The study was carried out to investigate the antibacterial activity of an endophytic fungal isolate, Lasiodiplodia pseudotheobromae IBRL OS-64 residing in leaves of a medicinal herb, Ocimum sanctum Linn. Qualitative screening of the antimicrobial activity was done using an agar plug assay and the results showed that the fungal isolate were able to inhibit all the 13 test bacteria. Three Gram-positive bacteria (MRSA ATCC 33591, Staphylococcus aureus and Streptococcus mutans) were the most susceptible species with the inhibition zones of ≥ 21 mm. The other 3 (B. cereus ATCC 10876, Bacillus subtilis IBRL A3 and Streptococcus agalactiae showed the inhibition zones of 11 to ≤20 mm of diameter. As for Gram-negative bacteria, Yersinia enterocolitis was the most susceptible to the fungal isolate with the size of inhibition zone of ≥ 21 mm, followed by Klebsiella pneumoniae ATCC 13883, Salmonella typhimurium and Shigella boydii ATCC 9207 with the inhibition zones of 11 to ≤ 20 mm, whereas Escherichia coli IBRL 0157, Proteus mirabilis and Pseudomonas aeruginosa (ATCC 27883 were the least susceptible with the inhibition zones of ≤ 10 mm. Quantitative screening using disc diffusion assay showed that the fungal ethyl acetate extract prepared from the fermentative broth (extracellular) exhibited better antibacterial activity compared to the methanolic extract prepared from the fungal biomass (intracellular). The results showed that the ethyl acetate extract exhibited antibacterial activity against all the thirteen test bacteria with the inhibition zone sizes of 20.0 ± 0.3 to 31.3 ± 1.2 mm in diameter for Gram positive bacteria and 10.31 ± 0.6 to 20.1 ± 0.6 mm in diameter for Gram negative bacteria. On the other hand, the methanolic extract only inhibited three Gram positive bacteria (MRSA ATCC 33591, S. aureus and Strep. mutants) with the inhibition zones of 9.0 ± 0.6 to 11.0 ±0.3 mm in diameter, whereas only one Gram negative (S. typhimurium) with the inhibition zone size of 13.3 ± 1.5 mm diameter. The MIC and MBC values of the ethyl acetate extract on Gram positive bacteria were in the ranged of 62.50 to 125.00 µg/mL and 62.50 to 500.00 µg/mL, respectively, whereas for the Gram negative bacteria, the MIC and MBC values were 125.00 to 250.00 µg/mL and 250.0 to 1000.00 µg/mL, respectively. On the other hand, the MIC and MBC values for methanolic extract against Gram positive bacteria were 250.00 to 500.00 µg/mL and against Gram negative bacteria was 1000.00 µg/mL, respectively. Both of the extracts exhibited bactericidal effects on test bacteria with the MBC/MIC ratio ≤ 4. Further detail of the effects of the ethyl acetate extract on the bacterial cells were observed from the SEM photomicrographs which revealed the severity of the morphological deterioration experienced by the extract treated cells were beyond repair, and the most possible mode of actions were by interrupting the cell wall biosynthesis and cell membrane permeability.
Key words: Keywords: Antibacterial activity, endophytic fungus, Lasiodiplodia pseudotheobromae, Ocimum sanctum Linn.
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