Study of faecal colonization by vanA-containing Enterococcus strains in healthy humans, pets, poultry and wild animals in Portugal

Patricia Poeta1, Daniela Costa1, Jorge Rodrigues1,2 and Carmen Torres3

1 Universidade de Trás-os-Montes e Alto Douro, Departamento de Ciências Veterinárias, Vila Real; 2 Centro de Estudos de Ciência Animal e Veterinária, Vila Real, Portugal; 3 Area de Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain


* Corresponding author. Tel: +34-941299750; Fax: +34-941299721; Email: carmen.torres{at}daa.unirioja.es

Keywords: E. durans , E. hirae , E. faecium , enterococci , VRE

Sir,

Enterococcus spp. are commensal bacteria of the intestinal microbiota in humans and animals, but over the last decade have also been considered as important nosocomial pathogens. The emergence of vancomycin-resistant enterococci (VRE) in Europe has been associated with the use of avoparcin as a growth promoter in food animals,1 until its ban in 1997 by the EU. There are reports of VRE in farm animals in different countries, including in Portugal,14 but fewer studies have addressed the occurrence of VRE in pets or wild animals.2,46 VRE have not previously been detected in pets or in wild animals in Portugal, but the prevalence of vancomycin-resistant Enterococcus faecium in some Portuguese hospitals seems to be relatively high.7 The aim of this study was to analyse the level of faecal colonization by vanA-containing enterococci in healthy pets, poultry and wild animals as well as in healthy humans in Portugal, 7 years after the banning of avoparcin use.

The presence of faecal VRE was investigated in 292 faecal samples recovered from healthy humans (n=73), healthy pets (39 dogs and 32 cats), poultry (n=76) and wild animals (n=72; including 12 birds of prey, nine owls, six foxes, six wild rabbits, four European genets, four forest wildcats, four salamanders, three storks, three magpies, three deer, three vipers, two wolves, two otters, two partridges, two badgers, one mouflon, one hedgehog, one pigeon, one ferret, one tortoise, one quail and one wild boar). Faecal samples from healthy pets and humans were collected during 2003 (one sample per individual, none of whom had been administered antibiotics in the previous 4 months). Pet samples were obtained in rural and urban areas from different veterinarian clinics (n=37), or from pets in people's homes (n=34). Poultry samples were collected during February–June 2004 in a slaughterhouse located in Northern Portugal; each sample corresponded to a different flock. Faecal samples from wild animals were collected during 2003–2004, and most of them were obtained in the Pêneda Gêres Natural Park and the rest from other natural areas of Portugal.

Faecal samples were diluted and sampled in Slanetz-Bartley agar plates supplemented with 4 mg/L vancomycin and were incubated for 48 h at 35°C. Colonies with typical enterococcal morphology were identified by biochemical tests and specific PCR for the different enterococcal species.4 Antibiotic susceptibility was tested for 11 antibiotics (vancomycin, teicoplanin, ampicillin, streptomycin, gentamicin, kanamycin, chloramphenicol, tetracycline, erythromycin, quinupristin/dalfopristin and ciprofloxacin) by the NCCLS disc diffusion method and, for vancomycin and teicoplanin, also by the NCCLS agar dilution method. High-level resistance was considered for aminoglycosides. Vancomycin resistance genes (vanA, vanB, vanC-1, vanC-2/3 and vanD) were sought by PCR in all isolates that showed resistance or reduced susceptibility to glycopeptides.4 Resistance genes for other antibiotics, including tet(M), tet(K), tet(L), erm(A), erm(B), erm(C), aph(3')-IIIa, ant(6)-Ia and catA were also sought by PCR.4

