5 nm. Results were expressed as mm of residues of carbonyl mg−1 protein and calculated using a molar extinction coefficient of 22 mol−1 cm−1 for aliphatic hydrazones (Witko-Sarsat et al., 1998). Proteus mirabilis suspensions were prepared from 18-h cultures at 35 °C in Trypticase Soya Broth (TSB). Aliquots of 5 mL of the sample were incubated with 0.5 mL of CIP or with PBS (control) for 2 h. Then, 1 mL of the samples
selleck chemicals llc or 1 mL of 50 μM chloramine T (standard) was treated with 50 μL of 1.16 M KI and 0.1 mL of acetic acid. The absorbance at 340 nm was applied to estimate the AOPP concentrations, which were expressed as μM L−1 of chloramine-T equivalents (Witko-Sarsat et al., 1998). CIP MIC was determined by the broth dilution method as outlined by the Clinical and Laboratory Standards Institute (CLSI), in the presence or absence of the antioxidants 10 mM GSH or 10 mM ascorbic acid in the culture medium. Statistical analysis was performed using anova, with P < 0.05 taken as statistically significant. The experiments were repeated at least three times, and the means and standard deviations were calculated. Four CRVs (1X, 1Y, 2X and 2Y) with
attained resistance (MICs of 16, 4, 8 and 4 μg mL−1 respectively) were obtained from two sensible clinical P. mirabilis S1 and S2, by repeated cultures with a sub-inhibitory concentration of CIP. The resistance frequency provoked by a sub-MIC concentration of CIP was 10−6 and this resistant population was evaluated Selleckchem DZNeP and compared with the respective parental sensible strains. The NBT assay showed
a smaller increase of ROS in CRVs with CIP than in parental strains (Fig. 1a). Moreover, oxidative stress cross-resistance to telluride was induced by successive subcultures in CIP (Fig. 1b), as 1X, 1Y, 2X and 2Y exhibited a three- to eight-fold decrease in ROS stimuli with enhanced survivability in the presence of telluride. Also, CRVs exhibited a smaller reduction of CFU mL−1 in the presence of this oxidant agent (8-, 11.8-, 1.5- and 1.1-fold decrease in 1X, 1Y, 2X and 2Y, respectively) Methamphetamine compared with sensitive parental strains (57.7-fold decrease in S1 and 25.7-fold decrease in S2). In addition, the MIC to telluride was still increased eight-fold in CRVs (data not shown). PCR amplification and direct sequencing of gyrA, gyrB and parC of P. mirabilis showed no mutations in any CRVs, thus demonstrating sequences unaltered from those occurring in the parental isolates and the P. mirabilis ATCC 29906 strain in the QRDR regions (Table 1). In contrast, mutations in GyrA, GyrB and ParC appeared in the codons for S83, E466 and S80-E84, respectively, in the CIP-resistant clinical isolate R3. The possible involvement of an active efflux mechanism in CIP resistance of P. mirabilis CRVs was evaluated (Fig. 2a,b). Previous antibiotic accumulation at the addition of CCCP appeared to be less in the CRVs than in sensitive parent strains.