Abstract:
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
