Induction and prediction modeling of the VBNC waterborne pathogens under chlorine disinfection in drinking water

Viable but non-culturable (VBNC) waterborne pathogens pose critical challenges to the microbial safety of drinking water due to their resistance to chlorine disinfection and resuscitation risk. This study investigated the induction and resuscitation potential of VBNC Escherichia coli ( E. coli ) and VBNC Pseudomonas aeruginosa ( P. aeruginosa ) under chlorine disinfection (0.5–4 mg/L) in drinking water, with resulting VBNC pathogen levels persisting at 10 3 –10 4 CFU/mL. At higher concentrations (2–4 mg/L), chlorine induced more P. aeruginosa to enter the VBNC state, while E. coli exhibited greater resuscitation potential. In contrast, lower chlorine concentrations induced a greater abundance of VBNC bacteria (10 6 –10 7 CFU/mL), which also corresponded to an increased resuscitation potential. Compared to their culturable state, chlorine-induced VBNC pathogens showed membrane contraction and reduced cell volume, along with upregulation of oxidative stress response genes ( katG , oxyR , and soxR ) by 1.8–5.1 folds, and the general stress response sigma factor ( rpoS ) by 4.4–9.7 folds. A three-dimensional dynamic model was developed based on the Chick-Watson law to accurately predict the formation of VBNC bacteria during chlorine disinfection (R 2 > 0.90). This study provides a theoretical and practical foundation for controlling microbial risks in drinking water through a model developed from the induction mechanisms of VBNC bacteria under chlorine disinfection. • More P. aeruginosa than E. coli enter the VBNC state during chlorine disinfection. • VBNC formation entails cell contraction and upregulated stress-resistance genes. • A three-dimensional model to predict VBNC bacterial counts in drinking water.