Abstract
A bacteriocin from Bacillus subtilis (MK733983) originated from ethnomedicinal plant was purified using Preparative RP-HPLC. The HPLC fraction eluted with 65% acetonitrile showed the highest antimicrobial activity with Mycobacterium smegmatis as an indicator. Its specific activity and purification fold increased by 70.5% and 44%, respectively, compared to the crude bacteriocin. The bacteriocin showed stability over a wide range of pH (3.0–8.0) and preservation (− 20 °C and 4 °C), also thermal stability up to 80 °C for 20 min. Its proteinaceous nature was confirmed with complete loss of activity on its treatment with Trypsin, Proteinase K, and α-Chymotrypsin. Nevertheless, the bacteriocin retained up to 45% activity with Papainase treatment and was unaffected by salivary Amylase. It maintained ~ 95% activity on UV exposure up to 3 h and its activity was augmented by ethyl alcohol and metal ions like Fe2+ and Mn2+. Most of the common organic solvents, general surfactants, preservatives, and detergents like Sulfobetaine-14, Deoxy-cholic-acid did not affect the bacteriocin’s action. Its molecular weight was estimated to be 3.4KDa by LC-ESI-MS/MS analysis. The bacteriocin is non-hemolytic and exhibited a broad inhibition spectrum with standard strains of Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Chromobacterium violaceum with MICs ranging 0.225 ± 0.02–0.55 ± 0.05 mg/mL. Scanning Electron Microscopy showed cell annihilation with pores in cell membranes of S. aureus and P. aeruginosa treated with the bacteriocin, implicating bactericidal mode of action. These promising results suggest that the bacteriocin is significant and has wide-ranging application prospects.
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All the data, text and results presented in this publication are authors own. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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References
Afsar T, Razak S, Khan MR, Mawash S, Almajwa A, Shabir M, Haq IU (2016) Evaluation of antioxidant, anti-hemolytic and anticancer activity of various solvent extracts of Acacia hydaspica R. Parker aerial parts. BMC Complement Altern Med 16:258. https://doi.org/10.1186/s12906-016-1240-8
Agrawal P, Miryala S, Varshney U (2015) Use of Mycobacterium smegmatis deficient in ADP ribosyl transferase as surrogate for Mycobacterium tuberculosis in drug testing and mutation analysis. PLoS ONE 10(4):e0122076. https://doi.org/10.1371/journal.pone.0122076
Aguilar-Pérez C, Gracia B, Rodrigues L, Vitoria A, Cebrián R, Deboosère N, Song O-R, Brodin P, Maqueda M, Aínsa JA (2018) Synergy between circular bacteriocin AS-48 and ethambutol against Mycobacterium tuberculosis. Antimicrob Agents Chemother 62:e00359-e418. https://doi.org/10.1128/AAC.00359-18
Anand TP, Bhat AW, Shouche YS, Roy U, Siddharth J, Sarma SP (2005) Antimicrobial activity of marine bacteria associated with sponges from the waters off the coast of South East India. Microbiol Res 161(2006):252–262. https://doi.org/10.1016/j.micres.2005.09.002
Aurea AE, Tanh LC, Mai LN, Philippe D (2011) Reverse-high performance liquid chromatography mechanism explained by polarization of stationary phase. CheM 1:62–79. https://doi.org/10.5618/chem.2011.v1.n1.8
Aurea AE, Roya S, Manolis NR, Philippe D (2014) An alternative to trial and error methodology in solid phase extraction: an original automated solid phase extraction procedure for analyzing PAHs and PAH-derivatives in soot. RSC Adv 4:33636. https://doi.org/10.1039/c4ra03214d
Balouiri M, Sadiki M, Ibnsouda SK (2016) Methods for in vitro evaluating antimicrobial activity: a review. J Pharmaceut Anal 6(2):71–79
Batista JH, da Neto JFN (2017) Chromobacterium violaceum pathogenicity: updates and insights from genome sequencing of novel chromobacterium species. Front Microbiol 8:2213. https://doi.org/10.3389/fmicb.2017.02213
Bizani D, Brandelli A (2002) Characterization of a bacteriocin produced by a newly isolated Bacillus sp. Strain 8 A. J Appl Microbiol 93(3):512–519. https://doi.org/10.1046/j.1365-2672.2002.01720.x
