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Environmental Microbiology (2013) 15(2), 548556

doi:10.1111/1462-2920.12002

Vancomycin-resistant enterococci in rooks (Corvus frugilegus) wintering throughout Europe

Veronika Oravcova,1* Anuradha Ghosh,4 Ludek Zurek,4 Jan Bardon,5,6 Sebastian Guenther,7 Alois Cizek2,3 and Ivan Literak1,3 1 Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, 2Department of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine, and 3CEITEC VFU, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic. 4 Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA. 5 State Veterinary Institute, Olomouc, Czech Republic. 6 Department of Microbiology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic. 7 Institute of Microbiology and Epizootics, Veterinary Faculty, Free University Berlin, Berlin, Germany. Summary This studys aims were to assess the prevalence of, and to characterize, vancomycin-resistant enterococci (VRE) from rooks (Corvus frugilegus) wintering in Europe during 2010/2011. Faeces samples were cultivated selectively for VRE and characterized. Pulsed-eld gel electrophoresis and multilocus sequence typing (MLST) were used to examine epidemiologic relationships of vanA-containing VRE. The vanA-carrying VRE were tested in vitro for mobility of vancomycin resistance traits. VRE were found in 62 (6%) of 1073 rook samples. Enterococcal species diversity comprised Enterococcus gallinarum (48 isolates), followed by E. faecium (9) and E. faecalis (5). Eight VRE harboured the vanA and ermB genes. Seven vanA-carrying VRE originated from the Czech Republic and one from Germany. All vanA-carrying VRE were identied as E. faecium. Based on MLST analysis, six vanA-positive isolates were grouped as ST92 type, one isolate belonged to ST121, and the remaining one was described as a novel type ST671. Seven out of eight isolates were able to transfer the
Received 11 June, 2012; revised 20 September, 2012; accepted 23 September, 2012. *For correspondence. E-mail oravcova.veronica@ gmail.com; Tel. (+420) 54156 2631; Fax (+420) 54156 2631.

vancomycin resistance trait via lter mating with a transfer rate of 8.95 3.25 10-7 transconjugants per donor. In conclusion, wintering rooks in some European countries may disseminate clinically important enterococci and pose a risk for environmental contamination. Introduction Enterococci comprise a part of the intestinal microbiota in mammals, insects, and birds, but they are also important opportunistic pathogens responsible for serious diseases and are among the most common causes of nosocomial infections and superinfections (Murray, 1998; Simjee et al., 2006). Enterococci, which have increasingly become resistant to many antibiotics, can be used as indicator organisms for antimicrobial resistance in Grampositive bacteria (Simjee et al., 2006). One of the clinically most important antimicrobial resistances in enterococci is that of glycopeptide resistance, especially to vancomycin. In Europe, the prevalence of vancomycin-resistant enterococci (VRE) in food animals has been linked to the use of avoparcin (a glycopeptide related to vancomycin) as a growth promoter in food animals (Bager et al., 1997). The use of avoparcin as a growth promoter was banned in the European Union in 1997, and consequently the prevalence of VRE has been reduced (van den Bogaard et al., 2000). Eight genes encoding glycopeptide resistance have been described in enterococci: vanA, vanB, vanC, vanD, vanE, vanG, vanL and vanM (Courvalin, 2006; Xu et al., 2010). Enterococci with plasmid-mediated glycopeptide resistance genes vanA, vanB and vanM are reservoirs for transmission of these genes via conjugation to other enterococci and to other Gram-positive cocci, especially to Staphylococcus aureus (Courvalin, 2006; Hegstad et al., 2010; Xu et al., 2010). Wild bird populations sympatric to areas inhabited by people and areas with high density of livestock have been colonized with antibiotic-resistant bacterial strains that probably have been selected by antibiotic practice in humans and domestic animals (Literak et al., 2010). Corvids and gulls feeding on garbage dumps in urbanized areas are frequently colonized with antibiotic-resistant strains of Escherichia coli, and they are considered to be important reservoirs and vectors of these isolates in the

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Vancomycin-resistant enterococci in wintering rooks

