淡水蛭弧菌的分离鉴定及其裂解大肠杆菌作用机制的初步研究
发布时间：2019-03-08 18:15
【摘要】： 蛭弧菌作为一种依靠裂解宿主菌使自身得以繁殖的寄生微生物,它可以裂解大多数科、属的革兰氏阴性细菌,尤其是对病原弧菌具有良好的裂解作用。本实验采用DNB双层平板法以Escherichia coli DH5α作为宿主菌从湖南省东江水域采集到淡水水样中筛选得到6株菌株(分别命名为HNAL,HNAS,HNBL,HNBS,HNCL和HNCS)。通过观测6株菌在DNB双层平板上形成的噬菌斑的形状,同时利用透射电子显微镜观察它们的形态,并对照《伯杰氏细菌鉴定手册》(第九版),最后进行16S rRNA基因和16S-23S rRNA ITS扩增鉴定、测序和系统进化学分析,可以鉴定这6株菌为蛭弧菌,同时经系统进化学分析认为菌株HNAL,HNAS,HNBL和HNBS属于蛭弧菌的一个新种(Bdellovibrio sp.nov)。我们系统研究了6株蛭弧菌对能导致水产养殖病害的30株致病或潜在致病菌的裂解谱。此外,我们首次以湖南淡水蛭弧菌(HNAS)为研究对象,先在电镜下初步观察蛭弧菌与大肠杆菌的作用方式,之后利用超薄切片技术结合透射电子显微镜,初步研究淡水蛭弧菌作用大肠杆菌的裂解机制。 本研究结果表明,6株淡水蛭弧菌对30株致病或潜在致病菌具有较好的裂解能力,HNAL,HNAS,HNBL,HNBS,HNCL和HNCS的裂解率分别为70%、73.3%、66.7%、66.7%、50%和60%,如果考虑不同蛭弧菌株间的协同作用,它们的裂解率则达93.3%;通过负染法对菌体染色,在电镜下观察到蛭弧菌HNAS以机械钻孔的形式侵染大肠杆菌;同时通过电镜观察比较,确定蛭弧菌与大肠杆菌最适浓度比为1:1;在电镜下观察蛭弧菌作用大肠杆菌过程的超微结构,根据所观测到电镜图可进一步确认该株淡水蛭弧菌是以机械钻孔的方式对宿主菌进行裂解的,同时观察到了蛭弧菌附着、侵染、蛭质体和释放的阶段。
[Abstract]:Bdellovibrio bdellovibrio, as a parasitic microorganism which can reproduce itself by lytic host bacteria, can cleave Gram-negative bacteria of most families and genera, especially for pathogenic Vibrio. In this experiment, six strains (named HNAL,HNAS,HNBL,HNBS,HNCL and HNCS)., respectively) were isolated from fresh water samples collected from Dongjiang waters of Hunan province by DNB double-layer flat plate method with Escherichia coli DH5 伪 as host bacteria. By observing the shape of the plaque formed on the double-layer plate of DNB, the morphology of the plaque was observed by transmission electron microscope (TEM), and compared with the Burger's Manual of bacterial Identification (ninth edition). Finally, 16s rRNA gene and 16S-23S rRNA ITS amplification, sequencing and phylogenetic analysis showed that the six strains were identified as Bdellovibrio spp. At the same time, the phylogenetic analysis showed that the strain HNAL,HNAS, was identified as Bdellovibrio bdellovibrio. HNBL and HNBS are new species of Bdellovibrio (Bdellovibrio sp.nov). We have systematically studied the cleavage spectra of 6 strains of Bdellovibrio to 30 pathogenic or potential pathogenic bacteria which can cause aquaculture diseases. In addition, for the first time, we studied the interaction of vermiculovibrio vermiculosus and Escherichia coli under electron microscope, and then we used ultrathin section technique combined with transmission electron microscope (TEM) to study the interaction of Vibrio pellicularis (HNAS) with E. coli. The cleavage mechanism of E. coli by Vibrio leech was studied. The results showed that 6 strains of Vibrio leech had good cleavage ability to 30 pathogenic or potential pathogenic bacteria. The cleavage rates of HNAL,HNAS,HNBL,HNBS,HNCL and HNCS were 70%, 73.3%, 66.7%, 66.7%, respectively, and the lytic rates were 70.0%, 73.3%, 66.7% and 66.7%, respectively. 50% and 60%, if the synergetic effects among different strains of vermiculose arc were considered, the cleavage rate of the strains was 93.3%. Bdellovibrio HNAS was observed to infect E. coli in the form of mechanical drilling by negative staining method, and the optimum concentration ratio of Bdellovibrio to E. coli was determined by electron microscope, and the optimal concentration ratio of Bdellovibrio to E. coli was determined by electron microscope. The electron microscope was used to observe the ultrastructure of E. coli induced by Bdellovibrio vermiculosus. It was further confirmed that the strain of Vibrio vibrio was lysed by mechanical drilling, and the attachment of Vibrio vermiculoides was observed at the same time. Infection, vermiculite and release stages.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378
