解淀粉芽孢杆菌AP193鱼类饲喂抗感染分子机制研究

Bacillus amyloliquefaciens showed efficient disease protection effect when used as probiotics in aquaculture. However, the underlying mechanisms are unclear. In this study, the associated mechanisms will be investigated in Bacillus amyloliquefaciens AP193, a strain isolated from pond sediment with good anti-infection effect in fish. Preliminary results have shown that difficidin, one of the three polyketide antibiotics produced by AP193 was the main antibiotic responsible for the in vitro antagonism of AP193 against pathogens, suggesting an important role of polyketide antibiotics in the disease protection effect of AP193, and the antibiotics-associated risk of AP193 as feed supplement probiotics. In this study, the contribution of antibiotics produced by AP193 to the anti-disease effect will be systematically studied by direct comparison of the disease protection effect of AP193 wildtype strain with knock-out mutants deficient in antibiotic(s) production in zebrafish against Aeromonas hydrophila, and by analysis of the expression of antibiotic production genes and the content of main antibiotics in the intestine of zebrafish. Further, the antibiotics-associated risk of AP193 will be evaluated by investigating the influence of main antibiotics on the intestinal microbiota of zebrafish, and the potential in inducing antibiotic-resistance in the intestinal bacteria. Lastly, the production of the main antibiotics in other aquaculture-use probiotic B. amyl. strains will be evaluated by bioinformatic techniques. This project will direct selection of aquaculture-use probiotic Bacillus strains and propose strategies for improvement of present ones.

解淀粉芽孢杆菌是一类抗病效应明确但机制尚且模糊的水产饲用益生菌。本项目以池塘底泥来源、抗病性能优良的解淀粉芽孢杆菌AP193为对象，前期工作表明AP193所生产3种聚酮类抗生素之一的difficidin主导其体外病原菌拮抗效应，提示AP193鱼体抗病力极可能与聚酮类抗生素生产相关，提示了巨大的饲用风险，迫切需要澄清。拟通过敲除AP193主要抗生素合成基因, 对比野生型与突变株鱼类饲喂（以斑马鱼为模型）抗 Aeromonas hydrophila感染力的差异, 结合 AP193主要抗生素消化道转录与合成分析，阐明AP193抗生素合成对其鱼类抗病力的贡献，并评估潜在主效应抗生素肠道菌群结构损伤和肠道代表菌株耐药性诱导的风险。在此基础上，结合生物信息学手段分析主要抗生素编码基因在其它解淀粉芽孢杆菌的分布。本项目实施将为水产饲用芽孢杆菌的筛选与优化提供指导。

本研究首先构建了斑马鱼的维氏气单胞菌(Aeromonas veronii)攻毒体系，并用该攻毒模型研究了AP193和抗生素合成基因敲除株作为饲用添加物对斑马鱼的保护作用，结果显示AP193可显著降低攻毒后斑马鱼死亡率，缺失聚酮类和非核糖体多肽的sfp基因敲除株饲喂组死亡率与AP193野生型相比有上升趋势，说明抗生素在AP193的抗病效应中起一定的作用。AP193可显著促进斑马鱼生长，表面活性肽（surfactin）对AP193的促生长功能起重要作用。通过对水产养殖业代表性的两种解淀粉芽孢杆菌B. amyloliquefaciens BCS01和B. amyloliquefaciens C3102的全基因组测序，发现两株菌都具有多种合成非核糖体多肽类和聚酮类抗生素的基因簇，提示在应用中的抗生素相关风险。最后构建了C3102的基因敲除体系，为后续进一步研究打下基础。本项目发表SCI论文4篇，中文期刊1篇，申请国家发明专利1项，毕业硕士研究生3名，博士研究生1名。