Dual-platform metagenomic surveillance distinguishes pathogen and resistome hotspots across agricultural and mixed-use watersheds

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 <i>Pseudomonas</i>, <i>Sphingomonas</i>, and <i>Massilia</i>. 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., <i>oxa</i>, <i>imp</i>, <i>sme</i>) 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.