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Manish Goswami, Suhas H. Mangoli and Narendra Jawali Different types of biochemical and physiological factors (including commonly used cellular anddietary antioxidants) affect the activity of therapeutic antibiotics. The findings from our lab haveshown that the presence of glutathione, N-acetylcysteine or ascorbic acid in the growth mediumdecreases the bacterial susceptibility towards fluoroquinolone and aminoglycoside antibiotics. Howeverthe mechanism behind the antioxidant mediated protection against these two groups of antibioticscould be different. In addition, presence of glutathione increased antibacterial activity of β-lactamantibiotics showing that glutathione could act as a differential antibiotic susceptibility modulator forbacteria. Our data therefore demonstrate that therapeutic effectiveness of antibiotic treatment couldbe modulated by the dietary intake and cellular level of these antioxidants.
In our lab, we are studying the effect of dietary andcellular antioxidants on antibacterial effect of Antibiotics are natural or synthetic compounds with commonly used antibiotics. Dietary supplements selective bactericidal or bacteriostatic effects that eliminate pathogens or slow their growth such that E (α-tocopherol), having antioxidant properties, are host defense mechanisms can clear the infection.
prescribed many a times by the physicians along The mechanisms of antibiotics actions are well with antibiotics during the course of treatment of studied, particularly in relation to their targets an infection. Besides antioxidants such as interactions. Majority of the commonly used N-acetylcysteine (NAC) are used as auxiliary antibiotics fall into following groups: DNA damage- medication in certain pathological conditions along causing agents, inhibitors of protein synthesis, with the antibiotic therapy (NAC is used as a inhibitors of cell wall biosynthesis and metabolic mucolytic agent in combination with clinically inhibitors. Antibiotics are weapons of choice in fight relevant antibiotics for treatment of lower respiratory against infectious bacterial diseases, however, their tract infection). Therefore it is important to overuse and misuse has contributed significantly to understand the effects of antioxidants on the the growing problem of antibiotic resistance and antibacterial action of commonly used antibiotics.
emergence of superbugs such as Methicillin We are actively working to understand how different Resistant Staphylococcus aureus (MRSA) and New antioxidants affect the action of diverse antibiotics Delhi Metallo-beta-lactamase-1 (NDM-1) isolates.
under in-vitro and in-vivo conditions and what are Therefore knowledge about molecular mechanism the mechanisms operating behind the observed of antibiotic action, related bacterial response and effect. The overall aim of our studies was to factors modulating antibiotic activity could be of understand the effect of commonly used antioxidants used for development of improved antibacterial on antibacterial efficacy of therapeutically relevant substances and therapeutic regimen, which could antibiotics. It was followed up by understanding help us in keeping pace with remarkable adaptability the role of reactive oxygen species (ROS) in the antibacterial action of antibiotics which displayed 42 I ISSUE NO. 323 I NOV. - DEC. 2011
reduced effectivenessin presence of the antioxidants.
We have used wild type E. coli K-12 strain MG1655as the model test organisms for our study. Duringthe course of our studies we have examined theeffect of a number of antioxidants viz. glutathione,ascorbic acid, histidine, mannitol, sodium pyruvateand α-ketoglutarate on susceptibility of MG1655against antibiotics such as streptomycin,chloramphenicol, tetracycline, ampicillin, penicillinand ciprofloxacin etc. After initial rounds ofstandardization 10 mM of antioxidant concentrationwas chosen for further studies (depending on theirmaximum achievable intra-cellular concentrationand toxic side effects). The major findings of ourstudies are abridged under following sub-headings: Fig. 1: Decreased susceptibility of MG1655 towards ciprofloxacin in the presence of 10 mM glutathione (GSH) or Ascorbic acid (ASC). C-1, C-2, C-3 and C-4correspond to 40, 200, 400 and 2000 ng ofciprofloxacin spotted on the Whatman disk.
