Antimicrobial resistant and extended‐spectrum β‐lactamase producing Escherichia coli in common wild bird species in Switzerland

Abstract A total of 294 fecal swabs from 294 wild birds in Switzerland were cultivated for antimicrobial resistant (AMR) Escherichia coli. Samples were also subcultivated to detect E. coli with extended‐spectrum β‐lactamases (ESBL), carbapenemases, and plasmid‐mediated aminoglycoside or colistin resistance, respectively. Samples from 17 (5.8%) of the birds contained 19 AMR E. coli, whereof 26.3% were multidrug resistant. Five (1.7%) ESBL‐producing E. coli were detected. The isolates harbored bla CTX‐M‐1 (two isolated from carrion crows and from one great spotted woodpecker, respectively), bla CTX‐M‐15 (originating from a grey heron), bla CTX‐M‐55 (from a carrion crow), and bla CTX‐M‐65 (from a common blackbird). Phylogenetic analysis assigned three isolates to commensal phylogroups A and B1, one to extraintestinal pathogenic group B2, and one to phylogroup F. Multilocus sequence typing identified sequence types (STs) that have been found previously in ESBL E. coli in wild birds (ST58, ST205, ST540). One isolate harboring bla CTX‐M‐55 was assigned to the recently emerged fluoroquinolone‐resistant, extraintestinal pathogenic E. coli clone ST1193. Wild birds have the potential to disperse AMR, including clinically important resistance genes, from anthropogenic‐influenced habitats to diverse areas, including vulnerable natural environments such as surface waters or mountain regions.

a feral pigeon yielded three E. coli strains with distinct resistance profiles (Table 1). Multidrug resistance (MDR), defined as resistance to three or more classes of antimicrobials was identified in 5 (26.3%) of the 19 resistant E. coli isolates, thus in 2% of the 256 isolated E. coli (Table 1).
The majority (13/76.5%) were nestlings, pulli, or juvenile birds (Table 1). Their AMR E. coli are likely acquired via parental transfer. Therefore, it cannot be excluded that the occurrence of AMR in adult birds, which are exposed to a diverse pool of AMR, may be higher than our data suggest.
Within the set of AMR E. coli, high rates of resistance to ampicillin (8/19 isolates), nalidixic acid ( By selective cultivation, five ESBL-producing E. coli were detected in samples from five (1.7%) of the 294 birds, including two carrion crows, a common blackbird, a great spotted woodpecker, and a grey heron (Table 2). Phylogenetic groups, multilocus sequence types (STs), and bla ESBL genes were determined by PCR and sequencing as described previously (Clermont, Christenson, Denamur, & Gordon, 2013;Wirth et al., 2006;Woodford, Fagan, & Ellington, 2006; ( Table 2). Two isolates belonging to commensal phylogroups A and B1 harbored bla CTX-M-1 . A further strain assigned to phylogroup B1 carried bla CTX-M-65 . One isolate belonged to extraintestinal pathogenic phylogenetic group B2 and carried bla CTX-M-55 . One isolate harboring bla CTX-M-15 was assigned to phylogenetic group F (Table 2).
This particular phylogroup is rarely identified, but of particular interest, since strains belonging to this group are associated with extraintestinal infections of companion animals and have been frequently detected in wild birds being treated in wildlife rehabilitation centers (Vangchhia et al., 2016). Overall, five different STs were detected among the ESBL E. coli ( Table 2). The majority of these, including ST58, ST205, ST540, and ST1722, have already been reported in ESBL-producing E. coli from waterfowl in Pakistan (Mohsin et al., 2017), gulls in Chile (Hernandez et al., 2013) Portugal (Guenther et al., 2011) andSweden (Atterby et al., 2017). Interestingly, E. coli China (Xia et al., 2017). Our results indicate that wild birds may contribute to the global dissemination of this important clonal lineage.
Using selective cultivation with colistin, two Enterobacter asburiae and one Enterobacter cancerogenus were found (data not shown).
Screening for plasmid-mediated colistin resistance genes mcr-1 through mcr-5 was undertaken by PCR (Rebelo et al., 2018), but TA B L E 1 Antimicrobial resistant Escherichia coli obtained by nonselective isolation from fecal swabs of wild birds birds frequently observed in urbanized areas, thus, the occurrence of these variants in wild birds is likely associated with proximity to human and livestock-associated settings, as well as exposure to contaminated surface waters (Guenther et al., 2012).
Reassuringly, bla ESBL genes were rare among the birds analyzed in this study, and no acquired carbapenemase genes, mcr genes, or transmissible aminoglycoside genes were detected. Our results contrast with data reported in a study that identified bla ESBL genes, the carbapenemase gene bla OXA-48 , and mcr in 8.7%, 0.8%, and 0.1%, respectively, of 28 species of wild birds in Spain (Oteo et al., 2018). However, caution should be used when comparing data obtained using different methods for screening for ESBL producers. In contrast to Oteo et al. Migratory birds in particular have been identified as potent disseminators of AMR, because they may cover long distances and impact diverse ecological niches by introducing new or emerging AMR (Agnew et al., 2015). Whilst the majority of the birds from this study were resident or short-distance migratory birds, they nonetheless have the potential to disperse AMR to diverse areas, including vulnerable environments such as surface waters or mountain regions.
Moreover, they can be considered indicators for the occurrence and distribution of AMR in the environment.

ACKNOWLEDG MENTS
We are grateful to the staff of the Swiss Ornithological Institute in Sempach, Switzerland for the collaboration. We thank Kira Schmitt for technical assistance and Marc Stevens for support in bioinformatics. This work was partly supported by the Swiss Federal Office of Public Health, Division Communicable Diseases.

CO N FLI C T O F I NTE R E S T S
None declared.

AUTH O R CO NTR I B UTI O N S
RS and BRV contributed to design of the study. PM performed sampling. KZ and PK performed laboratory analysis. KZ, SA, RS, BRV, and MN-I performed data analysis. KZ and MN-I drafted the manuscript. All authors contributed helpful comments and approved the final version of the manuscript.

E TH I C S S TATEM ENT
This study cobnformed to the legal requirements of Switzerland and in accordance with the guidelines of the Swiss Ornithological Institute.

DATA ACC E S S I B I L I T Y
All raw data generated during the current study are available from the corresponding author on reasonable request. Emergence and diversity of Salmonella enterica serovar Indiana isolates with concurrent resistance to ciprofloxacin and cefotaxime from patients and food-producing animals in China.