Characterization of the Antimicrobial Compound produced by the Soil Bacterium Brevibacillus laterosporus

Abstract

The resistance of pathogenic microbes to an increasing number of antimicrobial agents poses a serious threat to human health. Soil-dwelling bacteria have received great attention as a reservoir of new antibiotics because of large populations and great diversity. In this project, a new soil bacteria (named ZYD4) producing antimicrobial compound(s) was isolated from soil samples collected in the Rocky Mountain National Park. ZYD4 was identified as Brevibacillus laterosporus based on 16S rRNA gene sequence analysis. The antimicrobial compound(s) showed a broad spectrum of activity against 11 species of microorganism pathogens with clinical importance, including methicillin-resistance Staphylococcus aureus (MRSA). The specific substance was also proved to have strong inhibition on proliferation of melanoma and prostate cancer cells. We explored the effects of using different media on the production of the antimicrobial agent(s), and found that the inhibitory activity of culture supernatant reached a peak after incubating ZYD4 in LB for 96 hours at room temperature. The addition of 20% (v/v) supernatant reduced the growth of Escherichia coli in log phase by 70%-80%. This project used the AlamaeBlue Assay, a method of measuring the viability and multiplication of cells, to examine the inhibitory effects of the antimicrobial compound produced by ZYD4. The antimicrobial agents showed stability to alkalinic pH and heat, and resistance to proteinase K. The purification process involved ammonium sulphate precipitation/ethanol precipitation, Amberlite XAD-16 adsorption, n-butanol extraction, thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) on a C18 reverse phase column. Three possible substances were isolated from the ZYD4 bacteria culture. The molecular weights were identified by a Mass Spectrometer (MS) as 1263, 1499 and 357 Da. The physical and chemical properties indicate that the antimicrobial agents are highly likely to be peptides, except the one with the smallest molecular weight. The antimicrobial agents identified here could be effective candidates for future clinical research.