Identification of BSBL reservoirs and risk factors associated with BSBL inter-sectorial transfer will be important to control further selection and dissemination of BSBL producing bacteria. However, not much is known on the presence of BSBLs in zoos in Belgium. In this study, we investigated the faecal carriage of BSBL producing Enterobacteriaceae in zoo animals from 2 Belgian zoos. Presence of resistance encoding genes and clonality of BSBL producing isolates were investigated using whole genome sequencing and phylogenetic analysis.Fresh faecal droppings were collected from 38 various terrestrial mammals residing in two different zoos in Belgium. In total, thirty-five ceftiofur resistant isolates were obtained from faecal samples of 20 zoo mammals. The majority of the isolates belonged to the Enterobacteriaceae family (80%, 28/35), namely E. coli (93%, 26/28). Potential ESBL and AmpC producers were identified in 84.6% and 15.4% of the E. coli isolates (22/26 and 4/26, resp.). By using PCR, all E. coli isolates showed presence of bla gene(s), namely: 69.2% isolates carried the blaCTX-M-type gene (18/26), 26.9% isolates the blaTEM gene (7/26) and one isolate showed presence of both the blaCTX-M-type gene and the blaTEM gene. Sequence analysis revealed the following ESBLs: CTX-M-1 (n=19), TEM-1 (n=7) and TEM-214 (n=1). No blaCMY genes were found, despite presence of phenotypic AmpC producers. Similar results were obtained when analyzing the resistome of the E. coli isolates using CARD and Resfinder. While the presence of bla-genes detected by PCR was confirmed, additional bla-genes were found, namely: ampC (all isolates, except for isolate µ), DHA-1 (isolates E1-4), CTX-M-3 (isolates T1-3) and CTX-M-61 (isolate γ2). In general, the observed phenotypic resistance towards tetracyclines, fluoroquinolones, aminoglycosides, trimethoprim and/or sulphonamids could be linked with the presence of acquired resistance genes. Furthermore, acquired resistance genes towards macrolides, peptide antibiotics and elfamycins were detected in all E. coli isolates, as well as multiple genes encoding multidrug efflux pumps. All E. coli isolates showed presence of plasmids, but these encoded no antimicrobial resistance genes.
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