亚抑菌浓度莫匹罗星增强金黄色葡萄球菌生物膜形成机制的研究

Biofilm formation on Staphylococcus aureus (S.aureus) can causing life-threatening infections, while antibiotic were poorly controlled. In our previous study, biofilm formation were measured in 96-well microtiter plates using a biofilm assay and observed by confocal laser scanning microscopy. Compared with USA300, sub-MICs of mupirocin can significantly induce biofilm in S. aureus. And mechanism of biofilm formation was Polysaccharide intercellular adhesion-independent. The RNAs of USA300 and sub-MICs mupirocin USA300 were sequenced. Compared with USA300, the expression level of cidA of differentially expressed genes in sub-MICs mupirocin USA300 was significantly increased. Therefore, we speculate that sub-MICs mupirocin may enhance biofilm formation of S.aureus by cidA affecting released of extracellular DNA. To corroborate our speculation, we will construct USA300 ΔcidA and complementary strain of USA300 ΔcidA with plasmid containing cidA. Using quantitative experiment, initial adhesion and aggregation experimentsthe, in vitro static and dynamic biofilm models and in vivo catheter-associated infections to investigate sub-MICs of mupirocin whether increased biofilm formation of S. aureus through cidA enhanced eDNA. Based on the data of RNA sequencing obtained from previous study, the mechanism of cidA expression will be further studied. The findings of present study will be helpful for providing a basis for guiding the clinical avoidance of insufficient use of antibiotics to increase biofilm formation.

金黄色葡萄球菌（金葡菌）形成生物膜引起的感染威胁生命，而抗菌药物控制不佳。我们前期研究利用结晶紫半定量实验和激光共聚焦扫描电镜观察，发现与金葡菌USA300相比，亚抑菌浓度莫匹罗星处理组的生物膜形成能力明显增强，进一步研究发现生物膜形成增强的机制并非是胞间多糖黏附素（PIA）依赖途径；前期转录组数据分析发现，亚抑菌浓度莫匹罗星处理组差异表达基因的cidA表达量明显增强。我们推测亚抑菌浓度莫匹罗星可能通过cidA影响胞外游离DNA的释放而增强金葡菌生物膜的形成。为了验证该假设，拟构建cidA阴性突变株和回复株，利用定量实验、初始粘附和聚集实验、体外静态和动态生物膜模型及体内导管相关感染的小鼠模型研究亚抑菌浓度莫匹罗星是否可以通过cidA增加eDNA的释放而增强金葡菌生物膜的形成；挖掘前期转录组数据研究cidA基因的表达调控机制。为指导临床避免抗生素用量不足反而导致生物膜形成增强。

金黄色葡萄球菌形成的生物膜引起的感染严重威胁到生命。特别是亚抑菌浓度药物的使用还可能诱导生物膜的形成，但是其机制尚不清楚。.首先构建cidA基因阴性突变株和回补株，通过小鼠导管感染模型，在亚抑菌浓度莫匹罗星作用下，cidA基因阴性突变株的细菌数量显著低于野生株，提示亚抑菌浓度莫匹罗星cidA增强金黄色葡萄球菌生物膜的形成。检测eDNA的释放量以及自溶能力，提示亚抑菌浓度莫匹罗星cidA通过胞外游离DNA的释放增强金黄色葡萄球菌生物膜的形成。此外通过RT-PCR研究agrA和sarA对cidA的调控作用，提示cidA可能通过agr的调控影响金黄色葡萄球菌在亚抑菌浓度莫匹罗星的作用下增强生物膜的形成。通过EMSA研究CidA对自身的调控作用，结果发现CidA与cidA启动子不结合，表明对自身无调控作用。.我们还对莫匹罗星耐药的MRSA检测分子流行特点，发现高水平耐药的MRSA最常见的流行克隆株是ST764-II-t1084。