Characterization of Bacillus spp. isolated from the intestines of Rhynchocypris lagowskii as a potential probiotic and their effects on fish pathogens

Probiotics sourced from fish intestinal microbiota have a merit over other bacterial sources due to colonization ability and effective time. This study aimed to evaluate the bacilli isolated from the Rhynchocypris lagowskii intestines and their validity as a probiotic. Three isolates were selected (LSG 2–5, LSG 3–7, and LSG 3–8) and defined by morphological and 16S rRNA analysis as Bacillus velezensis, Bacillus aryabhattai, and Bacillus mojavensis, respectively. Results showed the strain tolerant abilities to gastrointestinal fluid, bile salt, pH, and temperature expotures. Additionally, all bacterial strains showed anti-pathogenic activity against at least four strains out of six tested pathogen strains (Staphylococcus aureus, Aeromonas hydrophila, Escherichia coli, Aeromonas veronii, Edwardsiella, and Aeromonas sobria). The bacterial strains also showed a high percentage of co-aggregation activity, more than 70%, with Aer. hydrophile, Staph. epidermidis, and Klebsiella aerogenes. At the same time, the results of competition, rejection, and substitution activity with Aer. hydrophila and Aer. veronii indicated the ability of the isolated strains to reduce the adhesion of pathogens to mucin. All strains showed safety properties, non-hemolytic, and sensitivity characteristics for most of tested antibiotics. In vivo test after injecting these strains into fish at various concentrations showed no side effects in the internal or external organs of fish compared to controls, proving that this is safe for these fish. Furthermore, the three strains produced lipase, amylase, and protease enzymes. The strains also showed bile salt hydrolase activity and biofilm formation, allowing them to tolerate stressful conditions. Conclusion: Based on these strains characteristics and features, they could be considered a promising candidate probiotic and can be used as an anti-pathogenic, especially in aquaculture.