Interspecies transmission of antimicrobial-resistant bacteria between wild birds and mammals in urban environment.

The transmission of antibiotic-resistant bacteria among wild animal species may hold significant epidemiological implications. However, this issue is seldom explored due to the perceived complexity of these systems, which discourages experimental investigation. To address this knowledge gap, we chose a configuration of birds and mammals coexisting in an urban green area as a research model: the rook Corvus frugilegus and the striped field mouse Apodemus agrarius. The indirect transmission of antimicrobial-resistant bacteria between these species is possible because rodents inhabiting rook colonies frequently come into contact with the birds' faeces and pellets. The study was conducted in two cities in eastern Poland (Central Europe) - Lublin and Chelm. Among 71 Escherichia (E.) coli isolates studied, 19.7% showed resistance to from one to six of the antibiotics tested, with much higher prevalence of antibiotic-resistant bacteria in the birds (32%) than in the rodents (7%). Whole genome sequencing was performed on 10 selected E. coli isolates representing similar resistance phenotypes. The following antimicrobial resistance genes were detected: blaTEM-1b, tet(A), tet(B), aph(6)-Id, aph(3'')-Ib, aadA1, aadA2, catA1, floR, cmlA, sul2, sul3, dfrA14, and dfrA2. Birds from the same city and also from both neighbouring cities shared E. coli bacteria with the same sequence types, whereas isolates detected in birds were not found to have been transferred to the mammalian population, despite close contact. This demonstrates that even intensive exposure to sources of these pathogens does not necessarily lead to effective transmission of antibiotic-resistant E. coli strains between birds and mammals. Further efforts should be dedicated to investigating actual transmission of antimicrobial-resistant bacteria in various ecological systems, including those that are crucial for public health, such as urban environments. This will facilitate the development of more accurate models for epidemiological threats and the formulation of well-balanced decisions regarding the coexistence of humans and urban wildlife.

Dispersal of Aphanoascus keratinophilus by the rook Corvus frugilegus during breeding in East Poland.

The process of dispersal of the potentially disease-causing, geophilic and keratinolytic fungal strain Aphanoascus keratinophilus (the perfect, sexual stage of Chrysosporium keratinophilum) by the rook Corvus frugilegus was studied. The source of A. keratinophilus strains was pellets of the rook, thus far not considered a carrier of this particular opportunistic pathogen. Pellets collected from breeding colonies of rooks were analysed in terms of the occurrence of keratinolytic fungi with the application of the native keratin bait method. Among the 83 rook pellets analysed, 24 (29%) were infected by keratinophilic fungi. Pure cultures of the fungi were identified to species based on traditional morphological features. Traditional mycological identification was verified by the PCR-RFLP molecular identification method as well as DNA sequencing. The obtained results showed the presence of 90 Aphanoascus keratinophilus strains, 6 Chrysosporium tropicum strains, and 3 Chrysosporium pannicola strains. The PCR melting profile (PCR-MP) method was used to identify intraspecies variations of the 90 analysed A. keratinophilus strains. The dispersal of genotypes and possible pathways of A. keratinophilus dispersal and infection via rook pellets were analysed.

Endless Forms: Within-Host Variation in the Structure of the West Nile Virus RNA Genome during Serial Passage in Bird Hosts.

RNA viruses are infamous for their high rates of mutation, which produce swarms of genetic variants within individual hosts. To date, analyses of intrahost genetic diversity have focused on the primary genome sequence. However, virus phenotypes are shaped not only by primary sequence but also by the secondary structures into which this sequence folds. Such structures enable viral replication, translation, and binding of small RNAs, yet within-host variation at the structural level has not been adequately explored. We characterized the structural diversity of the 5' untranslated region (UTR) of populations of West Nile virus (WNV) that had been subject to five serial passages in triplicate in each of three bird species. Viral genomes were sampled from host serum samples at each passage (n = 45 populations) and subjected to next-generation sequencing. For populations derived from passages 1, 3, and 5 (n = 9 populations), we predicted the impact of each mutation occurring at a frequency of ≥1% on the secondary structure of the 5' UTR. As expected, mutations in double-stranded (DS) regions of the 5' UTR stem structures caused structural changes of significantly greater magnitude than did mutations in single-stranded (SS) regions. Despite the greater impact of mutations in DS regions, mutations in DS and SS regions occurred at similar frequencies, with no evidence of enhanced selection against mutation in DS regions. In contrast, mutations in two regions that mediate genome cyclization and thereby regulate replication and translation, the 5' cyclization sequence and the UAR flanking stem (UFS), were suppressed in all three hosts.IMPORTANCE The enzymes that copy RNA genomes lack proofreading, and viruses that possess RNA genomes, such as West Nile virus, rapidly diversify into swarms of mutant lineages within a host. Intrahost variation of the primary genomic sequence of RNA viruses has been studied extensively because the extent of this variation shapes key virus phenotypes. However, RNA genomes also form complex secondary structures based on within-genome nucleotide complementarity, which are critical regulators of the cyclization of the virus genome that is necessary for efficient replication and translation. We sought to characterize variation in these secondary structures within populations of West Nile virus during serial passage in three bird species. Our study indicates that the intrahost population of West Nile virus is a diverse assortment of RNA secondary structures that should be considered in future analyses of intrahost viral diversity, but some regions that are critical for genome cyclization are conserved within hosts. Besides potential impacts on viral replication, structural diversity can influence the efficacy of small RNA antiviral therapies.

