AAC
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
AAC.00779-06v1
51/3/1119    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Goswami, M.
Right arrow Articles by Jawali, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Goswami, M.
Right arrow Articles by Jawali, N.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, March 2007, p. 1119-1122, Vol. 51, No. 3
0066-4804/07/$08.00+0     doi:10.1128/AAC.00779-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Effects of Glutathione and Ascorbic Acid on Streptomycin Sensitivity of Escherichia coli{triangledown}

Manish Goswami, Suhas H. Mangoli, and Narendra Jawali*

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

Received 29 June 2006/ Returned for modification 11 August 2006/ Accepted 19 December 2006


   ABSTRACT
Top
 Abstract
Text
 References
 
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.


   TEXT
Top
 Abstract
 Text
References
 
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, Mn-superoxide 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).


View this table:
[in this window]
[in a new window]

  		TABLE 1. Strains and plasmids used in this study

 
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 (1O2) and hydroxyl radicals (·OH), respectively, did not alter the streptomycin sensitivity, even at 25 mM concentrations (data not shown), suggesting that 1O2 and ·OH are not involved in the antibacterial action of streptomycin. In order to quantify the antioxidant-mediated protection, the MIC of streptomycin for MG1655 was determined, using the agar dilution method with and without the addition of 10 mM of either glutathione or ascorbic acid in the medium. The MICs increased 2.5-fold (from 8 µg/ml to 20 µg/ml) in the presence of ascorbic acid and >30-fold (from 8 µg/ml to >250 µg/ml) in the presence of glutathione, compared to that of the controls (Table 2), suggesting that the protective effect against streptomycin is more pronounced with glutathione than with ascorbic acid. The enhanced protective effect seen with glutathione might be due to the dependence of the protective ability of the given antioxidant on its reducing power (6) or to the contribution of glutathione metabolism proteins involved in detoxification pathways in prokaryotes (23). We also investigated whether this antioxidant-mediated protection phenomenon is specific to MG1655 or whether it can be seen across diverse E. coli K-12 strains. Our results showed that for the W3110, XL-1 Blue, and DH5{alpha} strains, the MICs of streptomycin increased >2.0-fold (from 4.0 µg/ml to >8.0 µg/ml) in the presence of 10 mM ascorbic acid, and the increases in the MICs in the presence of 10 mM glutathione were 25-, 35-, and 60-fold for the W3110, XL-1 Blue, and DH5{alpha} strains, respectively, suggesting that, irrespective of the genetic background, these antioxidants interfere with a step that is crucial for streptomycin to manifest its antibacterial action against E. coli strains.


Figure 1
View larger version (96K):
[in this window]
[in a new window]

  		FIG. 1. Decreased sensitivity of E. coli MG1655 to streptomycin in the presence of 10 mM glutathione or ascorbic acid. Points 1, 2, and 3 correspond to 0.5, 1.0, and 2.0 µg of streptomycin, respectively, spotted on the Whatman disk.

 

View this table:
[in this window]
[in a new window]

  		TABLE 2. Augmentation of intracellular ROS in MG1655 incubated with different antibiotics/chemicalsa

 
Whether the antioxidant-mediated protection against streptomycin can be observed after preexposure of E. coli cells to antioxidants rather than incubation of the antioxidants and streptomycin together in the culture medium was investigated. Results of the analysis showed that growing MG1655 cells with 10 mM of either glutathione or ascorbic acid did not increase the MICs of streptomycin compared to that of their control, indicating that glutathione can alter streptomycin sensitivity of E. coli only when it is present in the culture medium along with streptomycin. We also examined whether this protective phenotype against streptomycin is glutathione concentration dependent. Hence, we determined the MICs of streptomycin for MG1655 in the presence of various glutathione concentrations. The results showed that, in the presence of 2.5, 5.0, and 7.5 mM of glutathione, the MICs of streptomycin increased by at least two-, six- and ninefold, respectively (Fig. 2), implying that the protection phenomenon is indeed glutathione concentration dependent.


Figure 2
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 2. Effects of various glutathione concentrations on the MICs of streptomycin for MG1655. LB alone (with 0.0 mM glutathione) was used as the control here.

