Probiotic Bacillus tequilensis protected Micropterus salmoides against Aeromonas veronii infection by modulating digestion, immunity, and intestinal microbiota and metabolites

Micropterus salmoides , a freshwater fish of significant economic value, faces substantial challenges due to frequent infection by Aeromonas veronii and excessive antibiotic use. Bacillus tequilensis emerges as a promising antibiotic alternative, enhancing fish immunity and gut health. However, its specific effects on M. salmoides have not been fully clarified. This study examined the impact of different concentrations of B. tequilensis Bt-CO (0, 6.0 × 10 9 , 6.0 × 10 10 , and 6.0 × 10 11  CFU/kg) on juvenile M. salmoides , focusing on growth, survival, body indexes, immune and antioxidant responses, digestive enzymes, disease resistance, intestinal microbiota, and metabolism. Following an 8-week feeding trial, Bt-CO had no significant impact on the growth performance, hepatosomatic index, and condition factor of M. salmoides . However, at a concentration of 6.0 × 10 11  CFU/kg, it significantly enhanced immune indicators, glutathione peroxidase activity, digestive enzyme activities, and post- A. veronii infection survival rates of M. salmoides . Gut microbiota analysis revealed that Bt-CO enhanced beneficial bacteria while reducing harmful ones. Metabolomic analyses indicated that Bt-CO modulated intestinal metabolite levels, including SM(d16:1/24:1(15Z)), d -Threitol, and PC(18:1(9Z)/P-16:0), involving 25 metabolic pathways like d -Glutamine and D-glutamate metabolism. Correlation analysis highlighted significant links between specific gut bacteria and differential metabolites. Overall, this study suggests that incorporating Bt-CO at a concentration of 6.0 × 10 11  CFU/kg in the diet of juvenile M. salmoides could: (i) improve intestinal health by modulating microbial composition and metabolite profile in the intestine; and (ii) enhance fish digestive capacity, immunity, and disease resistance.