ANTI-MRSA OF THE ETHYL ACETATE CRUDE EXTRACT FROM LASIODIPLODIA PSEUDOTHEOBROMAE IBRL OS-64, AN ENDOPHYTIC FUNGUS ISOLATED FROM LEAF OF OCIMUM SANCTUM LINN
Objective: To investigate the effects of ethyl acetate crude extract of an endophytic fungus, L. pseudotheobromae IBRL OS-64 which was isolated from leaf of Ocimum sanctum against the growth of methicillin-resistant Staphylococcus aureus (MRSA)., a common pathogenic bacteria to human.
Methods: Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined. Time-kill assay was used to examine the effect of the extract on the MRSA growth profile. The effects of the extract on the ultrastructure of MRSA cells were analyzed by scanning electron microscopic (SEM) study.
Results: The results obtained from this study showed the fungal ethyl acetate crude extract exhibited a strong bactericidal effect on MRSA where the ratio of MBC/MIC was 2 and less than 4. The MIC and MBC values were 125.0 and 250.0 Âµg/ml, respectively. The time-kill study revealed that the bacteriocidal activity of the extract was both concentration and time-dependent. After 12 h treatment, the interaction of extract with MRSA cells resulted in the formation of dents, cavity or small dimples on the cell surface, indicating disintegration of the cell wall and cell membrane that resulting in leakage of their cytoplasmic contents, and ultimately cell death.
Conclusion: The ethyl acetate crude extract of L. pseudotheobromae IBRL OS-64 showed a significant anti-MRSA activity and principally affected the cell wall and the cell membrane of the growing MRSA cells. This is the first report on L. pseudotheobromae, an endophytic fungus isolated from medicinal herb, Ocimum sanctum Linn.
2. Basri DF, Jaffar N, Zin NM, Santhana-Raj L. Electron microscope study of gall extract from Quercus infectoria in combination with vancomycin against MRSA using post-antibiotic effect determination. Int J Pharmacol 2013;9:150-6.
3. Vimala S, Ilham MA, Rashih AA, Rohana S. Natureâ€™s choice to wellness: antioxidant vegetables/ulam. Siri Alam and Rimba 7. Forest Research Institute Malaysia (FRIM); 2003. p. 131.
4. Pattanayak P, Behera P, Das D, Panda SK. Ocimum sanctum Linn. A reservoir plant for therapeutic applications: an overview. Pharmacogn Rev 2010;4:95-105.
5. Jose B, Reddy LJ. Evaluation of antibacterial activity of the leaf and flower of essential oils of Gliricidia sepium from South India. Int J Appl Pharm 2010;2:20-2.
6. Moorthy K, Aravind A, Punitha T, Vinodhini R, Suresh M, Thajuddin N. In vitro screening of antimicrobial activity of Wrightia tinctoria (Roxb.) R. BR. Asian J Pharm Clin Res 2012;5:54-8.
7. Menghani E, Sudhanshu, Rao N, Mittal S. Evaluation of the anti-malarial and antioxidant effect of Parnassia nubicola methanolic extract. Int J Curr Pharm Res 2012;4:77-9.
8. Azevedo JL, Walter MJ, Pereira JD, Araujo WL. Endophytic microorganism: a review on insect control and recent advances on tropical plants. Electron J Biotechnol 2000;3:40-65.
9. Nagda V, Gajbhiye A, Kumar D. Isolation and characterization of endophytic fungi from Calotropis procera for their antioxidant activity. Asian J Pharm Clin Res 2017;10:254-8.
10. Faeth SH, Fagan WF. Fungal endophytes: common host plant symbionts but uncommon mutualists. Int Comp Biol 2002;42:360-8.
11. Sun JQ, Guo LD, Zang W, Ping WX, Chi DF. Diversity and ecological distribution of endophytic fungi associated with medical plants. Sci Chin Ser C 2008;51:751-9.
12. Castro-Medina F. First report on Lasiodiplodia pseudotheobromae causing trunk cankers in Acacia mangium in Venezuela. Plant Dis 2014;98:686.
13. Awan QN, Akgul DS. First report of Lasiodiplodia pseudotheobromae causing postharvest fruit rot of lemon in turkey. Plant Dis 2016;100:23-7.
14. Nogueira Junior AF, Santos RF, Pagenotto ACV, Sposito MB. First report of Lasiodiplodia pseudotheobromae causing fruit rot of persimmon in Brazil. New Dis Rep 2017;36:1.
15. Ismail AM, Cirvilleri G, Pollizzi G, Crous PW, Groenewald JZ, Lombard L. Lasiodiplodia species associated with dieback disease of mango (Mangifera indica) in Egypt. Australas Plant Pathol 2012;41:649-60.
16. Kwon JH, Choi O, Kang B, Lee Y, Park J, Kang PW, et al. Identification of Lasiodiplodia pseudotheobromae causing mango dieback in Korea. Can J Plant Pathol 2017;39:241-5.
17. Wei W, Jiang N, Mei YN, Chu YL, Ge HM, Song YC, et al. An antibacterial metabolite from Lasiodiplodia pseudotheobromae F2. Phytochemistry 2014;100:103-9.
