The Combination of Bacillus Subtilis wt55 with AiiO-AIO6 Improve the Resistance of Zebrafish to Aeromonas Veronii Infection by Simple Collocation Rather Than B. Subtilis Quorum-Quenching Recombinant Expression Strain

Abstract

Disease problems will seriously restrict the sustainable development of aquaculture, and the environmental-friendly prevention strategies are urgently needed. Probiotics and quorum-quenching enzyme are innovative strategies to control bacterial diseases. Firstly, the bacteriostatic activity of B. subtilis wt55 strain and quenching enzyme AiiO-AIO6 on the growth of A. veronii were tested in vitro, and the results showed that this two had different effects on A. veronii: wt55 inhibit the growth of A. veronii, but AiiO-AIO6 did not. Then, the synergistic effects of simple combination of B. subtilis wt55 and AiiO-AIO6 was evaluated next. The results showed this combination could improve the survival rate and significantly reduce the number of invasive A. veronii in gut after challenge compared to the other groups, corresponding to the lower intestinal alkaline phosphatase activity. In vitro co-culture experiments showed this combination could inhibit the growth of A. veronii. Direct immersion of germ-free zebrafish proved AiiO-AIO6 did not directly regulate the innate immune response of the host, but wt55 did it, and the combination of wt55 and AiiO-AIO6 could significantly reduce the expression of NF-κB and proinflammatory cytokine IL-1β, increase the expression of lysozyme gene. The gut microbiota induced by either experimental diet was transferred to germ-free zebrafish, and the results showed that intestinal microbiota also plays a regulatory role. The gut microbiota from combination group could significantly inhibit the expression of IL-1β and NF-κB, and increased the expression of TGF-β and lysozyme. Given the effectiveness of this combination, a B. subtilis quorum-quenching recombinant expression strain in which AiiO-AIO6 was surface displayed on the spores and secreted by vegetative cells was built. The results showed that the survival rate after challenge was lower than that of the group treated with AiiO-AIO6 or wt55 alone, and the expression of proinflammatory cytokine IL-1β and NF-κB were significantly higher. Our study demonstrated the effectiveness of B. subtilis and AiiO-AIO6 simple combination and established an efficient B. subtilis expression system.