The versatility of Bacillus tequilensis Bt-CO as an additive: Antagonizing pathogens, enhancing immunity, promoting intestinal health, and optimizing gut microbiota of tilapia.

Probiotics are gaining increasing attention as potential alternatives to antibiotics for enhancing the health and welfare of aquatic organisms. However, identifying and developing highly effective probiotic strains with superior intestinal colonization and antagonistic properties remains a significant challenge. This study identified a strain of Bacillus tequilensis, designated as Bt-CO, from coral in a tropical marine environment. Following an in vitro assessment of its antimicrobial activity and colonization properties, the potential of Bt-CO as an antibiotic alternative was evaluated through in vivo trials. A total of 720 fish (initial body weight 2.06 ± 0.01 g) were randomly distributed across four dietary treatments in triplicates, consisting of a basal diet (CON group), and the diet supplemented with 1 × 107 (T1 group), 1 × 108 (T2 group), or 1 × 109 colony-forming unit (CFU)/g (T3 group) of Bt-CO. After a feeding period of 4 weeks, fish were sampled or challenged with Aeromonas hydrophila. For the challenge, 30 fish in each tank were intramuscularly injected with 50 μL of A. hydrophila at a concentration of 5 × 106 CFU/mL. Tilapia survival was monitored for 20 d post-infection. The results demonstrated that Bt-CO possessed pronounced antagonistic effects against several important pathogenic bacteria affecting tilapia, particularly A. hydrophila. Bt-CO also demonstrated excellent physiological characteristics, including auto-aggregation, adhesion, biofilm formation, and exhibited remarkable tolerance to bile and gastrointestinal conditions. Notably, Bt-CO possessed colonization potential in the intestines of tilapia without negatively impacting the growth performance. Following a 4-week supplementation with Bt-CO at an optimal concentration of 1 × 108 CFU/g, tilapia displayed an increased resistance to A. hydrophila infection (P = 0.011), suggesting its potential to enhance disease resistance. Additionally, Bt-CO significantly augmented the hemocyte respiratory burst (P < 0.001), serum bactericidal activity (P = 0.002), complement C3 (P = 0.027), lysozyme (P < 0.001), alkaline phosphatase (P = 0.031), and superoxide dismutase (P = 0.003) levels in tilapia. In addition, Bt-CO supplementation modulated inflammatory responses by inhibiting pro-inflammatory cytokines such as interleukin-1β (P = 0.045) and tumor necrosis factor-α (P = 0.020) and promoting anti-inflammatory cytokines such as transforming growth factor-β (P = 0.013) and interleukin-10 (P = 0.021), concurrently enhancing the expressions of intestinal barrier related factors such as claudin-1 (P = 0.051) and claudin-3 (P = 0.014) thereby improving intestinal health. Microbiome analysis indicated that Bt-CO supplementation increased the diversity of gut microbiota, promoted beneficial microorganisms but suppressed pathogenic microorganisms within the intestines of tilapia. In conclusion, incorporating Bt-CO into the diet at 1 × 108 CFU/g shows significant probiotic potential and effectively enhances tilapia's disease resistance, which is likely attributed to its role in strengthening non-specific immunity and improving fish intestinal health and microecology.