Identification Of An Insertion Sequence, IS1203 Variant, In A Shiga Toxin 2 Gene Of Escherichia Coli O157:H7 In China

EHEC (Enterohaemorrhagic Escherichia coli,EHEC) represented byserotype O157:H7 have been closely associated with epidemic cases ofhemorrhagic colitis (HC), hemolytic uremic syndrome (HUS), andthrombotic thrombocytopenic purpura (TTP). Shiga toxins (Stxs) (alsoknown as Verotoxins) are high cytotoxic and important virulence factorsproduced by EHEC O157:H7. Stxs are classified into two major classes,Stx1 and Stx2. Stx1 and Stx2 have also includes several variantsrespectively. Stx types have close association with the development ofhuman diseases. The strains of EHEC O157:H7 was firstly isolated fromsporadic diarrheal patient in 1987 in China, outbreaks caused by EHECO157:H7 were observed in 1999 and 2000 in some areas of Jiangsu andAnhui provinces. During those years other strains of EHEC O157:H7 wascontinuously isolated from environments and animal reservoirs in otherregions in China. Epidemiology of EHEC O157:H7 in China should be paygreat attention and analyzed further. In order to understand the epidemiological behaviors of EHECO157:H7, we investigated the Shiga toxin genes of 289 strains of Shigatoxin-producing E. coli O157:H7 isolated from patients and animalsources from 1999 to 2002 in some regions in China (mainly fromoutbreaks of E. coli O157:H7 infections in Jiangsu and Anhui provinces),using polymerase chain reaction (PCR) amplifying the specific sequencesof these genes and determining the nucleotide sequences. We found novelstx2 genes in 3 of 289 E. coli O157:H7 strains, 2 designated 02F044 and99B113 of which isolated from the feces of sheep in Jiangsu province and 1designated 9 of which isolated from swine in Anhui province. Bydetermining the nucleotide sequences of theses genes and Blast comparing,we found that the three novel stx2 genes were inserted by a 1313-bpinserted fragment and the novel stx2 gene was 2554-bp instead of 1241-bpby corresponding to the wild-type stx2 gene. The three insertion fragmentsinclude a 1310-bp insertion sequence (IS) and a 3-bp direct repeat (DR) atthe target site of stx2 genes for generating upon insertion. The nucleotidesequences of the three 1310-bp IS are identical, except for the 3-bp DR atthe target site of stx2 genes from the 1313-bp fragments, and show 100%homology with that of IS1203v (insertion sequence 1203 variant)(stx2::IS1203v) from E. coli O157:H7 strains reported in 1999 in Japan.This was firstly reported to found three E. coli O157:H7 strains carryingstx2::IS1203v in China. In addition, the three IS1203v inserted in the stx2genes at the different sites respectively, the open reading frames (ORFs) ofIS1203v of one strain designated 02F044 and one strain designated 9 arecontrary to those of stx2 genes of the two strains, the ORFs of IS1203v ofanother strain designated 99B113 are the same as that of stx2 genes it . Theresults suggested that the changes in productivity and activity of Stx2 maybe due to insertion of IS1203v. The productivity and activity of Stx2 through Stx2 preparations fromthe three E. coli O157:H7 strains carrying stx2::IS1203v were examined bythe HeLa and Vero cells assays and by measuring lactate dehydrogenase(LDH) release from HeLa and Vero cells as an indicator of cytotoxicity. Wefound Stx2 preparations from the three E. coli O157:H7 strains producedcytopathic effect for HeLa and Vero cells. The results of measuring LDHrelease from cells showed that Stx2 preparations from the three E. coliO157:H7 strains (designated 02F044, 99b113 and 9) induced respectively25.16%,26.08%,26.07% LDH which were lower than 51.62% LDH of thecarrying stx2 gene strain as positive control from HeLa cells and 31.15%,30.31%,31.32% LDH which were lower than 40.53% LDH of the carryingstx2 gene strain as positive control from Vero cells in 18 hours. The resultssuggest that the productivity of Stx2 could be influenced by IS1203vinserting in stx2 genes. By statistical analysis of data of LDH values, Stx2preparations from the three carrying stx2::IS1203v E. coli O157:H7 strainsinduced lower LDH releases from HeLa cells than did only one carryingstx2 gene E. coli str...