Objective: To investigate the potential role of wild birds as fecal spreaders of enteropathogenic,enterohemorrhagic and Shiga-toxins producing Escherichia coli(E. coli),enteropathogenic E. coli,enterohemorrhagic E. co...Objective: To investigate the potential role of wild birds as fecal spreaders of enteropathogenic,enterohemorrhagic and Shiga-toxins producing Escherichia coli(E. coli),enteropathogenic E. coli,enterohemorrhagic E. coli and Shiga toxin-producing E. coli strains. Methods: Fecal samples collected from 121 wild birds of different orders and species were submitted to molecular analyses. In particular,eaeA encoding intimin,hlyA encoding for hemolysin,stx1 and stx2 genes encoding Shiga-toxins 1 and 2,respectively,were investigated. Results: Overall,21(17.35%) fecal samples resulted positive for at least one of the investigated genes. In detail,12(9.91%) samples were positive for eaeA,10(8.26%) for stx1,4(3.31%) for hylA and 1(0.83%) for stx2. An owl(Athene noctua) positive for the four investigated genes suggesting that it harbored a STEC strain. However,virulence genes characterizing EPEC,and EHEC strains were mainly found among seagulls,waterfowl and feral pigeons. Conclusions: Seagulls,waterfowl and feral pigeons,which frequently reach and contaminate rural,urban and peri-urban areas with their droppings,may be important sources of E. coli infection for other animals and humans.展开更多
Escherichia coli O157:H7 is a human pathogen that was first identified from a foodborne outbreak in 1982, and in the 25 years that followed, many new strains were identified and emerged in numerous outbreaks of human ...Escherichia coli O157:H7 is a human pathogen that was first identified from a foodborne outbreak in 1982, and in the 25 years that followed, many new strains were identified and emerged in numerous outbreaks of human disease. Extensive research has been conducted to identify virulence factor genes involved in the pathogenesis of E. coli O157:H7 and many genome sequences of E. coli O157:H7 strains have become available to the scientific community. Here, we provide a comprehensive overview of the research that has been conducted over the first 25 years to identify 394 known or putative virulence factor genes present in the genomes of E. coli O157:H7 strains. Finally, an examination of the conservation of these 394 virulence factor genes across additional genomes of E. coli O157:H7 is provided which summarizes the first 25 years and 13 genomes of this human pathogen.展开更多
目的:构建对肠上皮细胞粘附缺陷的肠出血性大肠埃希菌O157∶H7(EHEC O157)突变株,为体内、体外研究其粘附机制提供可用工具。方法:将pSC101和m in i-Tn5Km2分别通过转化和结合导入O157Sakai菌株中,筛选粘附力缺陷的转化子感染Caco-2细胞...目的:构建对肠上皮细胞粘附缺陷的肠出血性大肠埃希菌O157∶H7(EHEC O157)突变株,为体内、体外研究其粘附机制提供可用工具。方法:将pSC101和m in i-Tn5Km2分别通过转化和结合导入O157Sakai菌株中,筛选粘附力缺陷的转化子感染Caco-2细胞株,观察Caco-2单层细胞中的微菌落数量和m in i-Tn5Km2插入位点。结果:经诱变,共分离到3组粘附缺陷突变株,1组完全丧失了粘附力,2组粘附力减弱,3组的粘附力更弱。1组和3组拥有LEE岛上多基因的转座子插入,而2组m in i-Tn5Km2的插入位点在LEE岛之外。结论:O157Sakai的肠道粘附能力可能与LEE岛的III型分泌系统有关。展开更多
To construct and express the fusion protein Stx2B-IntiminC300 of EHEC 0157 :H7, and to further investigate its immunoprophyiactic potential, the gene of Stx2B (stx2b) from EHEC 0157:H7 chromosome was cloned into pMD18...To construct and express the fusion protein Stx2B-IntiminC300 of EHEC 0157 :H7, and to further investigate its immunoprophyiactic potential, the gene of Stx2B (stx2b) from EHEC 0157:H7 chromosome was cloned into pMD18-T vector. Thereafter, the amplified gene was cloned into prokaryotic expression plasmid pET-28a (+)-eaeC300, which was constructed previously. The recombinant pasmid pET-28a(+)-stx2b-eaeC300 was transformed into E. coli BL21(DE3). After inducement, the protein Stx2B-IntiminC300 was successfully expressed and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and N-terminal amino acid residual sequencing. To evaluate its immunoprophyiactic potential, it was primarily purified by ion-exchange chromatography and injected into 30 BALB/c mice with Al(OH)3 in the subscapular region. Ten days after the last booster vaccination, 20 mice were attacked with EHEC 0157: H7 lysate and the protective efficacy was observed. In the present study, the gene of Stx2B-IntiminC300 was successfully cloned into pET-28a (+) vector. The results of SDS-PAGE and Western blotting assay showed that the fusion protein was successfully expressed in the inclusion body form, accounting for 25% of total expression products, and its molecular weight was about 43 kDa. The result of the N-terminal amino acid residual sequencing showed that it was identical to that of the molecular designed. The purity was about 75 % after primary purification. Animal tests revealed that the fusion protein Stx2B-IntiminC300 has elicited high titer of protective antibody relatively. These results demonstrate that the fusion protein Stx2B-IntiminC300 is successfully expressed in prokaryotic expression system and shows certain immunoprophyiactic potential.展开更多
