Screening and indentification of antimicrobial producing actinomycetes from mangrove soil of the inner gulf of Thailand

Abstract

In the course of our investigation for actinomycetes strains from mangrove soils collected in Samut prakarn, Samut sakorn, Samut songkram and Phetchaburi provinces, the inner gulf of Thailand. Fifty isolates of actinomycetes were isolates which produced spores on aerial mycelium were identified as Streptomyces and 12 isolates which produced single non-motile spores were Micrmonospora based on their phenotypic characteristics. On the primary screening of antimicrobial activity, 28 isolates could inhibit Gram-positive bacteria, Staphylococcus aureus ATCC 6538P, Micrococcus Iuteus ATCC 9341 and Bacillus subtilis ATCC6633. Six isolates could inhibit Gram-negative bacteria, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 and 5 isolates could inhibit yeast, Candida albicans ATCC 10231. Seven of the active isolates as a representative strains were selected for analysis of 16S rDNA sequences, strains SAM2- 1, B15-4, D10-1, J8-1, SMP3-1 and D10-5 were closely related to Streptomyces badius NRRL B-2567T, S. caesius NBRC13376T, S albolongus NBRC13465T, S. parvulus NBRC13193T, S.cinnamocastaneus NBRC14278T, S. vayuensis N2T, and S. caesius NBRC13376T with 98.4, 97.4, 98.3, 94.7, 93.5, 98.5 and 95.9% 16S rDNA sequence similarity, respectively. The lower 16S rDNA similarity than 97.0% indicated that they should be recognized as the novel Streptomyces species. The tested strains of Streptomyces contained L-diaminopimelic acid in cell wall (cell wall type I) and Micromonospora strains contained meso-diaminopimelic acid in cell wall (cell wall type II). Major menaquinone of Streptomyces strains tested were MK-9(H6) and MK-9(H8). The range of G+C contents of the DNA was 69-74 mol% Eight isolates that showed good antimicrobial activity SAM2-1, SMP3-1, C10-6, B10-3-2, B10-3-4, C10-2, B10-3-1 and J15-1 were selected for secondary antibiotic screening and NMR spectroscopy analysis. In addition, Streptomyces sp. C10-6 was selected for antibiotic fermentation in Yeast extract-Malt extract broth (10 litres), and ethylacetate crude extract was partially purified by chromatographic technique to give 10 fractions. Fraction 8 showed the most significant antimicrobial activity to Gram positive and Gram negative bacteria at the concentration 1mg per 20 UL per disk.