Bizani D, Motta AS, Morrissy JA, Terra R, Souto AA, Brandelli A (2005) Antibacterial activity of cerein 8A, a bacteriocin-like peptide produced by Bacillus cereus. Int Microbiol 8:125–131
Boelaert M, Jacobs J (2014) Fatal chromobacterium violaceum bacteraemia in rural Bandundu, Democratic Republic of the Congo. New Microbes New Infect 3:21–23. https://doi.org/10.1016/j.nmni.2014.10.007
Cita YP, Suhermanto A, Radjasa OK, Sudharmono P (2017) Antibacterial activity of marine bacteria isolated from sponge Xestospongia testudinaria from Sorong. Papua Asian Pac J Trop Biomed 7(5):450–454. https://doi.org/10.1016/j.apjtb.2017.01.024
Gao FH, Abee T, Konings WN (1991) Mechanism of action of the peptide antibiotic nisin in liposomes and cytochrome c oxidase-containing proteoliposomes. Appl Environ Microbiol 57(8):2164–2170. https://doi.org/10.1128/AEM.57.8.2164-2170.1991
Ge J, Sun Y, Xin X, Wang Y, Ping W (2016) Purification and partial characterization of a novel bacteriocin synthesized by Lactobacillus paracasei HD1-7 isolated from Chinese Sauerkraut juice. Sci Rep 6:19366. https://doi.org/10.1038/srep19366
Gray EJ, Lee KD, Souleimanov AM, Falco MRD, Zhou X, Ly A, Charles TC, Driscoll BT, Smith DL (2006) A novel bacteriocin, thuricin 17, produced by plant growth promoting rhizobacteria strain Bacillus thuringiensis NEB17: isolation and classification. J Appl Microbiol 100(3):545–554. https://doi.org/10.1111/j.1365-2672.2006.02822.x
Gutsmann T (2016) Interaction between antimicrobial peptides and mycobacteria. Biochem Biophys Acta 1858:1034–1043. https://doi.org/10.1016/j.bbamem.2016.01.031
Hammami RA, Jaouadi B, Rebai A, Nesme X (2009) Optimization and biochemical characterization of a bacteriocin from a newly isolated Bacillus subtilis strain 14B for biocontrol of Agrobacterium spp. Strains Lett Appl Microbiol 48(2):253–260. https://doi.org/10.1111/j.1472-765X.2008.02524.x
Kassaa A, Hober I, Hamze D, Chihib NE, Drider D (2014) Antiviral potential of lactic acid bacteria and their bacteriocins. Probiotics Antimicrob Prot 6:177–185. https://doi.org/10.1007/s12602-014-9162-6
Lange-Starke A, Petereit A, Truyen U, Braun PG, Fehlhaber K, Albert T (2014) Antiviral potential of selected starter cultures, bacteriocins and D, L-Lactic Acid. Food Environ Virol 6(1):42–47. https://doi.org/10.1007/s12560-013-9135-z
Le CF, Fang CM, Sekaran SD (2017) Intracellular targeting mechanisms by antimicrobial peptides. Antimicrob Agents Chemother 61:e02340-e2416. https://doi.org/10.1128/AAC.02340-16
Lee SG, Chang HG (2018) Purification and characterization of mejucin, a new bacteriocin produced by Bacillus subtilis SN7. LWT Food Sci Technol 87:815. https://doi.org/10.1016/j.lwt.2017.08.044
Li XZ, Li Z, Hiroshi N (2004) Efflux pump-mediated intrinsic drug resistance in Mycobacterium smegmatis. Antimicrob Agents Chemother 48(7):2415–2423. https://doi.org/10.1128/AAC.48.7.2415-2423.2004
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275
Malini M, Savitha J (2012) Heat stable bacteriocin from Lactobacillus paracasei subsp. tolerans isolated from locally available cheese: an in vitro study. J Biotechnol Pharm Res 3(2):28–41
Naafs MAB (2018) The antimicrobial peptides: ready for clinical trials? Biomed J Sci Technol Res. https://doi.org/10.26717/BJSTR.2018.07.001536
Park YS, Yang YJ, Kim YB, Hong JH, Lee C (2002) Characterization of subtilein, a bacteriocin from Bacillus subtilis CAU131 (KCCM 10257). J Microbiol Biotechnol 12(2):228–234
Peng JY, Horng YB, Wu CH, Chang CY, Tsai PS, Jeng CR, Cheng YH, Chang HW (2019) Evaluation of antiviral activity of Bacillus licheniformis-fermented products against porcine epidemic diarrhea virus. AMB Express 9(1):191. https://doi.org/10.1186/s13568-019-0916-0
Pfalzgraff A, Brandenburg K, Weindl G (2018) Antimicrobial peptides and their therapeutic potential for bacterial skin infections and wounds. Front Pharmacol 9:281. https://doi.org/10.3389/fphar.2018.00281
Ramya R, Chalasani AG, Lal R, Roy U (2014) A broad-spectrum antimicrobial activity of Bacillus subtilis RLID 12.1. Sci World J. https://doi.org/10.1155/2014/968487