vanA-containing enterococci were detected in seven of 76 poultry samples (9.2%), in one dog of the 71 pet samples (1.4%) and in two of 73 human samples (2.7%). No vanA-containing enterococci were detected in the faecal samples of wild animals. Four of the VanA enterococci were identified as E. faecium (three from poultry, and the dog isolate), three as Enterococcus durans (from poultry) and three as Enterococcus hirae (one from poultry and two from humans). The characteristics of these strains and of the animals from which they were recovered are shown in Table 1. All vanA isolates showed high-level vancomycin (MIC ≥ 128 mg/L) and teicoplanin (MIC 16–64 mg/L) resistance; they were also resistant to tetracycline (n=10), and some also to erythromycin (n=7), high-level kanamycin (n=4), high-level streptomycin (n=2), and ampicillin and ciprofloxacin (n=1). The tet(M) gene, related to tetracycline resistance, was found in all 10 vanA isolates, in all of the cases associated with tet(L) (Table 1); erm(B) was demonstrated in the seven erythromycin-resistant vanA isolates; aph(3')-IIIa in the four that showed high-level kanamycin resistance; and ant(6)-Ia in the two that showed high-level streptomycin resistance. We were interested to discover the wide pattern of antibiotic resistance of the vanA-containing E. faecium strain recovered from a dog, which included resistance to: ampicillin, ciprofloxacin, erythromycin, tetracycline and high-level kanamycin. The owner of this dog had no identifiable link with hospitals.


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Table 1. Characteristics of vancomycin-resistant enterococci recovered from healthy animals and humans in Portugal

 
Enterococci with intrinsic vancomycin resistance (vanC-1 or vanC-2/3) were found in one (1.3%), four (5.6%) and two (2.8%) faecal samples from poultry, pets and wild animals, respectively (Table 1). All five VanC-1 isolates were tetracycline-resistant [containing tet(M), associated or not with tet(L)], three carried erm(B), two carried the ant(6)-Ia gene and one carried catA-mediated chloramphenicol resistance (Table 1). The two VanC-2/3 isolates did not show resistance to other antibiotics; both were recovered from wild animals.

In this study, we detected a relatively high percentage of colonization by vanA enterococcal strains in food animals, such as poultry. This is the first report of vanA-containing enterococci in healthy pets in Portugal, representing 1.4% of the analysed pets. Higher percentages of colonization by vanA enterococci have been reported in companion animals in Spain.4,5 It is interesting to note the absence of VanA enterococci in wild animals, this being the first time that this animal group has been included in this type of study in Portugal. Epidemiological studies in different animals should be continued to monitor the evolution and dissemination of VanA enterococci in different ecosystems.

Acknowledgements

P. P. was supported by a grant of Fundaçao Calouste Gulbenkian from Portugal.

References

1 . Bager, F., Madsen, M., Christensen, J. et al. (1997). Avoparcin used as a growth promoter is associated with the occurrence of vancomycin-resistant Enterococcus faecium on Danish poultry and pig farms. Preventive Veterinary Medicine 31, 95–112.[CrossRef][ISI][Medline]

2 . Devriese, L. A., Ieven, M., Goossens, H. et al. (1996). Presence of vancomycin-resistant enterococci in farm and pet animals. Antimicrobial Agents and Chemotherapy 40, 2285–7.[Abstract]

3 . Novais, C., Coque, T. M., Sousa, J. C. et al. (2002). The community and the environment: insights about recent epidemiology of Enterococcus resistance in Portugal. In Abstracts of the Forty-second Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 2002. Abstract 1117, p. 101. American Society for Microbiology, Washington, DC, USA.

4 . Torres, C., Tenorio, C., Portillo, A. et al. (2003). Intestinal colonization by vanA- or vanB2-containing enterococcal isolates of healthy animals in Spain. Microbial Drug Resistance 9, Suppl.1, S47–S52.[ISI][Medline]

5 . Herrero, I. A., Fernandez-Garayzabal, J. F., Moreno, M. A. et al. (2004). Dogs should be included in surveillance programs for vancomycin-resistant enterococci. Journal of Clinical Microbiology 42, 1384–5.[Free Full Text]

6 . Mallon, D. J. P., Corkill, J. E., Hazel, S. M. et al. (2002). Excretion of vancomycin-resistant enterococci by wild mammals. Emerging Infectious Diseases 8, 636–8.[ISI][Medline]

7 . Bouchillon, S. K., Johnson, B. M., Hoban, D. J. et al. (2004). Determining incidence of extended-spectrum ß-lactamase producing Enterobacteriaceae, vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus in 38 centres from 17 countries: the PEARLS study 2001–2002. International Journal of Antimicrobial Agents 24, 119–24.[CrossRef][ISI][Medline]





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