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Antimicrobial Agents and Chemotherapy, March 2006, p. 949-954, Vol. 50, No. 3
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.3.949-954.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Involvement of Reactive Oxygen Species in the Action of Ciprofloxacin against Escherichia coli

M. Goswami, S. H. Mangoli, and N. Jawali^*

Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India

Received 7 November 2005/ Returned for modification 11 November 2005/ Accepted 22 December 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The emergence of drug resistance against ß-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 the generation of "cellular poison" which ultimately leads to cell death (12, 14, 17).

Narrow-spectrum quinolones like nalidixic acid and oxolinic acid are used less often today because of their moderate gram-negative bacterial activity, minimal systemic distribution, and development of rapid resistance against them (29). Expanded-spectrum quinolones, such as norfloxacin and ciprofloxacin, marked the arrival of fluoroquinolones with better antibacterial coverage, since introduction of fluoro group at position 6 and piperazinyl side chain at position 7 of the quinolone ring expanded their gram-negative activity and broadened their spectrum to Pseudomonas (3). Further improvement in the chemical structure led to the development of fluoroquinolone derivatives that were more effective against gram-positive organisms and anaerobes as well (29).

Recently, a number of antibiotics, including ciprofloxacin, have been demonstrated to stimulate the production of reactive oxygen species (ROS) in bacterial cells (2, 6). Reactive oxygen species are reactive by-products formed by the partial reduction of molecular oxygen (32). Redox cycling of various chemical substances, including some antibiotics, affects the reactive oxygen species produced by cells during the oxidation process (10). Fluoroquinolones are known to induce the formation of singlet oxygen (¹O₂) and superoxide anion (O₂^–), which are responsible for the phototoxic effect of the fluoroquinolones (37). In addition, the two prominent side effects of aminoglycoside antibiotics, ototoxicity and nephrotoxicity, are also believed to involve ROS (9, 26). A number of diverse cellular processes that lead to cell death are also mediated through ROS (8, 11).

The enzymatic defense system against ROS comprises of specific enzymes, like superoxide dismutase, catalase, and peroxidase, which decrease the steady-state level of reactive oxygen (11, 15). Escherichia coli has three different superoxide dismutase (SOD) enzymes encoded by sodA, sodB, and sodC that metabolize O₂^–. sodA codes for an inducible cytosolic Mn-SOD (21, 36), sodB codes for Fe-SOD, which is constitutively expressed at basal levels during normal metabolic processes inside the cell (33, 38), and sodC codes for a periplasmic Cu-Zn-SOD, which takes care of the periplasmic and extracellular O₂^– (7, 18, 20). The product of the dismutation reaction of O₂^– is H₂O₂ (24), an important entity with a highly reactive nature and capable of damaging critical biomolecules. E. coli has two catalases, hydroperoxidase I (HPI) and HPII, involved in detoxification of intracellular H₂O₂ (23). HPI is encoded by katG, which is present during aerobic growth and transcriptionally controlled at different levels, and HPII is encoded by katE, which is induced during stationary phase (11). In addition, alkyl hydroperoxide reductase (ahpCF) provides an additional mechanism for scavenging H₂O₂ (35).

Even though ROS are reported to be induced by fluoroquinolones (1, 6), their role in the antibacterial action of these antibiotics is not clearly understood. Antioxidant-mediated reduction in antibiotic sensitivity would be an indication of the involvement of ROS in this process. Since dietary supplements, such as vitamin C (ascorbic acid) and vitamin E (-tocopherol), which have antioxidant properties, are sometimes prescribed along with antibiotics during the course of treatment of an infection, it is important to understand the effects of these antioxidants on the antibacterial action of these antibiotics. The aim of the present study was to investigate the role of ROS in the antibacterial action of fluoroquinolones. 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 levels of ROS in the cell. We examined the effects of antioxidant compounds, such as ascorbic acid, glutathione, histidine, mannitol, and sodium pyruvate, on the ciprofloxacin sensitivity of E. coli cells. Further, we studied the effects of mutations in oxidative stress defense genes, viz., superoxide dismutase (sodA, sodB, and sodC), catalase (katE and katG), and alkyl hydroperoxide reductase (ahpCF) on the ciprofloxacin sensitivity of E. coli cells. The effects of multicopy sod genes on ciprofloxacin sensitivity were also examined.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. Cultures were grown in Luria broth (LB) or plated in Luria agar. The sod mutants of strain MG1655 were constructed by P1-mediated transduction as previously described (25). Strains NJ01 and NJ02 were generated by transduction of sodA and sodB from strains JI130 and JI131, respectively, to MG1655. Plasmid transformations were performed using the CaCl₂ method as described by Sambrook et al. (34).

