Chlorination-driven redox metabolic reprogramming promotes bacterial persistence and cross-resistance in drinking water systems.

Chlorination is a cornerstone of drinking water disinfection; however, the persistent oxidative stress it imposes may inadvertently select for metabolically resilient microorganisms within distribution systems. In this study, we investigated the universally conserved metabolic mechanisms underlying chlorine tolerance in a representative environmental bacterial isolate recovered from a full-scale drinking water distribution network. By integrating transcriptomic profiling, targeted metabolomics, and constraint-based flux balance analysis (FBA) within a proof-of-concept framework, we identified a coordinated metabolic reprogramming in response to chlorine exposure. Specifically, chlorine stress promoted increased flux through central carbon metabolism, including glycolysis, the tricarboxylic acid (TCA) cycle, and the oxidative pentose phosphate pathway (PPP), collectively enhancing intracellular NAD(P)H regeneration and redox buffering capacity. Model-guided simulations further revealed a subset of redox-associated metabolic nodes that are critical for maintaining cofactor balance and energy homeostasis under oxidative stress. Experimental validation via targeted gene disruption confirmed the model predictions, with key knockouts (e.g., mdh, narG) resulting in up to an 85% reduction in NAD(P)H production, accompanied by significant declines in metabolic activity, chlorine tolerance, and cross-resistance to multiple antibiotics. Collectively, these findings define a chlorine-responsive metabolic network that mechanistically links disinfection-induced oxidative stress to bacterial persistence and antimicrobial cross-resistance, providing critical insights into microbial survival strategies in drinking water systems.