Isolation And Identification Of A Bovine Klebsiella Pneumoniae And Preliminary Analysis Of Its Genome

Klebsiella pneumoniae is a common opportunistic pathogen of Enterobacteriaceae,Klebsiella genera,also an important zoonotic pathogen,which rank only second to Escherichia coli and Pseudomonas aeruginosa.K.pneumoniae is widely distributed in nature and mainly in intestines,respiratory and urinary tract of human and animals,as well as water,soil and grain,causing meningitis,peritonitis,enteritis,pneumonia,ophthalmia and traumatic infections,even sepsis in severe cases.Recently,broad spectrum of drug resistance makes clinical treatment of the bacteria is still a huge challenge.Therefore,relevant research on K.pneumoniae will establish a basis for new prevention and control of K.pneumoniae infections effectively.This study took ill calf internal organization as epidemic materials which was mainly related to outbreak of diarrhea and respiratory symptoms from Yuzhong county,Lanzhou city,Gansu province,was identified by pathogen separation and identification,determining the infection was caused by K.pneumoniae.Based on this,the biological characteristics of the isolates were studied.Finally,the bioinformatics of the isolates was analyzed,which laid a foundation for the further study on the pathogenesis and immune mechanism of K.pneumoniae.The research contents and results mainly include the following several aspects.1.Isolation and identification of K.pneumoniae YZ strainBased on clinical comprehensive diagnosis,we isolated the pathogenic bacteria from epidemic materials,and completed its pathogenicity test,drug sensitivity test,biochemical test,16 S r RNA gene,khe gene,as well as amplification and sequence analysis of partial virulence genes.The results showed that all biochemical test of the isolate were consistent with K.pneumoniae in addition to H2 S test,both the 16 S r RNA and khe gene sequence of the isolate shared 99% homology with most sequence of K.pneumoniae from Genbank.Three different virulence genes of the bacteria in sequence homology with other strains of K.pneumoniae is above 95%.Based on this,the isolates was finally identified as K.pneumoniae and named YZ strain.2.Biological characteristics analysis of K.pneumoniae YZ strainOn the basis of the separation and identification,capsule staining characteristics,growth curve determination,detection of biofilm formation,determination of the median lethal dose and immune protective test of the isolates was completed.The results showed that the isolates cannot effectively form capsules;As can be seen from the growth curve,it got into phase delay in 0~4 h,entered Logarithmic phase after 4 h,10~18 h for stability and entered the decline phase after 18 h;Qualitative and quantitative determination of the bacterial biofilm showed that the isolates could form a certain biofilm;By the Kouffer method,the median lethal dose of isolates was1.38×108 CFU,showing its weak pathogenicity to other K.pneumoniae;Using different concentrations(1×107 cfu/m L~1×1010 cfu/m L)to make vaccine of the separation,There was no abnormal phenomenon after inoculated mice,explaining the vaccine was safety;The toxicity test showed when the concentration of vaccine got to1×1010 cfu/m L,the protection ratio reached 100%,showing good protection effect,and the serum titer in mice reach above 1:3 200.3.Genome analysis of K.pneumoniae YZ strainTo understand it's causes of capsule defects and pathogenic mechanism,the study completed the genomic framework and the sequencing results were analyzed.Results showed that the genome length of the bacterial was 5 336 336 bp,GC content was 57.444%,contained 120 Scaffold,166 Contig,80 t RNA,3 r RNA.Genome contains 5 205 genes,the wab and wac gene were not found in the four conserved genes associated with the transfer and surface assembly of the capsule polysaccharide in the strain.In addition,only found negative regulatory genes Fur,crp and Sug E in the modulators of different roles in capsule synthesis.In the virulence genes,not find rmp A,mag A and wab G,and there were some genes that were unknown functions in these predict genes.The results laid a foundation for the further study of the causes of capsule defects and can clearly explain its pathogenic mechanism.