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
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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。
Bdellovibrio is a kind of parasitic bacteria which can reproduce itslf by lysing gram negative host microoganisims, and especially Bdellovibrio have good lysis ability on vibrios. In our investigation on fresh specimen obtained from Dongjiang river in Hunan provience, six strains (named HNAL,HNAS,HNBL,HNBS,HNCL和HNCS respectively) which could prey on Escherichia coli DH5αwere isolated through DNB double layer agar plating technique. These six strains were primarily identified, according to“Bergey's manual identification of bacteria”(ninth edition), as Bdellovibrio spp. through observing the plaques formed in the DNB double layer agar plate and their morphological properties by transmission electron microscopy. The amplification, clone and phylogenetic analysis of 16S rRNA gene and 16S-23S rRNA ITS were utilized to further identify these strains and strain HNAL, HNAS, HNBL and HNBS should be placed in the genus Bdellovibrio as a member of a novel species for which we propose the name Bdellovibrio sp. nov. To step further, the lysis spectrums of the six Bdellovibrio strains to 30 pathogens or potential pathogens were systematically explored. Besides, we preliminarily studied the lytic mechanism of Bdellovibrio on E coli. With the help of Transmission Electron Microscope, we were to explore the interaction mode between strain HNAS and Escherichia coli, and we combined the thin action technology and TEM to primarily study on lysis mechanism of strain HNAS on Escherichia coli.
The lysis experiments on 30 pathogens or potential pathogens showed that individual HNAL, HNAS, HNBL, HNBS, HNCL和HNCS lysed 70%、73.3%、66.7%、66.7%、50% and 60% of the total bacteria, respectively. In combination, they lysed 93.3% of the bacteria tested. The specimen (mix of strain HNAS and Escherichia coli) was negtively stained and examined by Transmission Electron Microscope, and we discovered strain HNAS was using the method of mechanical drill hole to lyse the Escherichia coli. After comparing the results of figures of Transmission Electron Microscope at different concentration ratios, we make sure the optimum ratio between strain HNAS and Escherichia coli is 1:1. The ultrastructures of Bdellovbrio (HNAS) lysed Escherichia coli were explored by Transmission Electron Microscope and we further confirm that Bdellovbrio used the method of mechanical drill hole to lyse the Escherichia coli; meanwhile, we also could find the stages of attach, attack, bdelloplast and release.
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。
Bdellovibrio is a kind of parasitic bacteria which can reproduce itslf by lysing gram negative host microoganisims, and especially Bdellovibrio have good lysis ability on vibrios. In our investigation on fresh specimen obtained from Dongjiang river in Hunan provience, six strains (named HNAL,HNAS,HNBL,HNBS,HNCL和HNCS respectively) which could prey on Escherichia coli DH5αwere isolated through DNB double layer agar plating technique. These six strains were primarily identified, according to“Bergey's manual identification of bacteria”(ninth edition), as Bdellovibrio spp. through observing the plaques formed in the DNB double layer agar plate and their morphological properties by transmission electron microscopy. The amplification, clone and phylogenetic analysis of 16S rRNA gene and 16S-23S rRNA ITS were utilized to further identify these strains and strain HNAL, HNAS, HNBL and HNBS should be placed in the genus Bdellovibrio as a member of a novel species for which we propose the name Bdellovibrio sp. nov. To step further, the lysis spectrums of the six Bdellovibrio strains to 30 pathogens or potential pathogens were systematically explored. Besides, we preliminarily studied the lytic mechanism of Bdellovibrio on E coli. With the help of Transmission Electron Microscope, we were to explore the interaction mode between strain HNAS and Escherichia coli, and we combined the thin action technology and TEM to primarily study on lysis mechanism of strain HNAS on Escherichia coli.
The lysis experiments on 30 pathogens or potential pathogens showed that individual HNAL, HNAS, HNBL, HNBS, HNCL和HNCS lysed 70%、73.3%、66.7%、66.7%、50% and 60% of the total bacteria, respectively. In combination, they lysed 93.3% of the bacteria tested. The specimen (mix of strain HNAS and Escherichia coli) was negtively stained and examined by Transmission Electron Microscope, and we discovered strain HNAS was using the method of mechanical drill hole to lyse the Escherichia coli. After comparing the results of figures of Transmission Electron Microscope at different concentration ratios, we make sure the optimum ratio between strain HNAS and Escherichia coli is 1:1. The ultrastructures of Bdellovbrio (HNAS) lysed Escherichia coli were explored by Transmission Electron Microscope and we further confirm that Bdellovbrio used the method of mechanical drill hole to lyse the Escherichia coli; meanwhile, we also could find the stages of attach, attack, bdelloplast and release.
引文
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