Fleroxacin

The nine drugs tested (amitryptiline, atenolol, carbamazepine, fleroxacin, loperamide, norfloxacin, pefloxacin, propranolol and zolpidem) and HPLC grade solvents (acetonitrile, methanol and water) were purchased from Sigma-Aldrich (Barcelona, Spain). MDCK cell line was purchased from ATCC (USA) and MDCK-MDR1 cells were provided by Dr. Gottessman, MM (Nathional Institutes of Health, Bethesda). Pig brain homogenate was kindly supplied by a local slaughterhouse and fresh unfertilized chicken eggs were bought in a local supermarket. Table 2 shows the molecular properties of the nine drugs mentioned above.
Dulbecco’s modified Eagle’s medium (DMEM) with high content of glucose, L-glutamine, HEPES, MEM non-essential aminoacid, penicillin−streptomycin, trypsin-EDTA, Hank’s balanced salt solution (HBSS) and fetal bovine serum (FBS) for the cell culture of MDCK and MDCK-MDR1 cell lines were purchased from Sigma-Aldrich.

EA27 is a multidrug resistant clinical isolate of Klebsiella aerogenes exhibiting a mutation in the quinolone resistance-determining region, a porin deficient phenotype and an energy-dependent efflux^{9 (link),25 (link)}. EA294 is the acrA::Kan^r derivative strain of EA289 (not presented in this study), which itself is a kanamycin-sensitive derivative strain of EA27^{26 (link)}. EA3, EA117, KP 55, ARS100 and ARS108 are multidrug resistant clinical isolates of Klebsiella aerogenes (EA3, EA117) K. pneumoniae (KP55) and Escherichia coli (ARS100, ARS108) exhibiting a porin deficient phenotype and expression of the AcrB efflux pump^{27 (link),28} .
All strains were cultured in Mueller Hinton II broth (MHII) or Luria Broth (LB) at 37 °C. EA294 was grown in MHII or LB supplemented with kanamycin (50 µg.ml⁻¹). The quinolones ciprofloxacin (CIP), enrofloxacin (ENR), fleroxacin (FLE), nalidixic acid (NAL), norfloxacin (NOR), sparfloxacin (SPA), chloramphenicol and erythromycin were purchased from Sigma-Aldrich. Rosoxacin (ROS) was purchased from LGC Standards Ltd. The quinolones were dissolved in Milli-Q water supplemented with 0.1 to 0.4% (v/v) NaOH, chloramphenicol and erythromycin were dissolved in 50% ethanol. Physicochemical parameters of the studied quinolones are listed in Supplementary Fig. 1.