 
The genes encoding the enzymatic defense machinery involved in keeping the steady-state level of ROS under control mainly comprise superoxide dismutases (sod), catalases (kat), and alkylhydroperoxide reductase (ahpCF) in E. coli (22). We analyzed the effects of multicopy sod genes as well as the effects of mutations in sod genes on the streptomycin sensitivity of MG1655. Transformation of MG1655 with pDT1.5, pFeSOD, or pSodC2.3 (multicopy plasmids having sodA, sodB, or sodC) did not alter the MICs of streptomycin (data not shown). Similarly, the sodA knockout strain NJ01 also did not differ from its parent strain with respect to its streptomycin sensitivity, showing that O2– does not have a role in the antibacterial action of this antibiotic. The sodB and sodC knockout strains were not used in the study, since they have aminoglycoside resistance markers associated with them. We also analyzed the effects of mutations in genes encoding enzymes that metabolize H2O2, i.e., catalases (katE and katG) and alkylhydroperoxide reductase (ahpCF), on the streptomycin sensitivity of MG1655. We examined all single and possible multiple mutants for these three genes (listed in reference 20), but for none of these mutants was the MIC of streptomycin increased compared to that of their wild-type parent strain (data not shown), hence negating the involvement of H2O2 in the antibacterial action of streptomycin. This observation puts streptomycin in a class separate from that of ciprofloxacin, in spite of the fact that antioxidant-mediated protection could be seen against both the antibiotics. That is because the katG ahpCF double mutant (JI374) and the katE katG ahpCF triple mutant (JI377) strains have severely compromised H2O2 scavenging functions (20), sensitizing them to ciprofloxacin where ROS were shown to be involved in its antibacterial action (13).

Alterations in ROS levels on exposure of bacterial cells to diverse antibiotics were measured by the NBT reduction method, as described by Albesa et al. (1), to authenticate our genetic data about the involvement of ROS in the antibacterial actions of different antibiotics. The results of the NBT assays indicated that ROS were intracellular, because reduced NBT was found only in the pellets and not in the supernatants. Table 2 shows the mean values of the ratio of the intracellular concentration of ROS detected in the presence or absence of the antibiotic. For all the aminoglycosides, this ratio was ≤1; however, for ciprofloxacin, which is known to generate ROS (2, 13), this value was 1.1, consistent with the value of 1.2 reported by Albesa et al. (1). For another well-known oxidative stress-inducing agent, menadione (24), it was found to be >2, which confirmed that treatment of MG1655 with streptomycin or other aminoglycosides does not lead to induction of ROS. On the contrary, reduced ROS levels were detected in the presence of streptomycin, since the ratio of ROS detected with streptomycin to ROS detected without streptomycin was found to be 0.78, which is <1.0. This drop in ROS levels in the presence of streptomycin could be due to the utilization of ROS for the initiation of oxidation of malformed proteins (3, 16) generated inside the cells due to mistranslations caused by streptomycin (10). This statement is supported by a recent report (11) establishing that efficient induction of the heat shock regulon due to increased mistranslation (21) requires the oxidative modifications of proteins.

Glutathione-mediated protection against other aminoglycosides, viz., kanamycin, gentamicin, and spectinomycin (Table 3), suggested that a protective phenotype against all the aminoglycoside antibiotics tested in the study could be seen. These observations extend our previous finding that glutathione can give protection not only against fluoroquinolones (13) but also against the aminoglycoside group of antibiotics. It is important to note here that aminoglycosides such as streptomycin, kanamycin, and gentamicin bind to the 30S ribosomal subunit and interfere with protein synthesis by causing mistranslations leading to the accumulation of aberrant proteins (9). However, spectinomycin, unlike other aminoglycosides, does not induce mistranslation but interferes with the translocation step of bacterial translation (5, 9). The fact that glutathione decreased the sensitivity of E. coli to both streptomycin and spectinomycin rules out the possibility that glutathione offers protection by overcoming mistranslations produced during bacterial-protein synthesis.


View this table:
[in this window]
[in a new window]

  		TABLE 3. Susceptibility of MG1655 to aminoglycoside and nonaminoglycoside antibiotics in the presence and absence of 10 mM glutathionea

