Subminimal inhibitory concentrations of the disinfectant benzalkonium chloride select for a tolerant subpopulation of Escherichia coli with inheritable characteristics

Int J Mol Sci. 2012;13(4):4101-23. doi: 10.3390/ijms13044101. Epub 2012 Mar 28.


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.

Keywords: Escherichia coli; benzalkonium chloride; efflux pump; osmotic and oxidative stress response; quaternary ammonium compounds; selection of subpopulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Bacterial Outer Membrane Proteins / biosynthesis
  • Bacterial Outer Membrane Proteins / genetics
  • Base Sequence
  • Benzalkonium Compounds / pharmacology*
  • Detergents / pharmacology
  • Disinfectants / pharmacology
  • Drug Resistance, Bacterial / genetics*
  • Escherichia coli / drug effects*
  • Escherichia coli / genetics*
  • Escherichia coli / growth & development
  • Escherichia coli Proteins / biosynthesis
  • Escherichia coli Proteins / genetics*
  • Gene Expression Regulation, Bacterial
  • Gene Knockout Techniques
  • Microbial Sensitivity Tests
  • Multidrug Resistance-Associated Proteins / genetics*
  • Osmotic Pressure
  • Oxidative Stress / genetics
  • Point Mutation / genetics
  • Porins / biosynthesis
  • Porins / genetics
  • Ribosomal Proteins / genetics
  • Sequence Analysis, DNA


  • AcrB protein, E coli
  • Anti-Bacterial Agents
  • Bacterial Outer Membrane Proteins
  • Benzalkonium Compounds
  • Detergents
  • Disinfectants
  • Escherichia coli Proteins
  • Multidrug Resistance-Associated Proteins
  • OmpF protein
  • Porins
  • Ribosomal Proteins
  • ompW protein, E coli
  • ribosomal protein S1, E coli