ISOLATION OF POTENTIAL ANTIMICROBIAL METABOLITE FROM ENDOPHYTIC BACILLUS AMYLOLIQUEFACIENS DL06 OF CARNIVOROUS PLANT DROSERA BURMANNII VAHL.
Abstract
Objectives: Exploitation of bacterial endophytes for production of antimicrobial substances has led to the discovery of novel natural metabolites of diverse chemical nature. The present study focuses attention toward optimization of cultural conditions for production of antimicrobial compound(s) by an endophytic bacterium DL06 followed by its extraction and partial purification.
Methods: The leaf endophytic bacterium Bacillus amyloliquefaciens DL06 (GenBank Accession no. MK696415, Microbial Culture Collection Accession no. 4186) isolated from carnivorous plant Drosera burmannii has been identified as a potent producer of antimicrobial metabolite following agar cup assay against several test bacterial and fungal strains. Cultural conditions for production of antimicrobials were optimized by “one variable at a time” method. The active fraction was isolated and purified partially using solvent extraction, thin-layer chromatography, and high performance liquid chromatography (HPLC) analysis.
Results: B. amyloliquefaciens DL06 produced maximum antimicrobial compound in tryptic soy broth and Davis–Mingioli’s medium when grown under shake culture. Production of the antimicrobial metabolite has been optimized for the inoculum density, aeration, temperature, pH as well as carbon, and nitrogen sources. The antimicrobial metabolite was extracted from the cell-free culture filtrate in butanol and partially purified by silica gel column chromatography and HPLC.
Conclusions: The antimicrobial metabolite, tentatively identified as quercetin showed broad spectrum bioactivity affecting several fungi and a number of Gram-positive and Gram-negative bacteria.
Keywords:
Carnivorous plant, Drosera burmannii, Bacterial endophytes, Antimicrobial metabolite, Bacillus amyloliquefaciens
References
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4. Guteirrez RM, Gonzales AM, Ramirez AM. Compounds derived from endophytes: A review of phytochemistry and pharmacology. Curr Med Chem 2012;19:2992-3030.
5. Castillo UF, Strobel GA, Ford EJ, Hess WM, Porter H, Jensen HB, et al. Munumbicins, wide-spectrum antibiotics produced by Streptomyces NRRL 30562, endophytic on Kennedia nigriscans. Microbiology 2002;148:2675-85.
6. Xie J, Wu Y, Zhang T, Zhang M. New antimicrobial compounds produced by endophytic Penicillium janthinellum isolated from Panax notoginseng as potential inhibitors of Ftsz. Fitoterapia 2018;131:35-43.
7. Manganyi M, Regnier T, Tchatchouang CK, Bezuidenhout C. Antibacterial activity of endophytic fungi isolated from Sceletium tortuosum L. (Kougoed). Ann Microbiol 2019;69:659-63.
8. Jayatilake PL, Munasinghe H. Antimicrobial activity of cultivable endophytic and rhizospheric fungi associated with “Mile-a-Minute” Mikania cordata (Asteraceae). Biomed Res Int 2020;2020:5292571.
9. Sanusi SB, Abu Bakar MF, Mohamed M, Sabran SF, Mainasara MM. Ethnobotanical, phytochemical, and pharmacological properties of nepenthes species: A review. Asian J Pharm Clin Res 2017;10:16-9.
10. Madhavan V, Basnett H, Gurudeva MR, Yoganarasimhan SN. Pharmacognostical evaluation of Drosera burmannii Vahl (Droseraceae). Indian J Tradit Knowl 2009;8:326-333.
11. Buch F, Rott M, Rottloff S, Paetz C, Hilke I, Raessler M, Mithofer A. Secreted pitfall-trap fluid of carnivorous Nepenthes plants is unsuitable for microbial growth. Ann Bot 2013;111:375-83.
12. Lee JM, Tan WS, Ting AS. Revealing the antimicrobial and enzymatic potentials of culturable fungal endophytes from tropical pitcher plants (Nepenthes spp.). Mycosphere 2014;5:364-77.
