NAKAYAMA Keisuke Miyazaki Medical College, Faculty Of Medicine, Research Associate, 医学部, 助手 (10347057)
OHNISHI Makoto Miyazaki Medical College, Faculty of Medicine, Associate Professor, 医学部, 助教授 (10233214)
|Budget Amount *help
¥12,500,000 (Direct Cost: ¥12,500,000)
Fiscal Year 2002: ¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2001: ¥8,800,000 (Direct Cost: ¥8,800,000)
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.