# Integrated Phenotypic and Genomic Profiling of Antimicrobial Resistance and Virulence-Associated Determinants in Poultry-Derived Enterococcus spp. from Hungary

## TL;DR

This study examines how poultry-derived Enterococcus bacteria carry genes linked to antibiotic resistance and virulence, highlighting their potential role in public health and the need for targeted surveillance.

## Contribution

The study provides an integrated analysis of virulence and resistance genes in poultry-derived Enterococcus, emphasizing species-specific patterns and genotype-phenotype discrepancies.

## Key findings

- E. faecalis isolates showed a broader range of virulence genes compared to E. faecium.
- Acquired resistance genes were common and aligned with antimicrobial use in food production.
- Genotype-phenotype discrepancies were observed, suggesting other mechanisms influence resistance.

## Abstract

Enterococci are common gut bacteria in animals and humans, but some lineages can also cause difficult-to-treat infections. Their public health relevance increases when antimicrobial resistance and virulence-associated traits co-occur, because such combinations may support persistence, colonization, and onward dissemination across the animal–food–environment–human interface. Here, we investigated poultry-derived Enterococcus isolates using paired phenotypic microdilution susceptibility testing and whole-genome sequencing for a defined subset. We focused on two complementary genomic layers: (i) acquired antimicrobial resistance genes (resistome) and (ii) virulence-associated determinants (virulome). Analyses were performed in a species-stratified manner because Enterococcus faecalis and Enterococcus faecium differ in their typical virulence repertoires and population structures. In the sequenced subset, E. faecalis carried a broad set of virulence-associated genes linked to adhesion and biofilm-related functions, whereas E. faecium showed a more limited high-confidence virulence gene repertoire. Acquired resistance determinants were common and supported the observed multidrug non-susceptibility patterns. Importantly, we interpret virulence genes as genetic potential rather than proof of pathogenicity, because gene presence does not imply expression or clinical disease. Overall, our results highlight that poultry-associated enterococci can co-harbor determinants relevant to persistence and antimicrobial resistance, supporting their use as One Health surveillance indicators and motivating targeted functional follow-up where genotype–phenotype discordance is observed.

Background: Poultry-associated Enterococcus spp. are widespread commensals but may serve as One Health indicators when virulence-associated determinants and antimicrobial resistance co-occur. We characterized paired phenotypic and genomic profiles to delineate species-stratified virulome and resistome patterns. Methods: Isolates originated from a previously established poultry collection with MIC testing. Genotype–phenotype analyses were restricted to the whole-genome sequenced subset (n = 31). The acquired antimicrobial resistance genes were identified using the Comprehensive Antibiotic Resistance Database (CARD), and virulence-associated determinants were screened using the Virulence Factors Database (VFDB). Results were summarized as isolate-level presence/absence matrices and integrated with MIC-derived susceptible/intermediate/resistant categories. Results: The WGS subset comprised E. faecalis (n = 23) and E. faecium (n = 8) with diverse sequence types. Virulome architecture was strongly species-dependent: E. faecalis carried a broad repertoire of adhesion/biofilm-associated determinants, whereas E. faecium showed a limited set of high-confidence virulence-associated hits. Acquired resistance determinants were common across isolates, and resistome profiles displayed structured co-occurrence. Integrated analyses suggested only a modest overall association between virulence-gene burden and acquired resistome size, largely driven by species-level differences. Genotype–phenotype concordance was class-dependent, with incomplete alignment in several antimicrobial classes, consistent with mechanisms beyond the screened acquired gene set. The acquired resistance determinants detected in the WGS subset predominantly mapped to antimicrobial classes commonly used in food-producing animals (e.g., tetracyclines, macrolides, lincosamides, aminoglycosides, and phenicols), supporting interpretation in the context of production-associated antimicrobial selection rather than implying last-line clinical resistance by default. Conclusions: Poultry-derived enterococci may combine genetic features compatible with persistence/colonization and acquired antimicrobial resistance, with co-occurrence patterns shaped primarily by species/lineage background. These findings support risk-stratified One Health surveillance and targeted functional and mechanism-focused follow-up. This integrated virulome–resistome view highlights species-specific risk signatures in poultry-associated Enterococcus and identifies discordant high-level phenotypes that merit targeted mechanistic follow-up.