一株能抑制罗非鱼源无乳链球菌的反硝化芽孢杆菌的筛选鉴定

Identification of a denitrifying Bacillus strain with an antagonistic effect on Streptococcus agalactiae isolated from tilapia

从尼罗罗非鱼（Oreochromis niloticus）肠道内容物中分离筛选出具有反硝化能力并拮抗无乳链球菌（Streptococcus agalactiae）的芽孢杆菌好氧菌株，并对其中拮抗性最强的1株芽孢杆菌进行形态学、生理生化特性、16SrRNA基因序列鉴定，进一步研究其最适生长条件、水解淀粉和蛋白的能力，并进行菌株药物敏感试验及安全性检测。经鉴定，筛选出的菌株（命名为NY5）为蜡样芽孢杆菌（Bacillus cereus）。在反硝化性能检测培养基中接种1％的NY5菌液后，对50mg／L的亚硝酸盐氮12h去除效率达到100％；对本实验室保存的21株不同来源的无乳链球菌均有拮抗作用，平均抑菌圈直径为（26．67±3．00）mm。NY5在温度为25～40℃、盐度为0-40、pH为5-9的环境中生长较好。同时NY5还具有水解酪蛋白和淀粉的功能。NY5菌株对多数抗生素敏感，对少数检测抗生素（青霉素G、麦迪霉素、头孢唑啉等）耐药。在水体中NY5菌液浓度为2．0×10^7CFU／mL，以及在注射200μL2．O×10^6CFU／mL浓度NY5菌液的条件下，体重为（6．0±1．1）g的罗非鱼均未出现死亡及其他异常现象。综合上述结果，证明筛选出的NY5菌株为蜡样芽孢杆菌。

We isolated and screened aerobic denitrifying Bacillus stains with antagonistic effects on Streptococcus agalactiae from the intestinal contents of Nile tilapia （Oreochromis niloticus）. Antagonistic ability was detected using morphological, physiological and biochemical characteristics, and 16S rRNA gene sequencing. Optimal growth conditions and the ability to hydrolyze starch and protein were tested, and drug sensitivity and safety tests were performed. We isolated a strain called NY 5 that was identified as Bacillus cereus. A total of 50 mg/L nitrite- nitrogen was removed completely within 12 hours after inoculating 1% NY 5 bacterial liquid on denitrification performance testing medium. NY 5 had antagonistic effects on 21 strains of S. agalactiae preserved in our labora- tory from different sources. The mean diameter of the inhibition zone was （26.67±3.00） ram. NY 5 grew well at temperatures of 25-40℃, salinities of 040, and pH values of 5-9. NY 5 hydrolyzed casein and starch. NY 5 was sensitive to most of the antibiotics tested but was resistant to nine antibiotics such as Penicillin G, maddie mycin, and cefazolin, etc. Tilapia with mean body weight of （6.0 ± 1.1） g did not die or show any symptoms after exposure to 2.0 ± 10^7 CFU/mL NY 5 bacteria in the water or after injection with 200 μL of 2.0×10^6 CFU/mL NY 5. These re- sults suggest that the NY 5 strain is B. cereus. This study provides a useful probiotic source to effectively inhibit growth of S. agalactiae isolated from tilapia.