Project Summary Antimicrobial resistance is a major threat to public health worldwide. The Centers for Disease Control and Prevention has identified extended spectrum beta lactamase-producing Enterobacterales (ESBL-E) as a serious threat to public health, and these organisms cause clinical infections in humans and a variety of veterinary species, including dogs and cats. Therefore, it is essential to adopt a “One Health” approach to understand and combat antimicrobial resistance. In addition, fecal carriage of the same strain of ESBL-E. coli has been demonstrated to be shared between humans and pets in the same household. However, the relationship between clinical isolates of ESBL-E isolated from infections in pets and people on a population level is unclear. The aims of this study are to: 1) expand capacity of Virginia Tech Animal Laboratory Services to perform whole genome sequencing and sequence analysis, and 2) determine the relatedness of ESBL-E isolated from both of human and companion animal infections isolated over a similar time-period and in the same geographic locations by genome sequence analysis. We have identified 60 clinical ESBL-E isolated between 2018 and 2021 from humans and companion animals in southwestern Virginia and have partially characterized those isolates by identifying ESBL genes. We propose to perform short-read whole genome sequencing (WGS) on those isolates at Virginia Tech Animal Laboratory Services in order to improve our laboratory’s capacity to perform and interpret WGS. Sequencing data will be evaluated for resistance gene and multilocus sequence (MLST) analysis. Isolates of the same MSLT type will undergo single-nucleotide polymorphism analysis to determine the relatedness of the organisms. Monitoring of regionally-specific population dynamics of drug-resistant pathogens allows for implementation of locally-relevant mitigation strategies that improve animal and human health, thereby directly supporting the Vet-LIRN mission to safeguard human and animal health and to increase our laboratory’s capacity to respond to Vet-LIRN investigations and enhance the safety of human and animal foods.