Involvement of reactive oxygen species in the action of ciprofloxacin against Escherichia coli

Abstract

Ciprofloxacin is an important and commonly used member of the fluoroquinolone group of antibiotics. Ciprofloxacin inhibits DNA topoisomerase II and DNA topoisomerase IV activities, eventually leading to bacterial cell death. In addition, an increase of reactive oxygen species in the bacterial cells in response to ciprofloxacin has been shown. We investigated the role of reactive oxygen species in the antibacterial action of ciprofloxacin by studying the effects of different antioxidant compounds on ciprofloxacin susceptibility of Escherichia coli. Among the antioxidants checked, glutathione and ascorbic acid provided substantial protection against ciprofloxacin. The involvement of superoxide anion (O2-) and hydrogen peroxide (H2O2) in the antibacterial action of ciprofloxacin was analyzed using superoxide dismutase, catalase, and alkyl hydroperoxide reductase knockout strains of E. coli. The effects of multicopy sod genes on ciprofloxacin susceptibility of E. coli were also analyzed. On the basis of our results, we conclude that O2- and H2O2 may be involved in antibacterial action of ciprofloxacin. Our findings that glutathione gave protection against other fluoroquinolones and not against nonfluoroquinolone antibiotics imply that reactive oxygen species may have a similar role in the antibacterial action of all these fluoroquinolones and that glutathione-mediated protection is not a general phenomenon but specific to fluoroquinolones. These observations are of significance, as fluoroquinolones are important antibiotics with immense therapeutic value, and the effectiveness of treatment by these drugs may be affected by dietary intake and cellular levels of these antioxidants.

FIG. 1.

Decreased sensitivity of E. coli MG1655 against ciprofloxacin in the presence of 10 mM glutathione or ascorbic acid. C-1, C-2, C-3, and C-4 correspond to 40, 200, 400, and 2,000 ng of ciprofloxacin, respectively, spotted on the Whatman disk.

FIG. 2.

Effects of glutathione and ascorbic acid on the viable counts of strain MG1655 in the presence of various concentrations of ciprofloxacin. Ascorbic acid (Asc) (10 mM) and glutathione (GSH) (10 mM) were added to LB. LB alone was used as a control. Since a zero value cannot be plotted on a log scale, a numerical value of 1 was used whenever no CFU was obtained.

FIG. 3.

Roles of katE, katG, and ahpCF in ciprofloxacin sensitivity of strain MG1655 in terms of its viable counts. The numbers of surviving bacteria of different strains at various concentrations of ciprofloxacin were determined as described in Materials and Methods. MG1655 was the control.