The effects of antioxidants on the antibioticsusceptibility of MG1655 were first analyzed by the MG1655 in LB-agar in presence and absence of antibiotic disk diffusion method, in which zone of either antioxidant as per the clinical laborarory inhibition around the antibiotic disk placed on the standard institute (CLSI) guidelines and our results bacterial cell lawn reveals antibiotic susceptibility showed that the protective effect against of the bacteria under study. Reduction in the zone ciprofloxacin is more pronounced with GSH than of inhibition around the ciprofloxacin disks indicated for ASC. The MIC of ciprofloxacin increased 3 fold that the presence of 10 mM glutathione (GSH) or in the presence of ASC and >10 fold in the presence ascorbic acid (ASC) in the growth medium leads to of GSH as compared to the control (Table 1).
reduced ciprofloxacin susceptibility of MG1655 cells Ciprofloxacin is a representative member of (Fig. 1). The action of antibiotics such as fluoroquinolone group of antibiotics, which act by chloramphenicol and tetracycline was not visibly affected due to presence of these antioxidants. Other topoisomerase IV activities. It was further found that antioxidants, such as histidine, mannitol (scavengers GSH gives protection against other fluoroquinolones specific for singlet oxygen and hydroxyl radicals as well such as ofloxacin, norfloxacin and respectively), sodium pyruvate and α-ketoglutarate gatifloxacin (please see table 1 and reference 3 for did not alter MG1655 susceptibility towards any of the antibiotics (data not shown) suggesting thatonly nonspecific antioxidants (capable of neutralizing different types of ROS) having low redox potential ciprofloxacin could be through scavenging of ROS could provide protection against ciprofloxacin.
generated in the presence of antibiotic.
Consequently effect of mutations in oxidative stress Quantitative estimates of the protection offered by defense genes viz. superoxide dismutases (sodA, GSH and ASC were made by measuring minimum sodB & sodC), catalases (katE & katG) and alkyl inhibitory concentration (MIC) of ciprofloxacin for hydro peroxide reductase (ahpCF) on ciprofloxacin ISSUE NO. 323 I NOV. - DEC. 2011 I 43
Table 1: Susceptibility of MG1655 towards different exhibited slightly increased ciprofloxacin antibiotics in the presence and absence of 10 mM susceptibility (3). Furthermore E. coli cells having any of the multi-copy sod genes showed better survival in comparison to wild type parent strain at low ciprofloxacin concentration (3). Our genetic data therefore suggested that the antibacterial action of ciprofloxacin involves ROS, such as superoxide anions and hydrogen peroxide and antioxidant mediated protection against ciprofloxacin could be Like in case of ciprofloxacin, GSH and ASC were susceptibility of MG1655 was studied. These genes found to protect E. coli cells against streptomycin encode enzymatic defense system against ROS, as well. Streptomycin belongs to aminoglycoside regulating their intracellular steady-state level.
group of antibiotics, which act by interfering with Besides effect of multi-copy sod genes on bacterial protein synthesis machinery. GSH was ciprofloxacin susceptibility of MG1655 was also found more effective as compared to ASC, since examined. Among different single and multiple MIC of streptomycin increased by 2.5 fold in the mutants of katE, katG & ahpCF studied, katG ahpCF presence of ASC and more than 30 fold in presence double mutant (JI374) and the katE katG ahpCF of GSH as compared to the control (Table 1). Both triple mutant (JI377) strains having severely GSH and ASC were found to inhibit the antibacterial exhibited significantly increased ciprofloxacin kanamycin, gentamycin and spectinomycin, please susceptibility in comparison to MG1655 (Fig. 2).
see table #1 for details). During the course of our Similarly E. coli cells carrying sodC mutation also studies we also investigated whether this antioxidant-mediated protection against streptomycin is specificto MG1655 or it can be seen across diverse E. colistrains such as W3110, Xl-1 blue and DH5á. Ourresults showed that GSH and ASC were effectiveagainst streptomycin in the above mentioned strainsas well. It implies that, irrespective of the geneticbackground, GSH and ASC interfere with a stepthat is crucial for antibacterial action of streptomycinin E. coli.
Our genetic data suggested that unlike ciprofloxacin,mitigated streptomycin susceptibility of E. coli cellsis not due to antioxidant mediated scavenging ofROS. None of the single and multiple mutants of Fig. 2: Graph showing increased ciprofloxacin susceptibility in katG ahpCF double mutant (JI374) and streptomycin susceptibility. Similarly mutated or the katE katG ahpCF triple mutant (JI377) strains incomparison to the wild type parent strain MG1655.
multi-copy sod genes also failed to alter the bacterial 44 I ISSUE NO. 323 I NOV. - DEC. 2011
streptomycin susceptibility levels. Our genetic data both cysteine and NAC exhibited protection against was further corroborated by the NBT reduction antibacterial action of fluoroquinolones and values which demonstrated that streptomycin aminoglycosides (please see reference 2 for details).
treatment does not lead to induction of oxidative The extent of protection was found to be higher stress in E. coli, whereas ciprofloxacin indeed with NAC in comparison to cysteine. Apart from E.