Population Genetics and Characterization of Campylobacter jejuni Isolates from Western Jackdaws and Game Birds in Finland.

Poultry are considered a major reservoir and source of human campylobacteriosis, but the roles of environmental reservoirs, including wild birds, have not been assessed in depth. In this study, we isolated and characterized Campylobacter jejuni from western jackdaws (n = 91, 43%), mallard ducks (n = 82, 76%), and pheasants (n = 9, 9%). Most of the western jackdaw and mallard duck C. jejuni isolates represented multilocus sequence typing (MLST) sequence types (STs) that diverged from those previously isolated from human patients and various animal species, whereas all pheasant isolates represented ST-19, a common ST among human patients and other hosts worldwide. Whole-genome MLST revealed that mallard duck ST-2314 and pheasant ST-19 isolates represented bacterial clones that were genetically highly similar to human isolates detected previously. Further analyses revealed that in addition to a divergent ClonalFrame genealogy, certain genomic characteristics of the western jackdaw C. jejuni isolates, e.g., a novel cdtABC gene cluster and the type VI secretion system (T6SS), may affect their host specificity and virulence. Game birds may thus pose a risk for acquiring campylobacteriosis; therefore, hygienic measures during slaughter and meat handling warrant special attention.IMPORTANCE The roles of environmental reservoirs, including wild birds, in the molecular epidemiology of Campylobacter jejuni have not been assessed in depth. Our results showed that game birds may pose a risk for acquiring campylobacteriosis, because they had C. jejuni genomotypes highly similar to human isolates detected previously. Therefore, hygienic measures during slaughter and meat handling warrant special attention. On the contrary, a unique phylogeny was revealed for the western jackdaw isolates, and certain genomic characteristics identified among these isolates are hypothesized to affect their host specificity and virulence. Comparative genomics within sequence types (STs), using whole-genome multilocus sequence typing (wgMLST), and phylogenomics are efficient methods to analyze the genomic relationships of C. jejuni isolates.

Molecular characterization of plasmid-mediated AmpC beta-lactamase- and extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae among corvids (Corvus brachyrhynchos and Corvus corax) roosting in Canada.

This study evaluated the carriage of AmpC and extended-spectrum beta-lactamase (ESBL) genes and associated plasmids in faecal bacteria of Canadian corvids. Faecal samples from 449 birds in five roosting sites across Canada were analyzed using selective media, screening for AmpC and ESBL genes by PCR, and sequencing. Genomic relatedness was determined by PFGE and MLST. Plasmid mobility was studied by conjugation and transformation experiments, followed by plasmid typing. In total, 96 (21%, n = 449) cefotaxime-resistant Escherichia coli and three (0.7%) Klebsiella pneumoniae isolates were identified. ESBL genes blaCTX-M-1 (n = 3), blaCTX-M-14 (n = 2), blaCTX-M-32 (n = 2) and blaCTX-M-124 (n = 1) were detected in eight E. coli isolates, whereas blaSHV-2 (2) was found in two K. pneumoniae. E. coli isolates contained blaCMY-2 (n = 83) and blaCMY-42 (n = 1). The high genetic diversity of the isolates and presence of clinically important E. coli ST69 (n = 1), ST117 (n = 7) and ST131 (n = 1) was revealed. AmpC genes were predominantly carried by plasmids of incompatibility groups I1 (45 plasmids), A/C (10) and K (7). The plasmid IncI1/ST12 was most common and found in diverse E. coli STs in all sites. Highly diverse E. coli isolates containing AmpC and ESBL genes, including clinically important clones and emerging plasmids, are in circulation throughout Canadian wildlife.

