Prolonged Exposure to Environmentally Relevant Concentrations of Chlorine Induces Heritable Antimicrobial Resistance in Disinfection Residual Pseudomonas aeruginosa

Chlorination, a crucial step in pathogen control, raises concerns due to the potential residual chlorine presence during water treatment and sanitation. However, the consequences of prolonged exposure to environmentally relevant chlorine concentrations on antimicrobial resistance (AMR) evolution and its driving mechanism in bacteria remain unclear. Therefore, this study utilized a combination of phenotypic and genotypic analyses, revealing that chlorination at concentrations of 0.2–0.4 mg/L induced enduring cross-resistance to both chlorine and multiple antibiotics (β-lactams, tetracyclines, sulfonamides, and chloramphenicol) in Pseudomonas aeruginosa after 3 days of exposure. Both Escherichia coli and P. aeruginosa exhibited outer membrane (OM) damages, evidenced by adenosine triphosphate and reactive oxygen species, though P. aeruginosa displayed stepwise OM resilience over prolonged exposure. Transcriptomic analyses of resistant P. aeruginosa unveiled heightened metabolic activity and a reinforced OM barrier after exposure. Weighted gene coexpression network analysis highlighted the pivotal role of a fortified bacterial OM, featuring activated efflux systems and modified lipopolysaccharides, in developing cross-resistance. Overexpression and mutation in mexXY–OprM and muxABC–OpmB efflux systems, along with reduced membrane electronegativity, confirmed that hereditary genetic adaptation drove AMR evolution. This study provides valuable insights into potential strategies for mitigating AMR evolution under residual chlorine disinfection.