Subminimal Inhibitory Concentrations of the Disinfectant Benzalkonium Chloride Select for a Tolerant Subpopulation of Escherichia coli with Inheritable Characteristics

Abstract

Exposure of Escherichia coli to a subminimal inhibitory concentration (25% below MIC) of benzalkonium chloride (BC), an antimicrobial membrane-active agent commonly used in medical and food-processing environments, resulted in cell death and changes in cell morphology (filamentation). A small subpopulation (1–5% of the initial population) survived and regained similar morphology and growth rate as non-exposed cells. This subpopulation maintained tolerance to BC after serial transfers in medium without BC. To withstand BC during regrowth the cells up regulated a drug efflux associated gene (the acrB gene, member of the AcrAB-TolC efflux system) and changed expression of outer membrane porin genes ( ompFW ) and several genes involved in protecting the cell from the osmotic- and oxidative stress. Cells pre-exposed to osmotic- and oxidative stress (sodium chloride, salicylic acid and methyl viologen) showed higher tolerance to BC. A control and two selected isolates showing increased BC-tolerance after regrowth in BC was genome sequenced. No common point mutations were found in the BC- isolates but one point mutation in gene rpsA (Ribosomal protein S1) was observed in one of the isolates. The observed tolerance can therefore not solely be explained by the observed point mutation. The results indicate that there are several different mechanisms responsible for the regrowth of a tolerant subpopulation in BC, both BC-specific and general stress responses, and that sub-MIC of BC may select for phenotypic variants in a sensitive E. coli culture.

Loss of Function in Escherichia coli Exposed to Environmentally Relevant Concentrations of Benzalkonium Chloride

“…This result agrees with previous reports documenting the adaptive BAC insusceptibility in E. coli, where microarray analysis indicated the involvement of active efflux (43). However, in contrast to previous reports, significant upregulation of the AcrAB efflux system or the outer membrane protein TolC was not observed, and there was no significant increase in the transcription of stress response systems, such as those belonging to the SoxRS regulon, or the multiple antibiotic resistance operon MarA (43,50). An increase in the transcription of genes associated with the Mdt and Yhi efflux systems was observed, which could further explain the minor changes in BAC susceptibility.…”

Polymorphic Variation in Susceptibility and Metabolism of Triclosan-Resistant Mutants of Escherichia coli and Klebsiella pneumoniae Clinical Strains Obtained after Exposure to Biocides and Antibiotics

“…The effect of biocides in antibiotic resistance has been explored in different studies specifically focused on particular situations of cross-resistance or induction of resistance (2,23,(36)(37)(38)(39). In this study, we went one step further and comprehensively analyzed the high diversity of effects exerted by several biocides and antibiotics on the most relevant enterobacterial pathogenic species, E. coli and K. pneumoniae.…”

Comparison of Gene Expression Profiles of Uropathogenic Escherichia Coli CFT073 after Prolonged Exposure to Subinhibitory Concentrations of Different Biocides

“…In a hospital setting, exposure of bacteria to residual concentrations of biocides could occur for example when residues of a compound are left on a surface after disinfection. Such exposure could potentially lead to development of increased resistance towards the biocide in use, as well as cross-resistance towards other antimicrobials, including antibiotics [11][12][13][14][15]. The exposure of bacteria to subinhibitory biocide concentrations could also induce other responses.…”

“…Many studies have shown how the transcriptome of various pathogens becomes altered after treatment with low concentrations of biocides [12,[23][24][25][26][27][28]. However, most of these studies investigated short-term response to sublethal or subinhibitory concentrations of biocides.…”