Genomic and Transcriptomic Insights into How Bacteria Withstand High Concentrations of Benzalkonium Chloride Biocides

Benzalkonium chlorides (BAC) are commonly used biocides in broadspectrum disinfectant solutions. How microorganisms cope with BAC exposure remains poorly understood, despite its importance for disinfection and disinfectantinduced antibiotic resistance. To provide insights into these issues, we exposed two isolates of an opportunistic pathogen, Pseudomonas aeruginosa, to increasing concentrations of BAC. One isolate was preadapted to BAC, as it originated from a bioreactor fed with subinhibitory concentrations of BAC for 3 years, while the other originated from a bioreactor that received no BAC. Replicated populations of both isolates were able to survive high concentrations of BAC, up to 1,200 and 1,600 mg/liter for the non-and preadapted strains, respectively, exceeding typical application doses. Transcriptome sequencing (RNA-seq) analysis revealed upregulation of efflux pump genes and decreased expression of porins related to BAC transport as well as reduced growth rate. Increased expression of spermidine (a polycation) synthase genes and mutations in the pmrB (polymyxin resistance) gene, which cause a reduction in membrane negative charge, suggested that a major adaptation to exposure to the cationic surfactant BAC was to actively stabilize cell surface charge. Collectively, these results revealed that P. aeruginosa adapts to BAC exposure by a combination of mechanisms and provided genetic markers to monitor BAC-resistant organisms that may have applications in the practice of disinfection. IMPORTANCE BAC are widely used as biocides in disinfectant solutions, foodprocessing lines, domestic households, and health care facilities. Due to their wide use and mode of action, there has been rising concern that BAC may promote antibiotic resistance. Consistent with this idea, at least 40 outbreaks have been attributed to infection by disinfectant- and antibiotic-resistant pathogens such as P. aeruginosa. However, the underlying molecular mechanisms that bacteria use to deal with BAC exposure remain poorly elucidated. Elucidating these mechanisms may be important for monitoring and limiting the spread of disinfectant- resistant pathogens. Using an integrated approach that combined genomics and transcriptomics with physiological characterization of BAC-adapted isolates, this study provided a comprehensive understanding of the BAC resistance mechanisms in P. aeruginosa. Our findings also revealed potential genetic markers to detect and monitor the abundance of BAC-resistant pathogens across clinical or environmental settings. This work contributes new knowledge about high concentrations of benzalkonium chlorides disinfectants-resistance mechanisms at the whole-cell genomic and transcriptomic level.