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TABLE 1. List of the bacterial strains and plasmids used in the study

Antioxidants and antibiotics. Antioxidants were freshly prepared as mentioned below before use. Stock solutions (250 mM) of glutathione and ascorbic acid were prepared in sterile distilled water and 0.5 N NaOH, respectively, followed by filter sterilization through 0.22-µm membrane (Millipore) and added to the media prior to pouring the plates. Stock solutions of histidine, mannitol, and sodium pyruvate were prepared in sterile distilled water and were added to Luria agar media prior to autoclaving. The pH of the media in general was adjusted to 7.4 before sterilization. Wherever required, Luria agar was supplemented with antibiotics and antioxidants at the concentration indicated.

Growth conditions. Strains were grown at 37°C for 12 to 18 h in Luria broth (Bacto tryptone, 10 g/liter; yeast extract, 5 g/liter; and NaCl, 10 g/liter) medium. Overnight cultures were inoculated into fresh medium. Cells in midexponential phase (optical density at 600 nm of 0.5 to 0.6) were used for the determination of antibiotic sensitivity. Wherever required, chloramphenicol (Cam), kanamycin (Kan), spectinomycin (Spec), and tetracycline (Tet) were added to a final concentration of 12.5, 30, 120, and 12.5 µg/ml, respectively. For the maintenance of plasmids pDT1.5 and pSodC2.3, ampicillin (Amp) was used at a final concentration of 100 and 300 µg/ml, respectively.

Sensitivity to antibiotics. Three methods were used for measuring antibiotic sensitivity.

(i) Antibiotic disk diffusion method. The antibiotic disk diffusion method was used as a qualitative measure to appreciate the differences in antibiotic sensitivities due to various treatments. Overnight E. coli cultures were diluted (1:100) in LB and grown afresh at 37°C. Mid-exponential-phase cultures were used to prepare the lawns of cells by the Kirby-Bauer method (5). A known amount of antibiotic solution was spotted on 5.5-mm-diameter Whatman filter disks placed on the bacterial lawn (no closer than 30 mm from the center of the disk), and the plates were incubated overnight at 37°C.

After overnight incubation, plates were scanned at a resolution of 300 x 300 dots per inch against a black background using a flat-bed scanner. The diameter of the zone of complete inhibition (as judged by the unaided eye) was measured using ImageJ software (http://rsb.info.nih.gov/ij). All the experiments were repeated more than two times to check the reproducibility of the results. The data of one representative experiment are presented here. The mean values of three measurements of the diameter taken at different angles were reported. For the antibiotic concentration that showed no visible zone of inhibition, a diameter of 5.5 mm was recorded (since the diameter of the zone of inhibition includes the diameter of disk spotted). As we have used various antibiotic concentrations, all the diameters of zone of inhibition were not within the quality control range for the diameter of the zone of inhibition (8 to 12 mm).

(ii) MIC. Ciprofloxacin MIC was determined by the agar dilution method as outlined by the Clinical and Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Standards [NCCLS]) (27, 28). An inoculum of approximately 10⁴ to 10⁵ CFU (simultaneously determined by plating) per spot was applied to the agar by a micropipette delivering 10 µl per spot. The MIC was the lowest concentration of antimicrobial agent that prevented visible growth after 20 h of incubation at 37°C. A slight haze of growth was ignored.

