Indentification Of An Attenuated Enterohemorrhagic Escherichia Coli O157:H7 Strain

Enterohemorrhagic Escherichia coli serotype O157:H7 is a food-borne pathogen. It resides in gastrointestinal tract of ruminant and causes infection of human and animals through contaminant food and water. The spectrum of illnesses associated with EHEC O157:H7 infection include diarrhea, hemorrhagic colitis and severe complications such as hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. The outbreak of EHEC O157:H7 have been reported in many countries including China, which gathered global concerns extensively.In china, a case of EHEC O157:H7 infection was first reported in 1986. After which a continuing number of outbreak of infection due to O157:H7 have been reported in china. The first outbreak of O157:H7 occurred in Xuzhou city, Jiangsu province in 1999. An outbreak of O157:H7 infection occurred in Jiangsu and Anhui province in 2000, which led to 20,000 peoples infected and 177 patients died. In addition, the strains of O157:H7 have also been detected in many samples including feces of domestic animals and food productions. Considering the potential risk of O157:H7 outbreak, which caused the public health authorities have paid more attention to it.In this study we identified a strain of EHEC O157:H7 isolated from clinical patient by Jiangsu provincial center for disease control and prevention in 2000. (Code: 00B015)Methods and results:1. 00B015 was identified as a strain of EHEC O157:H7 through the experiments including the CT-SMAC agar plate culture, serological and biochemical tests.2. Balb/c mouse was selected as a model for EHEC O157:H7 infection. In brief, Balb/c mice were orally challenged with 1×1010 CFU of bacteria (00B015 and Sakai). Then the number of survivals was recorded daily. Meanwhile, to visualize the histopathological manifestations of colon and kidney in mice, Section and hematoxylin and eosin (HE) staining were carried out as usual, and the image were got by light microscope at designated magnification. The results showed that all mice inoculated with EHEC O157:H7 developed clinic symptoms after 2 days. During the 7-day observation period all mice died in group challenged with Sakai, but all mice survived in 00B015 group. In addition, the pathological lesions of colon and kidney in mice challenged with Sakai were more obvious than that in mice challenged with 00B015. These findings suggested that the 00B015 was virulence-attenuate and unable to cause death to mice potently.3. To detect the hemolysin and shiga toxins with defibered sheep blood agar and immune colloidal gold technique individually. Study showed that 00B015 can produce hemolysin and shiga toxin 24. HeLa cells were infected with EHEC O157:H7 Sakai and EHEC O157:H7 individually, and visualized by Giemsa staining and fluorescence actin staining (FAS). Then the numbers of microcolonies and actin polymerizations formed on the cells were counted and caculated. Results showed that the ability of 00B015 to attach to HeLa cells was similar to that of Sakai (P>0.05). However, Sakai induced stronger actin polymerizaitons in host cells than 00B015 (P<0.05).5. The main virulent gene related to A/E lesions were detected by PCR, results showed there is no mutation in genes detected except the tccp. Agarose gel electrophoresis showed that tccp of 00B015 is smaller than that of Sakai, alignment of amino acid sequence showed that the TCCP of 00B015 was nearly identical to that of Sakai, except for the missing of two PRRs.The interaction of pathogens and host play a key role in pathogenicity. The production of Shiga toxins (Stx) and the induction of attaching and effacing (A/E) lesions on intestinal epitheliums are the two most crucial elements of EHEC to trigger infectious diseases.Conclusions:1. 00B015 is an attenuated strain of EHEC O157:H7.2. This study showed that the degree of A/E lesions caused by 00B015 is weaker than Sakai, which induced attenuated virulence of 00B015.3. This work to identify the attenuated strain of EHEC O157:H7-00B015 has enormous potential significance for the study of pathogenic mechanism and vaccine exploration associated with O157:H7.In summary, Our results show that the virulence of EHEC O157:H7 is severely sensitive to its ability to induce A/E lesions, which suggested that A/E lesion plays a more central role in the virulence of EHEC O157:H7. This work contributes to further study on EHEC O157:H7 including epidemiology, pathogenic mechanism and vaccine exploration.