Genomics and Population Analyses Reveal High Inter- and Intraserovar Diversity in Freshwater

Freshwater can support the survival of the enteric pathogen , though temporal diversity in a large watershed has not been assessed. At 28 locations within the Susquehanna River basin, 10-liter samples were assessed in spring and summer over 2 years. prevalence was 49%, and increased river discharge was the main driver of presence. The amplicon-based sequencing tool, CRISPR-SeroSeq, was used to determine serovar population diversity and detected 25 different serovars, including up to 10 serovars from a single water sample. On average, there were three serovars per sample, and 80% of -positive samples contained more than one serovar. Serovars Give, Typhimurium, Thompson, and Infantis were identified throughout the watershed and over multiple collections. Seasonal differences were evident: serovar Give was abundant in the spring, whereas serovar Infantis was more frequently identified in the summer. Eight of the ten serovars most commonly associated with human illness were detected in this study. Crucially, six of these serovars often existed in the background, where they were masked by a more abundant serovar(s) in a sample. Serovars Enteritidis and Typhimurium, especially, were masked in 71 and 78% of samples where they were detected, respectively. Whole-genome sequencing-based phylogeny demonstrated that strains within the same serovar collected throughout the watershed were also very diverse. The Susquehanna River basin is the largest system where prevalence and serovar diversity have been temporally and spatially investigated, and this study reveals an extraordinary level of inter- and intraserovar diversity. is a leading cause of bacterial foodborne illness in the United States, and outbreaks linked to fresh produce are increasing. Understanding ecology in freshwater is of importance, especially where irrigation practices or recreational use occur. As the third largest river in the United States east of the Mississippi, the Susquehanna River is the largest freshwater contributor to the Chesapeake Bay, and it is the largest river system where diversity has been studied. Rainfall and subsequent high river discharge rates were the greatest indicators of presence in the Susquehanna and its tributaries. Several serovars were identified, including eight commonly associated with foodborne illness. Many clinically important serovars were present at a low frequency within individual samples and so could not be detected by conventional culture methods. The technologies employed here reveal an average of three serovars in a 10-liter sample of water and up to 10 serovars in a single sample.