Table 1. Prevalence of VRE in rooks wintering in Europe during 2010/2011. No. of samples 150 31 100 145 150 148 150 150 49 1073 No. of samples with VRE (%) 15 (10) 0 (0) 3 (3) 5 (3) 21 (14) 12 (8) 4 (3) 0 (0) 2 (4) 62 (6)

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Country Czech Republic France Germany Italy Poland, Gdynia Poland, Jaroslaw Serbia Spain Switzerland Total

No. of samples with E. gallinarum (%) 7 (5) 0 (0) 1 (1) 4 (3) 21 (14) 11 (7) 2 (1) 0 (0) 2 (4) 48 (4)

environment (Literak et al., 2010; Guenther et al., 2011). Recent studies have shown that enterococci resistant to antimicrobials occur also in wild mammals and birds that are not directly affected by antibiotic treatment (Drobni et al., 2009; Radimersky et al., 2010; Radhouani et al., 2010a,b; 2011; Silva et al., 2011). Also one previous study detected VRE with vanA genotype in migrating gulls in Sweden (Sellin et al., 2000). The rook (Corvus frugilegus) is a migrating, omnivorous, medium-sized corvid with Palearctic distribution (dos Anjos, 2009). Rooks winter mostly in lowlands and keep gregarious roosting places. They leave their roosting places during the day to search for food, usually within 1025 km around their roosting places. The origin of the large numbers of rooks wintering in both central and western parts of continental Europe is mostly in Eastern Europe Russia, Belarus and Ukraine. Wintering rooks in Central Europe can serve as reservoirs and vectors of E. coli and Salmonella strains resistant to older generation of antibiotics and potentially can transmit these

isolates over long distances during their migrations (Literak et al., 2007). The aims of this study were to analyse whether rooks wintering in Europe are potentially reservoirs of VRE and to characterize vanA-carrying enterococci. Given the scavenging diet of rooks in winter and the long distances travelled, it was postulated that rooks could be important vectors for the dissemination of antibiotic-resistant enterococci throughout Europe. Results Isolation and characterization of VRE Vancomycin-resistant enterococci were found in 62 (6%) of 1073 rook faecal samples from eight European countries (Fig. 1). Isolates were considered as VRE if they grew on SlanetzBartley agar supplemented with 16 mg l-1 vancomycin. The prevalence of VRE in rooks in individual countries differed signicantly (P < 0.001) (Table 1). Using matrix-assisted laser desorbtion
Fig. 1. Locations and numbers of samples collected.

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Fig. 2. Antibiotic resistance proles of 62 VRE revealed in rook faeces (n = 1073). TET, tetracycline; ERY, erythromycin; GN, gentamicin; AMP, ampicillin; C, chloramphenicol; CIP, ciprooxacin.

ionization time of ight mass spectrometry (MALDI-TOF) and sodA sequencing, the enterococcal isolates were identied as E. gallinarum (48 isolates; 77%), E. faecium (9; 15%), and E. faecalis (5; 8%). A total of 43 (71%) isolates displayed resistance to one or more antibiotic agents. The most prevalent resistance was to gentamicin (20 isolates), followed by tetracycline (19), erythromycin (18), ampicillin (13), chloramphenicol (4) and ciprooxacin (1). The percentages of VRE resistant to other antibiotics are depicted by enterococcal species in Fig. 2. Out of 62 isolates, 55 (89%) exhibited minimum inhibitory concentration (MIC) > 32 mg l-1, thus indicating vancomycin resistance (Facklam et al., 2002). The most frequent MIC for vancomycin was 128 mg l-1 (27 isolates). Eight of these isolates had MIC higher than 256 mg l-1 (one of them 2048 mg l-1), and all these were carrying the vanA gene (Table 2). Overall, resistance to vancomycin was mainly encoded by vanC1 (47 isolates) while one isolate harboured the vanC2 gene. VanA gene was carried by eight isolates, while no vanB gene was detected. Six isolates were negative to the genes conferring resistance to vancomycin (vanA, vanB, vanC1, vanC2/C3) tested. Resistance to tetracycline was associated primarily with the tet(M) gene (7 isolates), while the tet(L) gene was found in one isolate. Two isolates were found to carry both tet(M) and tet(L). One gentamicin-resistant isolate was positive for the gene aph(3)-IIIa. None of the isolates

