Background: Multidrug-resistant (MDR) Escherichia coli in intensive pig production represents a persistent animal health and One Health concern. Here, we integrated quantitative phenotypic susceptibility data with whole-genome sequencing (WGS) to characterize the resistome and its inferred genomic context (chromosomal vs. plasmid-predicted contigs and mobile genetic element (MGE)-proximal regions) in swine-associated MDR E. coli from Hungary. Methods: A total of 203 E. coli isolates from large-scale pig farms were tested by broth microdilution. Based on resistance-oriented screening from an extended-spectrum β-lactamase (ESBL)-screen-positive pool, 116 isolates were subjected to whole-genome sequencing (WGS) as a resistance-enriched subset. Resistance determinants were annotated using the Comprehensive Antibiotic Resistance Database (CARD). Results: Resistance-oriented screening indicated frequent β-lactamase activity and ESBL screening positivity (110/203 and 127/203 isolates, respectively), consistent with strong antimicrobial selection pressure in the source population. Across the full phenotypic panel, 78/203 isolates (38.4%) met the MDR definition (non-susceptible to ≥3 antimicrobial classes), with marked between-farm variation (p < 0.001) but no age-group effect (p = 0.75). Non-β-lactam minimum inhibitory concentration (MIC) distributions showed pronounced, site-dependent high-MIC “tails”, most notably for tetracyclines, trimethoprim–sulfamethoxazole, fluoroquinolones, and colistin. In the WGS cohort (n = 116), we detected 82 distinct resistance determinants (5433 total occurrences), featuring a conserved chromosomal backbone enriched for intrinsic multidrug resistance components and lipid A modification pathways, alongside common plasmid- and MGE-associated acquired ARG modules involving tetracycline (tetA/tetB), sulfonamide/trimethoprim (sul/dfrA), aminoglycoside-modifying enzymes, and phenicol determinants (floR/cat). High-priority mobile determinants were rare but present, including mcr-1 (3/116; plasmid-associated) and plasmid-mediated quinolone resistance qnrB5 (2/116). Conclusions: Importantly, mobility/context inferences are restricted to this ESBL-screen-enriched WGS subset. Swine-associated E. coli from Hungarian large-scale farms harbors complex resistance architectures shaped by co-selection of mobile ARG modules on top of a pervasive chromosomal resistance backbone. Mobility-aware surveillance and stewardship are warranted to mitigate dissemination risks at the animal–environment–human interface.

Background: Public catering is an underexplored One Health interface where structurally complex food-transport equipment may sustain reservoirs of antimicrobial-resistant bacteria. We investigated Escherichia coli from reusable institutional catering food-transport containers, focusing on a difficult-to-clean pressure-relief/ventilation valve compartment. Our objectives were to quantify phenotypic resistance using applied clinical breakpoints, assess inhibitor-synergy outcomes in ESBL confirmatory testing, and contextualize inhibitor-positive isolates by whole-genome sequencing (WGS). Methods: E. coli was isolated from containers sourced from 17 institutions and three central kitchens using ISO 16649-2. Minimum inhibitory concentrations (MICs) were determined by broth microdilution. Extended-spectrum β-lactamase (ESBL) confirmatory testing used cefotaxime/ceftazidime ± clavulanate; inhibitor positivity was defined as a ≥3 two-fold MIC decrease in the presence of clavulanate in isolates meeting CLSI screening thresholds. Inhibitor-positive isolates underwent WGS and CARD-based resistome profiling. Results: Resistance was most frequent to colistin (10, 10.8%), followed by doxycycline (8, 8.6%), florfenicol (7, 7.5%), enrofloxacin (4, 4.3%), and gentamicin (3, 3.2%). Third-generation cephalosporin resistance by clinical breakpoints was uncommon (cefotaxime: 2, 2.2%; ceftazidime: 1, 1.1%). Inhibitor-positive ESBL confirmatory phenotypes occurred in 30 isolates (32.3%), which were sequenced. WGS identified 45 resistance-associated genes across inhibitor-positive isolates but detected no classical ESBL genes; all carried chromosomal ampC/ampH alongside ubiquitous efflux-associated determinants. All WGS isolates belonged to phylogroup A, with serotype O154:H9 (20, 66.7%) and ST5549 (17, 56.7%) predominating. Conclusions: Institutional catering food-transport containers can harbor AMR E. coli, with colistin as the most frequent resistance phenotype and frequent inhibitor-positive ESBL confirmatory profiles that, in this set, were not explained by classical ESBL gene carriage. Integrating phenotype, WGS resistomics, and lineage structure supports targeted hygiene surveillance and risk-informed One Health monitoring in mass catering systems.

