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Molecular analysis of anteliotic-tolerance and stress response in adherent bacteria

粘附细菌抗逆耐受性和应激反应的分子分析

基本信息

批准号：
17591914
负责人：
ONO Tsuneko
金额：
$ 2.31万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17591914/
关键词：
antibiotic tolerance stress response rpoS adherent bacteria ppGppc rpoN pvdS vqsR quorum sensing 遺伝子発現 pyoverdine pvdS vqsR tolerance 抗菌薬 tcp gene Pseudomonas aeruginosa

项目摘要

In this project, we have studied about the molecular mechanism of antibiotic tolerance and stress response in adherent cells The following results werte obtained in this study.1. To assess the contribution of ppGpp in antibiotic tolerance to quinolone in Pseudomonas aeruginosa, knockout mutants of relA, spoT and dksA, were constructed and investigated for their antibiotic susceptibility to quinolones. The survival of the dksA and spoT mutants in the presence of ofloxacin and ciprofloxacin were shown to be approximately 20-180 and 10-40 times respectively, higher than the same for the wild type strain.The data suggest that elevated basal levels of ppGpp may be responsible for rendering these mutants tolerant to quinolones and expand the importance of ppGpp as an antimicrobial target in P. aeruginosa2. An rpoN mutant in Pseudomonas aeruginosa has been constructed and investigated its importance as a target for antibiotics. The stationary phase cells of the rpoN mutant displayed an approximately 15 times higher survival rate to quinolones and carbapenems than the wild-type cells. Real-time PCR revealed that in contrast to the wild-type strain, the stationary phase cells of the rpoN mutant grown without antibiotics had approximately 4-140 and 2-14-fold higher transcripts of pvdS and vqsR genes, respectively, than the wild type strain. In the presence of antibiotics, pvdS and vqsR transcripts were 400 and 5-fold, respectively, elevated in comparison to the wild-type. Flow cytometry assays using GFP reporter demonstrated increased expression of the vqsR gene in the rpoN mutant. A pvdS mutant of P. aeruginosa, deficient in pyoverdine production, was shown to be susceptible to biapenem.These findings suggest that rpoN is involved in the tolerance to antimicrobial agents in P. aeruginosa and its tolerant effect is partly dependent on increased pyoverdine production and vqsR gene expression.

本课题对贴壁细胞抗生素耐受和应激反应的分子机制进行了研究，取得了以下结果.为了评估ppGpp在铜绿假单胞菌对喹诺酮类抗生素耐药性中的作用，构建了relA、spoT和dksA的敲除突变体，并研究了它们对喹诺酮类抗生素的敏感性。dksA和spoT突变体在氧氟沙星和环丙沙星存在下的存活率分别约为野生型菌株的20-180倍和10-40倍，数据表明ppGpp基础水平的升高可能是导致这些突变体对喹诺酮类药物耐受的原因，并扩大了ppGpp作为铜绿假单胞菌中抗微生物靶标的重要性2。已经构建了铜绿假单胞菌中的rpoN突变体，并研究了其作为抗生素靶标的重要性。rpoN突变体的稳定期细胞对喹诺酮类和碳青霉烯类的存活率比野生型细胞高约15倍。实时PCR显示，与野生型菌株相比，在没有抗生素的情况下生长的rpoN突变体的稳定期细胞分别具有比野生型菌株高约4-140倍和2-14倍的pvdS和vqsR基因转录物。在抗生素的存在下，pvdS和vqsR转录分别为400和5倍，相比野生型升高。使用GFP报告基因的流式细胞术测定证明了rpoN突变体中vqsR基因的表达增加。铜绿假单胞菌pvdS突变株对比阿培南敏感，提示rpoN参与了铜绿假单胞菌对抗菌药物的耐受，其耐受效应部分依赖于绿脓菌荧光素的产生和vqsR基因表达的增加。