were found to carry the erm(A), mefA, tetK, tetO, aac(6)aph(2), ant(4)-Ia or pbp5 genes. Eight (13%) of 62 VRE carrying the vanA gene also harboured erm(B). Seven of these originated from the Czech Republic, thus indicating that 47% of VRE isolated from the Czech Republic (n = 15) were vanA-positive. The remaining one was isolated from Germany (33%, n = 3); it carried the tet(L) gene along with vanA and was phenotypically resistant to ampicillin and ciprooxacin. All of these vanA-carrying isolates were identied as E. faecium. Other characteristics of E. faecium with vanA gene All vanA-carrying E. faecium were phenotypically negative for gelatinase and b-haemolysis. All these isolates were also negative for those virulence genes tested: gelE, cylA, and esp. Clonal analysis was performed on vanAcarrying E. faecium strains using PFGE (pulsed-eld gel electrophoresis) and MLST (multilocus sequence typing). A PFGE dendrogram revealed that ve out of eight isolates (HP87, HP80, HP63, HP60 and HP40) originating in the Czech Republic were highly related based on the restriction patterns of both enzymes, with coefficient of similarity (CS) higher than 95%. Two other isolates (HP09 and HP91) from the Czech Republic appeared to be distantly related, with CS equal to 85% and 70% respectively. Another one from Germany (HD31) also showed high

Table 2. Minimal inhibitory concentrations of vancomycin in tested samples. MICvan No. of isolates Percentage (%) E. gallinarum E. faecium E. faecalis <8 2 3.2 0 1 1 8 4 6.4 2 0 2 16 1 1.6 0 0 1 32 1 1.6 0 0 1 64 9 14.5 9 0 0 128 27 43.5 27 0 0 256 10 16.1 10 0 0 512 2 3.2 0 2 0 1024 5 8.0 0 5 0 2048 1 1.6 0 1 0 Total 62 100 48 9 5

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Fig. 3. Clonality of E. faecium carrying vanA. Dendrogram based on ApaI and SmaI restriction patterns resolved by PFGE. Strain numbers and their associations with MLST types (STs) are indicated.

difference (CS = 70%). Relatedness of the samples is shown in Fig. 3. The MLST prole showed that six isolates (HP87, HP80, HP63, HP60, HP40 and HP09) belonged to ST92, which has 19 single-locus variants (SLVs) and 62 doublelocus variants (DLVs) and a subgroup. One of the isolates (HD31) was typed as ST121, which is closely related (SLV) to clonal complex 17 (ST17) (Fig. 4). Isolate HP91 was a novel ST671 that had no subgroup, and it was related to ST66. Relationships between sequence types (STs) are shown in Fig. 4. Intraspecies conjugal transfer of the vancomycin resistance trait was tested among vanA-carrying E. faecium by broth and lter conjugation assays. Seven out of eight

isolates could transfer the vancomycin resistance trait in lter mating with low frequency [8.95 10-7 transconjugants per donor (TD) and 2.31 10-6 transconjugants per recipient (TR)]. All transconjugants were positive for vanA and 82% of these also carried the ermB gene. MIC of vancomycin for these transconjugants was 2048 mg l-1. Discussion The ow of resistant microorganisms and resistance genes from livestock and humans to wildlife remains poorly understood, even though wild animals may act as reservoirs of resistance that can be amplied and spread

Fig. 4. Clustering of three E. faecium STs from rook faeces. Each ST is represented as a node, lines connect single locus variants. Numbers in larger font are highlighted STs found in our study. Numbers in smaller font represent STs related to our STs. The dark, oblong ellipse encloses the area of STs found in poultry.