13. Christina A, Christapher V, Bhore SJ. Endophytic bacteria as a source of novel antibiotics: An overview. Pharmacogn Rev 2013;7:11-6.
14. Chaudhuri M, Pal A, Paul AK. Investigation of biochemical properties and biological activities of bacteria endogenous to carnivorous plant Drosera burmannii Vahl. Int J Adv Biotechnol Res 2015;6:278-89.
15. Chaudhuri M, Paul AK, Pal A. Isolation and assessment of metabolic potentials of bacteria endophytic to carnivorous plants Drosera burmannii and Utricularia spp. Biosci Biotech Res Asia 2019;16:731-41.
16. Gerhardt P. Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology; 1994.
17. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disc method, Am J Clin Pathol 1966;45:493.
18. Difco Manual. Dehydrated Culture Media and Reagents for Microbiology. 10th ed. Detroit, Michigan: Difco Laboratories; 1984.
19. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acid Res 1994;22:4673-80.
20. Buchanan RE, Gibbons NE. Bergey’s Manual of Determinative Bacteriology. Baltimore: Williams and Wilkins; 1975.
21. Yu H, Zhang L, Li L, Zheng C, Guo L, Li W, et al. Recent developments and future prospects of antimicrobial metabolites produced by endophytes. Microbiol Res 2010;165:437-49.
22. Joseph B, Priya RM. Bioactive compounds from endophytes and their potential in pharmaceutical effect: A review. Am J Biochem Mol Biol 2011;1:291-309.
23. Ek-Ramos MJ, Gomez-Flores R, Orozco-Flores AA, Rodríguez- Padilla C, González-Ochoa G, Tamez-Guerra P. Bioactive products from plant-endophytic Gram-Positive bacteria. Front Microbiol 2019;10:463.
24. Glenn A, Bodri MS. Fungal endophyte diversity in Sarracenia. PLoS One 2012;7:1-7.
25. Quillium RS, Jones DL. Evidence of host-specificity of culturable fungal root endophytes form the carnivorous plant Pinguicula vulgaris. Mycol Prog 2012;11:583-585.
26. Naseem F, Kayang H. Fungal endophytes associated with Nepenthes khasiana Hook. F., an endemic plant of Meghalaya, India. Int J Curr Res Life Sci 2018;7:1907-12.
27. White JF Jr., Torres MS, Sullivan RF, Jabbour RE, Chen Q, Tadych M, et al. Occurrence of Bacillus amyloliquefciens as a systemic endophyte of Vanilla orchids. Microsc Res Tech 2014;77:1-12.
28. Chen YT, Yuan Q, Shan LT, Lin MA, Cheng DQ, Li CY. Antitumor activity of bacterial exopolysaccharides from the endophyte Bacillus amyloliquefaciens sp. isolated from Ophiopogon japonicus. Oncol Lett 2013;5:1787-92.
29. Li H, Soares MA, Torres MS, Bergen M, White JF Jr. Endophytic bacterium, Bacillus amyloliquefaciens, enhances ornamental host a resistance to diseases and insect pests. J Plant Interact 2015;10:224-9.
30. Wang X, Liang G. Control efficacy of an endophytic Bacillus amyloliquefaciens strain BZ6-1 against peanut bacterial wilt, Ralstonia solanacearum. Biomed Res Int 2014;2014:465435.
31. Yin X, Xu L, Xu L, Fan S, Liu Z, Zhang X. Evaluation of the efficacy of endophytic Bacillus amyloliquefaciens against Botryosphaeria dothidea and other phytopathogenic microorganisms. Afr J Microbiol Res 2011;5:340-5.
32. Das I, Panda M, Rath CC, Tayung K. Bioactivities of bacterial endophytes isolated from leaf tissues of Hyptis suaveolens against some clinically significant pathogens. J Appl Pharm Sci 2017;7:131-6.
33. Wang C, Wang Y. Endophytic Bacillus amyloliquefaciens YTB1407 elicits resistance against two fungal pathogens in sweet potato (Ipomoea batatas Lam.). J Plant Physiol 2020;253:154260.
