Dual-platform metagenomic surveillance distinguishes pathogen and resistome hotspots across agricultural and...

Freshwater systems embedded in agricultural landscapes serve as dynamic reservoirs and conduits for fecal-associated microbes, zoonotic pathogens, and antimicrobial resistance (ARG) and virulence factor (VF) genes. Yet factors that govern their densities and diversity remain a research challenge. From 2016 to 2021, we conducted a longitudinal water surveillance in an agriculturally dominated river basin in eastern Ontario, Canada; characterizing fecal-associated bacterial communities using 16S rRNA gene amplicon and shotgun metagenomic sequencing. Agricultural drainage ditches consistently harbored higher fecal-associated bacterial diversity with pronounced seasonal shifts; i.e., higher levels during larger flow periods in spring and fall. Elevated discharge was associated with enrichment of genera containing zoonotic or opportunistic pathogens, such as those in Pseudomonas, Sphingomonas, and Massilia. Conditionally rare taxa (CRTs), although typically low in abundance, accounted for ∼12.6% of all pathogen-associated genera and disproportionately contributed to community turnover, highlighting their role as transient reservoirs of microbial risk. Shotgun metagenomics detected 27 ARGs, primarily at mixed-use sites, and 14 VFs, mainly in agricultural ditches. Clinically relevant β-lactamase genes (e.g., oxa, imp, sme) co-occurred with metal-resistance operons, a pattern suggestive of possible co-selection, although selective agents were not directly measured. Although the prevalence of ARG and VF was low (<5% of samples), their ecological context indicates potential transmission pathways. Limited overlap in ARGs between short-read and metagenome-assembled genome (MAG)-based profiling reflects their complementary strength: gene-level sensitivity versus host-resolved analysis. Together, these findings demonstrate the utility of integrated amplicon and shotgun metagenomic surveillance for proactive One Health risk assessment in agricultural watersheds.