 
On the whole, our data signify that the mitigated sensitivity of E. coli cells to streptomycin is not due to antioxidant-mediated scavenging of reactive oxygen species, as is the case with ciprofloxacin. Hence, other likely possibilities which might lead to protection against streptomycin need to be considered. The first possibility, which is unique to streptomycin, is a simple chemical reaction between the thiol group of glutathione and the aldehyde group of streptomycin to form a thiohemiacetal adduct. However, further metabolic modification of the streptomycin moiety is required for its complete detoxification, since formation of the thiohemiacetal adduct is a reversible reaction. This proposition is supported by studies in which glutathione has been shown to work in a sacrificial mode (7). The second possibility is glutathione S-transferase (gst)-mediated biotransformation of antibiotics, which could be applicable to both streptomycin and ciprofloxacin, since the gst-mediated enzymatic reaction produces glutathione conjugates of electrophilic compounds (17, 23), including antibiotics like fosfomycin (23), in bacterial cells. Moreover, glutathione transferases having high affinity toward different antibiotics are reported to be present in more than one bacterial system (17). This possibility is further supported by the observations that mycobacteria-like Mycobacterium smegmatis do not produce glutathione but instead synthesize a close homolog of glutathione, termed mycothiol (18), and that mycothiol-deficient M. smegmatis mutants were found to be hypersensitive to antibiotics (18). Moreover, mycothiol is involved in electrophile detoxification by conjugating to antibiotics. This reaction is performed by a protein analogous to glutathione transferase, and targeted mutagenesis of an amidase gene, mca, which works downstream of the conjugation reaction, led to 10-fold-lower MIC of streptomycin (19). Although the above-mentioned two possibilities can explain the increased protective capability of glutathione in comparison to that of ascorbic acid, they do not account for the protection offered by ascorbic acid. One possibility valid for both glutathione and ascorbic acid could be the alteration of the redox potential of the bacterial translational machinery in the case of streptomycin, which is its primary site of action. A few reports on the effects of redox compounds on protein synthesis have appeared previously (14, 25). Studies are under way to test whether the above-mentioned possibilities hold true for E. coli. Another potential target for antioxidant-mediated protection against diverse antibiotics could be the alteration in the redox potential of the medium, which may lead to simulation of anoxic conditions, as observed in a few earlier studies (8, 12).

On the basis of our results, we conclude that the presence of glutathione and ascorbic acid rescues bacteria from the antibacterial actions of aminoglycosides by a mechanism that is still not completely understood. However, unlike that of ciprofloxacin, the antibacterial action of aminoglycosides does not involve reactive oxygen species, implying that the presence of certain antioxidants might promote drug resistance. This observation might have important implications for therapeutics, since dietary supplements such as vitamins C (ascorbic acid) and E ({alpha}-tocopherol) and cysteine-rich foodstuffs (cysteine is a precursor for biosynthesis of glutathione) having antioxidant properties are sometimes prescribed along with antibiotics during the course of treatment of an infection and, under such conditions, the therapeutic effectiveness of aminoglycoside-mediated treatment might be substantially reduced due to increased cellular levels of antioxidants. Hence, further investigations pertaining to the concomitant intake of antioxidants and aminoglycosides during the treatment of various infections are required.


   ACKNOWLEDGMENTS
 
We are grateful to James Imlay (University of Illinois) for providing the strains and plasmid related to this study. We thank A. V. S. S. N. Rao for his help in preparing and critically reading the manuscript. We also thank D. Rath and S. Uppal for valuable discussions related to the study.