34. Bhoonobtong A, Sodngan S, Boonlue S, Bunyatratchata W, MongkoitW. Antibiotics constituents of endophytic Bacillus amyloliquefaciens UD25 extracted from a medicinal plant, Memecylon edule Roxb. Chiang Mai J Sci 2015;44:788-99.
35. Zhu H, Pan Y. A novel antimicrobial protein of the endophytic Bacillus amyloliquefaciens and its control effect against Fusarium chlamydosporum. Biocontrol 2019;64:737-48.
36. Wo?nicka E, Ku?niar A, Nowak D, Nykiel E, Kopacz M, Gruszecka J, et al. Comparative study on the antibacterial activity of some flavonoids and their sulfonic derivatives. Acta Pol Pharm 2013;70:567-71.
37. Jaisinghani RN. Antibacterial properties of quercetin. Microbiol Res 2017;8:13-4.
38. da Silva CR, Neto BA, Campos RS, Figueiredo NS, Sampaio LS, Magalhaes HI, et al. Synergistic effect of the flavonoid catechin, quercetin or epigallocatechin gallate with fluconazole induces apoptosis in Candida tropicalis resistant to fluconazole. Antimicrob Agents Chemother 204;58:1468-78.
39. De Oliveira MR, Nabavi SM, Braidy N, Setzer WN, Ahmed T, Nabavi SF. Quercetin and the mitochondria: A mechanistic view. Biotechnol Adv 2016;34:532-49.
40. Rocha MF, Sales JA, da Rocha MG, Galdino LM, de Aguiar L, et al. Antifungal effects of the flavonoids kaempferol and quercetin: A possible alternative for the control of fungal biofilms. Biofouling 2019;35:320-8.
41. Ramadhan F, Mukarramah L, Oktavia FA, Yulian R, Annisyah NH, Asyiah IS. Flavonoids from endophytic bacteria of Cosmos caudatas Kunth. leaf as anticancer and antimicrobial. Asian J Pharm Clin Res 2018;11:200-4.
42. Bhardwaj A, Sharma D, Jadon N, Agarwal PK. Antimicrobial and phytochemical screening of endophytic fungi isolated from spikes of Pinus roxburghii. Arch Clin Microbiol 2015;6:1-9.
43. Danagoudar A, Joshi CG, Ravi SK, Kumar HG, Ramesh BN. Antioxidant and cytotoxic potential of endophytic fungi isolated from medicinal plant Tragia involucrata L. Pharmacogn Res 2018;10:188-94.
2. Demain AL. Biology of antibiotic formation. In: Vandamme EJ, editor. Biotechnology of Industrial Antibiotics. New York: Marcel Dekker Inc.; 1984. p. 33-42.
3. Stone JK, Bacon CW, White JF. An overview of endophytic microbes: Endophytism defined. In: Bacon CW, White JF, editors. Microbial Endophytes/ New York: Marcel Dekker; 2009. p. 3-29.
4. Guteirrez RM, Gonzales AM, Ramirez AM. Compounds derived from endophytes: A review of phytochemistry and pharmacology. Curr Med Chem 2012;19:2992-3030.
5. Castillo UF, Strobel GA, Ford EJ, Hess WM, Porter H, Jensen HB, et al. Munumbicins, wide-spectrum antibiotics produced by Streptomyces NRRL 30562, endophytic on Kennedia nigriscans. Microbiology 2002;148:2675-85.
6. Xie J, Wu Y, Zhang T, Zhang M. New antimicrobial compounds produced by endophytic Penicillium janthinellum isolated from Panax notoginseng as potential inhibitors of Ftsz. Fitoterapia 2018;131:35-43.
7. Manganyi M, Regnier T, Tchatchouang CK, Bezuidenhout C. Antibacterial activity of endophytic fungi isolated from Sceletium tortuosum L. (Kougoed). Ann Microbiol 2019;69:659-63.
8. Jayatilake PL, Munasinghe H. Antimicrobial activity of cultivable endophytic and rhizospheric fungi associated with “Mile-a-Minute” Mikania cordata (Asteraceae). Biomed Res Int 2020;2020:5292571.
