Chlorination Enhances Bacterial Invasion and Conjugative Transfer of Antibiotic Resistance Genes in Biofilms

Biofilms are widespread in water distribution systems and consist of bacterial cells with extensive cell-to-cell contact, a prerequisite for plasmid-mediated conjugative transfer of antibiotic resistance genes (ARGs). Although plasmid-mediated conjugative ARG transfer has been extensively studied, our understanding of how conjugation occurs within spatially structured bacterial biofilms and how chlorine disinfection influences the conjugation process remains limited. This study systematically investigated the effects of chlorine exposure on biofilm disruption, resistant bacteria invasion, and the conjugative transfer of ARGs in both monoculture and multispecies biofilms composed of Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa. Results showed that free chlorine significantly enhanced plasmid-mediated ARG transfer in biofilms at an initial dose of 5 mgCl/L. This could be due to the disruption of recipient biofilm structure, which facilitated donor colonization of the biofilms and close contact with the recipient bacteria. The hotspots for ARG conjugative transfer in the biofilms shifted from the surface (18 ± 2 μm) to the inner layer (27 ± 3 μm) under free chlorine exposure in the multispecies biofilm model. Moreover, a mathematical model was developed to simulate the long-term dynamics of gene transfer within biofilms under free chlorine exposure. The simulation results indicated that exposure to 5 mgCl/L promoted deeper colonization of donor cells and enhanced the dissemination of ARGs throughout the biofilm. Collectively, our findings provide a mechanistic link between biofilm structural disruption, bacterial invasion, and accelerated ARG horizontal transfer in biofilms under free chlorine exposure.