Mutation rate and efflux response of bacteria exposed to a novel antimicrobial iodo-thiocyanate complex

Antimicrobials, at sub-lethal concentrations, can act as selectors and promoters of resistance by increasing mutation rates. We measured the rate of Escherichia coli mutation from levofloxacin (LVX) sensitivity to resistance when it was grown under the near-lethal challenge of the novel biocidal iodo-thiocyanate complex (ITC). Another relevant factor affecting the emergence of antimicrobial resistance is the role of efflux pumps. Consequently, we evaluated whether ITC could potentially be a substrate for efflux pumps, and thus that efflux-mediated resistance could arise towards ITC.

The mutation rate was measured by fluctuation analysis, when multiple parallel E. coli cultures were grown in the absence and presence of ITC. Then the mutational events, which occurred independently in each culture, were scored by plating the fraction of the culture in LVX-selective solid media and compared with the total cell number. To detect if ITC is a substrate for efflux pumps, minimum inhibitory concentrations (MICs) were determined against Pseudomonas aeruginosa in the absence and presence of the efflux pump inhibitor (EPI).

We have found that the E. coli exposed to the near-lethal level of ITC had a slight, but not significant, increase in mutation rate compared with unexposed cultures. Furthermore, the presence of EPI decreased the MIC of ITC by a modest 2-fold, showing that ITC was not a target for efflux pumps.

ITC usage most likely will not promote resistance development via increased mutation rates, and efflux-mediated resistance emergence to it is less likely than for some other antimicrobials.