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. • Amplicon profiling resolves ecological gradients driven by land use. • Shotgun sequencing detects clinical pathogens missed by amplicon methods. • Conditionally rare taxa act as sentinels for pathogen persistence. • Agri-ditches and mixed-use sites show distinct virulence and resistome hotspots. • Dual-platform monitoring enables robust One Health risk surveillance.
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