本文编号：2437077
[Abstract]:Bdellovibrio bdellovibrio, as a parasitic microorganism which can reproduce itself by lytic host bacteria, can cleave Gram-negative bacteria of most families and genera, especially for pathogenic Vibrio. In this experiment, six strains (named HNAL,HNAS,HNBL,HNBS,HNCL and HNCS)., respectively) were isolated from fresh water samples collected from Dongjiang waters of Hunan province by DNB double-layer flat plate method with Escherichia coli DH5 伪 as host bacteria. By observing the shape of the plaque formed on the double-layer plate of DNB, the morphology of the plaque was observed by transmission electron microscope (TEM), and compared with the Burger's Manual of bacterial Identification (ninth edition). Finally, 16s rRNA gene and 16S-23S rRNA ITS amplification, sequencing and phylogenetic analysis showed that the six strains were identified as Bdellovibrio spp. At the same time, the phylogenetic analysis showed that the strain HNAL,HNAS, was identified as Bdellovibrio bdellovibrio. HNBL and HNBS are new species of Bdellovibrio (Bdellovibrio sp.nov). We have systematically studied the cleavage spectra of 6 strains of Bdellovibrio to 30 pathogenic or potential pathogenic bacteria which can cause aquaculture diseases. In addition, for the first time, we studied the interaction of vermiculovibrio vermiculosus and Escherichia coli under electron microscope, and then we used ultrathin section technique combined with transmission electron microscope (TEM) to study the interaction of Vibrio pellicularis (HNAS) with E. coli. The cleavage mechanism of E. coli by Vibrio leech was studied. The results showed that 6 strains of Vibrio leech had good cleavage ability to 30 pathogenic or potential pathogenic bacteria. The cleavage rates of HNAL,HNAS,HNBL,HNBS,HNCL and HNCS were 70%, 73.3%, 66.7%, 66.7%, respectively, and the lytic rates were 70.0%, 73.3%, 66.7% and 66.7%, respectively. 50% and 60%, if the synergetic effects among different strains of vermiculose arc were considered, the cleavage rate of the strains was 93.3%. Bdellovibrio HNAS was observed to infect E. coli in the form of mechanical drilling by negative staining method, and the optimum concentration ratio of Bdellovibrio to E. coli was determined by electron microscope, and the optimal concentration ratio of Bdellovibrio to E. coli was determined by electron microscope. The electron microscope was used to observe the ultrastructure of E. coli induced by Bdellovibrio vermiculosus. It was further confirmed that the strain of Vibrio vibrio was lysed by mechanical drilling, and the attachment of Vibrio vermiculoides was observed at the same time. Infection, vermiculite and release stages.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378
【引证文献】
相关硕士学位论文 前3条
1 鲁锋;蛭弧菌2GS的生物学特性分析及其控制罗非鱼片沙门氏菌生长的应用[D];华南理工大学;2011年
2 何伟杰;蛭弧菌HKM的生物学特性分析及其相关应用研究[D];华南理工大学;2011年
3 鲍星;蛭弧菌控制鲜切果蔬致病菌数量的研究[D];华南理工大学;2012年
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