J Med Microbiol 54 (2005), 965-967; DOI: 10.1099/jmm.0.45824-0
© 2005 Society for General Microbiology
ISSN 0022-2615
Occurrence of vancomycin-resistant enterococci in humans and animals in the Czech Republic between 2002 and 2004
Milan Kolar1,
Roman Pantucek2,
Jan Bardon3,
Luboslava Cekanova4,
Michaela Kesselova1,
Pavel Sauer1,
Iva Vagnerova1 and
Dagmar Koukalová1
1Institute of Microbiology, Faculty of Medicine, Palacky University, Hnevotinska 3, 775 15, Olomouc, Czech Republic 2Department of Genetics and Molecular Biology, Faculty of Science, Masaryk University, Brno, Czech Republic 3National Veterinary Institute, Olomouc, Czech Republic 4Mikrochem s.r.o., Olomouc, Czech Republic
Correspondence Milan Kolar kolar{at}fnol.cz
Received July 17, 2004
Accepted June 16, 2005
In the period between September 2002 and May 2004, a total of 6023 rectal swabs from humans in the Czech Republic were evaluated and 821 Enterococcus spp. strains were isolated. Nine strains (1.1 %) were identified as vancomycin-resistant enterococci (VRE). Two strains were VanA Enterococcus faecium, one strain was VanB Enterococcus faecalis and six strains were VanC Enterococcus casseliflavus. In total, 527 Enterococcus spp. strains were isolated from poultry breeds of which 11 (2.1 %) were VRE. Most (54.5 %) were identified as VanA E. faecium. Cluster analysis of SmaI-generated macrorestriction patterns showed high variability in both human and animal VRE strains and no relatedness between strains from the two sources.
Abbreviation: VRE, vancomycin-resistant enterococci.
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INTRODUCTION
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At present, the increase in bacterial resistance to antibiotics poses a major medical problem. Increasingly, new bacterial strains emerge, often with a high level of resistance; among these are vancomycin-resistant enterococci (VRE). VRE in healthy carriers have rarely been described. The presence of these strains in the European population might be caused by transmission from domestic and farm animals colonized as a consequence of using glycopeptide-containing feeds (Bates, 1997).
The aim of this study was to monitor VRE in the community and in poultry breeds in one region of the Czech Republic, including a molecular biological analysis of VRE isolated from humans and animals.
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METHODS
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Targeted screening of VRE occurrence in the community in the Olomouc region (population of 300 000) in the Czech Republic was performed during the period from September 2002 to May 2004. Only rectal swabs from healthy, non-hospitalized persons were examined. Each subject from the community provided only one rectal swab for the study. Samples of cloacal swabs from seven poultry farms localized in the same region were examined. The parental breeds produced the final crossbreed of Brown Hissex. In each poultry farm, a total of 150 samples of cloacal swabs was taken from 150 healthy layer hens, i.e. one sample per hen.
Each specimen was cultivated on blood agar (Becton Dickinson) with subsequent identification of the enterococci present according to the criteria established by Facklam & Collins (1989) and evaluation of biochemical properties using the En-coccus test (PLIVA-Lachema). Resistance to vancomycin and teicoplanin was determined by a standard broth microdilution in accordance with the National Committee for Clinical Laboratory Standards guidelines (NCCLS, 2003). Values of 4 µg vancomycin ml–1 and 8 µg teicoplanin ml–1 were used as breakpoints for resistance. Species identification and glycopeptide-resistance genotypes of vancomycin-resistant strains were confirmed by multiplex PCR as described previously (Dutka-Malen et al., 1995), using the primers vanA-F (5'-GGGAAAACGACAATTGC-3') and vanA-R (5'-GTA CAATGCGGCCGTTA-3'); vanB-F (5'-ATGGGAAGCCGATAGTC-3') and vanB-R (5'-GATTTCGTTCCTCGACC-3'); van C-1-F (5'-GGTATCAAGGAAACCTC-3') and vanC-1-R (5'-CTTCCGCCATCA TAGCT-3'); and vanC-2/vanC-3-F (5'-CTCCTACGATTCTCTTG-3') and vanC-2/vanC-3-R (5'-CGAGCAAGACCTTTAAG-3').
Susceptibility to linezolid and quinupristin/dalfopristin was assessed by Etest (AB Biodisk) in accordance with NCCLS (2003) guidelines.
