Phenotypic and genomic characterization of linezolid non-susceptibility in poultry-derived multidrug-resistant Enterococcus spp. from Hungary

Background Linezolid is a critically important oxazolidinone in human medicine, and the emergence of linezolid non-susceptibility in food-animal–associated enterococci raises One Health concerns across the food chain, animal reservoirs, and environmental circulation. Methods We analyzed poultry-derived Enterococcus spp. from healthy broiler chicken and turkey farms across seven Hungarian regions. From a total collection of 969 isolates, a resistance-enriched analytical subset ( n = 218) underwent broth microdilution testing; linezolid results were interpreted transparently using both clinical breakpoints (CLSI, Clinical and Laboratory Standards Institute) and epidemiological wild-type/non-wild-type classification (EUCAST, European Committee on Antimicrobial Susceptibility Testing), acknowledging their distinct intent and scope. Whole-genome sequencing was performed on a targeted subset ( n = 73) enriched for elevated or borderline linezolid minimum inhibitory concentrations to screen for transferable oxazolidinone resistance determinants and target-site variation. Results Linezolid minimum inhibitory concentrations ranged from 0.015 to 512 μg/mL, with a MIC 50 = 2 μg/mL and a 90th percentile (MIC 90 ) of 128 μg/mL. By clinical categorization, 139/218 (63.7%) isolates were susceptible, 37/218 (17.0%) intermediate, and 42/218 (19.3%) resistant, yielding 79/218 (36.2%) linezolid non-susceptible isolates in this enrichment-based dataset (not designed for prevalence inference). In the sequenced subset, we did not detect the major transferable oxazolidinone resistance genes optrA , poxtA , or cfr/cfr -like, while a 23S rRNA target-site substitution (G2576T) was identified in seven isolates, underscoring heterogeneous and sometimes non-transferable genomic correlates of elevated phenotypes. Conclusion Integrating phenotypic distributions with genome-informed screening provides a robust One Health framework to characterize linezolid non-susceptibility in poultry-derived multidrug-resistant Enterococcus , while highlighting the need for cautious inference under enrichment-based designs and for surveillance approaches that capture both canonical and under-resolved mechanisms.