Widely used benzalkonium chloride disinfectants can promote antibiotic resistance.

While misuse of antibiotics has clearly contributed to the emergence and proliferation of resistant bacterial pathogens with major health consequences, it remains less clear if the widespread use of disinfectants, a different class of biocides than antibiotics such as benzalkonium chlorides (BAC), has contributed to this problem. Here, we provide evidence that exposure to BAC co-selects for antibiotic-resistant bacteria, and describe the underlying genetic mechanisms. BAC-fed bioreactors inoculated with river sediment selected for several bacterial taxa, including the opportunistic pathogen Pseudomonas aeruginosa, that were more resistant to several antibiotics compared to their counterparts in a control (no BAC) bioreactor. Metagenomic analysis of the bioreactor microbial communities, confirmed by gene cloning experiments with the derived isolates, suggested that integrative and conjugative elements encoding a BAC efflux pump together with antibiotic resistance genes were responsible for these results. Further, exposure of the P. aeruginosa isolates to increasing concentrations of BAC selected for mutations in pmrB (polymyxin resistance) and physiological adaptations that contributed to higher tolerance to polymyxin B and other antibiotics. Physiological adaptations included, for example, the overexpression of mexCD-oprJ multidrug efflux pump genes when BAC was added in the growth medium at sub-inhibitory concentrations. Collectively, our results demonstrated that disinfectants can promote antibiotic resistance via several mechanisms, and highlight the need to remediate (degrade) disinfectants in non-target environments to further restrain the spread of antibiotic resistant bacteria.IMPORTANCE Benzalkonium chlorides (BAC) are broadly used biocides in disinfectant solutions. Disinfectants are widely used in food processing lines, domestic households, and pharmaceuticals products, and are typically designed to have a different mode of action than antibiotics in order to not interfere with the use of the latter. Whether or not exposure to BAC makes bacteria more resistant to antibiotics remains, however, an unresolved issue of obvious practical consequences for public health. Using an integrated approach that combined metagenomics of natural microbial communities with gene cloning experiments with isolates and experimental evolution assays, we show that the widely used benzalkonium chloride disinfectants can promote clinically-relevant antibiotic resistance. Therefore, more attention should be given to the usage of these disinfectants, and their fate in non-target environments should be more tightly monitored.