罗非鱼源无乳链球菌肠道拮抗芽孢杆菌的筛选及其生物学特性

Selection and characterization of intestinal Bacillus strain antagonistic against pathogenic Streptococcus agalactiae of tilapia

摘要:

[目的]从健康尼罗罗非鱼（Oreochromis niloticus）肠道中筛选一株对罗非鱼源无乳链球菌等病原菌具有拮抗功能的益生菌。[方法]取健康尼罗罗非鱼肠道，匀浆后进行10倍系列梯度稀释，然后涂布BHI平板，培养1-2 d，挑取单克隆菌落。采用点种法初步筛选对罗非鱼源无乳链球菌有拮抗作用的菌株，选取其中一株拮抗效果较好的菌株LF01，通过形态学、生理生化特征以及分子生物学分析，对LF01菌株进行鉴定。然后对LF01菌株的生长特性、水解淀粉和酪蛋白能力、药物敏感特性、抗菌谱和生物安全性进行测定和分析。[结果]根据菌落形态和生长时间的差异，从健康尼罗罗非鱼肠道中筛选出64株细菌，通过拮抗试验筛选出6株具有明显拮抗效果的菌株，其中LF01菌株的拮抗效果最好。根据LF01的形态、生理生化特征和gyrA基因的进化分析，确定该菌株为贝莱斯芽孢杆菌（Bacillus velezensis）。LF01菌株的最适生长温度为30℃，最适pH值为7，最适盐度为5‰，而且该菌株具有水解淀粉和酪蛋白的功能。药敏试验结果显示，LF01菌株对多数抗生素敏感，仅对杆菌肽耐药。拮抗试验结果显示LF01株对无乳链球菌、海豚链球菌、迟缓爱德华氏菌、鮰爱德华氏菌、嗜水气单胞菌、舒氏气单胞菌、维氏气单胞菌、简氏气单胞菌、鰤鱼诺卡氏菌等病原菌均具有拮抗作用，其中对鰤鱼诺卡氏菌的拮抗作用最强，平均抑菌圈直径达28.3 mm。生物安全试验表明，LF01菌株对尼罗罗非鱼、斑马鱼（Danio rerio）和乌鳢（Channa argus）等3种鱼均无致病性，具有良好的安全性。[结论]本研究筛选了一株贝莱斯芽孢杆菌LF01株，该菌的生物安全性良好，而且可拮抗常见的水产病原菌，具有防控多种水产经济动物疾病的潜力，应用前景十分广阔。

关键词:贝莱斯芽孢杆菌;无乳链球菌;点种法;抑菌;药敏试验

Abstract:

[Objective] The aim of this study was to screen a probiotic strain that antagonizes Streptococcus agalactiae isolated from the intestine of healthy Nile tilapia (Oreochromis niloticus). [Methods] The gut of healthy tilapia was collected, homogenized, and diluted by a 10-fold series. Then 0.3mL suspension was streaked on brain heart infusion plates and incubated for 1-2 d at 30℃, and then single colonies were selected for purification. Strains had antagonistic function against S. agalactiae were screened by the dot-inoculating method, and a strain named LF01 with a better antagonistic effect was selected. In this study, the strain LF01 was identified by physiological, biochemical characteristics and molecular biological analysis. In addition, some characteristics and biosafety of the strain LF01 were determined and analyzed. [Results] A total of 64 strains were isolated from the intestine of healthy tilapia and 6 strains with obvious antagonistic effects were obtained, with the antagonism of LF01 strain considered the best. Strain LF01 was identified as Bacillus velezensis. The best growth temperature, pH, and salinities of strain LF01 were 30℃, 7, and 5‰, respectively. Furthermore, the strain LF01 exhibited the ability to hydrolyze starch and casein. Strain LF01 was sensitive to most antibiotics and resistant only to bacitracin, and showed antimicrobial activity against a broad range of fish pathogens including Streptococcus agalactiae, Streptococcus iniae, Edwardsiella tarda, Edwardsiella ictaluri, Aeromonas hydrophila, Aeromonas schubertii, Aeromonas veronii, Aeromonas jandaei and Nocardia seriolae. Biosafety tests suggested that the strain LF01 was not pathogenic to Nile tilapia, Danio rerio, and Channa argus. [Conclusion] In this study, it was found that B. velezensis LF01 strain isolated from the intestine of healthy tilapia has good biosafety and could antagonize the common aquatic pathogens. It has the potential to prevent and control a variety of aquatic animal diseases, and has broad application prospects.

Key words:Bacillus velezensis;Streptococcus agalactiae;dot-inoculating method;inhibition;antibiotic sensitivity test