Prolonged Exposureto Environmentally Relevant Concentrationsof Chlorine Induces Heritable Antimicrobial Resistance in DisinfectionResidual <i>Pseudomonas aeruginosa</i>

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 <i>Pseudomonas aeruginosa</i> after 3 days of exposure. Both <i>Escherichia coli</i> and <i>P. aeruginosa</i> exhibited outer membrane (OM) damages, evidenced by adenosine triphosphate and reactive oxygen species, though <i>P. aeruginosa</i> displayed stepwise OM resilience over prolonged exposure. Transcriptomic analyses of resistant <i>P. aeruginosa</i> 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 <i>mex</i>XY–<i>Opr</i>M and <i>mux</i>ABC–<i>Opm</i>B 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.