Construction Of Stx Deletion Mutant And Detection Of EHEC 0157

The term "enterohemorrhagic E.coli" (EHEC) was originally coined to denote strains that cause Hemorrhagic Colitis(HC) and Hemolytic Uremic Syndrome (HUS), express Shiga toxin(Stx), cause attaching and effacing(ATE) lesions on epithelial cells, and possess a ca. 60-MDa plasmid. Although there are many serotypes of EHEC, serotype O157:H7 has been most frequently implicated in foodborne diseases.Most of the work on pathogenic factors of ?coli O157:H7 has focused on the Stx, which are encoded on a bacteriophage inserted into the chromosome. Additional virulence trait includes the ability to intimately adhere to the intestinal mucosa by an attaching and effacing mechanism. This property encoded by a 43kb pathogenicity island (PAI) termed the locus of enterocyte effacement (LEE). On the PAI the most important virulence gene is eae gene, which encodes intimin.In this study a Multiplex PCR method was developed for the rapid detection of genes encoding Shiga toxins 1 and 2 (stxl and stx2), intimin (eaeA) and H7 flagellin antigen (fliC). Four pair's primers were synthesized. The size of amplified product is different, so it is easy to distinguish the bands on agarose gel. PCR products of these genes (stx, eae, fliC) were generated only with DNAs from E coli O157:H7 simultaneously. The method includes enrichment before the PCR reaction. The enrichment culture medium was modified by adding antibiotics and bile salt for improving the sensibility of the Mul-PCR. The effects of different broth enrichment media and incubation durations on the sensitivity of E coli O157:H7 detection were evaluated on artificially contaminated water. Modified Trypticase soy broth containing 20 y g/ml vancomycin and 1.5mg/ml bile salt No.3 was found to be a promising enrichment protocol. After 5h enrichment positive detection of E. coli470157:H7 was demonstrated when >1 CFU/ml was present in the artificially contaminated water. This method is sensitive, specific and simple. Therefore it would be a practical protocol for detecting EHEC O157:H7 rapidly.In order to determine the effect of stx gene of EHEC O157:H7, stx deletion mutant was constructed. ^ stx gene was amplified by PCR and cloned into a suicide vector pCVD442, which containing the /7/r-dependent R6K replicon and sacB gene. The recombinant plasmid pCVD442:: A stx was transformed into a host cell SMlOp/'r, then conjugated from SM10(pCVD442:: Ascc) into EHEC O157:H7 EDL933W. Because a homologous recombination would take place between A stx and stx which located on the chromosome, the strain of EHEC O157:H7 stx deletion mutant was screened out.A mitomycin and nalidixic acid-treated mouse model was used to compare the pathogenicity of EHEC O157:H7 strain EDL933W with the mutant strain EDL933W( A stx). All mice inoculated with a dose of 4x108 of 933 W died in 4 days while only one of mice with 933 W( A stx) died at 5th day and others had some clinic symptoms but survived. The study demonstrates that the stx deletion mutation results in loss of toxicity to vero cell, but some pathogenicity still remains.