Sacco Hospital, Milan, were included into the study. Susceptibility to the drugs under evaluation was considered as a pre-requisite for the study. One isolate per patient was used in order to avoid inclusion of the same strain. All isolates were stored at -80°C in brain-heart infusion broth containing 10% (w/v) glycerol until use. Antibiotics Levofloxacin (sanofi-aventis, S.p.A. Milan, Italy); ciprofloxacin (Bayer Italia, S.p.A., Milan, Italy), and prulifloxacin
(Aziende Chimiche Riunite Angelini Francesco ACRAF S.p.A, S. Palomba-Pomezia, Italy) were used to prepare stock solutions at concentrations of 5120 mg/L. Plasma maximum and minimum concentrations (Cmax, Cmin) of each antimicrobial studied were chosen from those obtained at steady state in previously published selleck chemical studies after oral administration [28–31]. Thus, the Cmax were as following: levofloxacin 500 mg (5.29 mg/L); levofloxacin 750 mg (11.98 mg/L); ciprofloxacin 500 mg (2.11 mg/L); prulifloxacin 600 mg (2 mg/L) [28–31]. The tested plasma Cmin were respectively: 0.60 mg/L for levofloxacin 500 mg; 1.69 mg/L for levofloxacin 750 mg; 0.08 mg/L for ciprofloxacin 500 mg; 0.10 mg/L for prulifloxacin
600 mg [28–31]. Determination of MIC Momelotinib molecular weight Antibiotic susceptibilities to the study drugs were determined by the microdilution broth assay in accordance with CLSI approved standards . Since no CLSI breakpoints for prulifloxacin against E. coli and Klebsiella spp. were available, reduced susceptibility to this agent was defined as a MIC ≥ 4 mg/L . Resistance
to levofloxacin and ciprofloxacin was defined by MIC values ≥ 8 and 4 mg/L, respectively . Frequency of mutation Colonies from an overnight culture in Mueller Hinton agar were resuspended in brain heart infusion (BHI) broth at a load of about 1010 CFU/mL. An aliquot of 100 μL from the bacterial suspension was spread onto Mueller Hinton agar plates containing antibiotics at plasma Cmax and Cmin, as reported above. After incubation for 72 h, the frequency of mutation was calculated from the ratio between colonies grown on antibiotic-containing plates and the initial inoculum, determined by plating 100 μL of bacterial suspension, after proper dilution, onto Mueller Hinton agar plates. Five colonies from each antibiotic most containing plate were randomly selected and their MIC for the corresponding antibiotic was determined as described above. When MIC was higher than the tested concentration, as occurred for Cmin for some strains, so that colony counts was not possible because of extensive growth on plate surface, frequency of mutation was not calculated, but the MIC was equally determined. Multi-step selection of resistant bacteria The ability to select for antibiotic resistance was LY2874455 mw evaluated by performing serial subcultures on Mueller Hinton agar plates, containing a gradient ranging from Cmax to Cmin.