Molecular characterization of covalently closed circular plasmid DNA in local Streptomyces strains isolated from soil with detection of antibiotic production and antibacterial activity

Abstract

Various of Streptomyces species have two kinds of plasmids, circular plasmids (8 to 31 kb) and linear plasmids (12 to 1700 kb). Covalently closed circular (CCC) plasmids are profuse in the genus of Streptomyces and involved in production and resistance of antibiotics by genetic controlling. We collected fifty clinical soil samples from different regions in Al-Najaf Al-Ashraf province/Iraq. The samples included five from Al-Ghadeer Quarter, five from Al-Karama Quarter, 10 from Kufa University, five from Al-Ameer Quarter, four from Al-Forat Quarter, 10 from North Quarters and eleven from desert roads in Al-Najaf. Diluted samples were cultured on Yeast extract Malt extract (YEME) agar medium as a selective medium; then the presumptive Streptomyces colonies were subcultured on Tryptone Yeast extract (TYE) agar, then incubation at 37ᵒC for 7 days. Seven biochemical tests for identification of Streptomyces isolates these are: Catalase test, Oxidase test, Urase test, Kligler Iron Agar test (KIA), Simmon᾽s Citrate test, addition to MacConkey agar test and Mannitol Salt agar test. Five antibiotic discs were used for detection of antibiotic sensitivity of the Streptomyces isolates; these are: Tetracycline, Gentamycin, Vancomycin, Ampicillin, Erythromycin. The sensitivity of the antibiotics was observed by recorded the diameter of inhibition zone around the discs. Two test bacteria (Staphylococcus aureus and E. coli) were used for the determination of antibacterial activity.  Plasmid isolation was done by the alkaline lysis method. This method is characterized by the rapid isolation of DNA from Streptomyces. Then, detection of Plasmid DNA  occurred by using  agarose gel electrophoresis.