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ABSTRACTThe development of fluoroquinolone resistance in Escherichia colimay be associated with mutations in regulatory gene loci such as marRABthat 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 frommarRAB- or acrAB-inactivated E. coliK-12 strains having a single mutation in the quinolone-resistance-determining region of gyrA. Mutant 3-AG100MKX, obtained from a marknockout strain after two selection steps, showed enhanced expression of acrBin a reverse transcriptase PCR associated with insertion of IS186into the AcrAB repressor gene acrR. In vitro selection experiments with acrABknockout strains yielded MDR mutants after a single step. Enhanced efflux in these mutants was due to increased expression of acrEFand associated with insertion of IS2 into the upstream region ofacrEF, 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.

ABSTRACTFluoroquinolone-resistant mutants, selected from a wild-typeEscherichia coliK-12 strain and its Mar mutant by exposure to increasing levels of ofloxacin on solid medium, were analyzed by Northern (RNA) blot analysis, sequencing, and radiolabelled ciprofloxacin accumulation studies. Mutations in the target genegyrA(DNA gyrase), the regulatory gene marR, and additional, as yet unidentified genes (genes that probably affect efflux mediated by the multidrug efflux pump AcrAB) all contributed to fluoroquinolone resistance. Inactivation of the acrABlocus made all strains, including those with target gene mutations, hypersusceptible to fluoroquinolones and certain other unrelated drugs. These studies indicate that, in the absence of the AcrAB pump, gyrase mutations fail to produce clinically relevant levels of fluoroquinolone resistance.

Escherichia coliwith high-level fluoroquinolone resistance were isolated from feces and/or various body sites of 16 cancer patients who were on oral fluoroquinolone prophylaxis. Population analysis of fecal isolates in 11 patients showed that fluoroquinoloneresistantE. coliwas the only aerobic gram-negative bacillus present and exhibited a relatively homogenous fluoroquinolone MIC distribution. Molecular typing by pulsed field gel electrophoresis of chromosomal DNA digests or by random amplified polymorphic DNA fingerprinting confirmed the clonal nature of gastrointestinal tract colonization withE. coli. Genotyping of ten colonies picked from the same fecal culture demonstrated identical strains in four of four patients examined. Identical genotypes from the same patient were isolated over prolonged periods of time in 12 of 12 cases examined, with one patient (with the longest follow-up of 14 months) who lost his initial genotype and became persistently colonized with a new genotype. In the 11 patients who developed infection due to fluoroquinolone-resistantE. coli, molecular typing also indicated that fecal colonization was associated with, and presumably preceded infection due to an indistinguishable genotype of fluoroquinolone-resistantE. coli.