Wild corvid birds colonized with vancomycin-resistant Enterococcus faecium of human origin harbor epidemic vanA plasmids.

The most prevalent type of acquired vancomycin resistance in Enterococcus faecium (VREfm) is encoded by the vanA transposon Tn1546, mainly located on transferable plasmids. vanA plasmids have been characterized in VREfm from a variety of sources but not wild birds. The aim of this study was to analyse the genetic context of VREfm strains recovered from wild corvid birds and to compare their plasmid and strain characteristics with human strains. To achieve that, 75 VREfm isolates, including strains from wild birds recovered during wide surveillance studies performed in Europe, Canada and the United States (2010-2013), and clinical and wastewater strains from Czech Republic, a region lacking data about vanA plasmids, were analysed. Their population structure, presence of major putative virulence markers and characterization of vanA transposons and plasmids were established. VREfm from wild birds were mainly associated with major human lineages (ST18 and ST78) circulating in hospitals worldwide and were enriched in putative virulence markers that are highly associated with clinical E. faecium from human infections. They also carried plasmids of the same families usually found in the clinical setting [RCR, small theta plasmids, RepA_N (pRUM/pLG1) and Inc18]. The clinically widespread IS1251-carrying Tn1546 type "F" was predominant and Tn1546-vanA was mainly located on pRUM/Axe-Txe (USA) and Inc18- or pLG1-like (Europe) plasmids. VREfm from hospitals and wastewaters carried Tn1546-vanA in different plasmid types including mosaic pRUM-Inc18 plasmids, not identified in wild birds. This is the first characterization of vanA plasmids obtained from wild birds. A similar plasmid pool seems to exist in different clonal E. faecium backgrounds of humans and wild birds. The isolation of VREfm strains from wild birds that belong to human E. faecium adapted lineages and carry virulence genes, Tn1546 and plasmid variants widespread in the clinical setting is of concern and highlight their role as potential drivers of the global dissemination of vancomycin resistance.

Vancomycin-resistant enterococci with vanA gene in treated municipal wastewater and their association with human hospital strains.

Vancomycin-resistant enterococci (VRE) are pathogens of increasing medical importance. In Brno, Czech Republic, we collected 37 samples from the effluent of a wastewater treatment plant (WWTP), 21 surface swabs from hospital settings, and 59 fecal samples from hospitalized patients and staff. Moreover, we collected 284 gull cloacal swabs from the colony situated 35km downstream the WWTP. Samples were cultured selectively. Enterococci were identified using MALDI-TOF MS, phenotypically tested for susceptibility to antibiotics, and by PCR for occurrence of resistance and virulence genes. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were used to examine genotypic diversity. VRE carrying the vanA gene were found in 32 (86%, n=37) wastewater samples, from which we obtained 49 isolates: Enterococcus faecium (44) and Enterococcus gallinarum (2), Enterococcus casseliflavus (2), and Enterococcus raffinosus (1). From 33 (69%) of 48 inpatient stool samples, we obtained 39 vanA-carrying VRE, which belonged to E. faecium (33 isolates), Enterococcus faecalis (4), and Enterococcus raffinosus (2). Nearly one-third of the samples from hospital surfaces contained VRE with the vanA gene. VRE were not detected among gulls. Sixty-seven (84%, n=80) E. faecium isolates carried virulence genes hyl and/or esp. Virulence of E. faecalis was encoded by gelE, asa1, and cylA genes. A majority of the E. faecium isolates belonged to the clinically important sequence types ST17 (WWTP: 10 isolates; hospital: 4 isolates), ST18 (9;8), and ST78 (5;0). The remaining isolates belonged to ST555 (2;0), ST262 (1;6), ST273 (3;0), ST275 (1;0), ST549 (2;0), ST19 (0;1), ST323 (3;0), and ST884 (7;17). Clinically important enterococci carrying the vanA gene were almost continually detectable in the effluent of the WWTP, indicating insufficient removal of VRE during wastewater treatment and permanent shedding of these antibiotic resistant pathogens into the environment from this source. This represents a risk of their transmission to the environment.

Vancomycin-resistant enterococci with vanA and vanB genes in Australian gulls.

This study is revealing the possible dissemination of vancomycin-resistant enterococci (VRE) from humans into the wildlife. We studied silver gulls (Chroicocephalus novaehollandiae) in Australia as potential carriers and reservoirs of VRE with acquired vancomycin resistance. In New South Wales (Australia), we have found two multi-resistant isolates belonging to Enterococcus faecium (sequence type 341, vanB genotype) and Enterococcus dispar (vanA genotype). Based on our knowledge, this is the first report of VRE in Australian wildlife.

Vancomycin-resistant Enterococcus faecium with vanA gene isolated for the first time from wildlife in Slovakia.