increases the ROS levels in bacterial cells (4). It coli, NAC had profound effect on other bacteria implies that the mechanism by which antioxidant such as Klebsiella aerogens, Klebsiella pneumoniae, mediated protection is brought about against these and Pseudomonas aeruginosa in terms of their two classes of antibiotics is not same. Our data survival and antibiotic susceptibility modulation.
therefore also emphasized that other than ROS Besides, NAC also augmented the action of β-lactam scavenging process, additional and alternate antibiotics. Our studies therefore suggest that mechanisms operate behind antioxidant-mediated administration of aerosolized NAC as a mucolytic protection against various antibiotics.
agent during the course of antibiotic therapy forrespiratory tract infection could modulate the Glutathione can act as bacterial antibiotic outcome of therapeutic process depending on the target bacterial pathogen and antibiotic being usedfor the therapy. Taken together our data furtherindicates that GSH and its precursors in general act An important observation made during the course as crucial determinant of antibiotic activity against of our study was that GSH not only reduces theantibacterial action of above mentioned antibiotics but it also augments the efficacy of β-lactams suchas penicillin and ampicillin (1, 5), since E. coli cells were found to be more susceptible towards themin presence of GSH (Table 1). However, we foundthat this effect was specific to GSH, as the presenceof ASC did not make any difference to the antibioticsusceptibility of MG1655. Therefore our studiesdemonstrate that GSH can act as an importantmodulator of antibiotic susceptibility for bacteria(Fig. 3).
Fig. 3: Biochemical functions associated with glutathione inside bacterial cells (according to Masip et al., 2006). The function indicated in red color isassigned by us on the basis of results from our lab(1,5).
GSH is a naturally occurring tri-peptide (glutamicacid-cysteine-glycine) and its biosynthesis is limited On the basis of our studies it can be concluded that by the amino acid l-cysteine. N-acetylcysteine (NAC) antibacterial action of therapeutically relevant is acetylated derivative of the amino acid cysteine.
antibiotics could be either diminished or augmented It is used as an important antioxidant and precursor by the presence of antioxidants like GSH, NAC and of GSH in higher organisms. Since we have ASC. Our study adds to the knowledge that apart demonstrated that GSH is an important antibiotic from existence of other antibiotic activity modulation susceptibility modulator, therefore it is of interest factors, dietary and cellular antioxidants also play a to study the effect of GSH precursors on antibiotic critical role in determination of bacterial antibiotic susceptibility of bacteria. Consequently the effect susceptibility. Although detailed mechanism(s) of of cysteine and NAC supplementation on bacterial antioxidant mediated modulation of antibiotic antibiotic susceptibility was determined. Like GSH, activity are yet to be fully understood, these findings ISSUE NO. 323 I NOV. - DEC. 2011 I 45
are of immense value. Dietary supplements such as vitamin C (ASC), cystiene rich foodstuffs andauxiliary medication like NAC, which have antioxidant properties, are prescribed sometimes Glutathione mediated augmentation of beta along with antibiotics during the course of treatment of an infection. The therapeutic effectiveness of Escherichia coli. J. Antimicrob. Chemother.
antibiotic mediated treatment under such conditions might be altered due to increased dietary intakeand cellular levels of these antioxidants. Hence, further investigations surrounding the intake of antioxidants on antibacterial effect of different bacterial antibiotic susceptibility. Antimicrob.
antibiotics for treatment of various infections are warranted in future. Currently, we are working inthis direction to understand the real time effects of Goswami, M., Mangoli, S. H. and Jawali M.
antioxidant supplementation on antibiotic mediated 2006. Involvement of reactive oxygen species clearance of bacterial infection in an animal model Escherichia coli. Antimicrob. AgentsChemother. 50: 949–54.
We are grateful to different people in scientific glutathione and ascorbic acid on streptomycin community who have provided strains and plasmids sensitivity of Escherichia coli. Antimicrob.
related to this study. We also thank Dr S. K. Apte, Associate Director (B), BMG and Head MBD for hisconstant encouragement and support during the University. 2010. Studies on mechanism ofantioxidant mediated protection againstaminoglycoside antibiotics in Escherichia coli.
Masip, L., Veeravali, K., and Georgiou, G.
2006. The many faces of glutathione inbacteria. Antiox. Redox Signal. 8:753-62 Seaver, L. C. and Imlay, J. A. 2001. Alkylhydroperoxide reductase is the primaryscavenger of endogenous hydrogen peroxide in Escherichia coli. Bacterial. 183:7173-81.
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