Phenotypic and Genomic Characterization of Vancomycin Non-Susceptibility in Multidrug-Resistant Enterococcus spp....

Background: Vancomycin is a critically important antimicrobial in human medicine, and vancomycin-non-susceptible enterococci represent a One Health concern when animal reservoirs contribute to the wider resistance ecology. We aimed to characterize vancomycin non-susceptibility among poultry-derived Enterococcus spp. from Hungary, using a combined phenotypic-genomic approach. Methods: Following a phenotypic pre-screen with antimicrobials authorized for poultry, 218 isolates with elevated minimum inhibitory concentrations (MICs) were selected for extended broth microdilution testing including vancomycin. Vancomycin susceptibility was interpreted using Clinical and Laboratory Standards Institute (CLSI) clinical breakpoints and European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off values (ECOFFs). Whole-genome sequencing was performed on a targeted multidrug resistant (MDR) subset (n = 42), enriched for elevated or borderline vancomycin MICs and stratified by region and host species (chicken, turkey), and resistance determinants were annotated against the Comprehensive Antibiotic Resistance Database (CARD) using stringent similarity/coverage thresholds. Results: Among the 218 pre-screened isolates (126 from chickens; 92 from turkeys), 196 (89.9%) met MDR criteria. For vancomycin, 15.6% of isolates were resistant and 9.2% intermediate by CLSI, while EUCAST ECOFF classification placed 34.9% in the non-wild-type group. The vancomycin MIC distribution was right shifted, with high-end MICs observed. In the sequenced subset, vancomycin-associated determinants consistent with the vanC pathway (including regulatory and auxiliary components) were detected in five isolates. Beyond vancomycin-related determinants, the WGS subset harbored common resistance genes consistent with the observed multidrug-resistant phenotypes. Conclusions: Vancomycin non-susceptibility was detected among pre-screened poultry-derived Enterococcus isolates in Hungary, and genomic analysis revealed vanC-associated and other peptide antibiotic resistance signatures. These findings support targeted One Health surveillance integrating MIC distributions with genomic resistance determinants in food animal reservoirs.