Edwardsiella piscicida is an important fish pathogen that causes substantial economic losses. In order to understand its pathogenic mechanism, additional new virulence factors need to be identified. The bacterial thioredoxin system is a major disulfide reductase system, but its function is largely unknown in E. piscicida. In this study, we investigated the roles of the thioredoxin system in E. piscicida (named TrxB_Ep, TrxA_Ep, and TrxC_Ep, respectively) by constructing a correspondingly markerless in-frame mutant strain: ΔtrxB, ΔtrxA, and ΔtrxC, respectively. We found that (i) TrxB_Ep is confirmed as an intracellular protein, which is different from the prediction made by the Protter illustration; (ii) compared to the wild-type strain, ΔtrxB exhibits resistance against H₂O₂ stress but high sensitivity to thiol-specific diamide stress, while ΔtrxA and ΔtrxC are moderately sensitive to both H₂O₂ and diamide conditions; (iii) the deletions of trxB_Ep, trxA_Ep, and trxC_Ep damage E. piscicida’s flagella formation and motility, and trxB_Ep plays a decisive role; (iv) deletions of trxB_Ep, trxA_Ep, and trxC_Ep substantially abate bacterial resistance against host serum, especially trxB_Ep deletion; (v) trxA_Ep and trxC_Ep, but not trxB_Ep, are involved in bacterial survival and replication in phagocytes; (vi) the thioredoxin system participates in bacterial dissemination in host immune tissues. These findings indicate that the thioredoxin system of E. piscicida plays an important role in stress resistance and virulence, which provides insight into the pathogenic mechanism of E. piscicida.

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.