The Diversity Of Bdellovibrio Spp. In Porcine Gi Tract And Its Isolation,Characterization And Preliminary Study Of Predation Ability

In addition to prevent and treat disease in animal industry,antibiotics are commenly given as growth promoters to increase animal growth,improve feed effciency and reduce animal mortality from pathogen infection.The liberal and continuous use of antibiotics may contribute to some increasingly grave issues,such as generation of mutiple drug-resistant(MDR)bacteria and antibiotic drug residues in anmial products.As time passes,it not only affect the healthy and sustainable development of animal industry,but also emerge as a serious threat to human health and public safety.Bdellovibrio spp.are Gram-negative predators that feed on other Gram-negative bacteria.Numerous studies reported their potential role as alternative antibiotics to inhibit the Gram-negative bacteria.However,the majority of the studies relating to Bdellovibrio spp.are based on the isolates from water and soil.The isolates from animal GI tract and their predation ability are rarely involved.Therefore,this thesis intends to focus on the diversity and predation ability of Bdellovibrio spp.derived from animal GI tract.Experiments are conducted in the following two sections:In the first section,the experiment was conducted to investigate the distribution of Bdellovibrio spp.in porcine GI tract and its diversity in porcine stomach.It was not only the first time,to our knowledge,to study the Bellovibrio spp.in animal GI tract,but also to provide the theoretical basis for the isolation of Bdellovibrio bacteriovorus in porcine GI tract for the further study.In the present experiment,extract total bacterial DNA of digesta,which were collected from porcine stomach,duodenum,ileum,cecum and colon,for the specific amplicfication of Bdellovibrio spp.16S rDNA partial sequence;then detect the quantity of Bdellovibrio spp.in each intestianl segament by qPCR;and finally build the clone library of Bdellovibrio spp.16S rDNA sequence in porcine stomach to explore its diversity.It turned out that,all the five parts of the porcine GI tract contained specific amplification of Bdellovibrio spp.16S rDNA partial sequence;the quantity of Bdellovibrio spp.in the posterior GI tract(cecum,colon)were generally higher than in the anterior GI tract(stomach,duodenum),and there was a significant difference between the quantity of Bdellovibrio spp.in cecum(104.32 copies/g)and duodenun(102.77 copies/g)(P<0.05);5 different Bdellovibrio spp.16S rDNA sequences were acquired,which were designated as ST1?ST5,by restriction fragment length polymorphism for the sequences in porcine stomach clone library.Phylogenetic analysis indicated that ST1,ST5 and ST2,ST3 were belong to new species in Bacteriovorax spp.and Bdellovibrio spp.,respectively;and ST4 was in the same phylogenetic branch with Bdellovibrio starrii.In the second section,the experiment was conducted to isolate and purify Bdellovibrio bacteriovorus,which was able to form plaques in the double-layer agar plate,from porcine GI tract,and also to test its predation ability on two other Gram-negative bacteria(E.coli.and Salmonella).The objective of this experiment was to provide a new way for alleviating the increasingly grave MDR Gram-negative bacteria in animal industry.In the present experiment,Bdellovibrio bacteriovorus was isolated from digesta in porcine stomach,duodenum,ileum,cecum and colon at 37?,using Pseudomonas stutzeri as prey bacteria by double-layer agar plate technology.The results showed that the bacteria strain that can lyse the host cell can be isolated from all the five parts of the porcine GI tract.The isolate from porcine stomach with the best plaques-forming ability was chosed for the further study of purification and characterization.Through transmission electron microscope(TEM)and 16S rDNA sequence alignment,it was further confirmed that the isolate was Bdellovibrio bacteriovorus,which was in the same branch with Bdellovibrio bacteriovorus HD 100 in the phylogenetic tree.Then,use MDR E.coli and salmonella as prey bacteria,both concentrations are 106 cfu/mL,to detect the predation ability of isolate at 37?.The isolate had a predation ability for both two Gram-negative bacteria.The number of plaques were 114.5 ± 1.5 pfu/mL and 200.5 ± 2.0 pfu/mL for E.coli and Salmonella plates,respectively.Moreover,the isolate was more likely to prey Salmonella compared with E.coli(P<0.05).Through the preliminary study of isolation and characterization of Bdellovibrio bacteriovorus from porcine stomach and its predation ability on two major pathogens in animal industry,we built a strong foundation for its potential use as alternative antibiotics in animal industry.