Fluoroquinolone resistance of Escherichia coli at a cancer center: epidemiologic evolution and effects of discontinuing prophylactic fluoroquinolone use in neutropenic patients with leukemia.

The aim of the present study was to investigate the epidemiologic evolution of fluoroquinolone resistance of E. coli clinical isolates from patients admitted to a hematology-oncology service where fluoroquinolone prophylaxis during neutropenia was recommended as the standard of care for many years but was then discontinued in a trial conducted in patients with acute leukemia. Fluoroquinolones had been shown to decrease the incidence of gram-negative bacteremia in cancer patients with neutropenia, yet it was thought that the emergence of resistance in Escherichia coli and other gram-negative bacteria may have caused a progressive lack of efficacy of fluoroquinolone prophylaxis. Epidemiologic surveillance of fluoroquinolone resistance of E. coli clinical isolates at our cancer center since 1992 showed a continuing influx of new clones not previously observed in the population of cancer patients, an increase in the number of cancer patients per year colonized and/or infected by fluoroquinolone-resistant E. coli (1992-1994, 10-16 patients; 1995-1997, 24-27 patients), and a resistance rate of >50% among E. coli bloodstream isolates of hematology-oncology patients. A 6-month fluoroquinolone prophylaxis discontinuation intervention trial in 1998 suggested that despite increasing resistance among E. coli isolates, fluoroquinolone prophylaxis in acute leukemia patients was still effective in the prevention of gram-negative bacteremia (incidence rates, 8% during the pre-intervention period vs. 20% after discontinuation; p<0.01). The resumption of fluoroquinolone prophylaxis in acute leukemia patients thereafter decreased the incidence of gram-negative bacteremia to the pre-intervention level (9%; p=0.03), while the proportion of in vitro fluoroquinolone resistance in E. coli bacteremia isolates again increased (from 15% during the intervention period to >50% in the post-intervention period). Relative rates of resistance thus were a poor indicator of the potential clinical benefits associated with fluoroquinolone prophylaxis in cancer patients.

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

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.

Non-target gene mutations in the development of fluoroquinolone resistance in Escherichia coli.

Mutations in loci other than genes for the target topoisomerases of fluoroquinolones, gyrA and parC, may play a role in the development of fluoroquinolone resistance in Escherichia coli. A series of mutants with increasing resistance to ofloxacin was obtained from an E. coli K-12 strain and five clinical isolates. First-step mutants acquired a gyrA mutation. Second-step mutants reproducibly acquired a phenotype of multiple antibiotic resistance (Mar) and organic solvent tolerance and showed enhanced fluoroquinolone efflux. None of the second-step mutants showed additional topoisomerase mutations. All second-step mutants showed constitutive expression of marA and/or overexpressed soxS. In some third-step mutants, fluoroquinolone efflux was further enhanced compared to that for second-step mutants, even when the mutant had acquired additional topoisomerase mutations. Attempts to circumvent the second-step Mar mutation by induction of the mar locus with sodium salicylate and thus to select for pure topoisomerase mutants at the second step were not successful. At least in vitro, non-target gene mutations accumulate in second- and third-step mutants upon exposure to a fluoroquinolone and typically include, but do not appear to be limited to, mutations in the mar or sox regulons with consequent increased drug efflux.

Colonization and infection with fluoroquinolone-resistant Escherichia coli among cancer patients: clonal analysis.

Escherichia coli with 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 fluoroquinolone-resistant E. coli was 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 with E. 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-resistant E. coli, molecular typing also indicated that fecal colonization was associated with, and presumably preceded infection due to an indistinguishable genotype of fluoroquinolone-resistant E. coli.

Ineffectiveness of topoisomerase mutations in mediating clinically significant fluoroquinolone resistance in Escherichia coli in the absence of the AcrAB efflux pump.

Fluoroquinolone-resistant mutants, selected from a wild-type Escherichia coli K-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 gene gyrA (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 acrAB locus 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.