   FOOTNOTES
 
* 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.gov.in. Back

{triangledown} Published ahead of print on 8 January 2007. Back


   REFERENCES
Top
 Abstract
 Text
 References
 

  1. Albesa, I., M. C. Becerra, P. C. Battan, and P. L. Paez. 2004. Oxidative stress involved in the antibacterial action of different antibiotics. Biochem. Biophys. Res. Commun. 317:605-609.[CrossRef][Medline]
  2. Becerra, M. C., and I. Albesa. 2002. Oxidative stress induced by ciprofloxacin in Staphylococcus aureus. Biochem. Biophys. Res. Commun. 297:1003-1007.[CrossRef][Medline]
  3. Berlett, B. S., and E. R. Stadtman. 1997. Protein oxidation in aging, disease and oxidative stress. J. Biol. Chem. 272:20313-20316.[Free Full Text]
  4. Butler, J., and B. M. Hoey. 1993. Redox cycling drugs and DNA damage, p. 243-273. In B. Halliwell and O. I. Aruoma (ed.), DNA and free radicals. Ellis Horwood Ltd., West Sussex, England.
  5. Carter, A. P., W. M. Clemons, D. E. Broderson, R. J. Morgan-Warren, B. T. Wimberly, and V. Ramakrishnan. 2000. Functional insights from the structure of the 30S ribosomal subunit and its interaction with antibiotics. Nature 407:340-348.[CrossRef][Medline]
  6. Cheng, Z., and Y. Li. 2004. Reducing power: the measure of antioxidant activities of reductant compounds. Redox Rep. 9:213-217.[CrossRef][Medline]
  7. Chesney, J. A., J. W. Eaton, and J. R. Mahony, Jr. 1996. Bacterial glutathione: a sacrifice defense against chlorine compounds. J. Bacteriol. 178:2131-2135.[Abstract/Free Full Text]
  8. Cox, D. L., B. Riley, P. Chang, S. Sayahtaheri, S. Tassell, and J. Hevelone. 1990. Effects of molecular oxygen, oxidation-reduction potential, and antioxidants upon in vitro replication of Treponema pallidum subsp. pallidum. Appl. Environ. Microbiol. 56:3063-3072.[Abstract/Free Full Text]
  9. Davis, B. D. 1987. Mechanism of bactericidal action of aminoglycosides. Microbiol. Rev. 51:341-350.[Free Full Text]
  10. Dukan, S., A. Farewell, M. Ballesteros, F. Taddei, M. Radman, and T. Nystrom. 2000. Protein oxidation in response to increased transcriptional or translational errors. Proc. Natl. Acad. Sci. USA 97:5746-5749.[Abstract/Free Full Text]
  11. Fredriksson, A., M. Ballesteros, S. Dukan, and T. Nystrom. 2006. Induction of the heat shock regulon in response to increased mistranslation requires oxidative modification of the malformed proteins. Mol. Microbiol. 59:350-359.[CrossRef][Medline]
  12. George, S. M., and M. W. Peck. 1998. Redox potential affects the measured heat resistance of Escherichia coli O157:H7 independently of oxygen concentration. Lett. Appl. Microbiol. 27:313-317.[CrossRef][Medline]
  13. Goswami, M., S. H. Mangoli, and N. Jawali. 2006. Involvement of reactive oxygen species in the action of ciprofloxacin against Escherichia coli. Antimicrob. Agents Chemother. 50:949-954.[Abstract/Free Full Text]
  14. Kosowera, N. S., G. A. Vanderhoffa, and E. M. Kosowerb. 1961. Glutathione VIII. The effect of glutathione disulfide on initiation of protein synthesis. Biochim. Biophys. Acta 49:235-236.[CrossRef]
  15. Mingeot-Leclercq, M. P., Y. Glupczynski, and P. M. Tulkens. 1999. Aminoglycoside: activity and resistance. Antimicrob. Agents Chemother. 43:727-737.[Free Full Text]
  16. Nyström, T. 2005. Role of oxidative carbonylation in protein quality control and senescence. EMBO J. 24:1311-1317.[CrossRef][Medline]
  17. Piccolomini, R., C. D. Ilio, A. Aceto, N. Allocati, A. Faraone, L. Cellini, G. Ravagnan, and G. Federici. 1989. Glutathione transferase in bacteria: subunit composition and antigenic characterization. J. Gen. Microbiol. 135:3119-3125.[Medline]
  18. Rawat, M., G. L. Newton, M. Ko, G. J. Martinez, R. C. Fahey, and Y. Av-Gay. 2002. Mycothiol-deficient Mycobacterium smegmatis mutants are hypersensitive to alkylating agents, free radicals, and antibiotics. Antimicrob. Agents Chemother. 46:3348-3355.[Abstract/Free Full Text]
  19. Rawat, M., M. Uppal, G. Newton, M. Steffek, R. C. Fahey, and Y. Av-Gay. 2004. Targeted mutagenesis of the Mycobacterium smegmatis mca gene, encoding a mycothiol-dependent detoxification protein. J. Bacteriol. 186:6050-6058.[Abstract/Free Full Text]
  20. Seaver, L. C., and J. A. Imlay. 2001. Alkyl hydroperoxide reductase is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli. J. Bacteriol. 183:7173-7181.[Abstract/Free Full Text]
  21. Shaw, K. J., N. Miller, X. Liu, D. Learner, J. Wan, A. Bittner, and B. J. Morrow. 2003. Comparison of the changes in global gene expression of Escherichia coli induced by four bactericidal agents. J. Mol. Microbiol. Biotech. 5:105-122.
  22. Storz, G., and J. A. Imlay. 1999. Oxidative stress. Curr. Opin. Microbiol. 2:188-194.[CrossRef][Medline]
  23. Vuilleumier, S. 1997. Bacterial glutathione S-transferases: what are they good for? J. Bacteriol. 179:1431-1441.[Free Full Text]
  24. Yan, G., D. Haz, and J. Chen. 2006. Adaptive response of Bacillus sp. F26 to hydrogen peroxide and menadione. Curr. Microbiol. 52:238-242.[CrossRef][Medline]
  25. Zehavi-Willner, T., E. M. Kosower, T. Huntc, and N. S. Kosower. 1971. Glutathione V. The effect of the thiol-oxidizing agent diamide on initiation and translation in rabbit reticulocytes. Biochim. Biophys. Acta 228:245-251.[Medline]

Antimicrobial Agents and Chemotherapy, March 2007, p. 1119-1122, Vol. 51, No. 3
0066-4804/07/$08.00+0     doi:10.1128/AAC.00779-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
AAC.00779-06v1
51/3/1119    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Goswami, M.
Right arrow Articles by Jawali, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Goswami, M.
Right arrow Articles by Jawali, N.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Clin. Vaccine Immunol. Clin. Microbiol. Rev.
J. Clin. Microbiol. ALL ASM JOURNALS