Phenotypic and safety exploration of indigenous bacterial strains for probiotic applications in aquaculture.

Quorum-sensing (QS) interference is a promising antivirulence strategy in aquaculture. This study investigated six indigenous bacterial strains for their ability to degrade N-hexanoyl homoserine lactone, a key QS signaling molecule. All strains demonstrated significant QS interference, indicating potential as biocontrol agents. Furthermore, we assessed the key in vitro traits relevant to probiotic application. These included growth rate, pH tolerance and salinity resilience. MYUG presented the fastest growth, followed by KSNUG, PMUG01, LFUG, PMUG02, and HSNUG, representing promising establishment potential within production systems and in fish guts. All strains tolerated pH (3-9) and salinity (1%-4%), supporting their adaptability to aquaculture environments and dynamic GIT conditions. Glycan diversity analysis revealed distinct lectin-glycan interaction profiles, with HSNUG displaying the highest glycan diversity index, potentially enhancing its gut adhesion and colonization capacity. Functional-trait scoring based on these in vitro characteristics ranked the six strains' probiotic suitability as: KSNUG > LFUG > PMUG01 > MYUG > HSNUG > PMUG02. A sporulation assay showed that four Bacillaceae strains (HSNUG, LFUG, PMUG01, and PMUG02) formed spores, an advantageous trait for stability during probiotic feed formulation, storage and administration. Antibiotic susceptibility screening confirmed that PMUG01 and PMUG02 had no concerning patterns, while HSNUG and LFUG exhibited resistance to certain antibiotics, warranting further molecular analysis. Finally, biosafety and gut colonization assays in axenic zebrafish embryos confirmed the in vivo safety and gut colonization potential. Within the spore-forming group, additional trait-based scoring ranked probiotic-suitability as: LFUG > PMUG01 > PMUG02 > HSNUG. Overall, these findings support in vitro-based selection of safe and effective probiotic candidates for sustainable aquaculture.