Integrating multi-method approach reveals extensive antibiotic resistance dissemination from concentrated animal feeding operations to surface waters.

Concentrated animal feeding operations (CAFOs) are important sources of antimicrobial resistance (AMR), but how mixed livestock inputs and seasonality shape antibiotic resistance profiles in receiving surface waters remains uncertain. We integrated culture-based screening, qPCR, and shotgun metagenomics to assess AMR in surface waters influenced by dairy and mixed swine and dairy operations across seasonal campaigns. CAFO-impacted sites, which were shown to have much greater levels of multidrug resistance among purified Escherichia coli isolates in our previous study, had higher culturable E. coli than reference sites, and extended-spectrum beta-lactamase (ESBL)-producing E. coli were detected only at CAFO sites during spring. qPCR analysis showed significantly higher relative abundances of tetracycline (tetW) and macrolide (ermF) resistance genes at CAFO-impacted sites, with strong co-occurrence between the cattle fecal marker CowM3 and these antibiotic resistance genes (ARGs) (adjusted p < 0.05). Metagenomic profiling identified 619 unique ARG subtypes. CAFO-impacted sites contributed substantially greater resistance diversity, with 198 unique subtypes detected compared to 15 unique subtypes at reference sites. Seasonal shifts in metagenomic data were pronounced at dairy sites, including spring increases in tetracycline-, rifamycin-, and florfenicol-associated resistance. ESKAPE pathogens were detected only at CAFO-impacted sites, while Pseudomonas aeruginosa and Klebsiella pneumoniae were identified as putative ARG hosts. Across methods, culture and molecular approaches provided complementary information, with ESBL total coliforms correlating better with qPCR and metagenomic results (p < 0.005) then ESBL E. coli. By integrating phenotypic and molecular evidence, this study highlights seasonal windows of enhanced detectability and supports integrated One Health surveillance of AMR at agricultural-environment interfaces.