Disinfectant-microorganism interactions have posed increasing environmental and ecological concerns since the COVID-19 pandemic broke out. Previous studies have reported that disinfectant can promote horizontal transfer of antibiotic resistance genes. However, little is known about whether these disinfectants and in particular their combination could drive bacterial antimicrobial resistance (AMR) evolution and pathogenicity during long-term exposure. Here, we did 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 development of wild-type Escherichia coli. The results reveal that both disinfectants induced pan drug resistance to antibiotics and disinfectants. The coexistence of both disinfectants synergistically induced much higher resistance levels. We found that these disinfectants induced multiple mutations in antibiotic-related genes such as rpoS, crp, and fimE, primarily by stimulating excessive oxidative stress and cell membrane permeability. Phenotypic and transcriptomic analyses revealed that such resistance acquisition contributes to enhanced energy metabolism, biofilm formation, and cell motility. The acquired resistance mutations were associated with increased pathogenicity, as corroborated by enhanced bacterial invasion and intracellular survival in vitro, as well as a higher death rate of Galleria mellonella larvae in vivo. Our findings provide insight into the impacts of mixed disinfectants on the development of AMR evolution in the aquatic environment, and eventually the prevalence of virulence, thus broadening our understanding of both environmental and ecological risks associated with disinfectants.
 

antibiotic resistance, gene mutation, evolution, disinfectants, polyhexamethylene guanidine, benzalkonium chloride