The Molecular Epidemiology Of Enterohemorrhagic Escherichia Coli (EHEC) O157:H7

Escherichia coli O157:H7, one important enteropathogenic bacteria, is amain bacteria type of the enterohemorrhagic escherichia coli (EHEC). During the pastfew years, EHEC O157has been detected from the meat products in China frequently.The pathogenic bacteria may result in acute food poisoning and must be detectedaccording to the country regulation. It has different scales of outbreaks over the worldand its infection has a potential outbreak tendency, intensive pathogenicity, lethality, thetherapy may increase the pathogenetic condition by using antibiotics. The EHEC O157becomes a threat of public health in the whole world, so effective prevention and controlresearch about the bacteria is the current urgent problem. The author do some research tomaster the infection condition among the animals and the molecular features of thestrains, providing a reference for controlling the infection during humans and animals.1. Developing a treble PCR by using the three genes rfbE, fliC and eaeA ofEscherichia coli O157:H7to understand the prevalence condition of EHEC O157.Optimizing the annealing temperature, annealing time, primer concentration, Mg2+andthe cycles of the PCR and evaluate the sensitivity and specificity of the essay. Using themethod to detect the feces and the result shows the specificity is well and the sensitivityis103cfu/mL. The method is successful and the isolated strains of Escherichia coliO157:H7and O157:H? are detected, it will provide a new test method for the molecularepidemiological study of Escherichia coli O157:H7.2. To isolate and identify Escherichia coli O157:H7from2067samples of Henan,Gansu, Guangxi and Sichuan by using the common method for the isolation andidentification of the bacteria.6Escherichia coli O157:H7strains and3O157:H? strainsare detected, serological method, biochemical identification and molecular biologymethod are used to identified the isolated strains. And the result is accordant. Though thepositive rate is very low and there is not any outbreaks of Escherichia coli O157, thebacteria is still exist during the samples, it shows the bacteria still has a potential threat ofoutbreak and the supervision and precaution still need to be enhanced.3. To understand the phylogenetic relationship among Escherichia coli O157isolatedstrains more rationally and determine their evolution position and origin. Using the sequences published by Genbank to design primers for the housekeeping genes16S rRNA,gyrB, recA and the shiga toxin genes stx1, stx2. The genes are amplified and theirnucleotide sequences are determined, the cladogram for the phylogenetic analysis couldbe constructed. The result shows the Escherichia coli O157isolated strains and theEscherichia coli O157positive strains are near in the cladograms of the housekeepinggenes,2Escherichia coli O157:H? isolated strains do not have gene stx2and1Escherichia coli O157:H? isolated strain do not have gene stx. To analyse the origin bythe cladogram may conclude the origin of the isolated strains, it may provide a referencefor the molecular epidemiological study and origin.4. To develop a quick, sensitive and specific duplex SYBR GreenⅠ real timefluorescence quantitative PCR assay for detecting Escherichia coli O157:H7, whichcould detect the Escherichia coli O157:H7somatic antigen gene and the flagellar antigengene simultaneously. Two pairs of primers are designed according to the Escherichia coliO157:H7rfbE and fliC genes sequences published on GenBank, then the method isconstructed and optimized. The specificity, sensitivity and reproducibility of the methodare tested. The specificity test shows that only Escherichia coli O157:H7strains ispositive. The sensitivity of the assay is2.95×100copies/μL, which may be103timeshigher than the conventional PCR. The intra-assay and inter-assay variables of CV areless than2%. The duplex SYBR GreenⅠ real time fluorescence quantitative PCR assaydeveloped by present study Is sensitive, specific, reproducible, rapid and accurate. Itprovides a new identification method for the clinical diagnosis of Escherichia coliO157:H7and the food safety detection, while also for the molecular epidemiologicalstudy of the food origin diseases.5. To research the genotyping and molecular epidemiology of the Escherichia coliO157:H7by multiple-locus variable number of tandem repeat analysis (MLVA). Usingthe nucleic acid extraction, PCR, Agarose Gel Electrophoresis and the software ofBioNumerics(Version5.0) to type the O157:H7isolated strains.6variable number oftandem repeat (VNTR) loci are chosen to make cluster analysis and the result shows acertain polymorphic. The Escherichia coli O157:H7isolated strains are geneticpolymorphic, MLVA has a high discriminative typing power and is convenient to benetworking and can be compared among different labs. MLVA can contribute tounderstand the Escherichia coli O157:H7infection in epidemiologic investigation.