The Study Of Intestinal Bacterial Community Structure And Antibiotics Susceptibility Of Intestinal Bacterial Strains From The Farming Giant Salamander

Giant salamander is Second Class National-Level Protected Animal and the important special economic animals in China.Giant salamander is transitional species between fish and reptiles.Giant salamander have unique biological characteristics,environment and evolutionary status.Gut microbiota is very closely related to growth and development,nutrient absorption,metabolism,disease occur and immune regulation in animal.Giant salamander perhaps has special gut microbial structure,but little research focused on this topic at present.In this research we used PCR-DGGE technology,High-Throughput Sequencing and convention culture technology to study the intestinal bacteria community structure of giant salamander.30 antibiotics were used to test antibiotics drug sensitivity of bacteria isolated from giant salamander intestinal.This study provided a basis for revealing the relationship between bacterial community structure and healthy of giant salamander,preventing and controlling the disease in this animal.The results were as followed:1.Intestinal bacterial community structure analysis by PCR-DGGE technology.PCR-DGGE was used to analyze intestinal bacteria community structure and the DGGE fingerprint bands for dominant bacteria were obtained.The result showed that similarities of the bacteria structures in healthy giant salamanders gut were 42%,and the evenness of the bacteria community from these guts were closed.Shannon Diversity index showed a great abundance of intestinal bacteria species in these giant salamander.The intestinal bacterial community structure was stable in these giant salamander.The DGGE fingerprint bands were abundant and there were 56 detectable bands with different signal intensities.After recovering and cloning we found that 39 bands sequence were successful sequened.The core microbiota were Proteobacteria,Actinobacteria,Firmicutes and Bacteroidetes.The brightest bands were identified,which belong to Aquabacterium,Lacinutrix,Actinopolyspora,Arthrobacter,Serratia and Clostridium.2.Intestinal bacterial community structure analysis by High-Throughput SequencingTo insight giant salamander intestinal bacterial community structure we used High-Throughput Sequencing to analyze intestinal bacterial community structure in detail.The experimental result showed that an abundance of bacteria species existed in farming giant salamander intestinal.The core microbiota were Bacteroidetes,Proteobacteria,Firmicutes,Verrucomicrobia.The same result was found in different groups of the salamander.The intestinal bacteria of Bacteroidetes was the largest in number.The bacteria of Bacteroides was the dominate strain.Starvation treatment of the salamander led to a decrease of Proteobacteria and Fusobacteria and an increase of Firmicutes.The core microbiota had no changed.Analysis of ? diversity for different giant salamander intestinal samples,showed that bacteria species diversity,richness and evenness were decreased in a certain extent.Analysis of ? diversity for giant salamander intestinal samples.Revealing that there was very small differences in species diversity within same group.But the difference in species diversity was great between feeding group and starvation group.The composition of community structure was similar within feeding group.But the difference in the composition of community structure was obvious between feeding group and starving group.3.Isolating and identifying intestinal bacteria from farming giant salamander by conventional culture.The conventional culture technology was used to isolate and identify intestinal bacteria.The culture medium for isolation of the intestinal bacterial were LB medium,TSA medium,EE medium and BHI medium.The result showed that a total of 9 bacteria species were isolated,7 of which were Proteobacteria and 2 of which were Firmicutes.The intestinal bacteria from the farming healthy giant salamander included pathogen,opportunistic pathogen and probiotics bacteria.Bacillus bataviensis isolated from the intestinal was probiotic bacteria.The opportunistic pathogens included Aeromonas hydrophila,Citrobacter freundii,Edwardsiella ictaluri,Enterobacter aerogenes,Vibrio tubiashii,Proteus mirabilis and Enterococcus gilvus.Plesiomonas shigelloides was a pathogen.4.The results of antibiotic susceptibility to intestinal bacteria.30 antibiotics were used to study the susceptibility to intestinal bacteria from the farming giant salamander.The result showed that 9 intestinal bacteria were high sensitive to the most of antibiotics.The antibiotics in treatment of diseases had an obvious impact on the intestinal bacteria.Ceftazidime,Levofloxacin,Doxycycline,Prperacillin and Ceftriaxone inhibited most of opportunistic pathogen and pathogenic bacteria and had little affect to probiotic bacteria.These antibiotics improved the intestinal bacterial community structure.Above all,the study provided the following conclusion:Three different techniques were used to research intestinal bacteria community structure of giant salamander.conventional culture technology has many limitation,it can't reveal intestinal bacteria community structure.PCR-DGGE technology just identified some dominate bacteria sequences,the sequences from tiny amounts of intestinal bacteria would not be identified.High-Throughput Sequencing could identify dominant bacteria sequences and the sequences from tiny amounts of intestinal bacteria.So it provided reliable and comprehensive results to reveal intestinal bacteria community structure.The core microbiota were Bacteroidetes,Proteobacteria,Firmicutes,Verrucomicrobia.Bacteroides was the dominate bacteria.The susceptibility study showed 30 antibiotics drug had different susceptibility to intestinal bacteria.Antibiotic should be used reasonably to use them in production practice.The antibiotics,which are sensitive to opportunistic pathogen and pathogen and not to probiotics,should be selected.