Isolation And Identification Of Riemerella Anatipestifer And Analysis Of Multidrug-resistant Gene Clusters In Strain RCAD0122

Riemerella anatipestifer is an important pathogenic bacterium in ducks, and causes great damage to duck industry. Vaccine immunization and drug treatment are significant prevention and control methods for R.anatipestifer infection. But there are many different kinds of serotype in R. anatipestifer, and no cross-immunoprotection among them, which limits application of vaccine in preventing from R.anatipestifer infecting ducks. So drug becomes the main mean to prevent and control R.anatipestifer infection, but due to drug abuse, causes more and more serious resistance in R.anatipestifer, presents the phenomenon of multiple drug resistance. Researches show that resistant phenotype is result of resistance genes which bacteria contains. For understanding resistance molecular mechanism of R.anatipestifer, the research maked selection of clinical samples from various provinces, separated and identified bacterias, detected susceptibility to antibiotics cases of isolated strains, and got multiple drug-resistant strains. At last we used high-throughput sequencing and bioinformatics analysis for multiple drug-resistant strains, in order to study resistance molecular mechanism of R.anatipestifer preliminary. These studies laid a foundation for further in-depth study of R.anatipestifer resistance mechanism.First, dissected ill ducks from duck farms in eight provinces of Sichuan, Beijing, Guangdong, Hainan. Guangxi, Guizhou, Jiangsu and Jilin, and presented fibrinous exudate coated cover on heart, liver and meningeal congestion. Then picked live and brain of ducks, inoculated on the duck blood culture dish, and cultivated at 37? in CO2 conditions. According to the colony morphology and MacConkey Agar identification, we picked out suspected R.anatipestifer. Then got R.anatipestifer strains via biochemical and molecular biology identification. Second, detected susceptibility to antibiotics cases of isolated strains by drug susceptibility disc diffusion, and selected out twenty Multiple-resistant strains. Results of identification showed that fifteen strains of these clinical strains are R.anatipestifer, then detected MIC of 24 kinds drugs which belong to ten kinds of antibiotics by using mini broth dilution susceptibility. MIC value showed fifteen strains were all Multiple-resistant strains, and indicated RCAD0122 was most serious resistant to antibiotics.In order to study resistance molecular mechanism of R.anatipestifer preliminary, we extracted genomes of fifteen strains and sent samples for high-throughput sequencing by using Illumina HiSeq 2500, assembled and annotated, annotation method used Prokaryotic Genome Annotation Pipeline, then corrected by using annotation correction software for reducing occurrence of errors. We uploaded Fasta file of RCAD0122 to Comprehensive Antibiotic Resistance Database for identifying resistance gene. All of the resistance genes, upstream and downstream genes of these resistance genes were processed with sequence alignment in NCBI, and got all possible resistance genes in genomic of RCAD0122. Results indicated RCAD0122 contained resistant genes which was related to erythrocin, aminoglycosides, beta lactams, fluoroquinolone, lincosamide, sulfonamides, vancomycin, and several resistant efflux pumps. Finally, structured resistance gene structure according to the position and length of all related resistance genes in RCAD0122. We compared analysis drug-resistant genes, resistant phenotype of RCAD0122 and ATCC 11845, in order to study resistance molecular mechanism of R.anatipestifer preliminary.