Mechanisms for disinfection-driven conjugative spread of antibiotic resistance in drinking water biofilms

Abstract

The persistent use of antibiotics has exacerbated the spread of antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs) in aquatic environments, including drinking water systems. Pipeline biofilms, comprising over 95 % of microbial biomass, function as high-risk reservoirs for ARG accumulation and horizontal gene transfer (HGT). However, the roles of disinfection-induced biofilm aggregation in plasmid-mediated conjugation remains mechanistically obscure. This study investigated how sodium hypochlorite (NaClO), chlorine dioxide (ClO₂), and their combination affect ARB selection and ARG conjugation in multispecies drinking water biofilms at environmentally relevant chlorine concentrations (0–2.0 mg/L). Results showed that exposure to either NaClO or ClO₂ at 1.0 mg/L increased the relative abundance of ARB, while combined disinfection at 2.0 mg/L exerted selective pressure that further enhanced ARB enrichment. Sublethal chlorine levels increased conjugation frequencies within biofilms by 2.1- to 5.9-fold relative to untreated controls. Conjugation efficiency was positively correlated with bacterial surface colonization and metabolic activity (r = 0.95, p < 0.05). Mechanistic investigations revealed that chlorine exposure upregulated integrase genes, enriched potential ARG-hosting taxa, and preserved fimbrial structures, collectively facilitating donor-recipient interactions and promoting HGT. These findings elucidate the mechanistic pathways underlying disinfection-mediated horizontal gene transfer and provide critical insights into managing antibiotic resistance risks in drinking water systems.