Molecular epidemiology of extended-spectrum beta-lactamase-producing- species in East Tennessee dairy cattle farms.

Introduction: The rising prevalence of Extended-Spectrum Beta-Lactamase (ESBL)-producing species (spp.) poses a significant threat to human and animal health and environmental safety. To address this pressing issue, a comprehensive study was undertaken to elucidate the burden and dissemination mechanisms of ESBL- spp. in dairy cattle farms.

Methods: Fifty-seven species were isolated on CHROMagar™ ESBL plates and confirmed with MADLI-TOF MS and whole genome sequenced from 14 dairy farms.

Results And Discussion: Six families of beta-lactamase () ( , , , , and ) were detected in ESBL- spp. genomes. Most (73%) of isolates had the first three types of beta-lactamase genes, with being the most frequent, followed by . Most (93%) isolates harbored two or more bla genes. The isolates were genotypically MDR, with 26 distinct types of antibiotic resistance genes (ARGs) and point mutations in , , and genes. The genomes also harbored 22 different plasmid replicon types, including three novel IncFII. The IncFII and Col440I plasmids were the most frequent and were associated with and genes, respectively. Eighteen distinct sequence types (STs), including eight isolates with novel STs of , were detected. The most frequently occurring STs were ST353 ( = 8), ST469 ( = 6), and the novel ST7501 ( = 6). Clusters of ESBL- strains with identical STs, plasmids, and ARGs were detected in multiple farms, suggesting possible clonal expansion. The same ESBL variant was linked to identical plasmids in different STs in some farms, suggesting horizontal spread of the resistance gene. The high burden and dual spread mechanism of ESBL genes in species, combined with the emergence of novel sequence types, could swiftly increase the prevalence of ESBL- spp., posing significant risks to human, animal, and environmental health. Immediate action is needed to implement rigorous surveillance and control measures to mitigate this risk.