Browsing by Author "Patabedi, S.W.P.N.H."

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    Antibacterial activity of entomopathogenic fungi isolated from a beetle (Harmonia sp.) in Sri Lanka
    (Uva Wellassa University of Sri Lanka, 2021) Patabedi, S.W.P.N.H.; Ratnaweera, P.B.
    Majority of human pathogenic bacteria has become resistant to the exsisting antibiotics causing a human health crisis in the world. Thus discovering novel antibacterial drug leads has become an urgent concern. Entomopathogenic fungi (EPF) infect insects and kill or disable them. Previous research have shown entomopathogenic fungi exhibit various bioactivities. Thus, the objective of the current study was to isolate entomopathogenic fungi from a beetle, a Harmonia sp. in Sri Lanka and evaluvate their potential of producing antibacterial compounds. EPF were isolated from surface sterilized insect cadavers collected from Balangoda area in Sri Lanka. Isolated fungi were grown in potato dextrose agar and broth media, incubated close to sporulation, harvested and extracted into ethyl aceate. The obtained weights of the crude extracts of solid and liquid cultures were compared using ANOVA. Antibacterial activity of the crude extracts were evaluated using agar disc diffusion bioassays at 400 μg/disc, against two Gram-positive bacteria, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATTC 11778) and two Gram-negative, Escherichia coli (ATCC 35218) and Pseudomonas aeruginosa (ATCC 9027). The positive control used was Gentamycin and the negative control was methanol. Seven morphologically different EPF were isolated from Harmonia sp. The crude weights of the solid cultures were significantly higher than the crude weights of the liquid cultures (ANOVA, p < 0.05). This result showed fungi have preferred to synthesize more metabolites under solid culture conditions than liquid. Six out of the seven fungi showed antibacterial acitivities against at least one bacterium tested. EPF cultures BET 06, 08 and 10 showed high inhibition zones against the S. aureus and B. cereus. According to ANOVA there was a significant difference between the antibacterial activities of the crude extracts (p < 0.05). None of the fungal extracts showed activity against P. aeruginosa while BET 05, 06 and 08 showed mild activity against E. coli. According to the microscopic characteristics the fungi were tentatively identified as belong to Penicillium and Aspergillus species. A bioautography study reveled the presence of several bioactive compounds in BET 05, 06, 08 and 10 extracts. The results of this study showed that entomopathogenic fungi are potential sources for isolating antibacterial compounds. Keywords: Antibacterial; Entomopathogenic fungi; Harmonia sp.; Penicillium; Aspergillus
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    Isolation of Antibacterial Compounds from an Entomopathogenic Fungus Isolated from a Beetle, a Harmonia sp.
    (Uva Wellassa University of Sri Lanka, 2021) Abivarna, S.; Patabedi, S.W.P.N.H.; Ratnaweera, P.B.
    Antibiotic resistance of bacteria is a major human health concern in the world which requires research priorities. Therefore discovering new and effective antibacterial drug leads has become an urgent necessity. Entomopathogenic fungi act as parasites of insects which kill or disable them. However these fungi are known to synthesize various bioactive secondary metabolites. Thus, the objective of the current study was to isolate the antibacterial compounds from an entomopathogenic fungus isolated from a beetle (Harmonia sp.) in Sri Lanka. The fungus BET 05, which was microscopically identified as an Aspergillus sp. was grown in potato dextrose agar (PDA) and broth (PDB) media. After an incubation period of 06 days, both solid and liquid fungal cultures were extracted into ethyl acetate (EtOAc). The solid culture (30 large and 13 medium PDA dishes) yielded 804.8 mg of crude while the liquid culture (400 mL x 3) gave 436.7 mg. The solid and liquid crude extracts were subjected to an agar disc diffusion bioassay to determine the antibacterial activity and the inhibition zones diameters were 11.5 and 11.3 mm respectively, against the Gram positive Staphylococcus aureus (ATCC 25923) at 400 μg/disc concentration. The positive control used was Gentamycin, while the negative control was methanol. The main active compound in the extract was isolated using bioassay guided chromatographic techniques. The crude extract (436.7 mg) was first fractionated by solvent-solvent partitioning, using hexane and methanol/water (9:1), chloroform and methanol/water (6:4) and, EtOAc and water. The active chloroform fraction was purified by Sephadex LH20 size exclusion chromatography using methanol as the eluent. Fractions collected were grouped according to their TLC profiles and the combined fractions (A-F) were tested for antibacterial activity using a bioautography. The active fraction B was further purified by normal phase silica chromatography using 60:40 hexane: EtOAc to methanol solvent systems. A bioautography revealed, the combined fraction E contains main active compound while fraction C and G are also having some active compounds. Finally a preparative TLC was conducted using EtOAc as the mobile phase to get the main active compound purified (4.1 mg). This study reveals that entomopathogenic Aspergillus sp. (BET 05) is a potential producer of antibacterial compounds. Keywords: Antibacterial; Antibiotic resistance; Entomopathogenic; Aspergillus