Systematic citywide analysis reveals ecological connectivity of antimicrobial resistance genes across urban water systems

Antimicrobial resistance (AMR) in drinking water raises public health concerns, while its anthropogenic sources, transmission dynamics, and health risks remain poorly understood, hindering the development of effective strategies to reduce human exposure. Here we conduct a systematic investigation of anthropogenic contributions to AMR across urban water compartments in a megacity, combining metagenomics and culturomics. We identify 1,309 antibiotic resistance genes (ARGs), and tracking their dynamics across microbial communities and fecal Enterobacteriaceae isolates indicates that ecological connectivity establishes a cascading dissemination pathway: wastewater discharge promotes AMR accumulation in natural water bodies, facilitating its persistence in finished drinking water. Critical human-derived ARGs, primarily conferring resistance to beta-lactams and aminoglycosides, are enriched in clinically relevant pathogens. Further analysis reveals synergistic effects of biotic and abiotic drivers, including horizontal gene transfer (HGT), host proliferation, trace metals, disinfectants, and antibiotic residues, acting with connectivity to drive ARG proliferation. Mechanistic insights reveal that integron-mediated HGT captures and rearranges exogenous ARGs, thereby assembling multi-resistant genetic determinants along connected pathways. We establish a risk prioritization framework integrating dynamics, mobility, pathogenicity and clinical relevance to identify high-risk anthropogenic ARGs. These findings elucidate AMR transmission mechanisms via ecological connectivity, informing targeted interventions to disrupt transmission links and mitigate drinking water risks. Despite recent focus on resistance genes in urban wastewater, system-wide studies are still limited. Here, the authors provide genetic evidence across a citywide water system that anthropogenic antibiotic resistance genes might spread from sewage to natural waters and through urban water systems.