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

Abstract

Benzalkonium chlorides (BAC) are commonly used biocides in broad-spectrum disinfectant solutions. How microorganisms cope with BAC exposure remains poorly understood, despite its importance for disinfection and disinfectant-induced antibiotic resistance. To provide insights into these issues, we exposed two isolates of an opportunistic pathogen, , 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 (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 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. BAC are widely used as biocides in disinfectant solutions, food-processing 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 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 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.

Comparison of hydroalcoholic rubbing and conventional chlorhexidine scrubbing for aseptic skin preparation in dogs

“…The alcohol‐based product also contained propan‐2‐ol and benzalkonium chloride. As with other antiseptic products, there is increasing concern that benzalkonium chloride might promote antibiotic resistance . Use of an HA rub without any antiseptic, as in our study, should be unlikely to promote cross resistance to antibiotics because currently there is no known resistance to HA solutions.…”

Benzalkonium Chlorides: Uses, Regulatory Status, and Microbial Resistance

“…strains can naturally withstand the highest concentrations of BACs. Pseudomonas aeruginosa survives at up to 1,600 and 1,200 mg/liter BACs with or without a previous adaptation to the chemical, respectively (56). The MIC of BACs for the isolated strain Pseudomonas sp.…”

“…The mode of action of QACs, including BACs, involves the perturbation and disruption of the membrane bilayers by the alkyl chains and disruption of charge distribution of the membrane by the charged nitrogen (63). Accordingly, susceptibility to BACs may emerge through a combination of mechanisms (56), with many of those related to the cell membrane. The mechanisms proposed in the literature include changes in the overall membrane composition, downregulation of porins, overexpression or modification of efflux pumps, horizontal gene transfer of transposon elements and stress factors, biofilm formation, and biodegradation ( Fig.…”

“…A strain of E. coli with reduced susceptibility to BACs was shown to have a lipopolysaccharide composition diverse from that of the susceptible strain (64). Recently, it was suggested that Pseudomonas strains could partially adapt to BACs by stabilizing the membrane charge through the increase in polyamine synthesis gene expression and mutations in pmrB (56).…”

“…Another efflux pump, the PmpM of the multidrug and toxin extrusion (MATE) family, from P. aeruginosa, contributed to decreased susceptibility to BACs when expressed in a plasmid in E. coli (74). Accordingly, the exposure of Pseudomonas strains to BACs for a long time resulted in the overexpression of multidrug efflux pump genes (56). Mutations in the nfxB, a regulator for the Mex efflux system, as well as overexpression of both MexAB-OprM and MexCD-OprJ efflux systems and downregulation of mexR, a repressor of the Mex system, was also correlated to decreased sensitivity to BACs in P. aeruginosa (60).…”