Virulome-resistome convergence in swine-associated multidrug-resistantfrom Hungary: virulence marker profiles and zoonotic potential.

Abstract

BACKGROUND: Swine production can sustain dense reservoirs of multidrug-resistant (MDR)in which mobilizable resistance may co-occur with virulence modules relevant to animal health and One Health risk. We profiled the virulome of Hungarian swine-associated MDRand assessed whether key virulence signatures co-segregate with selected high-impact resistance flags. METHODS: In late 2023,isolates were obtained from four large-scale Hungarian pig farms through routine veterinary diagnostic/surveillance sampling. Phenotypic antimicrobial susceptibility testing was performed for the full collection ( = 203), including extended-spectrum-lactamase (ESBL) confirmation ( = 127). A whole-genome sequencing (WGS) subset ( = 116) underwent Illumina sequencing andassembly. Virulence genes were identified in silico using curated virulence databases with harmonized identity/coverage thresholds and summarized as gene prevalence and functional modules. Marker-based definitions were applied for extraintestinal pathogenic(ExPEC)-like and diarrheagenic(DEC)-like signatures. Acquired antimicrobial resistance genes were annotated using CARD/RGI, and focused analyses consideredextended-spectrum beta-lactamase (ESBL) genes and rare, high-consequence determinants (,). Associations between virulence markers and resistance flags were tested using Fisher's exact test. Rather than performing a comprehensive resistome analysis, we focused on selected high-impact resistance determinants (,,) and their co-carriage with virulence markers. RESULTS: Across 116 genomes, 208 distinct virulence-associated genes were detected; virulence gene load per genome was heterogeneous. Extraintestinal-associated iron acquisition modules were common, including aerobactin (/) in 31/116 (26.7%), yersiniabactin () in 27/116 (23.3%), and salmochelin () in 17/116 (14.7%). Toxin-associated determinants were frequent, within 33/116 (28.4%). DEC markers occurred in 33/116 (28.4%), includingin 23/116 (19.8%),in 6/116 (5.2%), predominantly enterohemorrhagic(EHEC)-like (and) profiles (5/116; 4.3%) andin 10/116 (8.6%);was present in 27/116 (23.3%). Stringent ExPEC-like criteria were met by 6/116 (5.2%), while 15/116 (12.9%) showed convergent profiles co-carrying aerobactin with at least one DEC marker.genes were detected in 24/116 (20.7%) and were enriched among aerobactin-positive isolates and DEC-marker-positive isolates; all EHEC-like isolates carried. Rare but critical determinants included(3/116; 2.6%) and(2/116; 1.7%). CONCLUSION: Hungarian swine-associated MDRshow virulome heterogeneity with frequent aerobactin/toxin modules and DEC markers.enrichment in these profiles indicates virulome-resistome convergence, supporting integrated One Health surveillance.