Genomic Analysis and Metabolite Profiling of Three Probiotic <i>Bacillus</i> Strains for Potential Application in Aquaculture

In this study, we investigated the genomic characteristics and bioactive metabolite profiles of three novel <i>Bacillus</i> strains that were isolated from the intestinal tracts of healthy carp (<i>Cyprinus carpio</i>). Strain identification and whole-genome sequencing were performed before analysis. Among the three novel strains, R1 and R4 were classified as <i>Bacillus subtilis</i>, and strain R5 was classified as <i>Bacillus velezensis</i>. Genome mining revealed a repertoire of secondary metabolites, including bacillaene, bacillibactin, bacilysin, difficidin, and fengycin, which likely contribute to the probiotic potential of these strains. The metabolite profiles were further characterized using matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry, with fengycins and bacillibactin specifically isolated and identified. Additionally, the presence of genetic markers associated with antimicrobial resistance was assessed, and these gene profiles were reconstructed. These <i>Bacillus</i> strains represent promising candidates for the development of novel feed additives for aquaculture.