Staphylococcus aureus thrives at animal-human-environment interfaces. A large-scale work from our group indicated that antimicrobial resistance (AMR) in commensal S. aureus strains from wild ungulates is associated with agricultural land cover and livestock farming, raising the hypothesis that AMR genes in wildlife strains may originate from different hosts, namely via exchange of mobile genetic elements (MGE). In this work, we generate the largest available dataset of S. aureus draft genomes from wild ungulates in Portugal and explore their mobilome, which can determine important traits such as AMR, virulence, and host specificity, to understand MGE exchange. Core genome multi-locus sequence typing based on 98 newly generated draft genomes and 101 publicly available genomes from Portugal demonstrated that the genomic relatedness of S. aureus from wild ungulates assigned to livestock-associated sequence types (ST) is greater compared to wild ungulate isolates assigned to human-associated STs. Screening of host specificity determinants disclosed the unexpected presence in wildlife of the immune evasion cluster encoded in φSa3 prophage, described as a human-specific virulence determinant. Additionally, two plasmids, pAVX and pETB, previously associated with avian species and humans, respectively, and the Tn553 transposon were detected. Both pETB and Tn553 encode penicillin resistance through blaZ. Pangenome analysis of wild ungulate isolates shows a core genome fraction of 2133 genes, with isolates assigned to ST72 and ST3224 being distinguished from the remaining by MGEs, although there is no reported role of these in adaptation to wildlife. AMR related gene clusters found in the shell genome are directly linked to resistance against penicillin, macrolides, fosfomycin, and aminoglycosides, and they represent mobile ARGs. Altogether, our findings support epidemiological interactions of human and non-human hosts at interfaces, with MGE exchange, including AMR determinants, associated with putative indirect movements of S. aureus among human and wildlife hosts that might be bridged by livestock.
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