海水养殖系统中37株枯草芽孢杆菌生理代谢、遗传特性及抑菌效果差异分析

Physiological metabolism, genetic characteristics, and bacteriostatic activity diversity of 37 Bacillus subtilis from mariculture system

摘要:

【背景】由水产致病菌导致的病害不断暴发，寻找安全有效的抗生素替代品是目前生产的迫切需求。人们通常过于关注益生菌效应，而对其安全性评价重视度不够。【目的】分析我国海水养殖系统中不同来源枯草芽孢杆菌菌株的表型及遗传特征，并寻找绿色安全且具有多重抑菌作用的菌株。【方法】以2009-2021年从我国海水养殖系统中分离的37株枯草芽孢杆菌为对象，利用纸片扩散法(K-B法)检测其对不同抗生素的抗性；利用培养基平板法测定淀粉酶、蛋白酶和溶血能力；通过PCR方法检测枯草芽孢杆菌溶血相关基因携带风险；采用牛津杯法测定其对副溶血弧菌、溶藻弧菌、爱德华氏菌、哈维氏弧菌、美人鱼发光杆菌和假交替单胞菌等6种病原菌的抑菌作用；并对候选益生性枯草芽孢杆菌的安全性进行评估。【结果】药敏检测结果显示，37株枯草芽孢杆菌对甲氧苄啶、吡哌酸、链霉素表现出强耐药性，对磺胺嘧啶表现出中等耐药，对头孢噻肟、环丙沙星、舒巴坦的耐药率低，对克拉霉素、诺氟沙星、氟苯尼考、氟甲喹、复方新诺明、四环素表现为完全敏感。蛋白酶、淀粉酶活性测试结果显示，37株枯草芽孢杆菌能不同程度地水解酪蛋白和淀粉。溶血性测试结果显示，37株枯草芽孢杆菌中有4株出现溶血现象，而8个溶血相关基因在37株枯草芽孢杆菌中均有检出，溶血表型与检测基因关联分析表明，产生溶血现象的菌株与其溶血基因携带间无直接相关性。抑菌试验分析表明，37株枯草芽孢杆菌均对2种及以上病原菌有抑制作用，对6种病原菌均具有良好抑菌作用的有2株(菌株Bs4和Bs7)。对凡纳滨对虾的安全试验表明，菌株Bs4对凡纳滨对虾具有高安全性，7 d对虾存活率为100%。【结论】通过对37株枯草芽孢杆菌生理代谢表型、遗传特性及病原拮抗特性进行比较分析，揭示了我国海水养殖系统中枯草芽孢杆菌具有多元化的表型及遗传特征，并筛选出一株生态安全且具有多重抑菌活性的益生性枯草芽孢杆菌，为水产养殖病害防控、开发抑菌类微生态制剂及水产养殖行业健康绿色发展提供了理论基础和技术支撑。

关键词:枯草芽孢杆菌;抗生素敏感性;溶血性;产酶能力;抑菌作用;生态安全

Abstract:

[Background] Diseases caused by aquatic pathogens continue to break out, and finding safe and effective alternatives to antibiotics is an urgent need. Probiotics are often influenced by interests and profit-making, whereas not enough attention has been paid to their safety evaluation. [Objective] To find green and safe probiotics with multiple bacteriostatic activities based on the phenotypic and genetic characteristics of Bacillus subtilis in China’s mariculture system. [Methods] Taking 37 B. subtilis isolated from China’s mariculture system from 2009 to 2021 as the research object, this study used K-B method to detect the resistance of B. subtilis to different antibiotics. The amylase, protease, and hemolytic capacity of B. subtilis were determined by medium plate method. Polymerase chain reaction (PCR) was used to detect the risk of carrying genes related to hemolysis in B. subtilis. The Oxford cup method was used to determine its bacteriostatic effects on six pathogens, including Vibrio parahaemolyticus, Vibrio algaelyticus, Edwardsiella tarda, Vibrio harveyi, Pseudoalteromonas sp., and Photobacterium damselae. The safety of candidate probiotic B. subtilis was evaluated. [Results] The results of drug susceptibility test showed that 37 isolates of B. subtilis showed strong resistance to trimethoprim, pipemidic acid, and streptomycin, moderate resistance to sulfadiazine, low resistance to cefotaxime, ciprofloxacin, and sulbactam, and complete sensitivity to clarithromycin, norfloxacin, florfenicol, flumequine, cotrimoxazole, and tetracycline. The test results of protease and amylase activity showed that 37 isolates of B. subtilis hydrolyzed casein and starch to varying degrees. The hemolytic test results showed that 4/37 isolates of B. subtilis had hemolytic phenomenon, while 8 hemolysis-related genes were detected in 37 isolates of B. subtilis. The analysis of the correlation between hemolytic phenotype and detection gene showed that there was no direct correlation between the strain that produced hemolysis and its hemolytic gene carrier. The analysis of bacteriostatic experiments showed that all isolates of B. subtilis had inhibitory effect on 2 or more pathogenic bacteria, and 2 isolates (strains Bs4 and Bs7) had good bacteriostatic effects on 6 pathogenic bacteria. Safety experiments on Litopenarus vanmamei showed that strain Bs4 had high safety against L. vanmamei, and the survival rate of 7 d was 100%. [Conclusion] Through the comparative analysis of the physiological metabolic phenotype, genetic characteristics, and pathogen antagonism characteristics of B. subtilis isolates, it is revealed that B. subtilis has diversified phenotypes and genetic characteristics in China’s mariculture system. An ecologically safe and probiotic B. subtilis with multiple bacteriostatic activities is screened out, which provides a theoretical basis and technical support for the prevention and control of aquaculture diseases, the development of bacteriostatic microecological preparations, and the healthy and green development of aquaculture industry.

Key words:Bacillus subtilis;antibiotic susceptibility;hemolytic;enzyme-producing capacity;bacteriostasis;ecological security