Treatment of Escherichia coli BW25113 with Subinhibitory Levels of Kanamycin Results in Antibiotic Cross-Resistance and TolC Up-regulation

To determine whether the efflux pump and outer membrane protein system, AcrAB-TolC, in Escherichia coli could be upregulated for antibiotic cross-resistance, we carried out several assays comparing cells that were primed with an initial kanamycin treatment and cells that were not pretreated. We specifically focused on the expression of the TolC outer membrane pore of the efflux complex. The impact of the TolC protein on antibiotic cross-resistance was observed by measuring the minimum inhibitory concentrations (MIC) of various antibiotics (ampicillin, streptomycin, tetracycline and kanamycin) for “wild type” (WT) E. coli BW25113 strains containing an intact tolC gene, with or without prior treatment with kanamycin at subinhibitory levels. The MIC results for the WT strain were then compared against values obtained by performing an assay with a transformed tolC deletion strain, JWKO5503. WT cells were found to have higher MIC values than the deletion strain. In addition, kanamycin pretreatment resulted in an overall increase in MIC of post-treatment antibiotics for both strains. The cell density of subinhibitory kanamycin pretreated and non-pretreated WT cells were also measured to further analyze the effect of pretreatment on antibiotic sensitivity, and faster growth was observed for pretreated cells. Results thus showed that exposure to non-lethal concentrations of kanamycin can infer cross-resistance. The specificity of the primers and expression of tolC in non-treated and treated cells were also observed using PCR. The presence of TolC in the wild-type strain and absence in the deletion strain were confirmed, and tolC upregulation may be responsible for antibiotic cross-resistance in E. coli WT cells.