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

## TL;DR

This study combines phenotypic and genomic analysis to understand resistance in E. coli from Hungarian pig farms, revealing complex resistance patterns and mobile genetic elements.

## Contribution

The study integrates phenotypic testing with whole-genome sequencing to uncover the genetic basis of resistance in E. coli from pig farms.

## Key findings

- 62.6% of isolates showed a strong clavulanate inhibitory effect.
- 40.5% of sequenced isolates contained classical ESBL genes like CTX-M and TEM, often on plasmid-like contigs.
- Many inhibitor-positive isolates lacked classical ESBL genes, indicating diverse resistance mechanisms.

## Abstract

Antimicrobial resistance is a growing One Health concern because resistant bacteria and their resistance genes can circulate between animals, humans, and the environment. In pig production, E. coli is a key indicator organism and an important reservoir for resistance traits, including extended-spectrum β-lactamases (ESBLs) that reduce the effectiveness of critically important cephalosporins. Here, we investigated multidrug-resistant E. coli from large-scale pig farms in Hungary using standardized phenotypic testing and whole-genome sequencing to clarify the genetic basis of cefotaxime (CTX)/clavulanic acid (CLA) inhibitor-positive phenotypes. Among 203 isolates, 62.6% showed a CTX/CLA inhibitor-positive phenotype with a strong clavulanate inhibitory effect. Whole-genome sequencing of 116 CTX/CLA inhibitor-positive isolates detected classical ESBL-associated β-lactamase gene families (CTX-M and/or TEM) in 40.5% of genomes, and these determinants were predominantly predicted to be located on contigs of putative plasmid origin, suggesting a potentially mobile genetic context, although horizontal transfer was not demonstrated in this study. Notably, many isolates with a CTX/CLA inhibitor-positive phenotype lacked these classical ESBL gene calls, highlighting that inhibitor-based phenotypes can reflect heterogeneous and complex resistance architectures. Our findings underscore the value of combining phenotypic methods with genome-resolved interpretation to strengthen AMR surveillance and to support targeted antimicrobial stewardship and biosecurity interventions in pig production.

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.