Occurrence of plasmid-mediated quinolone resistance genes in Escherichia coli and Klebsiella spp. recovered from Corvus brachyrhynchos and Corvus corax roosting in Canada.

The spread of antimicrobial resistance from human activity derived sources to natural habitats implicates wildlife as potential vectors of antimicrobial resistance transfer. Wild birds, including corvid species can disseminate mobile genetic resistance determinants through faeces. This study aimed to determine the occurrence of plasmid-mediated quinolone resistance (PMQR) genes in Escherichia coli and Klebsiella spp. isolates obtained from winter roosting sites of American crows (Corvus brachyrhynchos) and common ravens (Corvus corax) in Canada. Faecal swabs were collected at five roosting sites across Canada. Selective media isolation and multiplex PCR screening was utilized to identify PMQR genes followed by gene sequencing, pulse-field gel electrophoresis and multilocus sequence typing to characterize isolates. Despite the low prevalence of E. coli containing PMQR (1·3%, 6/449), qnrS1, qnrB19, qnrC, oqxAB and aac(6')-Ib-cr genes were found in five sequence types (ST), including E. coli ST 131. Conversely, one isolate of Klebsiella pneumoniae contained the plasmid-mediated resistance gene qnrB19. Five different K. pneumoniae STs were identified, including two novel types. The occurrence of PMQR genes and STs of public health significance in E. coli and Klebsiella pneumoniae recovered from corvids gives further evidence of the anthropogenic derived dissemination of antimicrobial resistance determinants at the human activity-wildlife-environment interface. SIGNIFICANCE AND IMPACT OF THE STUDY: This study examined large corvids as possible vector species for the dissemination of antimicrobial resistance in indicator and pathogenic bacteria as a means to assess the anthropogenic dissemination of plasmid-mediated quinolone resistance (PMQR) genes. Although rare, PMQR genes were found among corvid populations across Canada. The clinically important Escherichia coli strain ST131 containing aac(6')-Ib-cr gene along with a four-class phenotypic antimicrobial resistance (AMR) pattern as well as one Klebsiella pneumoniae strain containing a qnrB19 gene were identified in one geographical location. Corvids are a viable vector for the circulation of PMQR genes and clinically important clones in wide-ranging environments.

Differential Distribution of Salmonella Serovars and Campylobacter spp. Isolates in Free-Living Crows and Broiler Chickens in Aomori, Japan.

Salmonella and Campylobacter cause foodborne enteritis mainly via the consumption of raw/undercooked contaminated poultry meat and products. Broiler flocks are primarily colonized with these bacteria; however, the underlying etiology remains unclear. The present study was conducted in order to obtain further information on the prevalence and genotypic distribution of Salmonella and Campylobacter in free-living crows and broiler flocks in a region for 2 years, thereby facilitating estimations of the potential risk of transmission of C. jejuni from crows to broiler flocks. Salmonella serovars Bredeney and Derby were isolated from 8 and 3 out of 123 captured crows, respectively, both of which are not common in broiler chickens. Campylobacter were isolated from all 89 crows tested and C. jejuni was prevalent (85 crows). Pulsed field gel electrophoresis showed broad diversity in the crow isolates of C. jejuni. However, 3 crow isolates and 2 broiler isolates showing similar banding patterns were assigned to different sequence types in multi-locus sequence typing. These results indicate that crows do not share Salmonella serovars with broilers, and harbor various genotypes of C. jejuni that differ from those of broiler flocks. Thus, our results indicate that crows are not a potential vector of these bacteria to broiler flocks in this region.

Plasmid-mediated resistance to cephalosporins and quinolones in Escherichia coli from American crows in the USA.

