Alexandria Vingino

Project title: Surveillance for antibiotic resistant E. coli in the Salish Sea ecosystem and surrounding regions

Degree: MPH | Program: One Health (ONE) | Project type: Thesis/Dissertation
Completed in: 2021 | Faculty advisor: Marilyn C. Roberts


Antimicrobial resistant bacteria (ARB) can be shared between humans and animals through a common environment. The surveillance of ARB in the environment can inform us about contamination of shared ecosystems, like the Salish Sea, and how that contamination affects both the animals that rely on the ecosystem and humans who also live within it. Researchers detect ARB and their antibiotic resistance genes (ARG) by sampling animals and their environment. With this information, researchers can draw conclusions on the routes of transmission and plan interventions to reduce the spread of ARB. The objective of this study was to take a One Health approach to identifying E. coli in the Salish Sea assessing whether they were phenotypically and genotypically resistant to antibiotics and if so, which resistance genes they carried. The E. coli were isolated from samples of marine water, marine animals from the Salish Sea, river otters in a freshwater river watershed, and freshwater sources. The isolates were analyzed using both antimicrobial susceptibility testing and whole-genome sequencing (WGS). We collected marine water samples from North Puget Sound, Central Puget Sound, South Puget Sound, and the Strait of Juan de Fuca, freshwater from streams near marine beaches, and fecal samples from harbor porpoises (Phocoena phocoena), harbor seals (Phoca vituline), river otters (Lontra canadensis), and English sole (Parophrys vetulus). We isolated 551 E. coli colonies and characterized 305 isolates: 212 from marine water, 5 from freshwater, 3 from marine water by beaches, 52 from harbor seals, 7 from harbor porpoises, 24 from river otters, and 2 from English sole. Isolates were analyzed using minimum inhibitory concentrations (MIC): 20 (6.6%) were intermediate and 31 (10.2%) resistant to > 1 class of antibiotics. Whole genome sequence, sequence type (ST), resistance genes, and virulence factors were determined from sequence data. Using multilocus sequence typing (MLST), a total of 196 unique STs were identified. This included extra-intestinal pathogenic of E. coli (ExPEC)-associated STs (ST10, ST38, ST58, ST69, ST73, ST117, ST131, and ST405), which were identified in 37 isolates. The most commonly occurring ST was ExPEC-associated type, ST10 (n=12), and the least common ExPEC ST was ST405 (n=1). ResFinder was used to identify the genotypic antibiotic profile of resistant and intermediate E. coli isolates. Correlation between resistance phenotype and genotype varied by specific antibiotic. Isolates that were intermediate and resistant to tetracyclines were found to have the best correlation, with all 16 phenotypically resistant/intermediate isolates carrying either tet(A) or tet(B) or both tet genes. Intermediate and resistant isolates (n=51) were subjected to VirulenceFinder analysis for virulence factor characterization; gad (glutamate decarboxylase) was the most commonly identified virulence factor, appearing in 68% of isolates (n=35). This study found that in marine water only 7% of the E. coli were non-susceptible (resistant or intermediate resistant) to antibiotics. In contrast, non-susceptible E. coli accounted for 26.9% of the isolates from marine mammals and 70% of the E. coli from river otter stools, making them potential sentinels for antibiotic-resistant E. coli in the Salish Sea and other ecosystems. Monitoring of the marine mammal microbiome may lead to information about how antimicrobial resistant genes (AMR) persist in the local ecosystems.