文摘Objective: To investigate the potential role of wild birds as fecal spreaders of enteropathogenic,enterohemorrhagic and Shiga-toxins producing Escherichia coli(E. coli),enteropathogenic E. coli,enterohemorrhagic E. coli and Shiga toxin-producing E. coli strains. Methods: Fecal samples collected from 121 wild birds of different orders and species were submitted to molecular analyses. In particular,eaeA encoding intimin,hlyA encoding for hemolysin,stx1 and stx2 genes encoding Shiga-toxins 1 and 2,respectively,were investigated. Results: Overall,21(17.35%) fecal samples resulted positive for at least one of the investigated genes. In detail,12(9.91%) samples were positive for eaeA,10(8.26%) for stx1,4(3.31%) for hylA and 1(0.83%) for stx2. An owl(Athene noctua) positive for the four investigated genes suggesting that it harbored a STEC strain. However,virulence genes characterizing EPEC,and EHEC strains were mainly found among seagulls,waterfowl and feral pigeons. Conclusions: Seagulls,waterfowl and feral pigeons,which frequently reach and contaminate rural,urban and peri-urban areas with their droppings,may be important sources of E. coli infection for other animals and humans.
文摘Escherichia coli O157:H7 is a human pathogen that was first identified from a foodborne outbreak in 1982, and in the 25 years that followed, many new strains were identified and emerged in numerous outbreaks of human disease. Extensive research has been conducted to identify virulence factor genes involved in the pathogenesis of E. coli O157:H7 and many genome sequences of E. coli O157:H7 strains have become available to the scientific community. Here, we provide a comprehensive overview of the research that has been conducted over the first 25 years to identify 394 known or putative virulence factor genes present in the genomes of E. coli O157:H7 strains. Finally, an examination of the conservation of these 394 virulence factor genes across additional genomes of E. coli O157:H7 is provided which summarizes the first 25 years and 13 genomes of this human pathogen.
文摘目的:构建对肠上皮细胞粘附缺陷的肠出血性大肠埃希菌O157∶H7(EHEC O157)突变株,为体内、体外研究其粘附机制提供可用工具。方法:将pSC101和m in i-Tn5Km2分别通过转化和结合导入O157Sakai菌株中,筛选粘附力缺陷的转化子感染Caco-2细胞株,观察Caco-2单层细胞中的微菌落数量和m in i-Tn5Km2插入位点。结果:经诱变,共分离到3组粘附缺陷突变株,1组完全丧失了粘附力,2组粘附力减弱,3组的粘附力更弱。1组和3组拥有LEE岛上多基因的转座子插入,而2组m in i-Tn5Km2的插入位点在LEE岛之外。结论:O157Sakai的肠道粘附能力可能与LEE岛的III型分泌系统有关。
文摘To construct and express the fusion protein Stx2B-IntiminC300 of EHEC 0157 :H7, and to further investigate its immunoprophyiactic potential, the gene of Stx2B (stx2b) from EHEC 0157:H7 chromosome was cloned into pMD18-T vector. Thereafter, the amplified gene was cloned into prokaryotic expression plasmid pET-28a (+)-eaeC300, which was constructed previously. The recombinant pasmid pET-28a(+)-stx2b-eaeC300 was transformed into E. coli BL21(DE3). After inducement, the protein Stx2B-IntiminC300 was successfully expressed and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and N-terminal amino acid residual sequencing. To evaluate its immunoprophyiactic potential, it was primarily purified by ion-exchange chromatography and injected into 30 BALB/c mice with Al(OH)3 in the subscapular region. Ten days after the last booster vaccination, 20 mice were attacked with EHEC 0157: H7 lysate and the protective efficacy was observed. In the present study, the gene of Stx2B-IntiminC300 was successfully cloned into pET-28a (+) vector. The results of SDS-PAGE and Western blotting assay showed that the fusion protein was successfully expressed in the inclusion body form, accounting for 25% of total expression products, and its molecular weight was about 43 kDa. The result of the N-terminal amino acid residual sequencing showed that it was identical to that of the molecular designed. The purity was about 75 % after primary purification. Animal tests revealed that the fusion protein Stx2B-IntiminC300 has elicited high titer of protective antibody relatively. These results demonstrate that the fusion protein Stx2B-IntiminC300 is successfully expressed in prokaryotic expression system and shows certain immunoprophyiactic potential.