18. Cimmino A, Cinelli T, Masi M, Reveglia P, Silva MA, Mugnai L, et al. Phytotoxic lipophilic metabolites produced by grapevine strains of Lasiodiplodia species in Brazil. J Agric Food Chem 2017;65:1102-7.
19. Tong WY, Nurul Zaadah J, Nurhaida, Tan WN, Melati K, Latiffah Z, et al. Antimicrobial activity of Phomopsis sp. ED2 residing in medicinal plant Orthosiphon stamineus Benth. Annu Res Rev Biol 2014;4:1490-501.
20. Jorgensen JH, Ferraro MJ. Antimicrobial susceptibility testing: General principles and contemporary practices. Clin Infect Dis 1998;26:973-80.
21. Andrews JM. Determination of inhibitory concentration. J Antimicrob Chemother 2001;48:5-16.
22. Borges M, De-Ven MA, Van-Cutsem J. Structural degeneration of Aspergillus fumigatus after exposure to saperconazole. J Med Vet Mycol 1989;27:381-9.
23. Aksoy DY, Unal S. New antimicrobial agents for the treatment of Gram-positive bacterial infections. Clin Microbiol Infect 2008;14:411-20.
24. Nussbaum F, Brands M, Hinzen B, Weigand S, Habich D. Antibacterial natural products in medicinal chemistry-exodus or revival? Angewandte Chemie 2006;45:5072-129.
25. Xing YM, Chen J, Cui JL, Chen XM, Guo SX. Antimicrobial activity and biodiversity of endophytic fungi in Dendrobium devonianum and Dendrobium thyrsiflorum from vietnam. Curr Microbiol 2011;62:1218-24.
26. Zhao K, Penttinen P, Guan T, Xiao J, Chen Q, Xu J, et al. The diversity and anti-microbial activity of endophytic actinomycetes isolated from medicinal plants in Panxi Plateau, China. Curr Microbiol 2011;62:182â€“90.
27. Yang L, Yang C, Li C, Zhao Q, Liu L, Fang X, et al. Recent advances in biosynthesis of bioactive compounds in traditional Chinese medicinal plants. Sci Bull 2016;61:3-17.
28. Doughari JH. An overview of plant immunity. J Plant Pathol Microbiol 2015;6:322.
29. Kohlmeyer J, Kohlmeyer E. Marine mycology, the higher fungi. Mar Ecol 1979;1:103-4.
30. Levison ME. Pharmacodynamics of antibacterial drugs. Infect Dis Clin North Am 2000;14:281-91.
31. Nunart T, Chatsuwan T, Treyaprasert W. Time-kill study of the in vitro antimicrobial activity of tedizolid against methicillin-resistant Staphylococcus aureus. Thai J Pharm Sci 2017;41:25-9.
32. Tam VH, Schilling AN, Nikolaou M. Modelling timeâ€“kill studies to discern the pharmacodynamics of meropenem. J Antimicrob Chemother 2005;55:699-706.
33. Mueller M, Pena A, Derendorf H. Issues in pharmacokinetics and pharmacodynamics of anti-infective agents: kill curves versus MIC. Antimicrob Agents Chemother 2004;48:369-77.
34. Ingerman MJ, Pitsakis PG, Rosenberg AF, Levison ME. The importance of pharmacodynamics in determining the dosing interval in therapy for experimental Pseudomonas endocarditis in the rat. J Infect Dis 1986;153:707â€“14.
35. Nielsen EI, Anders Viberg A, Elisabeth Lowdin E, Otto Cars O, Mats O, Karlsson MO, et al. Semimechanistic pharmacokinetic/ pharmacodynamic model for assessment of activity of antibacterial agents from time-kill curve experiments. Antimicrob Agents Chemother 2007;51:128-36.
36. Hartmann M, Berditsch M, Hawecker J, Ardakani MF, Gerthsen D, Ulrich AS. Damage of the bacterial cell envelope by antimicrobial peptides gramicidin S and PGLa as revealed by transmission and scanning electron microscopy. Antimicrob Agents Chemother 2010;54:3132-42.
37. Bible AN, Khalsa Moyers GK, Mukherjee T, Green CS, Mishra P, Purcell A, et al. Metabolic adaptations of Azospirillum brasilense to oxygen stress by cell-to-cell clumping and flocculation. Appl Environ Microbiol 2015;81:8346-57.
38. Watanabe T, Ohashi K, Matsui K, Kubota T. Comparative studies of the bactericidal, morphological and post-antibiotic effects of arbekacin and vancomycin against methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother 1997;39:471-6.
39. Dubin DT, Hancock R, Davis BD. The sequence of some effects of streptomycin in Escherichia coli. Biochim Biophys Acta 1963;74:476-89.
40. Matsunaga K, Yamaki H, Nishimura T, Tanaka N. Inhibition of DNA replication initiation by aminoglycoside antibiotics. Antimicrob Agents Chemother 1986;30:468-74.
41. Ibrahim D, Chai Lee CC, Yenn TW, Zakaria L. Sheh-Hong L. Effect of the extract of endophytic fungus, Nigrospora sphaerica CL-OP 30, against the growth of methicillin-resistant Staphylococcus aureus (MRSA) and Klebsiella pneumoniae cells. Trop J Pharm Res 2015;14:2091-7.
42. Denyer SP. Mechanisms of action of biocides. Int Biodeterior Biodegradation 1990;26:89-100.