Fecal enterococci from wild roe deer in the Slovak area Zádiel: pathogenicity determinants without known antibiotic exposure

Wild ungulates live at the interface of natural ecosystems, livestock, and humans, yet their gut microbiota — and its capacity to harbor pathogenic bacteria — remains largely uncharacterized. This is especially true for enterococci, opportunistic pathogens whose virulence, biofilm-forming, and antimicrobial resistance traits are well studied in clinical and livestock settings but rarely examined in free-living wildlife. This study characterizes fecal enterococci from wild roe deer ( Capreolus capreolus ) to determine whether animals without known antibiotic exposure harbor enterococci carrying clinically relevant pathogenicity determinants. Twenty fecal samples were collected non-invasively in the Zádiel area of eastern Slovakia in spring 2024. Enterococci were isolated on M-Enterococcus agar and identified by MALDI-TOF MS. Twenty-five strains were taxonomically identified to the species level: Enterococcus mundtii ( n = 15), E. faecalis ( n = 9), and E. casseliflavus ( n = 1) — a composition dominated by plant- and environment-associated species. PCR screening detected multiple virulence-associated genes, with esp the most frequent (64%, predominantly in E. mundtii ), ccf and hyl (52% each), and gel and cpd (44% each); the cytolysin gene cylA was absent in all strains. All 25 strains exhibited a biofilm-positive phenotype on Congo red agar, and the biofilm-associated genes srtA , fsrA , bopD , ace , ica , and bap were widely distributed among them. The two dominant species differed markedly: 10 genes showed a significant species association (Fisher’s exact test with Benjamini–Hochberg correction, q < 0.05). Of these, eight ( gel , efaAfs , hyl , ccf , cpd , ace , bopD , srtA ) were enriched in E. faecalis and two ( esp, fsrA ) were enriched in E. mundtii , indicating two distinct genotypic signatures within the same host population. Resistance genes were also detected, including tetK (44%) and the vancomycin resistance gene vanA (12%), despite the absence of documented antibiotic exposure. Susceptibility testing was not performed, so these results reflect only gene carriage. These findings indicate that free-living roe deer harbor enterococci carrying determinants of pathogenicity, biofilm persistence, and antimicrobial resistance, and that wildlife warrants greater attention in studies of the environmental circulation of these traits. Therefore, continued monitoring of wild animal microbiota is warranted within a One Health framework.