Effect of exposure to chlorhexidine residues at “during use” concentrations on antimicrobial susceptibility profile, efflux, conjugative plasmid transfer, and metabolism of Escherichia coli

There is no standardized protocol to predict the concentration levels of microbicides that are left on surfaces as a result of the use of these products, and there is no standardized method to predict the potential risk that such levels pose to emerging antibacterial resistance. The ability to distinguish between selection and adaption processes for antimicrobial resistance in bacteria and the impact of different concentrations of microbicide exposure have not been fully investigated to date. This study considers the effect of exposure to a low concentration of chlorhexidine digluconate (CHX) on selected phenotypes of Escherichia coli and relates the findings to the risk of emerging antimicrobial resistance. A concentration of 0.006 mg/ml CHX is a realistic “during use” exposure concentration measured on surfaces. At this concentration, it was possible for CHX-susceptible bacteria to survive, adapt through metabolic alterations, exhibit a transient decrease in antimicrobial susceptibility, and express stable clinical cross-resistance to front-line antibiotics. Efflux activity was present naturally in tested isolates, and it increased in the presence of 0.00005 mg/ml CHX but ceased with 0.002 mg/ml CHX. Phenotypic microarray assays highlighted a difference in metabolic regulation at 0.00005 mg/ml and 0.002 mg/ml CHX; more changes occurred after growth with the latter concentration. Metabolic phenotype changes were observed for substrates involved with the metabolism of some amino acids, cofactors, and secondary metabolites. It was possible for one isolate to continue transferring ampicillin resistance in the presence of 0.00005 mg/ml CHX, whilst 0.002 mg/ml CHX prevented conjugative transfer. In conclusion, E. coli phenotype responses to CHX exposure are concentration dependent, with realistic residual CHX concentrations resulting in stable clinical cross-resistance to antibiotics