DNA isolation for PFGE was performed according to the method of Pantucek et al. (1996) with minor modifications of lysis conditions (Murray et al., 1990) in all VRE isolated. DNAs cleaved with SmaI restriction endonuclease (Roche Diagnostics) were separated by PFGE using the CHEF-DRII system (Bio-Rad) in 1.2 % (w/v) agarose gels at 14 °C in 1x TBE buffer. A constant voltage of 5 V cm–1 was applied with an increasing pulse time of 1–60 s over a period of 28 h. As size markers, concatemers of bacteriophage 
(Sigma) were used. Macrorestriction patterns of DNA isolated from individual strains were compared qualitatively. Digitized gel images were analysed using GelCompar 4.1 software (Applied Maths BVBA). Correlation coefficients of similarity between banding patterns and the unweighted pair-group method with arithmetic averages (UPGMA) algorithm were used for constructing the dendrogram.
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RESULTS AND DISCUSSION
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Altogether, 6023 rectal swabs from humans were collected and a total number of 821 enterococci were isolated. A total of 665 enterococci (80.9 %) were identified as Enterococcus faecalis strains, 62 enterococci (7.6 %) as Enterococcus faecium and 94 enterococci (11.5 %) were non-speciated (Enterococcus spp.). Resistance to vancomycin was observed in nine strains (1.1 %) isolated from nine individuals. Two strains were detected as VanA E. faecium, one strain was VanB E. faecalis and six strains were VanC Enterococcus casseliflavus.
A total of 527 Enterococcus spp. strains were isolated from poultry, of which 11 (2.1 %) were VRE. Six strains of VRE were identified as VanA E. faecium, four were VanB E. faecalis and one was VanB E. faecium. Details of VRE from the community and poultry breeds are given in Table 1.
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Table 1. Survey of VRE species and their genotypes isolated from humans or poultry breeds H1–H9, VRE of human origin; A1–A11, VRE of poultry origin.
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All isolated VRE were susceptible to linezolid; full susceptibility to quinupristin/dalfopristin was seen with E. faecium and E. casseliflavus strains, but not with E. faecalis (as would be expected).
The study compared nine VRE strains from humans in the community and 11 VRE strains of animal origin. The cluster analysis and visual inspection of SmaI-generated macrorestriction patterns showed high variability in both human and animal VRE strains. The distinguished groups of strains were in agreement with their classification into species. Fig. 1 shows the UPGMA tree. In nine different patterns obtained from humans in the community, high similarity was observed only between E. casseliflavus strains H4, H5, H7 and H8 (80–90 %). In macrorestriction profiles of animal VRE, three groups were differentiated. Two of them were formed by the highly related VanA phenotype strains of E. faecium A1–A3 and A4–A6, respectively. The isolates designated E. faecalis were clearly included in a separate group. The differences in banding patterns of strains from human and animal sources were substantial and the isolates found from either source were not related. A potential source of human VRE remains unclear, as all nine people from whose rectal swabs the VRE were isolated were not hospitalized in a health care facility in the period between September 2002 and May 2004. Previous hospital information is not known. Five people were administered no antibiotics and in four of them cotrimoxazole, penicillin V, amoxicillin, amoxicillin/clavulanic acid, cefuroxime and cephalexin were used to treat urinary and respiratory tract infections within the period mentioned above.
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Fig. 1. Dendrogram constructed from UPGMA linkage of similarity values and normalized SmaI macrorestriction patterns of PFGE fingerprints.
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In Europe, the rate of faecal carriage of VRE in the community varies from 2 to 28 % and vancomycin resistance is mostly VanA-mediated (Endtz et al., 1997; Jordens et al., 1994; Klare et al., 1995; Van der Auwera et al., 1996). The highest frequency of VRE in the human community in our study was found with E. casseliflavus. In VanC E. casseliflavus strains, genetic determinants of resistance to vancomycin are intrinsic and non-transferable (Arthur & Courvalin, 1993; Murray, 1997). In contrast, with VanA E. faecium and VanB E. faecalis strains, resistance is acquired, transferable and much more serious from an epidemiological point of view. The frequency of vancomycin-resistant strains in E. faecalis reached 0.2 %, and reached 3 % in E. faecium. Overall, the rate of faecal carriage of VRE in the community of the studied region was 1.1 %, which could be considered relatively low. In their work evaluating VRE prevalence in healthy, non-hospitalized persons in Switzerland, Balzereit-Scheuerlein & Stephan (2001) stated a 4.9 % prevalence of VRE carriers, mostly represented by E. faecium. In poultry breeds in the same region of the Czech Republic, the frequency of VRE reached 2.1 %.
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ACKNOWLEDGEMENTS
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The study was supported by the IGA, Ministry of Health, Czech Republic grant no. NI/7305-3 and 1A/8258-3.
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REFERENCES
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INTRODUCTION
METHODS