(iii) Survival curves. Overnight cultures were diluted (1:100) in LB medium and grown at 37°C. Cells from mid-exponential phase of growth were serially diluted and plated in duplicate on LB agar containing various concentrations of antibiotic with or without antioxidants. The number of colonies formed was counted and recorded after the agar plates were incubated overnight at 37°C. The count (log CFU/ml) was used as an estimate of bacterial viability. However, when the difference between two strains or treatments was less than 1 log unit, we represented the data in terms of % survival. In these cases, counts corresponding to the number of bacteria on LB agar without any antibiotics were taken as 100% survival for the respective culture.

Statistical analysis. The data reported are the average values from minimum of three experiments. Differences between two bacterial strains or treatments were analyzed by Student's t test. A P value of 0.05 was used as the cutoff for statistical significance.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of antioxidants on ciprofloxacin sensitivity of E. coli MG1655. The presence of 10 mM glutathione or ascorbic acid in the growth medium rendered MG1655 cells less susceptible to ciprofloxacin (Fig. 1) as seen by the antibiotic disk diffusion method. However, other antioxidants, such as histidine, mannitol, and sodium pyruvate, did not alter the ciprofloxacin sensitivity of MG1655 even at 25 mM concentration (data not shown). These results suggest that protection against the antibacterial action of ciprofloxacin is restricted to only a few antioxidants.

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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.

Quantification of antioxidant-mediated protection against ciprofloxacin sensitivity. Survival curves of strain MG1655 in the presence of various concentrations of ciprofloxacin were generated with and without the addition of a 10 mM concentration of either glutathione or ascorbic acid in the medium and were used to quantify the antioxidant-mediated protection. Our data that 50 and 100 ng/ml of ciprofloxacin decrease the number of CFU by more than 3 and 7 log units, respectively, indicated that MG1655 is highly sensitive to ciprofloxacin. Addition of glutathione to the medium gave complete protection up to 100 ng/ml of ciprofloxacin (Fig. 2). The survival was 90% in the presence of glutathione even at a 150-ng/ml concentration of ciprofloxacin (data not shown). In comparison, ascorbic acid gave 95% survival up to 25 ng/ml of ciprofloxacin and showed partial protection at higher concentrations (i.e., 51% survival at 50 ng/ml and 15% survival at 100 ng/ml). These data showed that the protective effect against ciprofloxacin is more pronounced with glutathione than for ascorbic acid.

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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.

Further quantitative estimates of the protection offered by glutathione and ascorbic acid against ciprofloxacin were made by measuring the MIC of ciprofloxacin for strain MG1655 in the presence and absence of either antioxidant. The MICs increased threefold (from 30 ng/ml to 90 ng/ml) in the presence of ascorbic acid and 10-fold (from 30 ng/ml to 300 ng/ml) in the presence of glutathione compared to the controls (data not shown).

Effects of glutathione on strain MG1655 sensitivity to other fluoroquinolone and nonfluoroquinolone antibiotics. Glutathione-mediated protection to MG1655 cells against some other fluoroquinolones, viz., norfloxacin, ofloxacin, and gatifloxacin, and nonfluoroquinolone antibiotics, such as ampicillin, chloramphenicol, and tetracycline, was investigated by the disk diffusion method. The diameters of the zone of inhibition were determined as described in Materials and Methods. For all the fluoroquinolones, the diameters of the zone of inhibition in the presence of 10 mM glutathione were lower than those of the corresponding controls (Table 2), suggesting that glutathione interfered with a step that is common among fluoroquinolones to bring about their antibacterial action. However, for all the nonfluoroquinolone antibiotics, the diameters of the zone of inhibition in the presence of 10 mM glutathione were not statistically different from the values for the corresponding controls (Table 3), suggesting that glutathione-mediated protection is not a general phenomenon but is specific to fluoroquinolones.

