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Intracellular oxidative damage due to antibiotics on gut bacteria reduced by glutathione oxidoreductase-derived antioxidant molecule GM15


The human gut consists of > 1000 different bacterial species for the smooth functioning of the gut. In normal conditions, the antioxidant system present in cells minimize the effects of reactive oxygen species. Upon exposure to antibiotics, there is a rise in ROS level which induces oxidative stress to the cells, ultimately killing the cells. Two broad-spectrum antibiotics, streptomycin and gentamicin at a concentration of 50 µM and 25 µM, were treated with Bacillus subtilis SRMIST201901 (MN726522) and B. cereus SRMIST201902 (MN726923); the treatment reduced the cell counts. Considering the bacterial defense property which relies on the antioxidant mechanism, in this study, we have reported an antioxidant peptide (GM15) derived from glutathione oxidoreductase of spirulina (or called cyanobacteria) Arthrospira platensis (Ap) which reduced the intracellular oxidative stress. Cellular ROS detection was confirmed by fluorescent-associated cell sorting (FACS) using the DCFDA dye. Resazurin dye test also confirmed the activity of peptide on the growth of the Bacillus sp. Based on the results obtained, it was concluded that there was a significant (P < 0.05) reduction in the intracellular oxidative stress on treating with GM15 peptide. Overall, the study indicates the influence of antioxidant peptide on the intracellular oxidative stress, leading to the development of an antioxidant drug from glutathione oxidoreductase of A. platensis against oxidative-related stresses.

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  1. Andrews JM (2001) Determination of minimum inhibitory concentrations. J Antimicrob Chemother 48:5–16

  2. Armstrong JS, Steinauer KK, Hornung B, Irish JM, Lecane P, Birrell GW, Peehl DM, Knox SJ (2002) Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line. Cell Death Differ 9:252–263

  3. Arockiaraj J, Sarasvathi E, Puganeshwaran V, Arunsingh SV, Rofina YO, Subha B (2011) Prophenoloxidase activating enzyme-III from giant freshwater prawn Macrobrachium rosenbergii: characterization, expression and specific enzyme activity. Mol Biol Rep 39:1377–1386

  4. Bela K, Horváth E, Gallé Á, Szabados L, Tari I, Csiszár J (2015) Plant glutathione peroxidases: emerging role of the antioxidant enzymes in plant development and stress responses. J Plant Physiol 176:192–201

  5. Dwyer DJ, Belenky PA, Yang JH, MacDonald IC, Martell JD, Takahashi N, Chana CTY, Lobritza MA, Braff D, Schwarz EG, Ye JD, Pati M, Vercruysse M, Ralifo PS, Allison KR, Khalil AS, Ting AY, Walker GC, Collins JJ (2014) Antibiotics induce redox-related physiological alterations as part of their lethality. Proc Natl Acad Sci USA 111:E2100–E2109

  6. Elshikh M, Ahmed S, Funston S, Dunlop P, McGaw M, Marchant R, Banat IM (2016) Resazurin-based 96-well plate microdilution method for the determination of minimum inhibitory concentration of biosurfactants. Biotechnol Lett 38:1015–1019

  7. Eruslanov E, Kusmartsev S (2010) Identification of ROS using oxidized DCFDA and flow-cytometry. Advanced protocols in oxidative stress II. Humana Press, Totowa, pp 57–72

  8. Gutteridge JM, Wilkins S (1983) Copper salt-dependent hydroxyl radical formation: damage to proteins acting as antioxidants. Biochim Biophys Acta 759:38–41

  9. He Y, Ingudam S, Reed S, Gehring A, Strobaugh TP, Irwin P (2016) Study on the mechanism of antibacterial action of magnesium oxide nanoparticles against foodborne pathogens. J Nanobiotechnol 14:54

  10. Kapusnik-Uner JE, Sande MA, Chambers HF (1996) Antimicrobial agents: tetracycline, chloramphenicol, erythromycin, and miscellaneous antibacterial agents. In: Hardman JG, Limberd LE (eds) Goodman and Gilman’s the pharmacological basis of therapeutics, 9th edn. McGraw-Hill, New York, pp 1124–1130

  11. Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ (2007) A common mechanism of cellular death induced by bactericidal antibiotics. Cell 130:797–810

  12. Kumaresan V, Bhatt P, Palanisamy R, Gnanam AJ, Pasupuleti M, Arockiaraj J (2014) A murrel cysteine protease, cathepsin L: bioinformatics characterization, gene expression and proteolytic activity. Biologia 69:395–406

  13. Kumaresan V, Gnanam AJ, Pasupuleti M, Arasu MV, Al-Dhabi NA, Harikrishnan R, Arockiaraj J (2015) Comparative analysis of CsCu/ZnSOD defense role by molecular characterization: gene expression-enzyme activity-protein level. Gene 564:53–62

  14. Kumaresan V, Nizam F, Ravichandran G, Viswanathan K, Palanisamy R, Prasanth B, Arasu MV, Al-Dhabi NA, Mala K, Arockiaraj J (2017) Transcriptome changes of blue-green algae, Arthrospira sp. in response to sulfate stress. Algal Res 23:96–103

  15. Kumaresan V, Sannasimuthu A, Arasu MV, Al-Dhabi NA, Arockiaraj J (2018) Molecular insight into the metabolic activities of a protein-rich micro alga, Arthrospira platensis by de novo transcriptome analysis. Mol Biol Rep 45:829–838

  16. Li Y, Jiang B, Zhang T, Mu W, Liu J (2008) Antioxidant and free radical-scavenging activities of chickpea protein hydrolysate (CPH). Food Chem 106:444–450