Corvids have been identified as an important vector of vancomycin-resistant enterococci (VRE) in several European countries. The aim of this study was to assess the prevalence of VRE in wildlife in Slovakia and to characterize vanA-carrying VRE. At the beginning of 2013, we collected 287 fecal samples of common raven (Corvus corax) in Petrovce and 99 fecal samples of rooks (Corvus frugilegus) in Kosice. Samples were cultured selectively on Slanetz-Bartley agar with vancomycin and screened for vanA, other resistance genes, and virulence genes. PCR mapping of Tn1546 carrying vanA gene was performed. Multilocus sequence typing and pulsed-field gel electrophoresis were used to examine the genotypic diversity of vanA-containing VRE. The mobility of vancomycin resistance traits was tested in vitro, using filter mating experiments. VRE with the vanA gene were found in 4 (1.4%) of 287 raven samples and in one (1%) of 99 rook samples. All 5 isolates belonged to Enterococcus faecium and were multiresistant with resistance to erythromycin encoded by the erm(B) gene, tetracycline (tet(M) and tet(L) genes), and ampicillin (mutations in C-terminal region of pbp5 gene). Isolates from Petrovce also were resistant to chloramphenicol. Virulence genes were not proven. The vanA gene was carried by Tn1546 types E (combined with insertion sequence IS1216) or F5 (IS1251). One isolate from a rook in Kosice belonged to ST (sequence type) 6 and the remaining four from ravens in Petrovce belonged to new ST917 (a single locus variant of ST18). All tested VRE were able to transfer the vancomycin resistance trait. In conclusion, we identified clinically important enterococci with the vanA gene in corvids in Slovakia.

Salmonella enterica resistant to antimicrobials in wastewater effluents and black-headed gulls in the Czech Republic, 2012.

We investigated the presence and epidemiological relatedness of Salmonella isolates from a wastewater treatment plant (WWTP) in Brno, Czech Republic and from nestlings of black-headed gulls (Chroicocephalus ridibundus) at the Nove Mlyny waterworks, situated 35 km downstream from the WWTP. During 2012, we collected 37 wastewater samples and 284 gull cloacal swabs. From wastewater samples, we obtained 89 Salmonella isolates belonging to 19 serotypes. At least one resistant strain was contained in 89% of those samples. Ten different serotypes of Salmonella were detected in 38 young gulls, among which 14 (37%) were resistant to antimicrobials. Wastewater isolates were mostly resistant to sulphonamides and tetracycline, gull isolates to tetracycline and ampicillin. We detected the occurrence of blaTEM-1,tet(A), tet(B), tet(G), sul1, sul2, sul3, floR and strA resistance genes. For the first time, we identified a class 1 integron with the dfrA12-orfF-aadA2 gene cassette in S. Infantis. Using pulsed-field gel electrophoresis, we confirmed the presence of identical clusters of S. Agona, S. Enteritidis PT8, S. Infantis and S. Senftenberg in wastewater and black-headed gulls, thus indicating the possibility of resistant Salmonella isolates spreading over long distances in the environment.

Prevalence, diversity and characterization of enterococci from three coraciiform birds.

Coraciiform birds hoopoe (Upupa epops), common kingfisher (Alcedo atthis) and European roller (Coracius garrulus) were examined for enterococci in their cloacae and uropygial glands. The enterococcal isolates were identified at the species level using several genomic and proteomic methods, screened for antibiotic susceptibility and genotyped by pulsed-field gel electrophoresis (PFGE). Clonality of isolates from the common kingfisher was also assessed by multi-locus sequence typing (MLST). Using selective media, putative enterococcal isolates (n = 117) were recovered from 74% (32 out of a total of 43) of the bird samples and 114 isolates were confirmed as enterococci. Overall, among the total of 6 different species detected, Enterococcus faecalis was dominant (59%) in all three bird species. The second most frequently isolated species was Enterococcus casseliflavus (32%). PFGE revealed great diversity of strains from different bird species and anatomic location. Closely related strains were found only from nestlings from the same nest. No genes conferring resistance to vancomycin (vanA, vanB, vanC1 and van C2/C3) or erythromycin (erm A, ermB and mefA/E) were detected. MLST analysis and eBURST clustering revealed that sequence types of E. faecalis from the common kingfisher were identical to those of isolates found previously in water, chickens, and humans.

First record of vancomycin-resistant Enterococcus faecium in Canadian wildlife.

In this study, we focused on spreading of vancomycin-resistant enterococci (VRE) to the environment. We studied that weather crows in Canada may be carriers and potentially reservoirs of VRE with vanA gene. We have found one multi-resistant isolate of Enterococcus faecium sequence type (ST) 448 with vanA gene on Prince Edward Island. This study is the first report of VRE in Canadian wildlife.

American crows as carriers of vancomycin-resistant enterococci with vanA gene.

We studied the vanA-carrying vancomycin-resistant enterococci (VRE) isolated from American crows in the United States during the winter 2011/2012. Faecal samples from crows were cultured selectively for VRE and characterized. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to examine epidemiological relationships of vanA-containing VRE. Isolates were tested in vitro for their ability to horizontally transfer the vancomycin resistance trait. VRE with the vanA gene were found in 15 (2.5%) of 590 crows samples, from which we obtained 22 different isolates. Enterococcal species were Enterococcus faecium (14) and E. faecalis (8). One, two and 19 isolates originated from Kansas, New York State and Massachusetts, respectively. Based on MLST analysis, E. faecium isolates were grouped as ST18 (6 isolates), ST555 (2), and novel types ST749 (1), ST750 (3), ST751 (1), ST752 (1). Enterococcus faecalis isolates belonged to ST6 (1), ST16 (3) and ST179 (4). All isolates were able to transfer the vancomycin resistance trait via filter mating with very high transfer range. Clinically important enterococci with the vanA gene occur in faeces of wild American crows throughout the United States. These migrating birds may contribute to the dissemination of VRE in environment over large distances. [Correction added after first online publication on 06 August 2013: The number of E. faecium ST752 isolate is now amended to '1', consistent with that shown in the 'Results' section and Figure 2.].

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

This study's 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-field 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 identified 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 vancomycin resistance trait via filter 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.