Santhi SS, Aranganathan V (2019) Bioprospecting of some ethnomedicinal plants for potential antimycobacterial bacteriocin like inhibitory substances (BLIS). Explor Anim Med Res 9(2):180–187
Sharma G, Dang S, Gupta S, Gabrani R (2018) Antibacterial activity, cytotoxicity, and the mechanism of action of bacteriocin from Bacillus subtilis GAS101. Med Princ Pract 27(2):186–192. https://doi.org/10.1159/000487306
Silva DS, Castro CC, Silva FS, Santanna V, Vargas GD, Lima MD, Fischer G, Brandelli A, Motta AS, Hübner SO (2014) Antiviral activity of a Bacillus sp. P34 peptide against pathogenic viruses of domestic animals. Braz J Microbiol 45(3):1089–1094
Sivaraj A, Sundar R, Manikkam R, Parthasarathy K, Rani U, Vanaja K (2018) Potential applications of lactic acid bacteria and bacteriocins in antimycobacterial therapy. Asian Pac J Trop Med 11(8):453–459. https://doi.org/10.4103/1995-7645.240080
Sohail M, Sultana Q, Rasool K, Sarwar S, Basit A, Khalid M (2015) Bacteremia prediction by inflammatory factors and recent trend in drug resistance of bacteria isolated from blood stream infection. J Inf Mol Biol 3(3):75–80. https://doi.org/10.14737/journal.jimb/2015/3.3.75.80
Sosunov V, Mischenko V, Eruslanov B, Svetoch E, Shakina Y, Stern N, Majorov K, Sorokoumova G, Selishcheva A, Apt A (2007) Antimycobacterial activity of bacteriocins and their complexes with liposomes. J Antimicrob Chemother 59(5):919–925. https://doi.org/10.1093/jac/dkm053
Soto PF (2014) Purification and characterization of an antimicrobial peptide produced by Bacillus sp. strain P7. Dissertation, University of Manchester.
Starosila D, Rybalko S, Varbanetz L, Ivanskaya N, Sorokulova I (2017) Anti-influenza activity of a Bacillus subtilis probiotic strain. Antimicrob Agents Chemother 61:e00539-e617. https://doi.org/10.1128/AAC.00539-17
Tagg JR, McGiven AR (1971) Assay system for bacteriocins. Appl Microbiol 21(5):943
Tiberi S, Muñoz-Torrico M, Duarte R, Dalcolmo M, D’Ambrosio L (2018) Migliori GB (2017) New drugs and perspectives for new anti-tuberculosis regimens. Pulmonology 24(2):86–98. https://doi.org/10.1016/j.rppnen.10.009
Wang X, Hu W, Zhu L, Yang Q (2017) Bacillus subtilis and surfactin inhibit the transmissible gastroenteritis virus from entering the intestinal epithelial cells. Biosci Rep 37(2):BSR20170082. https://doi.org/10.1042/BSR20170082
WHO (2017) Antibacterial agents in clinical development: an analysis of the antibacterial clinical development pipeline, including tuberculosis. Geneva: World Health Organization (WHO/EMP/IAU/2017.12). Licence: CC BY-NC-SA 3.0 IGO.CIP data are available at http://apps.who.int/iris. Accessed 20 Feb 2018
WHO (2020) Report https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance. Accessed 31 July 2020
Wolcott RD, Ehrlich GD (2008) Biofilms and chronic infections. JAMA 299(22):2682–2684. https://doi.org/10.1001/jama.299.22.2682
Wu J, Abbas HMK, Li J, Yuan Y, Liu Y, Wang G, Dong W (2020) Cell membrane-interrupting antimicrobial peptides from Isatis indigotica fortune isolated by a Bacillus subtilis expression system. Biomolecules. https://doi.org/10.3390/biom10010030
Zhang J, Yang Y, Yang H, Bu Y, Yi H, Zhang L, Han X, Ai L (2018) Purification and partial characterization of bacteriocin Lac-B23, a novel bacteriocin production by Lactobacillus plantarum J23, isolated from Chinese traditional fermented milk. Front Microbiol 9:2165. https://doi.org/10.3389/fmicb.2018.02165
Acknowledgements
The authors wish to express their sincere gratitude and recognition for assistance from Dr. Solomon (with HPLC) and Mr. Regan Charles (with SEM analysis) of Department of Biotechnology, Jain (Deemed to-be) University.
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Santhi Sudha, S., Aranganathan, V. Experimental elucidation of an antimycobacterial bacteriocin produced by ethnomedicinal plant-derived Bacillus subtilis (MK733983). Arch Microbiol 203, 1995–2006 (2021). https://doi.org/10.1007/s00203-020-02173-7
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DOI: https://doi.org/10.1007/s00203-020-02173-7