Atipathogenic bacterial activity of crystal proteins extracted from Bacillus subtilis isolated from the sheepfold in Oman

Summary

The increasing issue of antibiotic resistance is a pressing concern, prompting the search for sustainable antimicrobial resources. Bacillus subtilis, a Gram-positive bacterium found in soil and ruminants’ gastrointestinal tracts, is a promising candidate due to its crystal proteins. Oman, with its diverse agro-ecological zones and significant sheep farming industry, offers a unique reservoir for the isolation of Bacillus subtilis strains. The sheepfold environment provides an ideal setting for the bacterium to produce a wide range of metabolites, including crystal proteins with potential antibacterial activities. Previous studies have highlighted the antimicrobial capabilities of Bacillus spp. crystal proteins, primarily against phytopathogens and insect pests. However, their efficacy against bacterial pathogens relevant to human and animal health remains less explored. This research aims to bridge this gap by focusing on the isolation of Bacillus subtilis from sheepfolds in Oman and the subsequent extraction and characterization of its crystal proteins. The potential of these proteins to serve as a sustainable alternative to traditional antibiotics is of particular significance in the face of rising drug resistance and the global call for eco-friendly pest control and disease management strategies. Crystal proteins, produced during the sporulation phase of Bacillus subtilis, have been identified as potent antimicrobial agents. These proteins disrupt the cellular integrity of target pathogens, leading to cell lysis and death. Recent research suggests that Bacillus subtilis crystal proteins also possess significant antibacterial potential. Studies have shown that crystal proteins from Bacillus subtilis can inhibit the outer membrane of bacteria, leading to cell death. Kumar et al. (2014) also highlighted the broad-spectrum antibacterial potential of crystal proteins against multiple pathogens, including Listeria monocytogenes and Salmonella enterica. The study involved sampling and isolation of environmental samples, cultivation and induction of crystal protein production, extraction and purification of crystal proteins, concentration and storage, and antibacterial activity assay. The chosen pathogenic bacteria for testing included Staphylococcus aureus, Escherichia coli, Salmonella Typhimurium, Listeria monocytogenes, and Pseudomonas aeruginosa. The Disc Diffusion Method was used to apply crystal protein solutions of varying concentrations onto agar plates pre-inoculated with the pathogenic bacteria. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values of the crystal proteins against the selected bacteria were determined. The investigation of Bacillus subtilis strains from Oman or similar environments could contribute valuable insights into the search for new antibiotics, given the global interest in natural and sustainable antimicrobial solutions.

Objectives

1-   To isolate Bacillus subtilis strains from sheepfold environments using selective culturing techniques.

2-   To identify the isolated strains of Bacillus subtilis through molecular techniques such as 16S rRNA sequencing.

3-   To develop a protocol for the efficient extraction of crystal proteins from identified Bacillus subtilis strains.

4-   To characterize the extracted crystal proteins using techniques such as SDS-PAGE, mass spectrometry, and protein sequencing to determine their structure and composition.

5-   To assess the antipathogenic activity of the extracted crystal proteins against a panel of pathogenic bacteria using in vitro assays such as disc diffusion, and MIC determination.

6-   To determine the spectrum of antipathogenic activity and identify specific pathogens that are most susceptible to the crystal proteins.

7-   To investigate the potential application of these crystal proteins in developing novel antimicrobial agents for human medicine.

 Funding Agency:

MOHERI