A two-year study of Salmonella in four natural watersheds highlights the need for increased environmental Salmonella surveillance to close the One Health loop.

Salmonella enterica is a leading bacterial cause of foodborne illness, often transmitted through contaminated food and water. Improved food handling has led to considerable reductions of Salmonella contamination in meat and poultry products; this does not wholly contribute to decreased salmonellosis incidence, highlighting the need to define alternative reservoirs and transmission pathways. In this study, we collected samples from four distinct watersheds over 24 months to characterize Salmonella serovar diversity and utilized phylogenetic approaches, along with proximal land use analyses, to identify relationships between environmental reservoirs and hosts. Across 19 sites, including animal agriculture, suburban, and forested areas, 10 L water samples were collected (n = 456), and cultured for Salmonella, followed by whole genome sequencing of isolates and deep serotyping of positive samples. Overall prevalence was 69% (314/456), and generalized linear mixed models showed that compared to proximal land use, seasonal weather patterns, including precipitation and humidity, significantly influenced recovery and complexity. Antimicrobial resistance was detected in 11% (33/314) of isolates, with 21% (7/33) classified as multidrug resistant. CRISPR-SeroSeq identified 37 serovars, and multiserovar populations were detected in 89% (229/258) of positive samples with sequencing data, averaging 3.7 serovars/sample. Comparison with national food animal production monitoring showed limited serovar overlap, with serovar Rubislaw dominating water samples but absent in agricultural data sets. Collectively, these results demonstrate extensive serovar diversity within Salmonella populations in freshwater systems, including clinically relevant serovars, and emphasize the need to develop a robust surveillance platform for source attribution and, ultimately, prevention of future outbreaks.IMPORTANCEContaminated surface water significantly contributes to global Salmonella illnesses, marking a critical need to assess serovars present and determine environmental variables affecting the population dynamics in this reservoir. We found that complex multiserovar populations, often including pathogenic serovars, occur in surface water regardless of proximal land use. Notably, many aquatic serovars are not detected in animal agriculture monitoring and the phylogeny presented here shows isolates are more closely related to human clinical than animal-source isolates. However, limited serotyping data are available for alternative reservoirs of foodborne illness, namely, wildlife, which hinders source attribution. This study highlights a significant gap in understanding environmental Salmonella transmission and underscores the importance of a One Health surveillance approach to protect public health.