Occurrence, Seasonal Variation, and Microbial Drivers of Antibiotic Resistance Genes in a Residential Secondary Water Supply System

The widespread use of antibiotics has led to the persistence of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in drinking water systems, posing potential public health risks at the point of use. In this study, a residential secondary water supply system (SWSS) in eastern China was investigated over one year to characterize microbial communities, ARB and ARG occurrence, and their associations with water quality in bulk water and biofilms. Culture-based methods, flow cytometry, quantitative PCR, and high-throughput 16S rRNA and ITS sequencing were applied. Although conventional treatment removed 94.8% of total bacteria, significant microbial regrowth occurred during secondary distribution, with the highest heterotrophic plate counts observed in rooftop storage tanks (up to 4718 CFU/mL). ARG concentrations increased along the distribution line, and the class 1 integron intI1 was enriched in downstream locations, indicating enhanced horizontal gene transfer potential. Sulfonamide resistance genes dominated the resistome, accounting for more than 60% of total ARG abundance in water samples. Seasonally, ARG levels were higher in autumn and winter, coinciding with elevated disinfectant residuals and lower temperatures. Chlorine was negatively associated with total bacterial abundance, while positive correlations were observed with the relative abundance of several ARGs when normalized to bacterial biomass, suggesting selective pressure under oxidative stress. Turbidity and bacterial abundance were positively correlated with ARB, particularly sulfonamide-resistant bacteria. Biofilms exhibited more stable microbial communities and provided microhabitats that facilitated microbial persistence. Notably, fungal abundance showed strong positive correlations with multiple ARGs, implying that microbial interactions may indirectly contribute to ARG persistence in SWSSs. These findings highlight the role of secondary distribution conditions, disinfectant pressure, and microbial interactions in shaping resistance risks in residential water supply systems, and provide insights for improving microbial risk management at the point of consumption.