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Development of a new epidemiological method using patterns of exoproteins secreted from bacteria

利用细菌分泌的外蛋白模式开发新的流行病学方法

基本信息

批准号：
12557027
负责人：
OHTA Michio
金额：
$ 7.87万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

Discrimination of each strain of bacteria such as Staphylococcus aureus and group A streptococcus (GAS) is helpful for tracing a pathogenic strain and prevention of nosocomial infections. Although DNA-based typings have been generally used as epidemiological methods for discrimination, we developed two dimensional electrophoretic (2-DE) analysis of exoproteins secreted from bacteria as a new epidemiological method and we found that this method was well applicable for the discrimination of S.aureus and GAS strains. The conditions of culture, growth and extraction of exoproteins were identified to have reproducible results. The major results of this study were as follows ; the best condition was that exoproteins were extracted from early stationary culture with TCA and were quickly applied on 2-DE ; high degree of variety was observed in the 2-DE patterns of exoproteins in S.aureus and GAS ; spots of more than 100 exoproteins of S.aureus and 20 of GAS were identified by the determination of N-terminal amino acid sequences ; a specific spot pattern was identified for each coagulase type of MRSA and variation of the spots of super antigens were observed among the strains belonging to the same coagulase type ; a correlation was observed between the spot patterns and M antigen types in GAS strains ; the MRSA strains that caused NTED and SSSS showed unique toxin production patterns, respectively, observed by 2-DE ; we have accumulated 2-DE patterns of more number of strains that would be helpful for the construction of a 2-DE database of exoproteins.

区分金黄色葡萄球菌和A组链球菌(GAS)等细菌，有助于追踪病原菌和预防医院感染。虽然DNA分型已被普遍用作流行病学的鉴别方法，但我们发展了一种新的流行病学方法--细菌分泌外源蛋白的双向电泳法(2-DE)，并发现该方法非常适用于金黄色葡萄球菌和GAS菌株的鉴别。外源蛋白的培养、生长和提取条件经鉴定具有重复性。本研究的主要结果如下：最佳条件是用TCA从早期静止培养物中提取外源蛋白并快速应用于2-DE，金黄色葡萄球菌和GAS外源蛋白的2-DE图谱具有高度的多样性，通过N端氨基酸序列的测定鉴定出100多个金黄色葡萄球菌和20个GAS外源蛋白的斑点，MRSA的每种凝固酶类型都有一种特异的斑点图谱，属于同一凝固酶型的菌株之间存在超级抗原斑点的变异，GAS菌株的斑点图谱与M抗原类型存在相关性，GAS菌株的斑点图谱与M抗原类型存在相关性；用2-DE观察到引起NTT和SSSS的MRSA菌株分别表现出独特的产毒图谱；我们积累了更多菌株的2-DE图谱，这将有助于外源蛋白双向凝胶电泳数据库的构建。