Isolation, Identification, Culture Of Bdellovibrio Bacteriovorus And Its Inhibition Effects On Germ

Bdellovibrios are Gram negative bacteria. Bdellovibrios could attack other Gram negative bacteria, penetrate their periplasm, multiply in their cytoplasm, and finally burst their cell envelopes to start anew. They could form plaques in double-layer agar plates containing prey bacterium. Depended on this characteristic, 21 strains were isolated from 174 water samples. And then a strain (designated as BD5W) was selected from these isolates, which could grow very well at 37℃. Identification was confirmed by a PCR assay using Bdellovibrio Bacteriovorus-specific primers. Compared with the sequences on the GenBank, BD5W's partial 16S rDNA sequence showed the highest homology (99.4%) to B. Bacteriovorus HD 100 and B. Bacteriovorus HD 127, but showed a low homology (10.5%) to Bacteriovorax sp. EEA. Phylogenetic analysis of the sequences confirmed that Bdellovibrio genus were divided into two clades and BD5W strain belonged to Bdellovibrio Bacteriovorus clade.The results of antibiotic susceptibility test showed that BD5W strain was lowly sensitive to polymyxin B, and moderately sensitive to mydecamycin, acheomycin and lincomycin. When BD5W was cultivated at different temperature, its survival number was different. BD5W survical number remained 108pfu/mL with the temperature range of 30℃to 39℃. At 37℃and 38℃, plaques formed quickly in 23h and slowly at 12℃. BD5W couldn't grow at 4℃. OD600 of liquid medium containing prey bacterium reduced with Bdellovibrio Bacteriovorus growing. BD5W could grow at pH 4~9. Its optimal pH was 7. A series of tests showed that BD5W lysed prey (E.coi K88) quickly in 1/10 YT and 1/50 YT medium. If added 0.05M NaCl, 0.05mM KCl, 1mM MgCl2 and 1mM CaCl2 to the 1/50 YT diluted medium, the ability of Bdellovibrio Bacteriovorus BD5W was enhanced. At 23h, OD600 of the medium was reduced by 75.7%. But OD600 of the control group was reduced by 60.4% at 140h. The ability of Bdellovibrio Bacteriovorus BD5W was higher to heat-killed prey bacteria than to active bacteria in 1/100YT, 1/200YT and 1/500YT diluted medium. But the result was defferent in 1/10 YT and 1/50 YT diluted media.In vitro, Bdellovibrio Bacteriovorus BD5W could lyse E.coli K88, E.coli K99, S.typhimurium, S.choleraesuis, S.suis, Bordetella. But it couldn't lyse P.multocida and Staphylococcus aureus. The safe test was carried out through drenching Kunming mice with l07pfu/mL BD5W. All the mice of test group and control group were healthy. This result showed BD5W was safe to mice. The mice infected with S. typhimurium artificially were cured by 107pfu/mL and 105pfu/mL BD5W. The survival rate were 90% and 80%, respectively.The result showed that Bdellovibrio Bacteriovorus BD5W was effective to cure Salmonellosis.Bdellovibrio Bacteriovorus BD5W could lyse pathogenic bacteria. It could purify natural water systems and cure the animal diseases as a microbiol ecological agent.