Antimicrobial Potentials of Bacillus Subtilis Isolated from Soil and Water

Abstract

In this study, an attempt was made to extract antimicrobial substance from the soil isolate of Bacillus subtilis and explore its antimicrobial activity on some selected bacteria. The bacteria were typed using RAPD-PCR method to identify organism to strain level, using 16sr RNA A1A13 specific primers. Results revealed bands of different sizes identical in both strains of Bacillus subtilis. The antimicrobial potential of the soil isolate was determined by disc diffusion assay. The production of antimicrobial compound was recorded maximum at the late exponential growth phase. The filtered concentrate of the crude extract was used to determine antimicrobial activity on some selected gram positive and gram – negative bacteria. Partial purification of the crude extract was carried out using High pressure Liquid chromatography (HPLC), Zymogram analysis and TLC were performed to determine the preliminary biochemical nature. The molecular weight of the antimicrobial peptide was determined to be less than 2.5k Da. The presence of antibiotic Bacilysocin was discovered from the extract. The minimum inhibitory concentration MIC of the purified fraction showed (MIC) ranging from 0.5 to 10µg/ml for Staphylococcus aureus and Streptococcus pyogenes. The MIC for the gram–negative was 0ug/ml. The crude extracts showed antimicrobial activity only on gram positive selected bacteria strains, while the purified extract showed antimicrobial activity on both the gram-positive and gram-negative with more activity on the gram-positive. The zones of inhibition for crude extract 1 on Staphylococcus auerus were 8.0mm, 5.0mm, 3.0mm, 1.0mm; extract 2: 15.0mm, 10.0mm, 9.0mm, 7.0mm, and 6.0mm, 4.0mm, 3.0mm, 1.0mm respectively for extract 3. for Streptococcus pyogenes the zones of inhibition range from 2.0mm to 9.0mm, for extracts 1 and 2:10.0mm to 3.0m and10.0mm to 1.0mm for extract 3.for the gram negative test organisms, the zones of inhibition were 0.0mm for all the extracts tested against it.