Evaluation of probiotic properties and safety of a Bacillus strain for shrimp farming: Integrating in vitro testing, genomic analysis and in vivo validation.

This study evaluated the probiotic properties and safety of Bacillus subtilis subsp. inaquosorum BSXE-2102 through in vitro testing, genomic analysis, and in vivo validation in shrimp farming. In vitro assays revealed the potent antagonistic activity of BSXE-2102 against 8 aquatic pathogenic strains. Genomic analysis of BSXE-2102 unveiled multiple genes responsible for the biosynthesis of 12 secondary metabolites, indicating its potential in combating pathogens. The identification of stress tolerance and adhesion-related genes suggested that BSXE-2102 has the ability to survive and colonize the host environment. The presence of numerous genes involved in the metabolism of carbohydrates, lipids, and amino acids, as well as the biosynthesis of 13 amino acids, 5 organic acids, and 7 vitamins, highlighted its role in food digestion and nutrition provision. Additionally, genes related to immune stimulation were identified, underscoring its potential immunomodulatory effects. Safety assessments, including antibiotic resistance tests, hemolytic assays, and genomic analyses, indicated that BSXE-2102 was sensitive to all antibiotics tested and did not carry high-risk antibiotic resistance genes. The strain also exhibited no hemolytic activity and lacked high-risk virulence factor-coding genes. The in vivo feeding experiment demonstrated that dietary supplementation with BSXE-2102 (at concentrations of 1 × 105, 1 × 106, 1 × 107, and 1 × 108 CFU/g) improved growth performance, enhanced 8 serological immune parameters, upregulated the expression levels of 8 hepatopancreatic immune-related genes, and increased resistance against Vibrio parahaemolyticus of Penaeus vannamei to varying degrees. These results were consistent with the findings from in vitro assays and genomic analyses. Collectively, the in vitro, genomic, and in vivo findings supported the safe use of B. subtilis BSXE-2102 as a potential probiotic to enhance the growth performance, immune capacity, and disease resistance of P. vannamei. A supplementation concentration of 1 × 107-8 CFU/g for the strain in shrimp feed is recommended.