噬菌弧菌N1对淡水和海水养殖水体细菌群落影响PCR-DGGE分析

Effect of Bacteriovorax sp. N1 on the Bacterial Community in the Freshwater and Seawater Environment Using PCR-DGGE

【目的】研究噬菌弧菌Bacteriovorax sp.N1 在一个投放周期内对淡水和海水养殖环境中细菌、弧菌总数及细菌群落结构的影响。【方法】自市售微生态制剂分离蛭弧菌N1 并进行分子鉴定,测定其裂解效果,制备高浓度N1 菌液分别投放至淡水红鲤鱼(red carp)和海水仿刺参(Apostichopus japonicus)养殖水体,采用细菌平板计数法及PCR-变性梯度凝胶电泳(PCR-DGGE)技术分析48 h 内水体环境中细菌、弧菌总数及细菌群落结构变化。【结果】经鉴定蛭弧菌N1 为噬菌弧菌,其对大肠杆菌(Escherichia coli)、溶藻弧菌(Vibrio alginolyticus)、黄海希瓦氏菌(Shewanella smarisflavi)、灿烂弧菌(Vibrio splendidus)、哈氏弧菌(Vibrio harveyi)和金黄色葡萄球菌(Staphylococcus aureus)均有裂解效果。将噬菌弧菌N1 投放进淡水和海水养殖环境的12~24 h,其能显著降低两种环境中弧菌含量(P<0.05)。DGGE 分析显示添加噬菌弧菌N1 后淡水组优势菌群弧菌属(Vibrio,a1)和不可培养杆菌属(Uncultured bacterium, a5)在 12 h 以后含量有明显的减少,假单胞菌属(Pseudomonas, a3)和红杆菌科Shimia 属(Shimia, a6)菌含量增加。海水组优势菌群不可培养杆菌属(c2)菌在 12 h 时变成非优势菌群,而白杆菌属(Albirhodobacter,c1)增加成为优势菌群。噬菌弧菌N1 在水中含量在24 h 时降至最低。【结论】噬菌弧菌N1 对海水和淡水环境中的弧菌属和不可培养杆菌属菌群有明显的裂解作用导致其含量下降,但也使假单胞菌属(Pseudomonas),红杆菌科Shimia 属和白杆菌属(Albirhodobacter)菌群含量增加,但生物效应不明。为维持噬菌弧菌N1 对弧菌的控制,需要24h 左右重新补充投放。

【Objective】To study the effects of Bacteriovorax sp.N1 on the total number of bacteria, Vibrio and bacterial community structure within an addition cycle in seawater and freshwater aquaculture environments.【Method】 Bdellovibrio N1 was isolated from commercially available microecological preparations.Molecular identification was carried out and its lysis effect was preliminarily determined.In addition, high concentration of N1 strain was splashed evenly into freshwater tank and seawater tank containing the red carp Apostichopus japonicas respectively.The number of bacteria, Vibrio and structure of bacterial community in the water environment within 48 h were analyzed by plate counting and Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis(PCR-DGGE) respectively.【Result】The strain N1 was identified as Bacteriovorax sp..It had lysis effect on Escherichia coli, Vibrio alginolyticus, Shewanella smarisflavi, Vibrio splendidus, Vibrio harveyi and Staphylococcus aureus.When Bacteriovorax N1 was added to the fresh and marine culture environments for 12―24 hours, the content of Vibrio in both environments was significantly reduced(P<0.05).Results from DGGE showed that by adding Bacteriovorax sp.N1, the dominant Vibrio(a1) and uncultured bacterium(a5) counts decreased significantly after 12 h in freshwater group, but Pseudomonas(a3) and Shimia(a6) counts were increased.The dominant bacterium uncultured bacterium(c2) became a non-dominant species after 12 h in the seawater group, however, Albirhodobacter(c1) has become the new dominant bacterium.The content of Bacteriovorax sp.N1 decreased to the lowest level within 24 hours.【Conclusion】 Bacteriovorax sp.N1 can split Vibrio, uncultured bacterium, and also increase the number of microflora of Pseudomonas, Shimia and Albirhodobacter in freshwater and seawater aquaculture environment.At present, the biological effects of these newly increased microflora are not known.In order to control the outbreak of Vibrio in fish cluture, Bacteriovorax sp.N1 should be replenished in 24 hours.