JAC Advance Access originally published online on May 4, 2007
Journal of Antimicrobial Chemotherapy 2007 60(1):184-185; doi:10.1093/jac/dkm121
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Correspondence |
Glutathione-mediated augmentation of ß-lactam antibacterial activity against Escherichia coli
Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India
* Corresponding author. Tel: +91-22-25595078; Fax: +91-22-25505326; E-mail: enjay{at}barc.gov.in
Keywords: antioxidants , ascorbic acid , protection , susceptibility
We have previously established that antioxidants such as glutathione and ascorbic acid protect Escherichia coli against the antibacterial activity of fluoroquinolone and aminoglycoside groups of antibiotics.1,2 However, the mechanism by which the antioxidant-mediated protection is brought about against these two classes of antibiotics is not the same since our data signified that, unlike ciprofloxacin, reduced streptomycin susceptibility of E. coli cells is not due to antioxidant-mediated scavenging of reactive oxygen species.2 Here we report the effect of antioxidants on the antibacterial activity of commonly used and therapeutically relevant ß-lactam antibiotics.
The effect of glutathione (GSH) and ascorbic acid on the susceptibility of E. coli K-12 strain MG1655 to ampicillin and penicillin was investigated. MICs of these antibiotics in the presence and absence of 10 mM of these antioxidants were determined by agar dilution method as outlined by the CLSI (formerly the NCCLS).3 An inoculum of approximately 104–105 cfu (simultaneously determined by plating) per spot (in a volume of 10 µL) was applied to the agar plates with increasing antibiotic concentration. MIC was the lowest concentration of antimicrobial agent that prevented visible growth after 20 h of incubation at 37°C. The experiments were carried out at least twice and the representative results are mentioned here. We observed that the presence of 10 mM GSH made MG1655 cells more susceptible to these ß-lactams, because addition of GSH resulted in reduced MICs of ampicillin and penicillin from 8 and 64 mg/L to 4 and 48 mg/L, respectively. However, we found that this effect was specific to GSH, as the presence of ascorbic acid did not make any difference to the antibiotic susceptibility of MG1655.
Total intracellular glutathione (GSHin) is a sum of reduced glutathione (GSH) + oxidized glutathione (GSSG) amounts and bacterial cells can readily take up both GSH and GSSG, resulting in enhanced GSHin.4 As GSHin along with GSH to GSSG ratio are key regulators of various intracellular redox reactions, we investigated whether this glutathione-mediated phenotype is dependent on its redox status outside the cell. For this purpose the MICs of the above-mentioned ß-lactams were determined in the presence of 5 mM GSSG. The results showed that GSSG also decreased the MICs of the antibiotics to the same extent as observed with GSH, implying that glutathione-mediated augmentation of ß-lactam susceptibility is independent of its redox status outside the cell.
Gamma glutamyl-transepeptidase (ggt, EC 2.3.2.2 [EC] )-mediated cleavage of glutathione is required for the uptake of extracellular glutathione by bacterial cells.5 In addition, as ggt catalyses the transfer of the gamma glutamyl group of glutathione and related gamma-glutamyl amides to other amino acids and peptides (transpeptidation), it is likely to be involved in the transpeptidation step of bacterial cell wall synthesis. Considering that ß-lactams act at the transpeptidation step of cell wall synthesis, glutathione-mediated augmentation of ß-lactam antibacterial activity could be due to the competition between ggt-mediated cell wall synthesis and the glutathione uptake process. We investigated whether this possibility holds true by determining the MICs of ampicillin and penicillin for SH639, SH664 and SH673 strains (different ggt knockout strains of E. coli, a gift from Dr Hideyuki Suzuki) (see Suzuki et al.5 for details). However, we found that ggt knockout mutants did not differ from their parent strain in terms of ampicillin and penicillin susceptibility (data not shown), indicating that glutathione-mediated increased antibacterial activity of each ß-lactam is not arbitrated by the status of ggt in E. coli. Another possible mechanism for this enhanced antibacterial effect could be altered expression/regulation of cysB in the presence of glutathione (cysB is an ORF encoding a regulator for GSH transport) as cysB is known to be involved in determination of bacterial susceptibility to ß-lactams.6
Irrespective of which mechanisms operate behind the above-mentioned phenotype, our observations are of significance, revealing that glutathione cannot only reduce the antibacterial effect of certain antibiotics (as previously demonstrated by us), but also enhance the antibacterial activity of ß-lactam antibiotics. This study along with previous reports,1,2 reveal that glutathione differentially modulates the antibacterial activity of various groups of antibiotics. Additionally, since ß-lactams are important antibiotics having immense therapeutic value, further investigations surrounding the effect of glutathione on antibacterial activity of ß-lactams would be of interest in the future for development of improved treatment regimens for various infections.
None to declare.
Acknowledgements
We are grateful to Hideyuki Suzuki (Kyoto University, Japan) for providing the strains related to this study. We thank all the members of MMGES group for the valuable discussions related to the study. We also thank Dr S. K. Apte for his constant encouragement and support.
References
1
Goswami M, Mangoli SH, Jawali N. Involvement of reactive oxygen species in the action of ciprofloxacin against Escherichia coli. Antimicrob Agents Chemother (2006) 50:949–54.
2
Goswami M, Mangoli SH, Jawali N. Effects of glutathione and ascorbic acid on streptomycin sensitivity of Escherichia coli. Antimicrob Agents Chemother (2007) 51:1119–22.
3 National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Third Edition: Approved Standard M7-A3 (1997) Villanova, PA, USA: NCCLS.
4
Vergauwen B, Pauwels F, Vaneechoutte M, et al. Exogenous glutathione completes the defense against oxidative stress in Haemophilus influenzae. J Bacteriol (2003) 185:1572–81.
5
Suzuki H, Koyanagi T, Izuka S, et al. The yliA, -B, -C and -D genes of Escherichia coli K-12 encode a novel glutathione importer with an ATP binding cassette. J Bacteriol (2005) 187:5861–7.
6 Oppezzo OJ, Anton DA. Involvement of cysB and cysE genes in the sensitivity of Salmonella typhimurium to mecillinam. J Bacteriol (1995) 77:4524–7.
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