| Enterohermorrhagic Escherichia coli O157:H7 can cause hemorrhagic colitis (HC), thrombotic thrombocytopenia purpura (TTP) and life-threatening complications, hemolytic uremic syndrome (HUS). EHEC O157:H7 strains were first identified in 1982 and have since been rapidly becoming popular all over the world. During the period from April to August 1999, a massive outbreak of E. coli O157:H7 occurred in Jiangsu and Anhui Province of China, with 195 hospitalized patients who were clinically diagnosed as hemolytic-uremic syndrome (HUS) and 177 deaths. The sero-epidemiological study suggested that there were about 20,000 persons infected during the outbreak. This is the largest outbreak of human infections of E.coli on the world, resulted in 195 HUS patients and 177 deaths. However, the genetic charactics of the outbreak associated isolates have not been well understood.By using the 15-locus multilocus sequence typing (MLST) scheme (http://www.shigatox.net/mlst), we analyzed 124 strains of E.coli O157:H7 isolated in China in various years and from various geographic locations. The 15 locus MLST scheme differentiated the 124 isolates into five sequence types (STs).101 of 124 strains were typed as ST23, which included 1996 Japan outbreak associated strain Sakai. All of the 5 strains isolated fron patients during 1999 China outbreak were typed as ST96, a novel sequence type has not be reported. The ST96 has 16 strains, including 5 isolated from animals during 1999 outbreak, one isolated from patients in Shandong province in 1992 and 4 from food samples in Yunnan province in 2001. Interestingly, no strain of ST96 has been isolated after 2001 in China.Based on the sequences of 15 locus of MLST scheme, we found that 1999 China outbreak associated strain Xuzhou 21 was distantly related with1996 Japan outbreak associated strain Sakai,1992 and 2006 American outbreak associated strain EDL933 and TW14359. When whole genemo sequences were used to study the genetic ralatideness of those outbreak strains, we found that 1999 China outbreak associated strain Xuzhou21 was closed related with 1996 Japan outbreak associated strain Sakai. Since both of Sakai and Xuzhou 21 caused two largest outbreaks of human infections, their potential charactritics for causing big outbreak were further studied.When the organisms were exposed to acidic conditions at pH 3.0 for 2 hours, we found that the survival rate for EDL933 was 23.6%, significantly lower than 46.7% and 45.7% for Sakai and Xuzhou21 respectively. The difference has statistical significance. We found that the rpoS sequence of Sakai were identical to those of two co-called high acid resistant strains AF182102 and AF182107, which was associated with the high acid resistance. And, the rpoS sequence of EDL933 had a change at nucleotide 917, resulting in an early stop codon.We then measured the level of transcriptional of Shiga toxin 2 (stx2) gene in strains of Xuzhou21, EDL933 and Sakai using real-time quantitative PCR, which was demonstrated to be associated with development of HUS. The transcript levels of Sakai and Xuzhou21 were 3.7 and 7.5 times higher than that of EDL933. After induction of mitomycin C, the expressed level of stx2 in EDL933 was increased 4.5 times, but it was increased as high as 32.7,68.6 times in Sakai and Xuzhou21 respectively. When co-incubated with human peripheral blood mononuclear cells (PBMCs), it was found that the level of cytokines,IL-6 and IL-8 stimulated by strain EDL933,Sakai and Xuzhou21 were 207.45±11.08,234.84±7.87 and 315.34±23.71pg/ml for IL-1β,317.38±10.25,545.06±33.69 and 565.31±22.09 pg/ml for IL-6; and 2601.12±11.08,3568.51±123.97,4837.67±333.49 pg/ml for IL-8. In general, the level of expression of those 3 cytokines induced by Xuzhou21 is similar to that induced by Sakai, but is singnificantly higher than that induced by EDL933.In short, we found that the strains of E.coli O157:H7 associated with 1999 China outbreak belonged to a novel sequence type 96. Genome sequence analyziz indicated that the 1999 China outbreak associated strain Xuzhou21 was phylogenetically related with 1996 Japan outbreak associated strain Sakai, but distant from 1982 and 2006 American outbreak associated strain EDL933 and TW14359. And furthomore, we found that the two large outbreak associated strains Xuzhou21 and Sakai are more acid resistant, producing higher amount of Shiga toxin 2 and stimulated host cells to producing higher level of cytokines, namely IL-1 β, IL-6 and IL-8. Therefore, we proposed Sakai and Xuzhou 21 as the strains with potential to cause large outbreak.
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