Antimicrobial Susceptibility and Molecular Characterisation of <em>Escherichia coli</em> Strains Isolated from Raw Milk and Raw Milk Products in Ireland

Background/Objectives: Escherichia coli is a ubiquitous commensal organism in humans, animals, and the environment, but certain strains harbour virulence and antimicrobial resistance (AMR) determinants that can cause significant disease. Food-producing animals, including dairy cattle, may act as reservoirs for AMR E. coli, and raw milk and raw milk products can serve as potential exposure pathways to humans. However, data on the prevalence and genomic characteristics of AMR E. coli in raw milk in Ireland are limited. This study aimed to investigate the prevalence of commensal and clinically relevant AMR E. coli in raw milk and raw milk products in Ireland and to characterise their antimicrobial susceptibility and genetic characteristics. Methods: A total of 139 raw milk and raw milk product samples were collected and analysed for commensal E. coli and fluoroquinolone resistant, extended-spectrum β-lactamase (ESBL)/AmpC β-lactamase and carbapenemase producing E. coli. Antimicrobial resistance patterns were determined in line with EU surveillance guidelines based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines which use Minimum Inhibitory Concentration (MIC) breakpoints. Whole-genome sequencing (WGS) was conducted on selected isolates to identify AMR genes (ARG), virulence factors, plasmid replicons, efflux pump, disinfectant resistance genes, pathotypes, Multi Locus Sequence Types (MLST) and phylogenetic diversity. Results: Forty-seven E. coli isolates were recovered (33.8% isolation rate). Thirteen isolates exhibited resistance to between two and nine antimicrobials, with ten classified as multidrug resistant (MDR). The highest resistance frequencies were to ampicillin, sulfamethoxazole, trimethoprim and tetracycline. Four fluoroquinolone resistant isolates, one ESBL producer (blaCTX-M-3) and one AmpC promoter mutation were identified; no carbapenemase producers were detected. WGS revealed diverse sequence types, multiple virulence determinants, widespread pervasive efflux pump genes and limited presence of the disinfectant resistance gene qacEΔ1. Conclusions: Raw milk and raw milk products in Ireland can harbour antimicrobial-resistant E. coli, including MDR and potentially pathogenic strains, highlighting the need for ongoing surveillance within the dairy supply chain.