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552 V. Oravcova et al. in the environment over large areas, such as during the migration or seasonal movements of wild birds (Blanco et al., 2009). Previously, resistant Salmonella spp. and E. coli were found in rooks wintering in the Czech Republic (Literak et al., 2007). Consequently, we hypothesized that rooks also carry other enteric bacteria (i.e. enterococci). Rooks come into close contact with humans and animals, and they may contribute to the spread of these bacteria into the environment. Because of their migratory nature, they can spread the resistant bacteria over long distances (Literak et al., 2007). This study is the rst report of VRE in rooks. We detected VRE in rooks wintering in Europe, but the prevalence of VRE was different in individual countries. The highest prevalence of VRE in rooks was observed at one Polish location (14%) and in the Czech Republic (10%). Prevalence in others countries was low (Switzerland, Germany, Italy and Serbia), while no VRE were detected in Spain or France. The prevalence of VRE in wild animals recently has been documented in Portugal, where VRE occurred in buzzards, red foxes, seagulls and in wild birds from the Azores Archipelago (Radhouani et al., 2010a,b; 2011; Silva et al., 2011). In Europe, the prevalence of VRE in livestock has been linked to the use of avoparcin as a growth promoter in food animals (Bager et al., 1997); however, the use of avoparcin was banned in 1997 within the European Union (Commission Directive, 1997). Several studies have shown that VRE still occur in broilers, and they persist in some facilities even after cleaning and disinfection of the broiler houses (Borgen et al., 2000; Nilsson et al., 2009). For this reason, we can consider European broiler farms as a possible source of VRE for rooks. By contrast, in USA and in Canada, where avoparcin has never been approved for use in animals, VRE are rarely found in livestock or in the environment (Simjee et al., 2006; Lanthier et al., 2010). The VRE species most frequently isolated from rooks wintering in Europe was E. gallinarum, whereas E. faecium and E. faecalis were detected only in a minority of cases. This likely is due to the fact that E. gallinarum is intrinsically resistant (carrying the vanC1 gene) to vancomycin (Clark et al., 1998) while E. faecium and E. faecalis become vancomycin resistant by acquiring resistance mechanisms. Vancomycin-resistant enterococci in our study were often resistant to other antibiotics, as well, and especially to gentamicin, tetracycline and erythromycin. Similar results had been found among VRE in feral pigeons within the Czech Republic (Radimersky et al., 2010). In our study, the percentages of VRE isolates resistant to ampicillin, chloramphenicol and ciprooxacin were lower than those reported in birds from the Azores Archipelago (Silva et al., 2011). Nevertheless, the genes conferring these resistances in our study were identical to those found in other European wildlife studies (Radimersky et al., 2010; Radhouani et al., 2011; Silva et al., 2011). Acquired resistance to glycopeptides is mediated by various mechanisms, of which the vanA genotype is most frequently encountered in Central Europe (Aarestrup et al., 2002). The vanA gene cluster is typically located on transposons of the Tn1546 type, which can be integrated into conjugative plasmids (Arthur and Courvalin, 1993). The reservoir of this gene in humans is mostly E. faecium, while vancomycin-resistant E. faecalis isolates with these genes are rare (Willems et al., 2005; Willems and Bonten, 2007). Moreover, with the increase in nosocomial enterococcal infections, a partial replacement of E. faecalis (as causative agent) by E. faecium has occurred in European and US hospitals (Treitman et al., 2005; Top et al., 2007). This is of concern as most of the E. faecium are increasingly resistant to vancomycin and ampicillin (Arias and Murray, 2009). In this study, eight isolates of E. faecium with acquired resistance to vancomycin encoded by the vanA gene were detected. Seven of these originated from the Czech Republic and one from Germany. All of them carried also the ermB gene. A study on Danish pigs has shown that the persistence of VRE could be attributed to co-selection resulting from the use of macrolides, because genes encoding resistance to macrolides (ermB) and glycopeptides (vanA) are linked (Aarestrup, 2000). E. faecium isolates with the vanA and ermB genes have also been found in wild animals in Portugal (Poeta et al., 2005; Radhouani et al., 2010b). Such isolates from Portugal also carried virulence factors (Radhouani et al., 2010b; 2011), which is in contrast to our study as we detected no virulence genes esp, gelE or cylA in vanA-positive E. faecium. According to Cauwerts and colleagues (2007), 89% of ermB positive enterococci carried tet genes, but in our study this gure was only 25%. Based on MLST analysis, E. faecium disseminated in human hospitals in several parts of the world belong to the clonal complex 17 (CC-17) containing several subcomplexes (Willems et al., 2005; Freitas et al., 2009; Willems and van Schaik, 2009). We found that the vanApositive E. faecium isolate (HD31) from a rook in Germany belonged to ST121 and is a DLV of ST17. Moreover, it was resistant to ve different antibiotics (vancomycin, tetracycline, erythromycin, ampicillin and ciprooxacin). Therefore, we predict a human origin for the HD31 isolate found from the rook in Germany. ST121 has also been isolated from animals in Belgium, Italy and Denmark (De Leener et al., 2004; Bonora et al., 2007; Damborg et al., 2009). The other seven isolates belonging to ST92 (6 isolates) and ST671 (1 isolate) were indirectly linked to CC-17. The vanA-positive E. faecium isolate from rooks in the Czech Republic which was typed as a