9. Sanusi SB, Abu Bakar MF, Mohamed M, Sabran SF, Mainasara MM. Ethnobotanical, phytochemical, and pharmacological properties of nepenthes species: A review. Asian J Pharm Clin Res 2017;10:16-9.
10. Madhavan V, Basnett H, Gurudeva MR, Yoganarasimhan SN. Pharmacognostical evaluation of Drosera burmannii Vahl (Droseraceae). Indian J Tradit Knowl 2009;8:326-333.
11. Buch F, Rott M, Rottloff S, Paetz C, Hilke I, Raessler M, Mithofer A. Secreted pitfall-trap fluid of carnivorous Nepenthes plants is unsuitable for microbial growth. Ann Bot 2013;111:375-83.
12. Lee JM, Tan WS, Ting AS. Revealing the antimicrobial and enzymatic potentials of culturable fungal endophytes from tropical pitcher plants (Nepenthes spp.). Mycosphere 2014;5:364-77.
13. Christina A, Christapher V, Bhore SJ. Endophytic bacteria as a source of novel antibiotics: An overview. Pharmacogn Rev 2013;7:11-6.
14. Chaudhuri M, Pal A, Paul AK. Investigation of biochemical properties and biological activities of bacteria endogenous to carnivorous plant Drosera burmannii Vahl. Int J Adv Biotechnol Res 2015;6:278-89.
15. Chaudhuri M, Paul AK, Pal A. Isolation and assessment of metabolic potentials of bacteria endophytic to carnivorous plants Drosera burmannii and Utricularia spp. Biosci Biotech Res Asia 2019;16:731-41.
16. Gerhardt P. Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology; 1994.
17. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disc method, Am J Clin Pathol 1966;45:493.
18. Difco Manual. Dehydrated Culture Media and Reagents for Microbiology. 10th ed. Detroit, Michigan: Difco Laboratories; 1984.
19. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acid Res 1994;22:4673-80.
20. Buchanan RE, Gibbons NE. Bergey’s Manual of Determinative Bacteriology. Baltimore: Williams and Wilkins; 1975.
21. Yu H, Zhang L, Li L, Zheng C, Guo L, Li W, et al. Recent developments and future prospects of antimicrobial metabolites produced by endophytes. Microbiol Res 2010;165:437-49.
22. Joseph B, Priya RM. Bioactive compounds from endophytes and their potential in pharmaceutical effect: A review. Am J Biochem Mol Biol 2011;1:291-309.
23. Ek-Ramos MJ, Gomez-Flores R, Orozco-Flores AA, Rodríguez- Padilla C, González-Ochoa G, Tamez-Guerra P. Bioactive products from plant-endophytic Gram-Positive bacteria. Front Microbiol 2019;10:463.
24. Glenn A, Bodri MS. Fungal endophyte diversity in Sarracenia. PLoS One 2012;7:1-7.
25. Quillium RS, Jones DL. Evidence of host-specificity of culturable fungal root endophytes form the carnivorous plant Pinguicula vulgaris. Mycol Prog 2012;11:583-585.
26. Naseem F, Kayang H. Fungal endophytes associated with Nepenthes khasiana Hook. F., an endemic plant of Meghalaya, India. Int J Curr Res Life Sci 2018;7:1907-12.
27. White JF Jr., Torres MS, Sullivan RF, Jabbour RE, Chen Q, Tadych M, et al. Occurrence of Bacillus amyloliquefciens as a systemic endophyte of Vanilla orchids. Microsc Res Tech 2014;77:1-12.
28. Chen YT, Yuan Q, Shan LT, Lin MA, Cheng DQ, Li CY. Antitumor activity of bacterial exopolysaccharides from the endophyte Bacillus amyloliquefaciens sp. isolated from Ophiopogon japonicus. Oncol Lett 2013;5:1787-92.
29. Li H, Soares MA, Torres MS, Bergen M, White JF Jr. Endophytic bacterium, Bacillus amyloliquefaciens, enhances ornamental host a resistance to diseases and insect pests. J Plant Interact 2015;10:224-9.