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TABLE 2. Effects of antioxidants on the diameters of the zone of inhibition produced by fluoroquinolone antibiotics

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TABLE 3. Effects of antioxidants on the diameters of the zone of inhibition produced by nonfluoroquinolone antibiotics

Roles of katE, katG, and ahpCF in ciprofloxacin sensitivity of strain MG1655. H₂O₂ is an important molecule with a highly reactive nature and potential to react with various biomolecules inside the cell. We analyzed the effects of mutations in genes encoding enzymes that metabolize H₂O₂, i.e., katE, katG, and ahpCF, on the ciprofloxacin sensitivity of MG1655 cells. Mutating any of these genes independently did not alter the ciprofloxacin sensitivity of the MG1655 strain (data not shown). We examined all possible combinations of multiple mutations for these three genes. katG ahpCF double mutant (JI374) and katE katG ahpCF triple mutant (JI377) strains showed significant changes in ciprofloxacin sensitivity levels (Fig. 3). Except for these strains, all other combinations did not produce an observable effect on the ciprofloxacin sensitivity level (data not shown). Both JI374 and JI377 exhibited increased ciprofloxacin sensitivity than their parent strain MG1655 (e.g., the number of CFU had decreased by about 3 log units at 30-ng/ml ciprofloxacin concentration) with JI377 showing relatively higher sensitivity (Fig. 3). Both these mutants also showed a twofold decrease in the MIC for ciprofloxacin compared to their parent strain MG1655.

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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.

Effects of sod genes on ciprofloxacin sensitivity of strain MG1655. MG1655 transformants of plasmid pDT1.5, pFeSOD, or pSodC2.3 (having the sodA, sodB, or sodC gene, respectively) showed better survival at 10-ng/ml ciprofloxacin concentration; the increase in survival was 17% with pDT1.5 and 24% with pFeSOD or pSodC2.3 over that of MG1655 alone (Table 4). However, at 20- and 30-ng/ml ciprofloxacin concentrations, none of the plasmids decreased the ciprofloxacin susceptibility of MG1655. Superoxide dismutase knockout strains NJ01 (MG1655 sodA) and NJ02 (MG1655 sodB) did not differ significantly from their parent strain with respect to ciprofloxacin susceptibility (Table 4). However, the ciprofloxacin susceptibility of AS393 (AB1157 sodC) was found to be higher by 12% and 22% compared to AB1157 at 5- and 10-ng/ml ciprofloxacin concentrations, respectively (Table 5). This suggested that O₂^– might have a role in the antibacterial action of ciprofloxacin, particularly at low concentrations.

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TABLE 4. Effects of knocking out and presence of multicopy sod genes on the ciprofloxacin sensitivity of E. coli MG1655

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TABLE 5. Effect of knocking out sodC on the ciprofloxacin sensitivity of E. coli AB1157

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrates the role of reactive oxygen species in the antibacterial action of fluoroquinolones. This statement is supported by the observation that known ROS scavengers, such as glutathione and ascorbic acid, gave protection to E. coli MG1655 against ciprofloxacin. Two other independent reports, i.e., stimulation of ROS production by ciprofloxacin in E. coli, Enterococcus faecalis, and Staphylococcus aureus (2) and the presence of increased levels of ROS in ciprofloxacin-sensitive microorganisms (6) substantiate our findings. The inability of other antioxidants, viz., histidine, a ¹O₂ scavenger (16), mannitol, a known ·OH scavenger (13), and sodium pyruvate, to impart a protective phenotype suggests that only nonspecific scavengers having low redox potential could provide protection. The enhanced protective effect seen with glutathione in comparison to ascorbic acid might be due to the dependence of the protective ability on the redox potential of the given antioxidant for one electron reduction pathway (22). Such selectivity of the antioxidants for e protection against oxidative stress has recently been reported (22). The inability of glutathione to protect cells against the antibacterial action of nonfluoroquinolone antibiotics demonstrates that the glutathione-mediated protection is not a general phenomenon but is specific to fluoroquinolones.

