1 mL of the antibiotic and 0.5 mL of 1 mg mL−1 NBT for 30 min at 37 °C. Then, 0.1 mL of 0.1 M HCl was added and the tubes were centrifuged at 1500 g for 10 min, with the blue color of supernatants being measured at 575 nm (ROS extracellular). The separated pellets were treated with C646 0.6 mL dimethyl sulfoxide to extract the reduced NBT, and finally, 0.8 mL phosphate saline buffer was added and OD575 nm was determined (ROS intracellular). These studies were carried out with suspensions of S. aureus ATCC, supplemented with 10 mM or in the absence of glutathione, and incubated with ciprofloxacin (0.033,

0.5 and 32 μg mL−1) and gentamicin (0.125, 2 and 16 μg mL−1). Staphylococcus aureus 22 was incubated with ciprofloxacin (0.5, 32 and 2048 μg mL−1) and gentamicin (2, 16 and 2048 μg mL−1). Determinations were also made in the absence of antibiotics (control). The assays were performed at least in triplicate. Data were expressed as means ± SD BGB324 in vitro and analyzed using Student’s t-test. P<0.05 was accepted as the level of statistical significance. In S. aureus ATCC 29213 sensitive to the three antibiotics assayed, the values of MIC obtained for ciprofloxacin, gentamicin and chloramphenicol were

0.5, 2 and 4 μg mL−1, respectively. When the sensitivity to antibiotics was determined in the presence of glutathione, there were no significant changes in the MIC. In S. aureus 22, the values of MIC were 32, 2048 and 8 μg mL−1 for ciprofloxacin, gentamicin and chloramphenicol, respectively, and according to the CLSI breakpoint categorization, this strain was resistant to ciprofloxacin and gentamicin. In the presence of glutathione, the MIC values of ciprofloxacin and gentamicin were significantly

reduced. However, the addition of chloramphenicol and exogenous glutathione did not modify the susceptibility (Table 1). In the NBT assay, an increase of intracellular ROS with respect to the basal without ciprofloxacin was observed in the sensitive S. aureus ATCC 29213. This effect was dose-dependent, with the increase of extracellular ROS with ciprofloxacin being lower than intracellular ROS (Fig. 1a). The resistant cAMP S. aureus 22 had less stimuli of intracellular ROS than the sensitive strain, but showed a higher extracellular ROS than S. aureus ATCC (Fig. 1b). The oxidative stress associated with the increase in intracellular ROS was also observed with gentamicin in the sensitive strain S. aureus ATCC (Fig. 2a). No significant stimuli of intracellular ROS were found for resistant S. aureus 22, with 16 mg mL−1 of gentamicin being necessary to observe an increase in the extracellular ROS (Fig. 2b). In the presence of glutathione and ciprofloxacin, we noted more stimuli of intracellular ROS than with ciprofloxacin alone, with resistant S. aureus 22 exhibiting a higher oxidative stress than in sensitive S. aureus ATCC 29213. On the other hand, extracellular ROS decreased with exogenous glutathione in both strains.