Isolation of streptomyces from Sabah and screening for their antimicrobial activities

The objectives of this project were to isolate Streptomyces from tropical soil samples in Sabah; to screen for antimicrobial activities of Streptomyces against Escherichia coli 0125, Vibrio parahaemolyticus and Salmonella typhi; to characterize the Streptomyces possessing antimicrobial activities and lastly to identify the Streptomyces by 16S rDNA sequencing. The materials and methods involved the sampling of tropical soil samples of SST and FOR samples and were incubated at 31⁰C for four days after the addition of CaCO₃ at 1: 1 ratio. The soil were diluted with sterile distilled water and plated onto Streptomycetes agar (SA) containing cycloheximide at 30⁰C for several days. The Streptomyces were then spot-inoculated onto a fresh SA with grid and incubated at 30°C until sporulation. The plate was then being overlayed with soft agar containing target pathogens as stated above and incubated overnight. After the formation of inhibition zones, the Streptomyces was purified by restreaking the colony on fresh SA and were Gram-stained, KOH rapid test, and characterized. Then it was grown in broth and later stored in 20% glycerol stock at -80°C. Its temperature, pH, and NaCl growth range were also tested. The genotypic characterization involved the isolation of genomic DNA, the amplification of 16S rDNA gene using PCR the cloning of the gene using TOPO® TA cloning kit, restriction mapping of plasmid and sequencing of the gene by Sanger chain-termination method. A total of 523 isolates were successfully purified (218 isolates from SST and 305 isolates form FOR samples). Ten isolates exhibited antimicrobial activities (nine from SST samples and one from FOR samples). Four of these isolates were identified as Streptomyces, i.e. isolate SST2, No.2; SST4, No.11; SST5, No.14; and SST5, No.17. Further characterizations of the four showed they were different but stop short at identifying the species of the Streptomyces. Further genotypic characterization of isolate SST2, No.2 have shown that it has the 16S rDNA gene of 1576bp and similarity searches lead to the Streptomyces sp. 10-3 and Streptomyces sp. 10-2. It can be concluded that this project has achieved its main objectives while elucidating several new findings for future studies