In vitro Screening and Genome-wide Analysis of Probiotic Bacillus spp.from Locally Fermented Yogurt for Preliminary Probiotic Potential

The research was focused on extracting Bacillus spp. and genomic-wide analysis from locally fermented yogurt to assess their potential as probiotics through genetic characterization. Two bacterial strains, JF-5 and isolate JY-2 were isolated from total 25 yogurt samples. The samples were collected from markets in Rawalpindi and Islamabad. Isolation was carried out using BHI agar as culture medium. The tested isolates received morphological and biochemical examinations, biosafety testing and enzymatic evaluations to evaluate their preliminary probiotic-associated traits. The probiotic characteristics of both strains differed, although they shared Gram-positive morphology, together with rod-shaped features and exhibited catalase activity. Analyses demonstrated that isolate JF-5 possessed proteolytic and lipolytic capacities and showed resilience against bile and pH variations. However, isolate JY-2 displayed DNase activity in addition to hemolysis, making it unsuitable for use as a safe probiotic. Whole-genome sequencing was applied to the strain JF-5 as the next analysis step. The bacterial strain was defined as Bacillus altitudinis through genome sequencing, which showed a 3.77 Mbp genome size alongside 41.2% GC content and 3962 coding sequences. The antiSMASH analysis platform detected various biosynthetic gene clusters that can produce antibacterial and probiotic traits, such as lichenysin, bacilysin, fengycin and siderophore compounds. CRISPR -Cas systems and vancomycin resistance-related genes (vanG, vanY, vanT) were identified with analysis. These antimicrobial resistance determinants have important biosafety implications especially with regard to possible horizontal gene transfer. This is why the strain cannot be proposed to be implemented as probiotics without a careful study of its safety. Even though the genomic presence indicates environmental adaptability through the presence of stress response genes, sporulation genes, and nutrient assimilation genes, alone in the genome does not indicate functional probiotic efficacy. This investigation presents the first detailed genomic analysis of indigenous Pakistan-based probiotic Bacillus strains. Whole-genome sequencing were performed by MicrobesNG Lab, Birmingham, United Kingdom.