Biomolecular insights into opportunistic pathogens and a risk-threshold control strategy in rural household hot water...

Opportunistic pathogens (OPs) are typical biological indicators for public health monitoring in both freshwater and freshwater-derived engineered waters. This study investigated the OPs abundance and microbial communities in household hot water systems under small-central and urban-extension water supply modes in rural China. Quantitative polymerase chain reaction (qPCR) and 16S rRNA sequencing were employed to analyze the abundance of OPs (P. aeruginosa, E. coli, M. avium, and Acanthamoeba spp.) and microbial communities across different modes and seasons. Higher bacterial abundance (7.93 ± 0.42 log₁₀(gene copies/mL)) and turbidity (up to 2.2 NTU) were observed in the small-central mode, with Pseudomonadota and Deinococcota as the dominant phyla. However, Acanthamoeba spp. was enriched in the urban-extension mode, with abundance increasing significantly from 2.85 to 4.70 log₁₀(gene copies/mL) (p < 0.05), while Pseudomonadota and Actinomycetota were the dominant phyla. Seasonal variation had minimal impact on OPs abundance, particularly for M. avium and P. aeruginosa, due to stable warm temperatures and the absence of disinfectants in water heaters. The maximum permissible annual average living fraction (L_i) of P. aeruginosa, according to Quantitative Microbial Risk Assessment (QMRA) framework, were determined as 2.17 × 10^-4, and 2.17 × 10^-5 under WHO and U.S. EPA benchmarks, respectively. The lower QMRA-derived L_i threshold in the small-central mode required stricter control for OPs proliferation. This study integrated biomolecular OPs quantification with QMRA framework, providing insights into pathogenic risks control in household hot water systems in rural China.