American crow (Corvus brachyrhynchos) faeces were tested for Escherichia coli with plasmid-mediated quinolone resistance (PMQR), extended-spectrum beta-lactamases (ESBL) and AmpC beta-lactamases. A total of 590 faecal samples were collected at four roosting sites in the USA and cultivated on selective media. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were performed to assess clonality. Transferability of resistance genes was studied using conjugation and transformation bioassays. In total, 78 (13%, n = 590) cefotaxime-resistant isolates were obtained, of which 66 and 12 displayed AmpC and ESBL phenotypes, respectively. Fifty-four AmpC-producing isolates carried blaCMY-2 . Isolates producing ESBLs contained genes blaCTX-M-27 (5 isolates), blaCTX-M-15 (4), blaCTX-M-14 (2) and blaCTX-M-1 (1). Ninety isolates (15%, n = 590) with reduced susceptibility to ciprofloxacin were obtained, among which 14 harboured PMQR genes aac(6')-Ib-cr (4 isolates), qnrB19 (3), qnrS1 (2), qnrA1 (2), qnrB2 (1), qnrB6 (1) and qnrD3 (1). High genetic diversity was revealed by PFGE and MLST. Epidemiologically important E. coli clones (e.g., ST131, ST405) were identified. Plasmids carrying blaCMY-2 were assigned predominantly to IncA/C (8 plasmids), IncI1/ST23 (5) and IncI1/ST12 (3). The study demonstrates a widespread occurrence of E. coli with ESBL, AmpC and PMQR genes associated with clinically important multidrug-resistant clones and epidemic plasmids, in American crows.

Survey for Zoonotic Microsporidian Pathogens in Wild Living Urban Rooks (Corvus frugilegus).

Microsporidia are opportunistic pathogens in nature infecting all animal phyla. There is a potential risk of microsporidian spores transmission from urban rooks inhabiting some metropolitan cities to people through casual interactions. The aim of this study was to identify microsporidia species in the droppings of rooks in Wroclaw, Poland. A total of 15 collective sets of droppings were examined using nested-PCR method. Amplification of ITS rRNA gene revealed the presence of Enterocytozoon bieneusi D, Peru 6, and Encephalitozoon hellem 1A genotypes. This study indicates that excreta of urban rooks can be an important source of human infection with these pathogens.

Influence of Host Ecology and Behavior on Campylobacter jejuni Prevalence and Environmental Contamination Risk in a Synanthropic Wild Bird Species.

UNLABELLED: Campylobacter jejuni is a foodborne pathogen that often leads to human infections through the consumption of contaminated poultry. Wild birds may play a role in the transmission of C. jejuni by acting as reservoir hosts. Despite ample evidence that wild birds harbor C. jejuni, few studies have addressed the role of host ecology in transmission to domestic animals or humans. We tested the hypothesis that host social behavior and habitat play a major role in driving transmission risk. C. jejuni infection and host ecology were studied simultaneously in wild American crows (Corvus brachyrhynchos) in Davis, CA, over 3 years. We found that 178 of 337 samples tested were culture positive (53%), with infection varying by season and host age. Among adult crows, infection rates were highest during the winter, when migrants return and crows form large communal roosts. Nestlings had the highest risk of infection, and whole-genome sequencing supports the observation of direct transmission between nestlings. We deployed global positioning system (GPS) receivers to quantify habitat use by crows; space use was nonrandom, with crows preferentially occupying some habitats while avoiding others. This behavior drastically amplified the risk of environmental contamination from feces in specific locations. This study demonstrates that social behavior contributes to infection within species and that habitat use leads to a heterogeneous risk of cross-species transmission. IMPORTANCE: Campylobacter jejuni is the most common cause of gastroenteritis in industrialized countries. Despite efforts to reduce the colonization of poultry flocks and eventual infection of humans, the incidence of human C. jejuni infection has remained high. Because wild birds can harbor strains of C. jejuni that eventually infect humans, there has long been speculation that wild birds might act as an important reservoir in the C. jejuni infection cycle. We simultaneously studied infection prevalence, social behavior, and movement ecology in wild American crows (Corvus brachyrhynchos). We found that social behavior contributed to patterns of infection and that movement behavior resulted in some areas having a high risk of transmission while others had a low risk. The incorporation of ecological data into studies of C. jejuni in wild birds has the potential to resolve when and how wild birds contribute to domestic animal and human C. jejuni infection, leading to better control of initial poultry contamination.

Vancomycin resistant Enterococcus spp. from crows and their environment in metropolitan Washington State, USA: Is there a correlation between VRE positive crows and the environment?

