Enhanced expression of the multidrug efflux pumps AcrAB and AcrEF associated with insertion element transposition in Escherichia coli mutants Selected with a fluoroquinolone

Abstract

The development of fluoroquinolone resistance in Escherichia coli may be associated with mutations in regulatory gene loci such as marRAB that lead to increased multidrug efflux, presumably through activation of expression of the AcrAB multidrug efflux pump. We found that multidrug-resistant (MDR) phenotypes with enhanced efflux can also be selected by fluoroquinolones from marRAB- or acrAB-inactivated E. coli K-12 strains having a single mutation in the quinolone-resistance-determining region of gyrA. Mutant 3-AG100MKX, obtained from a mar knockout strain after two selection steps, showed enhanced expression of acrB in a reverse transcriptase PCR associated with insertion of IS186 into the AcrAB repressor gene acrR. In vitro selection experiments with acrAB knockout strains yielded MDR mutants after a single step. Enhanced efflux in these mutants was due to increased expression of acrEF and associated with insertion of IS2 into the upstream region of acrEF, presumably creating a hybrid promoter. These observations confirm the importance of efflux-associated nontarget gene mutations and indicate that transposition of genetic elements may have a role in the development of fluoroquinolone resistance in E. coli.

FIG. 1

Intracellular fluoroquinolone concentration and expression of the efflux pump genes acrB and acrE in E. coli mutants selected by ofloxacin. (Graphs) Steady-state intracellular concentrations of ofloxacin (external concentration, 10 μg/ml) in the absence of CCCP, measured spectrofluorometrically and expressed as percentages of that of the parental strain. (Gels) RT-PCR of acrB and acrE and of gapA (coding for d-glyceraldehyde-3-phosphate-dehydrogenase; used as the standard). (A) mar knockout mutants 1-AG100MKX (lane 1), 2-AG100MKX (lane 2), and 3-AG100MKX (lane 3). (B) acrAB knockout mutants 1-AG100AK (lane 1), 2-AG100AKX (lane 2), and 3-AG100AKX (lane 3).

FIG. 2

Schematic diagram of the integration site of the IS elements IS186 (upper panel) and IS2 (lower panel) in the genes coding for the E. coli multidrug efflux pumps AcrAB and AcrEF. Arrows indicate the binding sites of the primers used for PCR and nucleotide sequencing. P_H designates a potential hybrid promoter created by the integration of IS2.