Aquaculture Laboratory, Department of Zoology, The University of Burdwan, Burdwan 713104, West Bengal, India.
Fisheries Laboratory, Department of Zoology, Visva-Bharati University, Santiniketan 7131235, West Bengal, India; Department of Biochemistry, University of Calcutta, Kolkata 7000019, India.
Microb Pathog. 2019 May;130:146-155. doi: 10.1016/j.micpath.2019.02.028. Epub 2019 Feb 28.
Application of antibiotics to combat bacterial diseases in fish has been criticized due to likely emergence of drug resistance. Therefore, investigation of new bioactive compounds from natural sources has been taken into account. This study was designed to purify and characterize the bioactive compound in the cell free supernatant (CFSs) of autochthonous gut bacteria (Bacillus methylotrophicus KU556164, B. amyloliquefaciens KU556165, Pseudomonas fluorescens KU556166 and B. licheniformis KU556167) isolated from rohu, Labeo rohita. CFSs were antagonistic to fish pathogenic Aeromonas spp., moderately thermo-tolerant and active in wide range of pH (5-11). Antibacterial activity of the CFSs was reduced by the action of proteases (e.g., Proteinase K and Trypsin), indicating proteinaceous nature of the bioactive compound like the bacteriocins. Three-step purification procedure resulted in recovery of 16.97%, 18.04%, 33.33% and 6.38% activity of the antimicrobial protein produced by B. methylotrophicus, B. amyloliquefaciens, P. fluorescens and B. licheniformis, respectively. Purification at each step revealed decrease in protein content with gradual increase in the specific activity of the antimicrobial protein. The purified antibacterial compound ranged between 18.2 and 25.6 kDa. Identification through MALDI-TOF MS/MS and database search through Mascot search engine predicted that the bactericidal compound belonged to either alkaline proteases, or, transcriptional regulator and some hypothetical proteins. Apart from potential technological application of the antibacterial compound, the present study might show promise for application of gut-associated bacteriocinogenic bacteria to control diseases in fish caused by pathogenic bacteria.
抗生素在鱼类细菌性疾病防治中的应用因可能产生耐药性而受到批评。因此，人们考虑从天然来源中寻找新的生物活性化合物。本研究旨在从罗非鱼（Labeo rohita）肠道原籍菌（甲基营养芽孢杆菌 KU556164、解淀粉芽孢杆菌 KU556165、荧光假单胞菌 KU556166 和地衣芽孢杆菌 KU556167）中提取、分离并鉴定胞外产物（CFSs）中的活性化合物。CFSs 对鱼类病原菌气单胞菌具有拮抗作用，具有中等耐热性和较宽 pH 值范围（5-11）活性。CFSs 的抑菌活性可被蛋白酶（如蛋白酶 K 和胰蛋白酶）削弱，表明该活性化合物具有蛋白质性质，类似于细菌素。三步纯化程序使甲基营养芽孢杆菌、解淀粉芽孢杆菌、荧光假单胞菌和地衣芽孢杆菌产生的抗菌蛋白的回收率分别达到 16.97%、18.04%、33.33%和 6.38%。在每个步骤的纯化过程中，抗菌蛋白的比活逐渐增加，而蛋白含量逐渐降低。纯化后的抗菌化合物的分子量在 18.2-25.6 kDa 之间。基质辅助激光解吸电离飞行时间质谱/质谱联用（MALDI-TOF MS/MS）和 Mascot 搜索引擎数据库搜索分析预测，杀菌化合物属于碱性蛋白酶、转录调控因子或一些假定蛋白。除了抗菌化合物具有潜在的技术应用价值外，本研究还可能为利用肠道相关产细菌素的细菌来控制鱼类病原菌引起的疾病提供新的思路。