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Vancomycin-resistant enterococci in wintering rooks novel ST (ST671) is closely related to a group of STs originating in poultry (see Fig. 4, http://efaecium.mlst.net/ portable/portable.xls, accessed 17 April 2012). Most (6 from 8) of the tested isolates belonged to ST92 that were detected in samples from hospitalized human patients in Sweden and the Netherlands (http://efaecium.mlst.net/ portable/portable.xls, accessed 17 April 2012). In addition, ve of these isolates showed high similarity in PFGE analysis (CS 95%), and it is probable, therefore, that they originated from the same source. Spreading of these isolates among rooks also cannot be ruled out. Several poultry farms are located in the surroundings of Prerov, which is the area from which rook faeces were collected in the Czech Republic. These farms may be potential sources of VRE, due to the previous use of avoparcin as a growth promoter in poultry. We speculate that poultry comprise an important source for colonization of rooks in the Czech Republic with vanA-positive E. faecium. More studies are warranted to elucidate how vanApositive E. faecium isolates are transmitted to wild animals, including rooks. The spread of hospital associated E. faecium lineages in the environment should be also considered. In the past two decades, multi-drug resistant E. faecium has emerged as a serious nosocomial pathogen in human hospitals (Willems and van Schaik, 2009). BourgeoisNicolaos and colleagues (2006) described intra- as well as interspecies transfer of the vancomycin resistance traits from E. faecium with an intraspecies transfer frequency of 10-4 transconjugants per donor cell in lter mating. We detected low transfer frequency in lter mating, thus indicating the potential for horizontal transfer of vanA and ermB among E. faecium. In conclusion, wintering omnivorous rooks in Europe were colonized with VRE, some of which (E. faecium) carried the vanA gene and were also capable of horizontal transfer of vancomycin resistance. Rooks may disseminate clinically important enterococci and other bacteria over long distances and pose a risk for environmental contamination. Experimental procedures Sampling and transport of samples to laboratory
Samples of rook faeces were collected from roosting places throughout Europe during winter 2010/2011 (Fig. 1). The roosts were used by hundreds to thousands of rooks, together with various number of other corvids such as jackdaws (C. monedula) or crows (C. corone, C. cornix). Faecal samples were collected at each location only once. They were picked up individually in the morning from a large plastic lm placed overnight on the ground beneath the roosting place. Rooks had been dropping on the lm during the evening when they arrived to the roosting place and in early morning when leaving the location. Samples were collected

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in the Czech Republic (Prerov, 4928N, 1727E, 12 December 2010, 150 samples), France (Pire sur Seiche, 4800N, 125W, 8 April 2011, 31 samples), Germany (Wilhelmshaven, 5332N, 804E, 5 February 2011, 100 samples), Italy (San Benedetto Po, 452N, 1055E, 27 February 2011, 150 samples), Poland (Gdynia and near vicinity, 5431N, 1833E, 27 February 2011, 150 samples), Poland (Jaroslaw, 5000N, 2241E, 9 February 2011, 150 samples), Spain (La Baneza, 4218N, 554W, 4 February 2011, 150 samples, sedentary population), Serbia (Novi Sad, 4514N, 1951E, 17 January 2011, 150 samples), and Switzerland (Bern, 4656N, 726E, 8 February 2011, 49 samples). Samples from fresh faeces were taken in the eld by cotton swabs, dipped in Amies Transport Medium (Coban, Italy) and transported to the laboratory. In total, we collected 1073 samples.

