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

Objective:Enterohemorrhagic Escherichia coli(EHEC) O157, an emerging pathogen, causes severe hemorrhagic colitis and the life-threatening extraintestinal complication of hemolytic uremic syndrome (HUS) of patients. EHEC O157:H7 was first recognized as a cause of illness in 1982 during an outbreak of severe bloody diarrhea in USA. The EHEC O157 becomes a threaten of public health in the whole world. Moreover, people is susceptible to the infection of EHEC O157, especially the children, which are more likely to have severe symptoms, including kidney failure, and die. Making EHEC O157 as a biological weapon is available. The establishment of a good and reliable platform for epidemiological analysis will be invaluable for further investigations of E. coli O157:H7 infections and prevention, particularly in the investigation of the special class of STs being liable to appear in humans other than from food, animals and the environment.Recently, new routes of infection, including direct contact with animals, and survival in novel food vehicles, particularly fresh produce, have become major sources of disease cases and have contributed to widespread epidemics. This changing epidemiology is also influenced by the genetic variation and''relentless evolution''of the O157 pathogen population. As the population of EHEC O157 strains has increased in frequency and spread geographically, it has genetically diversified. Studies of EHEC O157 strains'sequence have indicated that genotypic diversity is largely attributable to the insertions, deletions, and duplications of variable sizes of DNA fragments of virulent factors. The importance of the Epidemiological study of E. coli O157:H7 was to determine to what extent of risk strains may be transmitted to and infect humans. This report provides more insight into the molecular epidemiological characteristic of this bacterium in China.The E. coli O157:H7 strains were identified systematicly by serologic test, Colony morphology, Biochemical identification, and 16s rRNA sequence. After estabalished the strains database, the virulent factors, such as stx1/2,hlyA and tccP were detected. Combined with Multilocus sequence typing (MLST) for investigating populations and population dynamics on a global scale using neutral or slowly accumulating genetic variation in housekeeping genes (aspc, icdA, uidA,clpX,mdh, fadD ,lysp ). To assess the genetic diversity and variability of EHEC O157:H7, and to to determine the molecular relatedness among these strains, which may provide insights into the microevolutionary history of genome divergence and to help understand sources and routes of transmission of this infection.Results:1. EHEC O157:H7 isolates were obtained from strain databases built by JS Province Center for Disease prevention and Control during the year 1999 to 2007, as well as from CQ municipality during 2005 to the 2007, China. MLST analysis and determination of diversity in the virluence agent :2. seven fragments of the housekeeping genes'allelic loci of E. coli O157:H7 were selected for the MLST scheme. There are aspc,icdA,uidA,clpX,mdh,fadD and lysp. There're 7 STs and most had not been described previously at the EcMLST database, that were ST836,ST837,ST838,ST839,ST840,ST841 and ST842. BURST (Based Upon Related Sequence Types) algorithm showed that these 7 STs could be clustered in 2 groups3. The presence of stx1, stx2, hlyA and TccP repeat-fragments was were identified by PCR. Only 1 (99G144) isolates carried stx1 genes, whereas all possessed stx2 genes. All strains possessed the large 60-MDa pO157 virulence plasmid and were PCR positive for the enterohemolysin gene (hlyA) (100%). The sequence results of the 527bp hlyA gene are all the same. PCR results detecter 3 kinds of tccp gene repeat-types. Among study strains there are that 13 (12.4%) isolates contained 3 repeats-fragments, 6 (5.7%) contained 4 repeats, and 86 (82.0% ) contained 5 repeats. Isolates from humans are mainly contained five repeats (100%) and animals posed 70%。4. The phylogenetic tree was built by blast our STs with other databases for the population genetic analyses of isolates. The ST836 may be the ancestor of these 105 strains, which was related with the ST66 from Sakai (Japan,1996), a genotype from the ST69 (EDL933, 1982). It infers that the E.coli O157:H7 caused outbreak in the USA(EDL933) have gene transferred, such as DNA insertion/deletion and recombination, and may lead to the outbreak in Japan (Sakai,1996), which caused the after outbreaks in China in 1999.5. Diversity of STs with different isolated hosts: The most common STs in the animals was ST837, followed by ST836. Four strains including 3 specimens from humans were the ST841 andT the ST842 were only in the human specimen, although the human species were small.Conclusion:This report provides more insight into the molecular epidemiological characteristic and could provide systematic data on the population genetic evolution of E. coli O157:H7 in China. The establishment of a good and reliable platform for epidemiological analysis will be invaluable for further investigations of E. coli O157:H7 infections and prevention, particularly in the investigation of the special class of STs being liable to appear in humans other than from food, animals and the environment.