Assessment of Sublethal Chlorine Disinfection on Bacterial Pathogenicity Potential Through Gastrointestinal Digestion and Human Cell Invasion Models

• Chlorine at sublethal doses could promote bacterial regrowth. • Surviving bacteria from gastric conditions can proliferate in intestinal conditions • Spore-forming bacteria could survive in chlorine and gastrointestinal conditions. • Chlorine-stressed bacteria had an enhanced ability to invade human cells. Although most bacteria in water can be effectively inactivated after chlorination during the wastewater treatment process, some may survive and spread waterborne diseases around the world. Their capacity to adapt to stressful conditions may make them even more hazardous. Herein, we investigated the tolerance of chlorine-stressed bacteria under simulated gastrointestinal (GI) conditions and their ability to invade human intestinal epithelial Caco-2 cells to understand the potential human health risk associated with chlorine-treated wastewater discharges. Chlorine-stressed bacterial samples were prepared using bacterial culture from secondary effluent after 30 minutes of exposure to 0.02 mg/L, 0.09 mg/L, and 0.6 mg/L of free chlorine. Remarkably, while 0.6 mg/L of free chlorine was insufficient for bacterial inactivation, free chlorine concentrations of 0.02-0.09 mg/L significantly promoted bacterial regrowth. Lysinibacillus, Klebsiella , and Morganella have shown low pH adaptability in simulated gastric conditions. Those genera were not observed in gastric conditions in samples with a higher chlorine dose of 0.6 mg/L, but more regrowth of resistant genera occurred in the simulated intestinal condition. Spore-forming bacteria survived even after chlorination and simulated gastric digestion. After disinfection with sublethal chlorine doses of 0.02 and 0.09 mg/L, surviving bacterial populations showed an enhanced ability to invade Caco-2 cells. Escherichia and Bacillus are the most dominant intracellular bacteria, suggesting their superior ability to invade Caco-2 cells. The study emphasized the importance of sufficient chlorination in minimizing microbial risks during wastewater management, reuse, and disposal.