  17. Liu Y, Liu X, Qu Y, Wang X, Li L, Zhao X (2012) Inhibitors of reactive oxygen species accumulation delay and/or reduce the lethality of several antistaphylococcal agents. Antimicrob Agent Chemother 56:6048–6050

  18. Maynard A, Butler NL, Ito T, da Silva AJ, Murai M, Chen T, Koffas MAG, Miyoshi H, Barquera B (2019) Antibiotic korormicin A kills bacteria by producing reactive oxygen species. J Bacteriol 201:e00718–e818

  19. Melander RJ, Zurawski DV, Melander C (2017) Narrow-spectrum antibacterial agents. Medchemcomm 9:12–21

  20. Mittler R (2017) ROS are good. Trend. Plant Sci 22:11–19

  21. Natividad JM, Verdu EF (2013) Modulation of intestinal barrier by intestinal microbiota: pathological and therapeutic implications. Pharmacol Res 69:42–51

  22. Ong IM, Gonzalez JG, McIlwain SJ, Sawin EA, Schoen AJ, Adluru N, Alexander AL, John-Paul JY (2018) Gut microbiome populations are associated with structure-specific changes in white matter architecture. Transl Psychiatry 8:6

  23. Palomino JC, Martin A, Camacho M, Guerra H, Swings J, Portaels F (2002) Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob Agent Chemother 46:2720–2722

  24. Priuska EM, Schacht J (1995) Formation of free radicals by gentamicin and iron and evidence for an iron/gentamicin complex. Biochem Pharmacol 50:1749–1752

  25. Rahman SMK (2015) Probiotic properties analysis of isolated lactic acid bacteria from buffalo milk. Arch Clin Microbiol 7:1

  26. Rajilić-Stojanović M, de Vos WM (2014) The first 1000 cultured species of the human gastrointestinal microbiota. FEMS Microbiol Rev 38:996–1047

  27. Ravichandran G, Kumaresan V, Mahesh A, Dhayalan A, Arshad A, Arasu MV, Al-Dhabi NA, Pasupuleti M, Arockiaraj J (2018) Bactericidal and fungistatic activity of peptide derived from GH18 domain of prawn chitinase 3 and its immunological functions during biological stress. Intl J Biol Macromol 106:1014–1022

  28. Rowland I, Gibson G, Heinken A, Scott K, Swann J, Thiele I, Tuohy K (2018) Gut microbiota functions: metabolism of nutrients and other food components. Euro J Nutr 57:1–24

  29. Sannasimuthu A, Arockiaraj J (2019) Intracellular free radical scavenging activity and protective role of mammalian cells by antioxidant peptide from thioredoxin disulfide reductase of Arthrospira platensis. J Funct Food 61:103513

  30. Sannasimuthu A, Kumaresan V, Pasupuleti M, Paray BA, Al-Sadoon MK, Arockiaraj J (2018) Radical scavenging property of a novel peptide derived from C-terminal SOD domain of superoxide dismutase enzyme in Arthrospira platensis. Algal Res 35:519–529

  31. Sannasimuthu A, Kumaresan V, Anilkumar S, Pasupuleti M, Ganesh MR, Kanchana M, Paray BA, Al-Sadoon MK, Albeshr MF, Arockiaraj J (2019) Design and characterization of a novel Arthrospira platensis glutathione oxido-reductase-derived antioxidant peptide GM15 and its potent anti-cancer activity via caspase-9. Free Rad Biol Med 135:198–209

  32. Sato H, Feix JB (2006) Peptide–membrane interactions and mechanisms of membrane destruction by amphipathic α-helical antimicrobial peptides. Biochim Biophys Acta 1758:1245–1256

  33. Sharma NYS (2016) Reactive oxygen species, oxidative stress and ROS scavenging system in plants. J Chem Pharm Res 8:595–604

  34. Sharma P, Jha AB, Dubey RS, Pessarakli M (2012) Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J Bot 2012:217037

  35. Sies H (1997) Oxidative stress: oxidants and antioxidants. Exp Physiol 82:291–295

  36. Song J, Kang SM, Lee WT, Park KA, Lee KM, Lee JE (2014) Glutathione protects brain endothelial cells from hydrogen peroxide-induced oxidative stress by increasing nrf2 expression. Expy Neurobiol 23:93–103

  37. Wu D, Yotnda P (2011) Production and detection of reactive oxygen species (ROS) in cancers. J Vis Exp 57:e3357

  38. Zaid AA, Hammad DM, Sharaf EM (2015) Antioxidant and anticancer activity of Spirulina platensis water extracts. Intl J Pharmacol 11:846–851

  39. Zhang YJ, Li S, Gan RY, Zhou T, Xu DP, Li HB (2015) Impacts of gut bacteria on human health and diseases. Intl J Mol Sci 16:7493–7519

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The authors would like to extend their sincere appreciation to the Researchers Supporting Project Number (RSP-2019/144), King Saud University, Riyadh, Saudi Arabia.

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Correspondence to Jesu Arockiaraj.

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The authors declare that they have no conflict of interest.

Human and animal rights

This experiment does not contain any human or animal participants. We have performed the experiment as per the SRM Institute of Science and Technology (SRMIST) research guidelines and regulations. All the experimental protocols were approved by the research committee of SRMIST.

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Communicated by Erko Stackebrandt.

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Sannasimuthu, A., Sharma, D., Paray, B.A. et al. Intracellular oxidative damage due to antibiotics on gut bacteria reduced by glutathione oxidoreductase-derived antioxidant molecule GM15. Arch Microbiol (2020). https://doi.org/10.1007/s00203-020-01825-y

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  • Glutathione oxidoreductase
  • Bacillus cereus
  • Bacillus subtilis
  • Arthrospira platensis
  • Gentamicin
  • Streptomycin