Title: N-Acetylcysteine (NAC) Mediated Modulation of Bacterial Antibiotic

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1 AAC Accepts, published online ahead of print on June 00 Antimicrob. Agents Chemother. doi:0./aac Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Title: N-Acetylcysteine (NAC) Mediated Modulation of Bacterial Antibiotic Susceptibility Authors: Manish Goswami and Narendra Jawali * Address: Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 0005, India. Correspondent Footnote: * Dr. Narendra Jawali, Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 0005, India. Tel: (+9) ; Fax: (+9) Keywords: N-acetylcysteine, Glutathione, Thiol, Antibacterial, Aminoglycoside, Downloaded from on December 9, 0 by guest 5 Fluroquinolone

2 N-acetylcysteine (NAC) acts as a precursor for glutathione biosynthesis (, ), in addition of performing several other biological functions in mammals and bacteria (, 5). As per our earlier studies (3, ), glutathione is an important modulator of antibiotic activity in bacteria; consequently it is of interest to study the effect of NAC on bacterial antibiotic susceptibility. It becomes even more important since NAC is supplied as a mucolytic agent in combination with antibiotics during treatment of lower respiratory tract infection (). Here we report the effect of NAC on various antibiotics against different bacterial strains including opportunistic respiratory pathogens like Klebsiella and Pseudomonas. The E. coli strains were same as used in one of our earlier study (). Pseudomonas and Klebsiella strains were obtained from National Collection of Industrial Microorganisms (NCIM, Pune, India). Minimum inhibitory concentrations (MIC) of antibiotics were determined in Mueller Hinton agar as outlined by the Clinical Laboratory Standards Institute (CLSI) (7). The effect of NAC on bacterial susceptibility to 0 antibiotics, belonging to different groups, was studied by measuring their MIC in presence and absence of 0 mm NAC in the medium. Antibiotic susceptibility of 3 Escherichia coli, Klebsiella and Pseudomonas strain (See table # ) was determined for this purpose. As commonly used therapeutic concentration of NAC ranges between to 0 mm per day, for management of severe respiratory disorders (), the concentration of 0 mm NAC was chosen for this study. Presence of 0 mm NAC did not affect the growth of these bacterial strains, with an exception of K. pneumoniae (NCIM No. 3) which failed to grow on plate containing NAC. Further analysis revealed that even as low as.5 mm NAC was inhibitory for growth of K. pneumoniae strain. The effect of NAC, on MIC of antibiotics Downloaded from on December 9, 0 by guest

3 for remaining strains was studied, which is mentioned in table. In case of fluoroquinolones, MIC of both ciprofloxacin and ofloxacin increased substantially for all E. coli and K. aerogens strains and moderately for P. aeruginosa in presence of NAC (table ). Similarly presence of NAC led to significant increase in MIC of aminonoglycosides like streptomycin, kanamycin and spectinomycin against all the bacterial strains tested. These observations suggest that presence of NAC gives protection against wide variety of the aminoglycoside and fluroquinolone antibiotics. On the lines of aminoglycosides and fluoroquinolones, NAC provided substantial protection against erythromycin as well, for all the strains. However NAC did not alter the MIC of chloremphenicol and tetracycline for any bacterial strain suggesting that action of these antibiotics is not affected by the presence of NAC. Presence of NAC did not alter the MIC of penicillin against K. aerogens or any of the E. coli strains although it led to reduction in MIC of penicillin against P. aeruginosa. In addition NAC reduced the MIC of ampicillin for all the strains except K. aerogens. Overall we could see a more profound augmentation of ampicillin activity in comparison to penicillin by NAC for most of the strains. We therefore conclude that presence of NAC can either reduce the antibacterial activity of aminoglycosides, fluoroquinolones and erythromycin or enhance the efficacy of -lactams against several bacterial strains. Besides, presence of NAC can be detrimental for bacteria like K. pneumoniae. Our current study therefore demonstrates that NAC is an important modulator of antibiotic activity. Although specific mechanisms of antibiotic susceptibility modulation are not yet clear these findings along with our earlier data (3, ) suggest that thiols are important determinant of antibiotic activity against bacteria. This Proposition is additionally supported by an earlier independent 3 Downloaded from on December 9, 0 by guest

4 report where l-cysteine has been shown to be involved in alteration of antibiotic activity against Clostridium sp. (6). Our results further suggest that administration of aerosolized NAC as a mucolytic agent during antibiotic therapy of the respiratory tract infection could modulate the outcome of therapeutic process depending on the target bacterial pathogen and antibiotic being used for the therapy. Therefore further clinical studies are required to establish the clear-cut relation between aerosolized NAC and the antibiotics being used for therapeutic purpose against diverse bacterial pathogens. Acknowledgement: We want to thank Dr S. K. Apte for his constant encouragement and support during the period of this work. Downloaded from on December 9, 0 by guest

5 00 Table:. Effect of NAC on susceptibility of diverse bacterial strains to different antibiotics MIC (in g/ml) for diverse bacterial strains* P. aeruginosa K. aerogens MG655 W30 DH5 (NCIM No. 509) (NCIM No. ) Antibiotic Control + NAC # Control + NAC # Control + NAC # Control + NAC # Control + NAC # Ciprofloxacin Ofloxacin Streptomycin Kanamycin Spectinomycin Erythromycin Chloremphenicol Tetracycline Ampicillin 0 Penicillin Downloaded from on December 9, 0 by guest

6 Footnotes to the Table:. * : Antibiotic susceptibility data of K. pneumoniae (NCIM No. 3) is not included in the table as presence of NAC led to complete growth inhibition for K. pneumoniae # : Final NAC concentration was 0 mm. Downloaded from on December 9, 0 by guest 5 6

7 6 References Anonymous. N-Acetylcysteine. Alternative Medicine Review. Oct 000 Online Issue.. Fraternale, A., M. Paoletti, A. Casabianca, J. Oiry, P. Clayette, J. Vogel, J. Cinatl Jr, A. Palamara, R. Sgarbanti, E. Garaci, E. Millo, U. Benatti and M. Magnani 000. Antiviral and immunomodulatory properties of new proglutathione (GSH) molecules. Curr. Med. Chem. 3: Goswami M. and N. Jawali Glutathione mediated augmentation of beta lactam antibacterial activity against Eschirichia coli. J. Antimicrob. Chemother.60:-5.. Goswami, M., S. H. Mangoli and N. Jawali Effect of glutathione and ascorbic acid on streptomycin sensitivity of Escherichia coli. Antimicrob. Agents Chemother. 5: Marchese, A., M. Bozzolasco, L. Gualco, E. A. Debbia, G. C. Schito and A. M. Schito Effect of fosfomycin alone and in combination with N- acetylcysteine on E. coli biofilms. Int. J. Antimicrob. Agents. : Markowitz, S. M. and D. S. Williams. 95. Effect of l-cysteine on the activity of penicillin antibiotics against Clostridium difficile. Antimicrob. Agents Chemother. 7: National Committee for Clinical Laboratory Standards Methods for dilutuion antimicrobial susceptibility tests for bacteria that grow aerobically, 3rd ed. Approved standard M7-A3. National Committee for Clinical Laboratory Standards. Villanova, Pa. Downloaded from on December 9, 0 by guest 7

8 Zuin, R., Palamidese, A., Negrin, R., Catozzo, L., Scarda, A. and M. Balbinot High-Dose N-Acetylcysteine in Patients with exacerbations of chronic obstructive pulmonary disease. Clin. Drug Invest. 5(6): Downloaded from on December 9, 0 by guest

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