Distribution, Host Association, and Dissemination of Antibiotic Resistance Genes in the Water-Receiving Area of the Middle Route of the South-to-North Water Diversion Project

Rapid urbanization has created heterogeneous aquatic niches in megacities; however, how functionally distinct water bodies shape the environmental resistome remains poorly understood. To elucidate this, we analyzed 30 surface-water metagenomes from four waterbody types in Beijing during summer and autumn. A total of 1 380 antibiotic resistance genes (ARGs) subtypes belonging to 28 classes were identified. Our results demonstrated that urban rivers sustained the highest average ARG burden (average of 0.536 copies per cell), whereas relatively isolated urban lakes exhibited the lowest (0.199 copies per cell). Notably, the receiving waters linked to inter-basin diversion maintained an intermediate ARG load (0.328 copies per cell), but displayed the strongest coupling between ARGs and mobile genetic elements (MGEs) ( R 2 = 0.965). This indicated an elevated mobilization potential at these hydrological interfaces. Seasonal succession displayed a significant reduction in ARG abundance (from 0.441 to 0.227 copies per cell), and the characteristic spatial distribution patterns remained consistent across sampling campaigns, with community turnover acting as the primary driver of resistome variation. Variance partitioning analysis (VPA) and partial least squares path modeling (PLS-PM) revealed that ARG variation was jointly shaped by microbial composition, MGEs, and environmental factors with high explanatory power ( R 2 = 0.91~0.97). In summer, the shared effect of the microbiome and MGE composition accounted for the largest explained fraction (25.69%), and PLS-PM highlighted MGEs as the strongest direct predictor of the resistome (path coefficient = 0.76). Genomic binning identified that the resistome was phylogenetically concentrated within Pseudomonadota, with Enterobacter being a core high-risk host harboring co-localized ARGs and virulence factors. Thus, urban water networks act as connected yet selective resistome environments, while providing a foundational ecological framework for understanding the transport and dissemination of antimicrobial resistance and informing risk-oriented management of complex megacity aquatic systems.