University of Washington ABSTRACT Whole Genome analysis of extraintestinal pathogenic Escherichia coli (ExPEC) isolated from the endangered Southern Resident Killer Whales (SRKW; Orcinus orca) Daira Melendez Chair of Supervisory Committee: Marilyn C. Roberts Department of Environmental and Occupational Health Sciences With current evidence of increased chemical contamination and antimicrobial resistance (AMR) in environmental samples from the Salish Sea, it is important to examine its resident wild life to further investigate the detrimental effects of these anthropogenic pressures. Surveillance of antibiotic resistant bacteria in marine mammals adds to our understanding of the health of the ecosystem, as well as the spread of AMR in these susceptible environments. To date, few studies have examined the prevalence of pathogenic microbes in wild cetacean species, specifically the critically endangered Southern Resident Killer Whales (SRKW; Orcinus orca). This population is known for their intimate connection to the Salish Sea, and act as sentinels for its vulnerable marine ecosystem. The objective of this study was to utilize next-generation sequencing tools to characterize and analyze nine Escherichia coli isolates collected from fecal samples of these critically at-risk orcas. For this study, we performed whole-genome sequencing (WGS) and de-novo assembly of each isolate to ascertain strain lineage by individual strain sequence type (ST), clonotype (C:H), antimicrobial resistance and virulence profile. By multi-locus sequence typing (MLST), all isolates belonged to either extraintestinal pathogenic E. coli (ExPEC) clonal lineage ST73 (8/9) or ST127 (1/9), lineages often associated with human and animal urinary tract infections. Clonotyping using fumC and fimH alleles showed further divergence in these clones with ST73 isolates belonging to the C24:H10 clade, and the ST127 to C14:H2. All eight ST73 clones carried multiple AMR genes including aadA, sul1 and tet(B), coding for resistance to aminoglycoside, sulfonamide, and tetracycline, respectively. Conjugative transfer of resistance tet(B) gene was observed for three of these nine isolates suggesting presence of mobile genetic elements. The ST127 isolate did not carry any resistance genes. Sixteen virulence-associated genes identified include: adhesins (iha, papC, sfaS), toxins (sat, vat, pic, hlyA, cnf1), siderophores (iroN, ireA, iutA, fyuA), serum survival/protectins (iss, ompT), capsule (kpsM), and pathogenicity island marker (malX). This study helps to advance our understanding of the dissemination of antimicrobial resistant E. coli in the Salish Sea, and demonstrates the need for increased surveillance efforts for any microbial factors potentially impacting the health of the SRKW. Additionally, it supports the use of next generation sequencing tools for increased and high-resolution insight into the genomic landscape of indicator bacteria for endangered species and other wildlife. Antimicrobial resistance is a global and emerging threat, affecting human, animal, and environment health. This study showed that the SRKW can carry antibiotic resistant, potentially pathogenic strains of E.coli. Possible sources include contamination of the orca’s environment and/or food. While it is unknown if these isolates cause disease in the SRKW which could contribute to the ongoing decline of this critically endangered population.