姜黄素抑制阪崎肠杆菌生物被膜形成及作用机制研究

Enterobacter sakazakii is an opportunistic foodborne pathogen that contaminates powdered infant formula, causing a rare but life-threatening form of neonatal meningitis, bacteremia, and necrotizing enterocolitis. Biofilm formation of E. sakazakii enhances the resistance of cells to environmental stress and provides protection against a variety of environmental stress such as antibiotics, disinfectants, osmotic stress, heat and starvation, thus increasing the risk of human infections. Recent studies concerning E. sakazakii biofilm formation focused on the ability to produce biofilm in food-processing environments and contaminated food products. However, in general, little information is available on the inhibition of E. sakazakii biofilm formation. In our previous work, inhibitory activity of curcumin (a plant-derived natura product) on the biofilm formation of E. sakazakii was preliminary confirmed. The overall goals of this study are to investigate the effect and mechanism of curcumin against biofilm formation of E. sakazakii. To achieve these goals, the following aims are outlined: Aims 1. Investigate the efficacy of curcumin for inhibition biofilm of E. sakazakii using the microtiter plate assay. Aims 2. Observe the biofilm structure of E. sakazakii under scanning electronic microscopy (SEM) and confocal laser scanning microscope (CLSM) when treated with curcumin. Aims 3. Figure out the mechanism of curcumin against biofilm formation of E. sakazakii using real-time PCR (qPCR）and differential proteomics technologies. These efforts will be helpful for the application of curcumin on the inhibition of E. sakazakii biofilm formation in food industries.

阪崎肠杆菌(E. sakazakii)是一种能通过污染婴幼儿配方奶粉导致新生儿脑膜炎、菌血症和坏死性小肠结肠炎等疾病的食源性条件致病菌。阪崎肠杆菌生物被膜的形成提高了该菌对外界环境的抗逆性，增强了该菌对抗生素、消毒剂、渗透胁迫、热和饥饿等外界环境压力的适应能力，因而增加了人类感染阪崎肠杆菌的风险。目前的研究多集中于阪崎肠杆菌能够在食品加工环境及被污染的食品中形成生物被膜，而关于如何抑制该菌生物被膜形成的研究却鲜有报道。前期研究结果初步证实了一种植物来源的天然活性物质— —姜黄素具有抑制阪崎肠杆菌生物被膜形成的能力。本课题拟通过微量板定量检测法，扫描电镜和激光共聚焦扫描显微镜技术，系统的研究姜黄素对阪崎肠杆菌生物被膜形成的抑制作用，并通过qPCR和差异蛋白质组学技术，探索姜黄素抑制阪崎肠杆菌生物被膜形成的作用机制，为食品工业中姜黄素抑制阪崎肠杆菌生物被膜形成的应用提供理论与技术支撑。

细菌生物被膜是一种由非游离细胞所分泌的胞外基质所组成的细菌菌落。生物被膜的存在提高了细菌对外界环境的抗逆性，一旦形成将很难清除并且会持续不断地向食品级环境中释放细菌，因而可能会导致潜在的公共卫生风险。本项目研究了姜黄素抑制克罗诺杆菌生物被膜的形成及作用机制。首先通过生理生化鉴定和分子生物学方法从谷类食品、食品香辛料、干制菌类及环境中分离得到了99株克罗诺杆菌分离株，并通过O抗原分型、多位点序列分型技术对其进行了鉴定。其次通过微量板定量检测法，扫描电镜和激光共聚焦扫描显微镜技术，系统的研究姜黄素对克罗诺杆菌生物被膜形成的抑制作用。最后通过实时荧光定量PCR方法，初步探索了姜黄素抑制克罗诺杆菌生物被膜形成的作用机制。 结果发现姜黄素对克罗诺杆菌的抑制效果呈剂量依赖型。姜黄素对克罗诺杆菌分离株XZCRO007 的最小抑菌浓度为512 μg/mL。浓度为128 μg/mL的姜黄素处理6h、12h、24h和48后，对克罗诺杆菌 XZCRO007 菌株生物膜的抑制率分别为 65.2%、68.5%、70.8%和51.6%。扫描电镜结果显示姜黄素处理24h 后， 细菌生物被膜开始解体，而未处理组的生物被膜与基体结合紧密。qPCR 结果表明姜黄素黄可能通过下调克罗诺杆菌生物膜相关基因的表达来抑制其生物膜的形成。