Urbanization shapes the landscape of antibiotic resistance genes and water quality microbial markers across an urban watershed.

Urbanization alters watershed hydrology, land use, and water quality, potentially influencing the distribution of antibiotic resistance genes (ARGs) and microbial contaminants in urban rivers. However, watershed-scale relationships among urbanization, microbial water quality markers, and environmental antimicrobial resistance remain incompletely understood. This study investigated spatial patterns and differences among sampling campaigns in ARGs and microbial water quality markers across the Rouge River watershed in southeastern Michigan, USA, by integrating high-throughput quantitative PCR (qPCR) arrays with watershed-scale geospatial characteristics, physicochemical measurements, and multivariate analyses. Water samples were collected from six sites spanning an urbanization gradient during three sampling campaigns (2023, 2024, and 2026). Detection profiles from arrays targeting 83 ARGs and 50 microbial water quality markers were evaluated using non-metric multidimensional scaling (NMDS), redundancy analysis (RDA), and exploratory Pearson correlations. Microbial water quality marker composition exhibited substantial variation among sites and sampling campaigns but was not significantly associated with the measured watershed characteristics or physicochemical variables. In contrast, ARG composition showed exploratory associations with several measured watershed and physicochemical gradients in the RDA, although the overall relationship was not statistically significant after accounting for repeated observations within sites. β-Lactam resistance genes comprised the largest proportion of detected ARGs, whereas tetracycline resistance genes exhibited the highest normalized detection frequency. Notably, the midstream Inkster site exhibited the greatest ARG richness in both 2024 and 2026 despite having an intermediate proportion of developed land. This repeated pattern occurred under contrasting antecedent precipitation conditions, suggesting that developed land and recent rainfall alone are unlikely to account for the observed ARG hotspot. These findings demonstrate that microbial water quality markers and ARGs exhibit distinct spatial patterns within the same urban watershed and provide new insight into the spatial complexity of environmental antimicrobial resistance in freshwater systems. Together, they underscore the value of integrated molecular, environmental, and geospatial approaches for watershed-scale AMR surveillance within a One Health framework.