Vancomycin-resistant enterococci [VRE] have been isolated from municipal, hospital and agricultural wastewater, recreational beaches, wild animals, birds and food animals around the world. In this study, American crows (Corvus brachyrhynchos) from sewage treatment plants (WWTP), dairy farms, and a large roost in a restored wetland with corresponding environmental samples were cultured for VRE. A total of 245 samples [156 crows, 89 environmental] were collected and screened for acquired vanA, vanB and/or intrinsic vanC1 genes. Samples were enriched overnight in BHI supplemented with 20µg/mL aztreonam, 4µg/mL vancomycin and plated on m-Enterococcus agar media supplemented with 6µg/mL vancomycin. Selected colonies were grown on BHI media supplemented with 18µg/mL vancomycin. Of these, 24.5% of the crow and 55% the environmental/cow samples were VRE positive as defined by Enterococcus spp. able to grow on media supplemented with 18µg/mL vancomycin. A total of 122 VRE isolates, 43 crow and 79 environmental isolates were screened, identified to species level using 16S sequencing and further characterized. Four vanA E. faecium and multiple vanC1 E. gallinarum were identified from crows isolated from three sites. E. faecium vanA and E. gallinarum vanC1 along with other Enterococcus spp. carrying vanA, vanB, vanC1 were isolated from three environments. All enterococci were multidrug resistant. Crows were more likely to carry vanA E. faecium than either the cow feces or wetland waters/soils. Comparing E. gallinarum vanC1 from crows and their environment would be useful in determining whether crows share VRE strains with their environment.

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.

High Prevalence and Genetic Diversity of Campylobacter jejuni in Wild Crows and Pigeons.

The occurrence, seasonal variation and genetic diversity of Campylobacter spp. in pigeons and crows over a 1-year period were evaluated. Campylobacter spp. were isolated from 166 (34.6 %) out of 480 wild bird faecal samples. The occurrence of Campylobacter spp. in faecal samples was higher among crows (39.2 %) than pigeons (30.0 %), (P < 0.05). Campylobacter jejuni was the most common species detected among wild bird faecal samples (98.2 %). Meanwhile, Campylobacter coli prevalence in wild bird faecal samples was low-6 %. The Simpson's diversity index of C. jejuni flaA RFLP types was lower in pigeons (D = 0.88) compared with C. jejuni isolates detected in crows (D = 0.97). Obtained results revealed that C. jejuni are widely prevalent among crows and pigeons, indicating these wild birds as potential infection sources to humans. Further studies are required to determine crows and pigeons role in zoonotic transmission of Campylobacter.

WEST NILE VIRUS-RELATED TRENDS IN AVIAN MORTALITY IN CALIFORNIA, USA, 2003-12.

West Nile virus (WNV) is an arbovirus transmitted enzootically by Culex mosquitoes among avian hosts. Since 2000, the California Dead Bird Surveillance Program (DBSP) has tracked avian mortality reported by the public on a telephone hotline and website and measured the prevalence of WNV infection in dead birds. We summarize herein WNV prevalence in dead birds tested and variation of WNV transmission over time and space with the use of DBSP data from 2003 to 2012. Prevalence among dead birds was highest in 2004, 2008, and 2012. This pattern was similar to peak WNV infection years for mosquitoes but not to human WNV incidence. Although American Crows (Corvus brachyrhynchos) were most frequently reported and tested, this species ranked third in infection prevalence (44%) after Yellow-billed Magpies (Pica nuttalli; 62%) and Western Scrub-Jays (Aphelocoma californica; 48%). Overall prevalence in American Robin (Turdus migratorius), House Finch (Haemorhous mexicanus), and House Sparrow (Passer domesticus) carcasses ranged from 18% to 22%. Corvid WNV prevalence was highest in South Coast, Bay/Delta, Sacramento, and San Joaquin valleys, and Klamath/North Coast bioregions, overlapping areas of elevated WNV activity in other surveillance measurements. Bioregional analysis revealed the avian species most likely to be reported and found positive in each bioregion. Our results may be useful to WNV surveillance and control efforts and provide insight into bird population trends in California.

Virulence characterization of Campylobacter jejuni isolated from resident wild birds in Tokachi area, Japan.

The prevalence of Campylobacter jejuni in wild birds is a potential hazard for human and animal health. The aim of this study was to establish the prevalence of C. jejuni in wild birds in Tokachi area, Hokkaido, Japan and investigate their virulence in vitro. In total, 173 cloacal swabs from individual wild birds were collected for the detection of Campylobacter spp. Thirty four samples (19.7%) were positive for Campylobacter of which 94.1% (32/34 samples) were C. jejuni. Additionally, one C. coli and one C. fetus were isolated. Seven C. jejuni isolates (one from crows and the other from pigeons) had important virulence genes including all three CDT genes (cdtA, cdtB and cdtC) and flaA, flaB, ciaB and cadF, and the other isolates were lacking cdtA gene. Further studies on in vitro virulence-associated phenotypes, such as motility assay on soft agar and invasion assay in Caco-2 cells, were performed. The wild bird C. jejuni isolates adhered and invaded human cells. Although the numbers of viable intracellular bacteria of wild bird isolates were lower than a type strain NCTC11168, they persisted at 48-hr and underwent replication in host cells.