Our finding that glutathione gave protection against other fluoroquinolones as well implies that reactive oxygen species may have a similar role in the antibacterial action of all these fluoroquinolones. These observations are in contrast to those of Alba et al. (1) who, on the basis of unaltered in vitro bactericidal effect of norfloxacin by the presence of ß-carotene, suggested that ROS do not have a role to play in the bactericidal effect of fluoroquinolones. However, it is important to note here that E. coli is a noncarotenogenic microorganism (4) without any reported transport protein for ß-carotene. Hence, the observations of Alba et al. could be attributed to the inefficient transport of ß-carotene across the E. coli cell membrane due to its lipophilic nature and the absence of specific transporters for it. On the other hand, compounds such as glutathione and ascorbic acid can readily cross the cell membrane because of their hydrophilic nature, low molecular weight, and presence of specific transporters for these antioxidants on the cell membrane (31, 39), which enables them to manifest their antioxidant action in the cytosol.

Our observation that intact katG or ahpCF is required by dividing E. coli cells for protection against the antibacterial action of ciprofloxacin confirms the involvement of oxidative stress in this phenotype, and the presence of wild-type katE alone is not sufficient to protect the dividing cells against the H₂O₂-mediated antibacterial action of ciprofloxacin. It is important to note here that both ahpCF and katG lie under the control of the oxyR regulon that plays an important role in overcoming the oxidative stress caused by H₂O₂ (11, 36). JI374 cells that have severely compromised H₂O₂ scavenging function (35) show increased ciprofloxacin sensitivity. Further increased ciprofloxacin sensitivity of JI377 demonstrates that the complete elimination of the H₂O₂ scavenging function in E. coli makes cells hypersensitive to ciprofloxacin. Mutations of kat genes in combination with ahpCF could alter the ciprofloxacin sensitivity of the cells, implying that ahpCF has an equally important role in scavenging of endogenous H₂O₂, which is in agreement with the findings of Seaver and Imlay (35).

The presence of the multiple H₂O₂ scavenging activities ensures that the remaining functional H₂O₂ metabolizing activities protect the cells from ciprofloxacin when ahpCF alone is knocked out or when one or both of the catalases are mutated in E. coli. An unaltered ciprofloxacin sensitivity of a strain carrying mutations in katE and ahpCF is in line with the findings of Seaver and Imlay (35) that mutations in katE and ahpCF together do not hamper the H₂O₂ detoxification ability of the cells.

Superoxide dismutases present in the cell are sufficient to take care of the significant increase of superoxide anions inside the cell (15, 36) that are generated as the by-products of normal metabolic processes. The modulation of ciprofloxacin sensitivity either by the multiple copies of sod genes or by sod mutants shows that superoxide anions also have a role in the antibacterial action of this antibiotic (Table 2 and 3). All the sod transformants showed better survival in comparison to strain MG1655 (Table 2) at low ciprofloxacin concentration (10 ng/ml). The unaltered ciprofloxacin sensitivity of sodA and sodB mutant derivatives (NJ01 and NJ02, respectively) may be due to the redundancy of cytosolic superoxide dismutase activities where the absence of one activity can be compensated by the presence of the other one. On the other hand, the slightly increased ciprofloxacin sensitivity of a sodC mutant derivative (AS393) at a low antibiotic concentration shows the distinctive importance of sodC, as it encodes the sole superoxide dismutase activity present in the periplasm of E. coli. However, the concentration dependence of this effect needs further characterization.

On the basis of our results, we conclude that the antibacterial action of fluoroquinolones involves reactive oxygen species, such as superoxide anions and hydrogen peroxide. However, the exact mechanism of this phenomenon is yet to be worked out. We have shown that the presence of antioxidants rescues bacteria against the antibacterial action of fluoroquinolones. These observations are of significance, as fluoroquinolones are important antibiotics with immense therapeutic value, and further investigations surrounding the intake of antioxidants on the effects of fluoroquinolones for the treatment of infections caused by E. coli are warranted in the future.

 
We are grateful to James Imlay for providing the strains and plasmids related to this study. We also thank A. V. S. S. N. Rao, D. Rath, and S. Uppal for valuable discussion and critical reading of the manuscript.

 
* Corresponding author. Mailing address: Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India. Phone: (91) 22-25595078. Fax: (91) 22-25505326. E-mail: enjay{at}apsara.barc.ernet.in.

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Antimicrobial Agents and Chemotherapy, March 2006, p. 949-954, Vol. 50, No. 3
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.3.949-954.2006
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