Integrated Phenotypic and Genomic Characterization of Cefotaxime/Clavulanic Acid Inhibitor-Positive Multidrug-Resistant Escherichia coli from Large-Scale Pig Farms in Hungary.

Background: Extended-spectrum β-lactamase (ESBL)-producing E. coli are a major One Health concern because they compromise critically important cephalosporins and may spread via mobile genetic elements, including plasmids and transposon-associated modules, within food-animal production systems. Objectives: The aim of this study was to characterize cefotaxime (CTX)/clavulanic acid (CLA) inhibitor-positive phenotype profiles in pig-associated multidrug-resistant (MDR) E. coli and resolve their genetic basis using whole-genome sequencing, with emphasis on ESBL determinants and their predicted mobility context. Methods: MDR E. coli isolates (n = 203) from four large-scale pig farms in Hungary were tested by broth microdilution minimum inhibitory concentration (MIC) determination and Clinical and Laboratory Standards Institute (CLSI) inhibitor-based ESBL confirmation using cefotaxime with/without clavulanic acid. CTX/CLA inhibitor-positive isolates (inhibitor-positive phenotype) were subjected to whole-genome sequencing (WGS; n = 116) and resistome profiling; antimicrobial resistance genes (ARGs) were called against the Comprehensive Antibiotic Resistance Database (CARD) and mobility context was inferred using plasmid-origin and MGE-proximity prediction. Results: Overall, 127/203 isolates (62.6%) showed a CTX/CLA inhibitor-positive phenotype with a pronounced inhibitory effect (median cefotaxime/cefotaxime-clavulanate ratio: 33.3). In the sequenced subset (n = 116), 5427 ARG hits were identified (82 unique ARGs in the export), including frequent acquired determinants affecting tetracyclines, sulfonamides/trimethoprim, aminoglycosides, and phenicols; plasmid-mediated quinolone resistance (qnrB5) and mobilizable colistin resistance (mcr-1) were detected at low frequency. Classical β-lactamase genes were detected, including CTX-M (ESBL genes) and TEM alleles. CTX-M and/or TEM were detected in 47/116 genomes (40.5%), dominated by CTX-M-32 (11.2%) and TEM-1 (23.3%); detected ESBL determinants were predominantly predicted to be located on contigs predicted to be of plasmid origin, with a subset showing signatures consistent with transposition-associated mobilization. Conclusion: The high burden of inhibitor-positive phenotype, together with an inferred plasmid-/MGE-associated context for a subset of ESBL genes, and substantial phenotype-genotype heterogeneity, supports integrated phenotypic-genomic surveillance to refine AMR risk assessment and guide targeted stewardship and biosecurity interventions in pig production.