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

## TL;DR

Agricultural watersheds host diverse microbes and antibiotic resistance genes, with dual metagenomic methods revealing seasonal and land-use patterns in pathogen and resistance hotspots.

## Contribution

Integrating amplicon and shotgun metagenomics reveals distinct pathogen and resistome dynamics in agricultural watersheds.

## Key findings

- Agricultural ditches show higher fecal bacterial diversity with seasonal shifts and enrichment of zoonotic pathogens.
- Shotgun sequencing detects 27 ARGs and 14 VFs, with distinct hotspots in mixed-use and agricultural sites.
- Conditionally rare taxa disproportionately contribute to microbial risk and community turnover.

## Abstract

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.

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•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.

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.