| Enterohemorrhagic Escherichia coli(EHEC) is a subset of pathogenic Escherichia coli that can cause diarrhea or hemorrhagic colitis in humans. E. coli O157︰H7 is the most common serotype. E. coli O157︰H7 was first recognized as an important zoonotic enteric pathogen in 1982 in the United States, and since then, it has been isolated in many countries around the world, most frequently in the United States, Netherlands, Canada, Japan, and the United Kingdom. In China, there are more than ten provinces that have been detected the pathogens in food, poultry, insects and diarrhea patients. The disease has become one of the important public health problems in the world. In this paper, multiplex PCR method was established, and used to detect the animal-derived E. coli O157︰H7 in Henan Province. Moreover, the pathogenic mechanism of E. coli O157︰H7 was investigated too.1.Two pairs of primers were designed according to the rfb E and fli C genes of E. coli O157︰H7. We performed the multiplex PCR in one amplificated system. The system was optimized and the specificity and sensitivity of this system were evaluated, it was used to detect the clinical isolates. The result showed that the multiplex PCR method was successful to amplify the 327 bp and 247 bp fragments of rfb E and fli C gene respectively. Sensitivity of the method was 102 cfu/m L of bacteria samples. A rapid, specific, and sensitive multiplex PCR method for the E. coli O157︰H7 has been established.2. In order to investigate the prevalenc of E. coli O157︰H7 that existed in animals and its food in Henan, 1460 samples of animal manure, meat and their products were collected. The established multiplex PCR method was used to detect E. coli O157 ︰ H7, and the biological characteristics, virulence genes and drug resistances of the animal-derived isolates were analyzed. The results showed that 34(2.3%) E. coli O157︰H7 were isolated. The detection rate of intimin(eae A) and hemolysin(hyl A) gene were 100%, Shiga toxin(Stx1and Stx2) was 14.7% and 52.9% respectively. The results of drug resistance showed that the isolates were mainly sensitive to quinological and chloramphenicol drugs. The isolates displayed high-level resistance to tetracyclines, with 70.6% to doxycycline and 76.5 % to tetracycline. Our results complemented the epidemiological data of E. coli O157︰H7, which laid the foundation of effective monitoring about E. coli O157︰H7 in Henan province.3. In order to study the pathogenesis of E. coli O157︰H7,the IPEC-J2 cells was used as the experimental model in vitro, animal-derived E. coli O157︰H7 was infected the cell line, then the bacterial adhesion ability, the cytokine expression, and cell apoptosis were analysed by using Host33258 and Annexin V-FITC/PI staining method, real-time PCR after E. coli O157︰H7 infecting IPEC-J2 cells. The results showed that the bacteria could adhere to IPEC-J2 cells and the adhesion rate was 30.04%. The cell apoptosis happened after half an hour of the infection, the total rate of apoptosis increased with time lasted. The infection of E. coli O157︰H7 could increase the expression of inflammatory cytokines of IL-1β, IL-12(P35), IL-12(P40), IL-6, IL-17, and IL-18 significantly, but the expressive level of IL- 13 was lower after infection. And the infection could up-regulate the expression of apoptosis related cytokines of TNF-α、TNFR2、Bcl-2、Bcl-xl、Fas L、p53、Caspase-8 and PBR in the m RNA level. These results laid the foundation for study of the pathogenic mechanism of the E. coli O157︰H7 in the further. |
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