Selective isolation of VRE

Samples were placed overnight in buffered peptone water (Oxoid, UK) at 37C and then cultivated for enterococci on SlanetzBartley agar (Oxoid, UK) with 16 mg l-1 vancomycin and incubated for 48 h at 37C. Individual colonies with enterococcal morphology were selected, puried and conrmed to genus level by Gram staining, potassium hydroxide test, catalase production, and detection of pyrrolidonyl arylamidase PYRAtest (Lachema, Czech Republic). The identied enterococcal isolates were inoculated onto Columbia agar (Oxoid, UK) with 5% of sheeps blood, then incubated for 24 h at 37C. Further, all isolates were identied using MALDI-TOF MS (Microex LT, Bruker Daltonics, Brehmen, Germany), and results were evaluated using the software FlexControlmicroex v 3.3 and Maldi Biotyper v 3.0. The identities of these isolates were also conrmed by sodA gene sequencing (Poyart et al., 2000; Frolkova et al., 2012), whereby the PCR products were puried using Gel/ PCR Fragments Extraction Kit (Geneaid Biotech, Taiwan), were sequenced, and the identities of sequences were then determined by BLAST search of the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed 25 February 2012).

Antibiotic sensitivity testing and detection of genes conferring resistance by PCR

Minimum inhibitory concentration for vancomycin was determined on MuellerHinton agar (Oxoid, UK) supplemented with vancomycin (concentration range: 1256 mg l-1). Isolates with MIC > 256 mg l-1 were tested by broth microdilution technique using MuellerHinton broth (BBL, USA) with vancomycin concentration up to 2048 mg l-1. The results were interpreted according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2008; 2010). Antibiotic sensitivity of enterococcal isolates was determined using disc diffusion method following CLSI guidelines (CLSI, 2008). Seven different antibiotics were tested (mg per disc): tetracycline (30), vancomycin (30), erythromycin (15), gentamicin (120), ampicillin (10), chloramphenicol (30), and ciprooxacin (5). For PCR detection of antibiotic resistance genes, template DNA was prepared by alkaline lysis of the enterococcal cells.

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Antibiotic-resistant isolates were tested for tet(M), tet(O), tet(K) and tet(L) coding tetracycline resistance; vanA, vanB, vanC1 and vanC2/C3 for vancomycin resistance; ermA, ermB and mefA/E for erythromycin; ant(4)-Ia, aac(6)aph(2) and aph(3)-IIIa for gentamicin; and pbp5 for ampicillin, as described previously (Radimersky et al., 2010). database in a single diagram as a snapshot of E. faecium clonal diversity, and the STs were further described as SLVs versus DLVs.

Horizontal gene transfer

VanA-positive E. faecium isolates were tested in vitro for mobility of vancomycin resistance traits using broth and lter mating assays (Ike et al., 1998; Tendolkar et al., 2006). Enterococcus faecium strain 38-13 (Qi et al., 2006) (spectinomycin, MIC = 250 mg ml-1), was used as recipient. Both assays were performed with a donor-to-recipient ratio of 1:10. After allowing mating for 4 h in broth and 16 h on lter, the mixed culture was dilution plated on brainheart infusion agar (Difco, USA) supplemented with vancomycin (32 mg l-1) or spectinomycin (250 mg l-1) or both, then incubated for 2448 h at 37C. The transfer frequency for each isolate was calculated as the number of transconjugants per donor colony-forming units (T/D) or as transconjugants per recipient (T/R). The transconjugants were tested for the respective resistance genes (vanA and ermB) by PCR and also conrmed for phenotypic expression of the resistance traits by determination of MIC for vancomycin, as described above.

