antibiotics on the faecal enterococci of healthy young chickens. Journal of Applied Bacteriology 64, 57–64.[ISI][Medline]

31 . Kaukas, A., Hinton, M. & Linton, A. H. (1986). Changes in the faecal enterococcal population of young chickens and its effect on the incidence of resistance to certain antibiotics. Letters in Applied Microbiology 2, 5–8.[ISI]

32 . Kaukas, A., Hinton, M. & Linton, A. H. (1986). The speciation and biotyping of enterococcal isolates from chickens. Letters in Applied Microbiology 3, 113–6.[ISI]

33 . Jensen, L. B. (1998). Differences in the occurrence of two base pair variants of Tn1546 from vancomycin-resistant enterococci from humans, pigs, and poultry. Antimicrobial Agents and Chemotherapy 42, 2463–4.[Free Full Text]

34 . Woodford, N., Adebiyi, A. M., Palepou, M. F. & Cookson, B. D. (1998). Diversity of VanA glycopeptide resistance elements in enterococci from humans and nonhuman sources. Antimicrobial Agents and Chemotherapy 42, 502–8.[Abstract/Free Full Text]

35 . Simonsen, G. S., Haaheim, H., Dahl, K. H., Kruse, H., Lovseth, A., Olsvik, O. et al. (1998). Transmission of VanA-type vancomycin-resistant enterococci and vanA resistance elements between chicken and humans at avoparcin-exposed farms. Microbial Drug Resistance Mechanisms Epidemiology and Disease 4, 313–8.

36 . Jensen, L. B., Ahrens, P., Dons, L., Jones, R. N., Hammerum, A. M. & Aarestrup, F. M. (1998). Molecular analysis of Tn1546 in Enterococcus faecium isolated from animals and humans. Journal of Clinical Microbiology 36, 437–42.[Abstract/Free Full Text]

37 . Aarestrup, F. M. (1995). Occurrence of glycopeptide resistance among Enterococcus faecium isolates from conventional and ecological poultry farms. Microbial Drug Resistance 1, 255–7.[ISI][Medline]

38 . Descheemaeker, P. R. M., Chapelle, S., Devriese, L. A., Butaye, P., Vandamme, P. & Goossens, H. (1999). Comparison of glycopeptide-resistant Enterococcus faecium isolates and glycopeptide resistance genes of human and animal origins. Antimicrobial Agents and Chemotherapy 43, 2032–7.[Abstract/Free Full Text]

39 . Butaye, P., Devriese, L. A., Goossens, H., Ieven, M. & Haesebrouck, F. (1999). Enterococci with acquired vancomycin resistance in pigs and chickens of different age groups. Antimicrobial Agents and Chemotherapy 43, 365–6.[Abstract/Free Full Text]

Received 12 July 2001; returned 24 September 2001; revised 12 November 2001; accepted 30 November 2001