The endophytic fungi isolated from leaves of Ocimum sanctum Linn. of different ages were examined for antimicrobial activity. The agar plug diffusion assay was used for primary screening. A total of 148 fungal endophytes were successfully isolated and cultured but only 134 of them (90.5%) exhibited inhibitory activity towards at least one test microorganisms. Moreover, the colonization rate indicated that the old leaves were frequently and densely colonized by endophytes. The results suggested that healthy leaves at older stages of growth can be a potential source for the isolation of endophytic fungi with antimicrobial properties. The ethyl acetate extract prepared from the fermentative broth exhibited better antimicrobial activity and it suggested the antimicrobial activity of the isolates was affected by the culture medium. A better antimicrobial activity was observed in the yeast extract sucrose broth as compared to malt extract broth. Significant improvements in the antimicrobial activity of the crude extract were observed after addition of water extract of the host plant in the culture medium.

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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 µg/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.

Citation: M.M.J. Taufiq and I. Darah, Effects of Cultural Conditions in Enhancing the Production of Anti-MRSA Activity of Lasiodiplodia pseudotheobromae IBRL OS-64, an Endophytic Fungus Isolated from Leaf of Ocimum sanctum L. in Submerged Fermentation System, J Pure Appl Microbiol., 2019; 13(4):Abstract The effects of cultural conditions on the production of anti-MRSA activity of the endophytic fungal isolate, Lasiodiplodia pseudotheobromae IBRL OS-64 isolated from the leaf of a local medicinal herb, Ocimum sanctum Linn. was studied. The highest anti-MRSA activity of 50.0±0.1 U/mL and 1.46±0.1 g/L of fungal growth were obtained after incorporating all the improved cultural conditions which consisted of yeast extract sucrose broth supplemented with host plant extract, with initial medium pH of 6, 2 mycelial plugs of 4 days old seed culture, cultivation temperature of 30°C and cultivated for 16 days under static conditions without the presence of light. After improvement of cultural conditions, a significant increment of anti-MRSA activity of about 57.57% (2.4 folds) and a slight increment of fungal growth of about 8. 15% (1.1 folds) were obtained compared to the cultural condition before improvement. Indeed the improvement of cultural conditions greatly affected the anti-MRSA activity and growth production by L. pseudotheobromae IBRL OS-64 isolate.