Comparative Genomics and In Vitro Experiments Provide Insight into the Adaptation and Probiotic Properties of Shouchella clausii

<i>Shouchella clausii</i> (<i>S. clausii</i>) has been marketed as an important commercial probiotic, displaying significant therapeutic effects on antibiotic-associated diarrhea and providing benefits to humans. This study aimed to explore the distribution, adaptation, and probiotic properties of <i>S. clausii</i>. Based on 16S rRNA gene analysis, 43 strains of <i>S. clausii</i> were isolated from 317 soil samples in China. Based on the genomic index of Average Nucleotide Identity (ANI) results, 41 strains were confirmed as <i>S. clausii</i>, while two strains, FJAT-45399 and FJAT-45335, were identified as potential novel species distinct from <i>S. clausii</i>. Combined phenotypic and genomic predictions indicated that <i>S. clausii</i> could survive under harsh conditions. Comparative genomics revealed that these isolates possess antibiotic resistance genes, as well as capabilities for bacteriocin and folate production, while lacking toxins and hemolytic activity. Hemolysis tests indicated that strain FJAT-41761 exhibited non-pathogenic γ-hemolytic activity, while also demonstrating resistance to multiple antibiotics, consistent with probiotic characteristics. These findings suggest that strain FJAT-41761 is safe and holds potential as a future probiotic.