Prevalence, characterization and antibiotic resistance of Salmonella isolates in large corvid species of europe and north America between 2010 and 2013.

It is well understood that Salmonella is carried by animals and in majority of cases as asymptomatic hosts. Surveillance efforts have focused on the role of agriculture and contamination points along the food chain as the main source of human infection; however, very little attention has been paid to the contribution of wildlife in the dissemination of Salmonella and what effect anthropogenic sources have on the circulation of antibiotic resistant Salmonella serovars in wildlife species. A purposive survey was taken of large corvids roosting yearly between November and March in Europe and North America. Two thousand and seven hundred and seventy-eight corvid faecal specimens from 11 countries were submitted for Salmonella spp. culture testing. Presumptive positive isolates were further serotyped, susceptibility tested and analysed for antibiotic resistance genes. Overall, 1.40% (39/2778) (CI = 1.01, 1.90) of samples were positive for Salmonella spp. Salmonella Enteritidis was the most prevalent serovar followed by S. Infantis, S. Montevideo and S. Typhimurium. No significant difference (P > 0.05) was found in the proportion of Salmonella recovered in Europe versus North America. The most variability of serovars within a site was in Kansas, USA with five different serovars recovered. European sites were significantly more likely to yield Salmonella resistant to more than one antibiotic (OR 71.5, P < 0.001, CI = 3.77, 1358) than North American sites, where no resistance was found. Resistance to nalidixic acid, a quinolone, was recovered in nine isolates from four serovars in four different sites across Europe. Large corvids contribute to the transmission and dissemination of Salmonella and resistance genes between human and animal populations and across great distances. This information adds to the knowledge base of zoonotic pathogen prevalence and antibiotic resistance ecology in wild birds.

Plasmid-mediated resistance to cephalosporins and fluoroquinolones in various Escherichia coli sequence types isolated from rooks wintering in Europe.

Extended-spectrum-beta-lactamase (ESBL)-producing, AmpC beta-lactamase-producing, and plasmid-mediated quinolone resistance (PMQR) gene-positive strains of Escherichia coli were investigated in wintering rooks (Corvus frugilegus) from eight European countries. Fecal samples (n = 1,073) from rooks wintering in the Czech Republic, France, Germany, Italy, Poland, Serbia, Spain, and Switzerland were examined. Resistant isolates obtained from selective cultivation were screened for ESBL, AmpC, and PMQR genes by PCR and sequencing. Pulsed-field gel electrophoresis and multilocus sequence typing were performed to reveal their clonal relatedness. In total, from the 1,073 samples, 152 (14%) cefotaxime-resistant E. coli isolates and 355 (33%) E. coli isolates with reduced susceptibility to ciprofloxacin were found. Eighty-two (54%) of these cefotaxime-resistant E. coli isolates carried the following ESBL genes: blaCTX-M-1 (n = 39 isolates), blaCTX-M-15 (n = 25), blaCTX-M-24 (n = 4), blaTEM-52 (n = 4), blaCTX-M-14 (n = 2), blaCTX-M-55 (n = 2), blaSHV-12 (n = 2), blaCTX-M-8 (n = 1), blaCTX-M-25 (n = 1), blaCTX-M-28 (n = 1), and an unspecified gene (n = 1). Forty-seven (31%) cefotaxime-resistant E. coli isolates carried the blaCMY-2 AmpC beta-lactamase gene. Sixty-two (17%) of the E. coli isolates with reduced susceptibility to ciprofloxacin were positive for the PMQR genes qnrS1 (n = 54), qnrB19 (n = 4), qnrS1 and qnrB19 (n = 2), qnrS2 (n = 1), and aac(6')-Ib-cr (n = 1). Eleven isolates from the Czech Republic (n = 8) and Serbia (n = 3) were identified to be CTX-M-15-producing E. coli clone B2-O25b-ST131 isolates. Ninety-one different sequence types (STs) among 191 ESBL-producing, AmpC-producing, and PMQR gene-positive E. coli isolates were determined, with ST58 (n = 15), ST10 (n = 14), and ST131 (n = 12) predominating. The widespread occurrence of highly diverse ESBL- and AmpC-producing and PMQR gene-positive E. coli isolates, including the clinically important multiresistant ST69, ST95, ST117, ST131, and ST405 clones, was demonstrated in rooks wintering in various European countries.

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.].