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 (O 2 ؊ ) and hydrogen peroxide (H 2 O 2 ) 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 O 2 ؊ and H 2 O 2 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.The emergence of drug resistance against ␤-lactam and aminoglycoside antibiotics resulted in the discovery of newer classes of synthetic antibiotics, including quinolones. Since the discovery of the first quinolone, nalidixic acid, various structural and chemical modifications resulted in expanded-spectrum and broad-spectrum quinolones (reviewed in references 3 and 29). These drugs are well absorbed following oral administration, with moderate to excellent bioavailability, and they show rapid bactericidal effect against susceptible organisms and cover a broad antibacterial spectrum (29). Since these drugs consist of carboxyl and amine groups along with other chemical functionalities, their acid-base behavior is influenced by the physiochemical properties of the solvent and their antibacterial activity is pH dependent (30). The mechanism of antibacterial action of quinolones is not completely understood; however, it has been proposed that the initial event is the inhibition of DNA synthesis by interference with the nick sealing activity of DNA topoisomerase II (DNA gyrase) and DNA topoisomerase IV. In the presence of these antibiotics, the enzyme is trapped on the DNA, resulting in the formation of quinolone-enzyme-DNA complexes, and the subsequent release of DNA ends from this complex leads to

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Resveratrol (5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol), a redox active phytoalexin with a large number of beneficial activities is also known for antibacterial property. However the mechanism of action of resveratrol against bacteria remains unknown. Due to its extensive redox property it was envisaged if reactive oxygen species (ROS) generation by resveratrol could be a reason behind its antibacterial activity. Employing Escherichia coli as a model organism we have evaluated the role of diffusible reactive oxygen species in the events leading to inhibition of this organism by resveratrol. Evidence for the role of ROS in E. coli treated with resveratrol was investigated by direct quantification of ROS by flow cytometry, supplementation with ROS scavengers, depletion of intracellular glutathione, employing mutants devoid of enzymatic antioxidant defences, induction of adaptive response prior to resveratrol challenge and monitoring oxidative stress response elements oxyR, soxS and soxR upon resveratrol treatment. Resveratrol treatment did not result in scavengable ROS generation in E. coli cells. However, evidence towards membrane damage was obtained by potassium leakage (atomic absorption spectrometry) and propidium iodide uptake (flow cytometry and microscopy) as an early event. Based on the comprehensive evidences this study concludes for the first time the antibacterial property of resveratrol against E. coli does not progress via the diffusible ROS but is mediated by site-specific oxidative damage to the cell membrane as the primary event.

We examined the effects of antioxidants and the role of reactive oxygen species (ROS) on the antibacterial action of aminoglycosides in Escherichia coli. We concluded that reduced streptomycin sensitivity in the presence of glutathione and ascorbic acid is not due to the antioxidant-mediated scavenging of ROS.Aminoglycosides like streptomycin, gentamicin, etc., are highly potent, broad-spectrum antibiotics (15), crucial for the treatment of various infections and prophylaxes in special situations. The mechanism of antibacterial action of aminoglycosides is not completely understood. However, it has been established that these antibiotics act primarily by impairing bacterial protein synthesis through binding to the 30S ribosomal subunit (5,9,15).Recent studies have shown that some antibiotics stimulate the induction of reactive oxygen species (ROS) in different bacterial species (1, 2). Redox cycling of some of the antibiotics affects the formation of ROS during the oxidation process (4). We have recently established the involvement of ROS in the antibacterial action of ciprofloxacin (13). Since two of the major side effects of the aminoglycosides, ototoxicity and nephrotoxicity, are believed to be mediated through ROS (15), the role of ROS in their antibacterial actions is worth investigating.The aim of the present study was to investigate whether antioxidants alter the aminoglycoside sensitivity of E. coli and, if they do, to further identify the role of ROS in the antibacterial action of aminoglycosides. This was undertaken by supplementing the growth medium with antioxidants and by introducing mutations in genes whose products are known to reduce the steady-state level of ROS in the cell, namely, Mnsuperoxide dismutase (sodA), catalases (katE and katG), and alkylhydroperoxide reductase (ahpCF). The effects of multicopy sod genes on streptomycin sensitivity were also examined, and the changes in ROS levels in the presence of these antibiotics were measured using the nitroblue tetrazolium (NBT) reduction method.The bacterial strains and plasmids used in this study are listed in Table 1. The preparation and handling of the antioxidant solutions, microbial-culture growth conditions, and antibiotic susceptibility-testing methods were essentially the same as those previously described by Goswami et al. (13).The effects of antioxidants on the streptomycin sensitivity of E. coli strain MG1655 were analyzed by the antibiotic disk diffusion method. Reduction in the zone of inhibition around the antibiotic disk indicated that the presence of 10 mM glutathione or ascorbic acid in the growth medium reduced the sensitivity of MG1655 cells to streptomycin (Fig. 1). However, other antioxidants, such as histidine and mannitol, which act as specific scavengers for singlet oxygen ( 1 O 2 ) and hydroxyl radicals ( ⅐ OH), respectively, did not alter the streptomycin sensitivity, even at 25 mM concentrations (data not shown), suggesting that 1 O 2 and ⅐ OH are not involved in the antibacterial action of streptomycin. In order to q...