Background/Objectives: Probiotic feed additives are increasingly used in livestock production as antimicrobial-sparing tools, yet viable microbial products should not introduce clinically relevant antimicrobial resistance genes (ARGs) into the intestinal resistome. This study evaluated farm-animal probiotic products using an integrated phenotypic, metagenomic and mobilome-aware safety framework. Methods: Seven commercially available products intended for poultry, pigs, cattle or horses were assessed using product metadata, culture-based recovery, broth microdilution minimum inhibitory concentration (MIC) profiling and Illumina short-read sequencing as a screening-level resistome approach. Reads were quality controlled, assembled, screened using the Comprehensive Antibiotic Research Database (CARD)/Resistance Gene Identifier (RGI) workflow and interrogated for plasmid-, phage- and insertion sequence/mobile genetic element-associated genomic context. Results: MIC profiles were generated for viable bacterial isolates representing Enterococcus faecium, Pediococcus acidilactici, Pediococcus pentosaceus and Bacillus subtilis. One labelled Lactobacillus plantarum component was not recovered as viable culture, and one labelled P. acidilactici component was recorded as P. pentosaceus. Sequencing-based resistome screening identified 30 antimicrobial resistance (AMR)-associated CARD antibiotic-resistant organism (ARO) hits belonging to 13 determinants across six ARG-positive coded products, while one coded product had no retained CARD/RGI hit. Profiles were dominated by recurrent Enterococcus-associated background determinants, including aac(6′)-Ii, msrC and eatAv. Plasmid prediction was positive for five hits, whereas no iMGE- or phage-associated ARG context was detected. No vanA/vanB, mcr, optrA, poxtA, cfr, extended-spectrum β-lactamase (ESBL) or carbapenemase gene was detected. Conclusions: The investigated products did not show evidence of high-priority mobile ARG carriage. Nevertheless, AMR-associated determinants and occasional predicted mobile contexts support routine integrated MIC-sequencing-based resistome–mobilome assessment of veterinary probiotic products. Because short-read assemblies do not fully resolve plasmid architecture or transferability, mobile-context predictions should be considered screening-level indicators requiring confirmatory long-read or functional testing for higher-priority findings.

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.

Background/Objectives: Regional antimicrobial-resistance surveillance can be strengthened by linking phenotypic susceptibility data with genomic evidence. We characterized chicken-associated commensal bacteria from Northern Hungary and examined resistance-gene evidence in available Escherichia coli assemblies. Methods: Broth-microdilution minimum inhibitory concentration (MIC) data were available for 166 bacterial isolates collected in 2022–2023. Among the 69 E. coli isolates in this collection, 24 had corresponding draft whole-genome assemblies, defined here as assembled contig sets that had not been resolved into closed genomes; these assemblies were linked to the MIC records of the same isolates. Analyses included MIC distributions, descriptive correlations, principal component analysis, assembly-quality assessment, resistance-gene profiling, and sequence-context analysis. Results: The collection comprised 26 Staphylococcus spp., 71 Enterococcus spp., and 69 E. coli isolates. In E. coli, ceftriaxone MIC₅₀ and MIC₉₀ values were 0.031 and 512 µg/mL, respectively. Five assemblies contained bla_CTX-M-1-labelled intervals that were identical across 876 bp and corresponded to ceftriaxone MICs of 32–1024 µg/mL. In E. coli isolate 1316, seven resistance-gene calls co-occurred within a 12,933-bp region of one contig. Assembly quality varied substantially, and the draft assembly of E. coli isolate 832 was unsuitable for reliable gene-negative interpretation. Conclusions: The dataset showed heterogeneous susceptibility profiles and identifiable resistance-gene contexts. These findings support isolate-linked phenotypic and genomic surveillance while avoiding population-level, transmission, or clinical resistance classifications unsupported by the available data.

Background: The surveillance of non-clinical bacteria from food-producing animals can reveal antimicrobial selection patterns without conditioning sampling on clinical disease, but categorical resistance estimates are difficult to compare when organism-specific breakpoints are incomplete. We aimed to characterize antimicrobial MIC distributions in chicken-associated Staphylococcus spp., Enterococcus spp., and Escherichia coli from Central Hungary and to determine whether E. coli MIC profiles formed structured covariance networks. Methods: The dataset comprised 133 isolates collected in 2022–2023 (28 Staphylococcus spp., 59 Enterococcus spp., and 46 E. coli). MIC distributions were summarized using MIC₅₀/MIC₉₀, and E. coli MICs were analyzed by Spearman correlation with Benjamini–Hochberg correction. Historical threshold classification was retained only as a sensitivity analysis, and nine E. coli genomes provided contextual genomic information. Results: Staphylococcus spp. showed lower amoxicillin MIC₅₀/MIC₉₀ values (0.25/1 µg/mL) than Enterococcus spp. (1/256 µg/mL), illustrating marked cross-genus differences. In E. coli, MIC₅₀/MIC₉₀ values included 16/512 µg/mL for ceftriaxone, 128/256 µg/mL for neomycin, 32/128 µg/mL for enrofloxacin, and 32/>512 µg/mL for trimethoprim–sulfamethoxazole. Ten associations met |ρ| ≥ 0.40 and adjusted q < 0.05, forming two connected components that persisted after sensitivity analyses for upper-bound accumulation and the amoxicillin–amoxicillin/clavulanic acid relationship. The nine genomes showed heterogeneous resistance-associated gene calls. Conclusions: Continuous MIC analysis revealed phenotypic covariance not captured by categorical summaries alone and identified region-specific targets for stewardship-oriented follow-up. Inference remains isolate-level rather than farm-adjusted, and the genomic panel is not representative of the phenotypic cohort; longitudinal sampling linked to antimicrobial-use data and larger matched phenotype–genotype datasets are therefore warranted.