30. Wang X, Liang G. Control efficacy of an endophytic Bacillus amyloliquefaciens strain BZ6-1 against peanut bacterial wilt, Ralstonia solanacearum. Biomed Res Int 2014;2014:465435.
31. Yin X, Xu L, Xu L, Fan S, Liu Z, Zhang X. Evaluation of the efficacy of endophytic Bacillus amyloliquefaciens against Botryosphaeria dothidea and other phytopathogenic microorganisms. Afr J Microbiol Res 2011;5:340-5.
32. Das I, Panda M, Rath CC, Tayung K. Bioactivities of bacterial endophytes isolated from leaf tissues of Hyptis suaveolens against some clinically significant pathogens. J Appl Pharm Sci 2017;7:131-6.
33. Wang C, Wang Y. Endophytic Bacillus amyloliquefaciens YTB1407 elicits resistance against two fungal pathogens in sweet potato (Ipomoea batatas Lam.). J Plant Physiol 2020;253:154260.
34. Bhoonobtong A, Sodngan S, Boonlue S, Bunyatratchata W, MongkoitW. Antibiotics constituents of endophytic Bacillus amyloliquefaciens UD25 extracted from a medicinal plant, Memecylon edule Roxb. Chiang Mai J Sci 2015;44:788-99.
35. Zhu H, Pan Y. A novel antimicrobial protein of the endophytic Bacillus amyloliquefaciens and its control effect against Fusarium chlamydosporum. Biocontrol 2019;64:737-48.
36. Wo?nicka E, Ku?niar A, Nowak D, Nykiel E, Kopacz M, Gruszecka J, et al. Comparative study on the antibacterial activity of some flavonoids and their sulfonic derivatives. Acta Pol Pharm 2013;70:567-71.
37. Jaisinghani RN. Antibacterial properties of quercetin. Microbiol Res 2017;8:13-4.
38. da Silva CR, Neto BA, Campos RS, Figueiredo NS, Sampaio LS, Magalhaes HI, et al. Synergistic effect of the flavonoid catechin, quercetin or epigallocatechin gallate with fluconazole induces apoptosis in Candida tropicalis resistant to fluconazole. Antimicrob Agents Chemother 204;58:1468-78.
39. De Oliveira MR, Nabavi SM, Braidy N, Setzer WN, Ahmed T, Nabavi SF. Quercetin and the mitochondria: A mechanistic view. Biotechnol Adv 2016;34:532-49.
40. Rocha MF, Sales JA, da Rocha MG, Galdino LM, de Aguiar L, et al. Antifungal effects of the flavonoids kaempferol and quercetin: A possible alternative for the control of fungal biofilms. Biofouling 2019;35:320-8.
41. Ramadhan F, Mukarramah L, Oktavia FA, Yulian R, Annisyah NH, Asyiah IS. Flavonoids from endophytic bacteria of Cosmos caudatas Kunth. leaf as anticancer and antimicrobial. Asian J Pharm Clin Res 2018;11:200-4.
42. Bhardwaj A, Sharma D, Jadon N, Agarwal PK. Antimicrobial and phytochemical screening of endophytic fungi isolated from spikes of Pinus roxburghii. Arch Clin Microbiol 2015;6:1-9.
43. Danagoudar A, Joshi CG, Ravi SK, Kumar HG, Ramesh BN. Antioxidant and cytotoxic potential of endophytic fungi isolated from medicinal plant Tragia involucrata L. Pharmacogn Res 2018;10:188-94.
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CHAUDHURI, M., P. AK, and A. PAL. “ISOLATION OF POTENTIAL ANTIMICROBIAL METABOLITE FROM ENDOPHYTIC BACILLUS AMYLOLIQUEFACIENS DL06 OF CARNIVOROUS PLANT DROSERA BURMANNII VAHL.”. Asian Journal of Pharmaceutical and Clinical Research, Vol. 14, no. 1, Jan. 2021, pp. 163-70, doi:10.22159/ajpcr.2021.v14i1.40187.
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