乙酰丙酸与十二烷基硫酸钠协同抑制金黄色葡萄球菌生物膜机理研究

Biofilm formation of Staphylococcus aureus is a high risk to food hygiene and safety since it not only releases daughter cells to the surroundings continuously contaminating environment but has high resistance to commonly used sanitizers. The emergence of drug resistant strains, especially, raises the concern. In recent years, combination of levulinic acid (LVA) and sodium dodecyl sulfate (SDS) has shown considerable promise as synergism between the two compounds is observed in the case of their antimicrobial efficacy targeting both planktonic cells and biofilms. Levulinic acid has been designated as Generally Recognized as Safe (GRAS) by the United States Food and Drug Administration, and SDS is a multipurpose food additive. Therefore，this study plans to firstly determine the antimicrobial mechanism of the combination on Staphylococcus aureus biofilms, then find the different gene expression and protein profiles of Staphylococcus aureus at different stages of biofilms treated with LVA + SDS through transcriptomic and proteomic approaches. The functional role of key target gene will be identified, which will contribute to understanding the antimicrobial mechanism of LVA + SDS on the Staphylococcus aureus cells in biofilms. Finally, Real-time dynamics of the action of the biocide in the biofilms will be identified and its antibiofilm model will be calculated based on the predictive food microbiology software. These results will provide a theoretical basis for develop a more potent method to mitigate the impact of biofilms.

金黄色葡萄球菌生物膜的形成对食品卫生与安全是巨大的威胁，因为其不仅可以释放子细菌进入周围环境造成持续污染而且对传统消毒液具有很高的抗性。耐药菌株的出现加重了公共卫生担忧。近年来发现的乙酰丙酸（LVA）与十二烷基硫酸钠（SDS）混合液对生物膜的消除效果良好。乙酰丙酸被美国食品药品监督管理局认定为GRAS安全原料，而十二烷基硫酸钠是合法的食品添加剂。因此，本课题拟以金黄色葡萄球菌为研究对象，从确定LVA+SDS对金黄色葡萄球菌生物膜的抑制能力入手，通过转录组学和蛋白组学分析技术研究生物膜中金黄色葡萄球菌在不同生长阶段被LVA+SDS处理后参与表达的差异基因和蛋白，并根据关键差异基因在突变体中的差异表达明确LVA+SDS抑制生物膜中金黄色葡萄球菌的机理。最后采用实时观测两种成分在生物膜结构中的动态来构建其协同抑制金黄色葡萄球菌生物膜的模型。研究结果将为全面有效的抑制生物膜奠定基础。

本研究为揭示乙酰丙酸（LVA）与十二烷基硫酸钠（SDS）协同杀菌机制而开展了以下工作：首先，以金黄色葡萄球菌悬浮态及其生物膜为对象，通过时间-致死曲线明确了两者之间的协同杀菌作用。其次，通过微观实时观测技术发现，两者联用能加强其生物膜内渗透作用，不仅能除去生物膜基质（EPS），还能有效杀死生物膜内金葡菌；两者作用的最佳时间在施加消毒液后70 s内，后随着时间延长杀灭效果趋缓。再次，利用转录组与蛋白组学技术对基因表达情况进行分析，两者联用引起转录翻译水平显著变化的基因主要涉及参与金黄色葡萄球菌生物膜形成和毒力的基因，包括表面蛋白（如clfA、clfB）、粘附素（如MSCRAMMs家族）、蛋白酶（如sceD, mgt）、荚膜多糖（cap）、转录调节因子（如sarA）等。本项目采用微观形态学与分子生物学技术揭示了LVA与SDS协同杀菌作用的机理，为研发新一代消毒液提供了理论基础。本研究发表SCI文章一篇，投稿一篇，另一篇还在撰写中。培养学生4名，完成预期目标。