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

本项目旨在揭示大肠杆菌O157的基因组多样性，并开发基于基因组多样性的O157感染分子流行病学研究新工具。首先从1998年日本分离的1796株O157菌株中筛选出8株具有不同xbal消化模式的O157菌株。接下来，我们根据O157 Sakai的基因组序列，制备了一套PCR引物，扩增了覆盖O157全基因组的560个DNA片段（片段平均长度约为10 Kb）。以Sakai菌株为对照，采用长链PCR和引物对8株O157进行全基因组结构分析。通过全基因组PCR扫描（Whole Genome PCR Scanning， WGPS），我们可以识别出所有具有任何结构多态性的基因组区域，这表明WGPS是一种强大的近缘基因组比较分析技术。数据还表明，在O157菌株中存在出乎意料的高度基因组多样性。特别是，包括stx转导噬菌体在内的前噬菌体区域表现出广泛的多样性。通过WGPS分析，还鉴定了约30个通过常规PCR检测到的具有小尺寸结构多态性的基因组区域。选取其中的9个靶区，构建多重PCR体系。由于stx1， stx2和eae基因也被纳入靶标，因此该体系最终由12个PCR反应组成。初步试验结果表明，该系统可将51株菌株划分为21种类型，为流行病学研究提供了一种有用的工具，但仍需进一步改进。此外，我们对部分O157基因进行了比较和功能分析，发现脲酶基因特异性分布于肠出血性大肠杆菌菌株中，并且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.