Integrated Multi-Omics Characterization of Bacillus altitudinis B16 as a Potential Probiotic in Aquaculture

This study presents the detailed characterization of Bacillus altitudinis B16 (= VTCC 910232) through phenotypic, genomic, and metabolomic analyses, highlighting its potential for aquaculture applications. Strain B16 exhibited a non-hemolytic profile, strong biofilm formation, moderate thermotolerance, growth across pH 5.0-9.0, tolerance to up to 10% NaCl, and susceptibility to most tested antibiotics. The extracellular ethyl acetate extract showed notable in vitro anti-Vibrio activity against five species (14.7-21.3 mm inhibition zones) and an anti-inflammatory effect in RAW 264.7 macrophages (IC₅₀ = 33.9 µg/mL), with low cytotoxicity (> 91% viability). Metabolomic profiling tentatively identified 25 metabolites, predominantly bioactive diketopiperazines, together with putative anthramycin-like and mazethramycin-like compounds. Genome analysis (3.79 Mb; GC content 41.5%; 3,905 coding sequences; 73 transfer RNAs) revealed biosynthetic gene clusters for surfactin and bacilysin, likely involved in antimicrobial compound biosynthesis. B16 also harbored numerous genes involved in stress response and environmental adaptation, particularly those in transport systems and signal transduction pathways. No plasmid was confidently identified, and the genome harbored two genomic islands lacking virulence- or antibiotic resistance-associated genes. Collectively, the integrated phenotypic, metabolomic, and genomic analyses revealed the biosynthetic potential and adaptive traits of strain B16, including multiple putatively identified metabolites with reported bioactivities, suggesting its promise as a biocontrol agent for aquaculture.