Virulence factors
VanA-containing isolates were tested for several tentative virulence factors. Multiplex PCR was performed to screen the identied isolates for gelE, cylA and esp (Vankerckhoven et al., 2004). Phenotypic screening of the virulence traits was performed following the method previously described by Macovei and Zurek (2006). Isolates were tested for gelatinase activity on Todd Hewitt agar (BBL, USA) with 1.5% skim milk, as well as cytolysin expression by b-haemolysis on Columbia blood agar base (Difco, USA) with 5% human blood (Rockland Immunochemicals, USA). Isolates having complete clearance zones around the colonies (b-haemolysis) were considered positive for cytolysin expression (Gilmore et al., 2002).

Pulsed-eld gel electrophoresis

The PFGE was carried out to provide information on the relatedness of vanA-containing E. faecium isolates. The restriction endonucleases ApaI and SmaI (Promega, USA) were used for digestion. The digested DNA plugs were placed on 1% SeaKem gold agarose (Lonza, USA) and electrophoresed in a contour-clamped homogeneous electric eld apparatus (Bio-Rad, USA) with initial-to-nal switch times ranging from 1 to 20 s at 200 V for 17.5 h. Gel was stained with ethidium bromide and photographed under UV transillumination. Analysis was performed using BioNumerics (Applied Maths, Belgium).

Acknowledgements
The authors thank Frank Borleis, Francisco de la Calzada, Dragan Fabijan, Sebastian Franco, Lisa Guardone, Susanne Homma, Josef Hordowski, Ruben Gonzales Janez, Jiri Klimes, Adam Konecny, Tomas Lang, Cyrille Lejas, Benito Fuertes Marcos, Zuzana Markova, Wlodzimierz Meissner, Radim Petro, Jakub Prochazka, Marko Sciban, Marie Slavikova, Eva Suchanova, Marco Tucakov, and Jiri Vojtech for excellent cooperation in the eld or in the laboratory. We appreciate assistance from Dr Janetta Top (University Medical Centre Utrecht, The Netherlands) in accessing the E. faecium MLST database and assigning new ST. This study was funded by project CEITEC Central European Institute of Technology (CZ.1.05/1.1.00/02.0068) from European Regional Development Fund.

Multilocus sequence typing

Enterococcus faecium carrying the vanA gene were characterized by MLST (Homan et al., 2002). For this purpose, internal 400600 bp fragments of seven housekeeping genes (adk, atpA, ddl, gdh, gyd, purK and pstS) were amplied according to the standard protocol (http://efaecium. mlst.net/misc/info.asp, accessed 10 February 2012) using Maxima Hot Start PCR Master Mix (Fermentas, Canada). PCR products were puried using DNA Clean and Concentrator kit (Zymo Research Corp., USA) and sequenced. Sequences were edited, aligned, and compared with the reference set of alleles using CodonCode Aligner v. 2.0.4. MLST proles were submitted to the MLST database (http://efaecium.mlst.net, accessed 16 February 2012) and assigned their MLST STs. The combination of seven alleles obtained for each isolate gave a specic ST (Homan et al., 2002). Clustering of STs was performed using the eBURST ver. 3 algorithm implemented as a Java applet at http:// eburst.mlst.net (accessed 22 February 2012), and detailed guidance in its use is available at this website. A description of the algorithm is given by Feil and colleagues (2004). eBURST clustering displayed all STs from a large MLST

Conicts of interest The authors have no conicts of interest to declare. References

Aarestrup, F.M. (2000) Characterization of glycopeptideresistant Enterococcus faecium (GRE) from broilers and pigs in Denmark: genetic evidence that persistence of GRE in pig herds is associated with coselection by resistance to macrolides. J Clin Microbiol 7: 27742777. Aarestrup, F.M., Butaye, P., and Witte, W. (2002) Nonhuman reservoirs of enterococci. In The Enterococci: Pathogenesis, Molecular Biology, and Antibiotic Resistance. Gilmore, M.S. (ed.). Washington, DC, USA: ASM Press, pp. 5599. dos Anjos, L. (2009) Family Corvidae (crows). In Handbook of the Birds of the World. Vol 14: Bush-Shrikes to Old World

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Vancomycin-resistant enterococci in wintering rooks

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