Screening and Characterization of Bioactive Actinomycetes and Their Products From Nepal Against Multidrug Resistant Bacteria and Fungi

Abstract

Actinomycetes are versatile producers of diverse secondary metabolites including antibiotics. New therapeutics of microbial origin is essential to combat multidrug resistant human pathogens. This descriptive study was conducted to isolate, screen and characterize bioactive actinomycetes and their anti-microbial product along with optimization of cultural conditions for better metabolite production. A total of 240 soil and 48 water samples were collected from different parts of Nepal and actinomycetes were selectively isolated, primarily screened by perpendicular streak method and secondary screening was performed by agar well diffusion technique against selected bacteria and fungi. The minimum inhibitory concentration of ethyl acetate extract was carried out by tube dilution method. The screened actinomycetes strains were characterized by cultural, morphological, biochemical and 16SrRNA sequencing. Optimization of fermentation was carried out by cultivating the screened strains separately at different nutritional and cultural conditions. Characterization of ethyl acetate extract was performed by FT IR and LC-MS method. Out of 320 actinomycetes isolates, 120 (37.5%) were found bioactive against one or more test microbes. Among them only 4(3.3%) were inhibitory against all test organisms. In secondary screening, the most potent strain A3 showed highest activity against C. albicans with zone of inhibition of 41.33± 1.5mm and lowest minimum inhibitory concentration of 2.5mg/ml against C. albicans and E. coli. Phenotypic characteristics predicted that strains A 3, D 2 , P 4 and J 1 were Nocardiosis prasina, Streptomyces violarus, S. krainskii and S. tsusimaensis respectively and the most potent strain A 3 was genotypically characterized as N. prasina. Optimization study revealed that starch, casein and potassium nitrate supported maximum bio mass and metabolite production in A 3 , P 4 and J 1 strains. Likewise, incubation temperature of 30 v 0 with pH 7-8 for 7-8days incubation time were found optimum for all screened strains. FT-IR and LC-MS analysis revealed that the ethyl acetate extracts of all the strains contained diverse functional groups and compounds having molecular mass (m/z) ranging from 106 to 986. Key words: Antibiotics, Multidrug Resistant, FT-IR, LC-MS, Actinomycetes.