Escherichia coli contain a high level of genetic diversity and are generally associated with the guts of warm-blooded animals but have also been isolated from secondary habitats outside hosts. We used E. coli isolates from previous in situ microcosm experiments conducted under actual beach conditions and performed population-level genomic analysis to identify accessory genes associated with survival within the beach sand environment. E. coli strains capable of surviving had been selected for by seeding isolates originating from sand, sewage, and gull waste (n = 528; 176 from each source) into sand, which was sealed in microcosm chambers and buried for 45 days in the backshore beach of Lake Michigan. In the current work, survival-associated genes were identified by comparing the pangenome of viable E. coli populations at the end of the microcosm experiment with the original isolate collection and identifying loci enriched in the out put samples. We found that environmental survival was associated with a wide variety of genetic factors, with the majority corresponding to metabolism enzymes and transport proteins. Of the 414 unique functions identified, most were present across E. coli phylogroups, except B2 which is often associated with human pathogens. Gene modules that were enriched in surviving populations included a betaine biosynthesis pathway, which produces an osmoprotectant, and the GABA (gamma-aminobutyrate) biosynthesis pathway, which aids in pH homeostasis and nutrient use versatility. Overall, these results demonstrate that the genetic flexibility within this species allows for survival in the environment for extended periods. IMPORTANCE Escherichia coli is commonly used as an indicator of recent fecal pollution in recreational water despite its known ability to survive in secondary environments, such as beach sand. These long-term survivors from sand reservoirs can be introduced into the water column through wave action or runoff during precipitation events, thereby impacting the perception of local water quality. Current beach monitoring methods cannot differentiate long-term environmental survivors from E. coli derived from recent fecal input, resulting in inaccurate monitoring results and unnecessary beach closures. This work identified the genetic factors that are associated with long-term survivors, providing insight into the mechanistic basis for E. coli accumulation in beach sand. A greater understanding of the intrinsic ability of E. coli to survive long-term and conditions that promote such survival will provide evidence of the limitations of beach water quality assessments using this indicator.
Keywords: E. coli; beach; beach sand; genomics; metagenomics; sand; survival.