Abstract
Spore-former Bacillus species have gained attention as potential probiotics due to their ability to endure environmental stress conditions, and their capability to synthesize beneficial compounds. Probiotics are live microorganisms which when administered in adequate amounts, confer health benefits to the host. In this study, a bacterial endophyte Bacillus paranthracis strain MHSD3 isolated from a medicinal plant Pellaea calomelanos, was characterized using physiological, biochemical, and molecular techniques. Strain MHSD3 was analyzed for in vitro and in silico probiotic properties. Gas chromatography-mass spectrometry was used to identify and characterize secondary metabolites secreted by the bacterial endophyte. To further verify strain MSHD3 as a potential probiotic, its bacteriocin-like inhibitory substances (BLIS) were isolated, analyzed and identified with liquid chromatography-mass spectrometry and assessed for antibacterial effect against various bacterial pathogens. For phenotypic characteristics, B. paranthracis strain MHSD3 was closely related to Bacillus paranthracis Mn5T. Based on genome analysis, strain MHSD3 had an average nucleotide identity value of 97.6 % and a digital DNA-DNA hybridization similarity value of 79 % with B. paranthracis Mn5T. The phenotypic and genotypic characteristics support strain MHSD3 as a subspecies of B. paranthracis Mn5T. The genome analysis revealed the presence of acid and bile tolerance genes, adhesions genes and bacteriocin genes. For the in vitro assays, B. paranthracis strain MHSD3 was tolerant to acidic, bile salts and gastric juice conditions. Furthermore, strain MHSD3 exhibited a decent auto-aggregation, cell hydrophobicity and substantial co-aggregative properties. Strain MHSD3 showed antibacterial activity against various Gram-negative and Gram-positive pathogenic strains. The analysis of cell-free extracts of B. paranthracis strain MHSD3 presented compounds with several biological properties which include heptacosane, hexadecane which possess antimicrobial properties, and acetic acid, tridecanoic acid all with antibacterial, antioxidant, and anti-cancer, and properties. Furthermore, BLIS were stable in broad ranges of pH and temperature. The BLIS were stable when treated with various enzymes and chemicals but were sensitive to chloroform. The BLIS were effective against both Gram-positive and Gram-negative bacteria. Thus, B. paranthracis strain MHSD3 is a promising potential probiotic.
Keywords: Antibacterial activity, Bacillus paranthracis, Bacterial Endophytes, Bacteriocins, In silico, In vitro, Probiotic, Secondary metabolites, Whole genome sequence.