Isolation, Identification And Lysing Characteristics Of Bdellovibrio From Fresh Water

Bdellovibrio and Like Organisms are small-sized parasitic bacteria which livedby preying on other bacteria. Bdellovibrio has physiological characteristics ofparasitic and lysis of bacteria, especially they can lyse lots of pathogenicgram-negative bacteria. In this study, using Pseudomonas stutzeri as prey bacteriathrough double-layer agar plate technology,6strains of bacteria were isolated fromwater samples and sludge samples from Chinese Soft-shell Turtle-breeding pond inShanghai Pudong New Area and Fu Xing Island Canal in Shanghai Yangpu Area. Thesix strains of bacteria were named as AS212，JS221，JS223，JS413，JS422and N322respectively.These six strains of bacteria were firstly identified by morphological observation,including the morphology of plaque formed in the double-layer agar plate and theirmorphological properties in different stage of life by transmission electronmicroscopy. According to―Bergey’s manual of systematic bacteriology‖(the secondedition), the morphology of plaque formed in the double-layer agar plate andmorphological properties by transmission electron microscopy of these6strains ofbacteria corresponded with the typical characteristic of Bdellovibrio.16S rRNA genesof these6strains of bacteria were amplified, and the sequences obtained comparedwith16S rRNA sequences of relative bacteria in GenBank. Phylogenetic analysis wasutilized to further identify these strains. Secondly, one of the six strains, AS212strainwas chosen for further study. We detected the growth condition and lysis effect indifferent salinity, temperature and pH conditions. The prey range of AS212to31pathogen or potential pathogen strains from different sources were systematicallyexplored. The elimination effect on the bacteria in aquaculture water of AS212wasanalyzed. AS212strain was compared with two disinfectants, BCDMH and TCCAwhich are commonly used in aquaculture, to eliminate the bacteria in water. Finally,analog application of AS212in Litopenaeus vannamei larval rearing was conducted.We detected the total number of bacteria and Bdellovibiro in the Litopenaeus vannamei larval rearing water after AS212was added. Furthermore, the effect ofAS212on water quality and survival rate of Litopenaeus vannamei was researched.The results of this study showed that the plaques formed by these6strains ofbacteria were all round, transparent and had regular edge. The shape of these6strainsof bacteria was short-rod and they had a single, polar and sheathed flagellumrespectively observed by Transmission Electron Microscopy. We preliminaryidentified these6strains of bacteria as Bdellovibrio by morphological identification.16S rRNA gene sequence and phylogenetic analysis were conducted, and these6strains of bacteria were further identified as Bdellovibrio.Strain JS413should be placed in the genus Bacteriovorax, and the species wasundetermined. Strain N322should be placed in the genus Bdellovibrio, and speciesBdellovibrio bacteriovorus. Strain AS212, JS221, JS223and JS422had a closehomologous relationship with Bdellovibrio sp. HEA（AY294216）, and the similaritywas90%. They had a far homologous relationship with Bdellovibrio bacteriovorusHD100T（AJ292759）, and the similarity was just80%. Thus, combination with thephylogenetic analysis, they should be placed in the genus Bdellovibrio as a member ofa novel species for which we propose the name Bdellovibrio sp. nov.The result of lysis capacity of AS212showed that AS212could not only lyse G-bacteria including E. coli, Aeromonas hydrophila, Vibrios, etc., but also lyse G+bacteria including Streptococcus agalactiae, Staphylococcus aureus, Bacillus, etc.The rate of eliminating the bacteria in aquaculture water of AS212was more excellentthan two disinfectants, BCDMH and TCCA. The result of analog application ofAS212in Litopenaeus vannamei larval rearing showed that, the total number ofbacteria was obviously decreased and the number of Bdellovibrio was increasedfirstly and decreased later, but finally formed a dynamic equilibrium with the bacteriain the water. There was no significant effect on the pH, NH4+-N、NO3--N、NO2--N.Thus, it showed that there was no effect of AS212on improving the water quality inLitopenaeus vannamei larval rearing. However, adding AS212strain in shrimp rearingwater could improve the survival rate of larvae of Litopenaeus vannamei, it showedthat AS212had a protective effect on the larvae.