Effect and mechanism of residual-chlorine on the horizontal transfer of antibiotic resistance genes of chlorine resistant bacteria in reclaimed water

Chlorine disinfection inactivates most microbes in reclaimed water, but chlorine resistant bacteria (CRB) persist, threatening water safety and spreading antibiotic resistance genes (ARGs). ARG proliferation in reclaimed water systems risks public health, as dissemination via irrigation or urban reuse may enable environmental transmission to humans, exacerbating global antibiotic resistance. One hundred and fifty-two strains of CRB were isolated from reclaimed water in this study, and the detection rate of ARGs in those CRB was 100%, the detection rate for blTEM was 100%, followed by sul3 and tetG. Macrogenomic analysis revealed that Proteobacteria, Actinobacteria, and Bacteroidetes are the dominant CRB in reclaimed water. Long-term induction with the minimum inhibitory concentration (MIC) of NaClO enhanced the resistance of CRB to both Amp and NaClO. The EPS of CRB increased 1.30- to 2.04-fold, and the elevated surface hydrophobicity may serve as a co-resistance mechanism. EPS hindered disinfectant/antibiotic penetration, while hydrophobicity reduced hydrophilic molecule adhesion and promoted bacterial aggregation, both of which contribute to the enhanced resistance of CRB. Residual chlorine dose-dependently enhances ARG conjugation via ROS-SOS, ATP, and EPS pathways, unveiling novel CRB mechanisms and urging revised disinfection to mitigate ARG spread.