Probiotic Characterization and Safety Assessment of Marine Bacillus Isolates from Diverse Biotopes

In modern aquaculture, the search for alternatives to antibiotics requires probiotic selection strategies integrating functional efficacy and biological safety. This study presents a scoring–heatmap approach to identify probiotic candidates from different marine biotopes and evaluate the impact of isolation source on probiotic selection. A total of 61 bacterial strains were isolated from the skin mucus and intestine of mullet (Chelon labrosus), the intestine of gilthead seabream (Sparus aurata) and seawater from Sfax. Among them, nine strains exhibited strong antibacterial activity against Lactococcus garvieae, Vibrio anguillarum, Vibrio harveyi, Aeromonas hydrophila, and Photobacterium damselae subsp. damselae and were subjected to hierarchical evaluation combining functional properties and critical safety criteria. Bacillus subtilis D6 and Bacillus pumilus D9 and EM7 emerged as the most promising candidates, achieving the highest scores due to broad-spectrum pathogen inhibition, remarkable adhesion capacity, hydrophobicity above 70%, and auto-aggregation above 90% after 24 h. Molecular analyses confirmed the absence of enterotoxins and virulence genes, while in vivo assays in European seabass (Dicentrarchus labrax) revealed no mortality or apparent clinical signs in fish injected with D6, D9, and EM7 during the 10-day post-injection period. The observed differences suggest a potential association between ecological origin and probiotic characteristics, warranting further investigation. Bacillus subtilis D6 and Bacillus pumilus D9 and EM7 emerged as promising potential probiotic candidates for sustainable aquaculture.