BackgroundLinezolid is a critically important oxazolidinone antibiotic used in human medicine. Although linezolid is not licensed for use in food-producing animals, the use of florfenicol in veterinary medicine co-selects for oxazolidinone resistance genes.ObjectiveThis study aimed to assess the occurrence ofcfr, optrA, andpoxtAin florfenicol-resistant isolates from beef cattle and veal calves from different herds in Switzerland.MethodsA total of 618 cecal samples taken from beef cattle and veal calves at slaughter originating from 199 herds were cultured after an enrichment step on a selective medium containing 10 mg/L florfenicol. Isolates were screened by PCR forcfr, optrA, andpoxtAwhich are genes known to confer resistance to oxazolidinones and phenicols. One isolate per PCR-positive species and herd was selected for antimicrobial susceptibility testing (AST) and whole-genome sequencing (WGS).ResultsOverall, 105 florfenicol-resistant isolates were obtained from 99 (16%) of the samples, corresponding to 4% of the beef cattle herds and 24% of the veal calf herds. Screening by PCR revealed the presence ofoptrAin 95 (90%) andpoxtAin 22 (21%) of the isolates. None of the isolates containedcfr. Isolates included for AST and WGS analysis wereEnterococcus(E.)faecalis(n= 14),E. faecium(n= 12),E. dispar(n= 1),E. durans(n= 2),E. gallinarum(n= 1),Vagococcus(V.)lutrae(n= 2),Aerococcus(A.)urinaeequi(n= 1), andCompanilactobacillus(C.)farciminis(n= 1). Thirteen isolates exhibited phenotypic linezolid resistance. Three novel OptrA variants were identified. Multilocus sequence typing identified fourE. faeciumST18 belonging to hospital-associated clade A1. There was a difference in the replicon profile amongoptrA-andpoxtA-harboring plasmids, with rep9 (RepA_N) plasmids dominating inoptrA-harboringE. faecalisand rep2 (Inc18) and rep29 (Rep_3) plasmids inpoxtA-carryingE. faecium.ConclusionBeef cattle and veal calves are reservoirs for enterococci with acquired linezolid resistance genesoptrAandpoxtA. The presence ofE. faeciumST18 highlights the zoonotic potential of some bovine isolates. The dispersal of clinically relevant oxazolidinone resistance genes throughout a wide variety of species includingEnterococcusspp.,V. lutrae, A. urinaeequi, and the probioticC. farciminisin food-producing animals is a public health concern.