Comparative genome analysis & enterohemorrhagic Escherichia coli O157 and its clinical application.

本研究首先从1998年日本分离的1796株大肠杆菌O157中筛选出8株XbaI酶切图谱不同的O157菌株，并对其基因组多样性进行了分析，为O157感染的分子流行病学研究提供新的工具。其次，根据O157酒井的基因组序列，我们制备了一组PCR引物，扩增了覆盖O157全基因组的560个DNA片段（片段的平均长度约为10 Kb）。利用长链PCR技术和引物组，以日本酒井株为参照，对8株O157菌株的全基因组结构进行了分析。通过这种我们称之为全基因组PCR扫描（Whole Genome PCR Scanning，WGPS）的分析，我们可以识别所有具有结构多态性的基因组区域，这表明WGPS是一种强大的技术，用于密切相关基因组的比较分析。这些数据还表明，O157菌株之间存在着出乎意料的高度基因组多样性。通过WGPS分析，还鉴定出约30个基因组区域，这些基因组区域表现出可通过常规PCR检测到的小尺寸结构多态性。从其中选取9个靶区，构建了多重PCR体系。由于stx1、stx2和eae基因也被包括作为靶标，因此该系统最终由12个PCR反应组成。初步鉴定结果表明，该系统可将51株肠出血性大肠杆菌分为21型，为流行病学研究提供了一个有用的工具，但仍需进一步完善.此外，我们还对部分O157基因进行了比较和功能分析，发现尿素酶基因在肠出血性大肠杆菌中具有特异性分布.大肠杆菌，和toxB基因参与上皮细胞粘附。

The aims of the project were to reveal the genomic diversity of Escherichia coli O157, and to develop a new tool for molecular epidemiological studies of O157 infection based on the genomic diversity.We first selected eight O157 strains that exhibited diverse Xbal-digestion patterns from 1796 strains isolated in Japan in 1998. Next, based on the genome sequence of O157 Sakai, we prepared a set of PCR primers to amplify 560 DNA segments covering the whole genome of O157 (the average length of segments was about 10 Kb). By using long PCR and the primer set, we analyzed the whole genome structures of the eight O157 strains using the Sakai strain as a reference. By this analysis which we named Whole Genome PCR Scanning (WGPS), we could identify all the genomic regions with any structural polymorphism, indicating that WGPS is a powerful technique for the comparative analysis of closely related genomes. The data also indicate that an unexpectedly high degree of genomic diversity exists among O157 strains. In particular, prophage regions, including Stx-transducing phages, exhibited extensive diversities.By the WGPS analysis, about 30 genomic regions exhibiting small-size structural polymorphisms detectable by conventional PCR were also identified. Selecting 9 target regions among them, we constructed a multiplex PCR system. Since the stx1, stx2 and eae genes were also included as targets, the system was finally composed of 12 PCR reactions. In our preliminary examination, this system classified 51 strains into 21 types, suggesting that the system could be developed as a useful tool for the epidemiological studies, but some improvement is still required.In addition, we performed comparative and functional analyses of some O157 genes, and found that urease genes are specifically distributed to strains belonging to enterohemorrhagic E. coli, and that the toxB gene is involved in epithelial cell adherence.