Dual Disinfectants Synergistically Drive Adaptation and Evolution toward Higher Antimicrobial Resistance and Pathogenicity in <i>Escherichia coli</i>

Numerous disinfectants have been discharged into the environment through anthropogenic activities, leading to ubiquitous coexistence of multiple disinfectants in the real environment. Previous studies reported that individual disinfectants induce the emergence of antimicrobial resistance (AMR); however, the impacts of the coexposure to multiple disinfectants on the development of AMR and its toxicity associated with bacterial pathogenicity are still unclear. Here, we conducted a 30-day stepwise exposure experiment to investigate the impacts of the commonly used disinfectants polyhexamethylene guanidine (PHMG), benzalkonium chloride (BAC), and their combination on AMR and pathogenicity of Wild-Type <i>Escherichia coli</i>. Results reveal that the coexistence of dual disinfectants dramatically led to higher levels of AMR (2.0- to 14.8-fold) and pathogenicity (8.7-fold) in the evolved strains. Dual disinfectant-induced bacterial evolution is primarily driven by the alteration in the expression of key functional genes, such as those involved in biofilm formation and cell motility. In contrast, genetic mutations may not account for the increased AMR and pathogenicity in combination of dual disinfectant treatment, as similar gene mutations are responsible for both dual and single disinfectant stress. The evolved strains induced by disinfectant combination, characterized with transcriptional regulation, exhibited a synergistic or additive response on AMR and pathogenicity, which was further confirmed by a toxicology prediction model. This study demonstrated that dual disinfectants synergistically enhanced AMR and the associated pathogenicity, advancing our understanding of the risks posed by coexistence of multiple chemical pollutants in the environment.