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

比较基因组分析

基本信息

批准号：
13470061
负责人：
HAYASHI Tetsuya
金额：
$ 8万
依托单位：
Miyazaki Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470061/
关键词：
enterohemorrhagic Escherichia coli O157 genome diversity bacteriophage PCR strain-discrimination system

项目摘要

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.

该项目的目的是揭示大肠杆菌 O157 的基因组多样性，并开发一种基于基因组多样性的 O157 感染分子流行病学研究的新工具。我们首先选择了 1796 年以来表现出不同 Xbal 消化模式的 8 个 O157 菌株1998年在日本分离出菌株。接下来，根据O157 Sakai的基因组序列，我们制备了一套PCR引物来扩增560个DNA片段覆盖了O157的整个基因组（片段的平均长度约为10 Kb）。通过长PCR和引物组，我们以Sakai菌株为参考，分析了8个O157菌株的全基因组结构。通过这种我们命名为全基因组 PCR 扫描 (WGPS) 的分析，我们可以识别具有任何结构多态性的所有基因组区域，这表明 WGPS 是一种用于对密切相关的基因组进行比较分析的强大技术。数据还表明 O157 菌株之间存在出人意料的高度基因组多样性。特别是，包括Stx转导噬菌体在内的原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个基因组区域，这些区域表现出可通过常规PCR检测到的小尺寸结构多态性。选择其中9个目标区域，构建多重PCR体系。由于stx1、stx2和eae基因也作为靶标，该系统最终由12个PCR反应组成。在我们的初步检查中，该系统将51株菌株分为21个类型，表明该系统可以开发为流行病学研究的有用工具，但仍需要一些改进。此外，我们对一些O157基因进行了比较和功能分析，并发现脲酶基因特异性分布于肠出血性大肠杆菌菌株，并且toxB基因参与上皮细胞粘附。