Chlorination promotes antibiotic resistance dissemination via conjugative transfer and stress response in reclaimed...

Chlorination is widely applied in municipal wastewater treatment for pathogen inactivation; however, it may inadvertently induce bacterial stress responses and promote the spread of antibiotic resistance genes (ARGs), posing potential environmental risks. The mechanisms underlying chlorination-enhanced horizontal ARG transfer in reclaimed water remain unclear. To address this knowledge gap, we investigated resistance evolution and horizontal transfer in reclaimed water following chlorination. Chlorination (0.5-5.0 mg/L) increased the absolute abundance of antibiotic-resistant bacteria by 1.38-4.93 log units during regrowth. At 3.0 mg/L chlorine with a 3-day regrowth, the bacterial community was profoundly reshaped, with dominant phyla shifting from Proteobacteria, Patescibacteria, and Bacteroidota in the control to a predominance of Proteobacteria (96.31%). Sul1 expression was upregulated 9.95-fold and ARG conjugative transfer increased by 13.2-fold. These changes were accompanied by significant upregulation of genes associated with resistance spread and stress responses, including efflux pump genes (acrD, ermA, tolC), outer membrane protein gene (ompA), and dormancy regulator gene (rpoS). Collectively, these findings demonstrate that sub-lethal chlorination facilitates ARG dissemination in reclaimed water by inducing bacterial stress responses and conjugation, highlighting the need for optimized disinfection strategies to reduce the environmental spread of antibiotic resistance.