Isolation And Characterization Of Bdellovibrio Sp. From Fresh Water And Preliminary Investigation Into The Lysis Mechanism Of Bdellovibrio On Escherichia Coli

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.