Abstract：
The indiscriminate use of antibiotics in aquaculture has led to the emergence of resistance; hence, eco-friendly, host-specific alternatives to mitigate bacterial infections have become imminent. In this study, bacteria that could possibly serve as probiotics were isolated and evaluated for their efficacy with in vitro experiments and in vivo zebrafish gut model. One isolate from each of the 23 rohu fish (Labeo rohita) was shortlisted after preliminary screening of several isolates and tested for their ability to inhibit two important warm water bacterial fish pathogens, Aeromonas hydrophila, and Edwardsiella tarda. An isolate (RODK28110C3) that showed broad-spectrum inhibitory activity against a battery of different isolates of the two fish pathogens included in this study and maintained in our repository was selected for further characterization. The culture was identified phenotypically as Bacillus subtilis and confirmed by 16S rDNA sequencing. The isolate was able to hydrolyze fish feed constituents that include starch, protein, and cellulose. Further in vitro tests ensured that the potential isolate with probiotic attributes could tolerate different gut conditions, which included a range of pH, salinity, and varying concentrations of bile salt. Exposure of 4 days post fertilization zebrafish embryos to the RFP-tagged isolate confirmed the colonization of B. subtilis in the gut of the zebrafish embryo, which is an important attribute of a probiotic. The isolate was able to inhibit both A. hydrophila and E. tarda in gnotobiotic zebrafish embryo in triplicate. The study demonstrates the probiotic characteristics of the B. subtilis isolated from L. rohita and its ability to inhibit A. hydrophila and E. tarda using in vitro conditions and in the zebrafish gut and could serve as an effective alternative to antibiotics in aquaculture.
摘要：
在水产养殖中滥用抗生素导致耐药性的出现；因此，环保,宿主特异性的替代方案，以减轻细菌感染已成为迫在眉睫。在这项研究中,分离可能作为益生菌的细菌，并通过体外实验和体内斑马鱼肠道模型评估其功效。在初步筛选了几种分离物后，从23种罗湖鱼（Labeorohita）中每种分离物入围，并测试了它们抑制两种重要的温水细菌性鱼病原体的能力，嗜水气单胞菌,还有Edwardsiellatarda.选择了一种分离株（RODK28110C3），该分离株对本研究中包含的两种鱼类病原体的不同分离株具有广谱抑制活性，并保存在我们的存储库中。将培养物表型鉴定为枯草芽孢杆菌，并通过16SrDNA测序确认。分离物能够水解鱼饲料成分，包括淀粉，蛋白质，和纤维素。进一步的体外测试确保具有益生菌属性的潜在分离株可以耐受不同的肠道条件，其中包括一系列的pH值，盐度,和不同浓度的胆汁盐。受精后4天斑马鱼胚胎暴露于RFP标记的分离株证实了枯草芽孢杆菌在斑马鱼胚胎肠道中的定植，这是益生菌的重要属性。该分离物一式三份能够抑制生齿斑马鱼胚胎中的嗜水A和E.tarda。该研究证明了从L.rohita分离的枯草芽孢杆菌的益生菌特性及其在体外条件下和斑马鱼肠道中抑制嗜水蛋白A和E.tarda的能力，并且可以作为水产养殖中抗生素的有效替代品。
