真菌和细菌混合生物膜的形成机制和治疗策略研究

Microbial biofilms formed on implanted medical devices are a common cause of fatal hospital-acquired infections (HAI), due to its extreme resistance to antibiotics and human immune system. Candida albicans is the leading fungal pathogen causing severe systemic infections in hospitalized patients, and often co-exists in polymicrobial biofilms with bacterial pathogens such as Staphylococcus aureus and Staphylococcus epidermidis. Device-associated infections caused by polymicrobial biofilms are resistant to currently available therapies and has a very high mortality. Little is known about mechanisms of C. albicans-bacterial polymicrobial biofilm formation. And there is a great need to develop evidence-based strategies to treat these deadly polymicrobial biofilm infections. We have recently uncovered an important role for the C. albicans biofilm extracellular matrix (ECM) material in enabling mixed biofilm formation with S. aureus. As ECM is implicated in both biofilm drug resistance and immune evasion mechanisms, a good understanding of the detailed role of biofilm ECM in the formation of fungal-bacterial polymicrobial biofilms will allow for the identification of credible targets to guide future strategies that can be used to treat polymicrobial biofilm infections. Using comprehensive approaches combining microbiology, molecular biology, biochemistry and an in vivo animal model we will understand the mechanisms by which C. albicans and other bacterial pathogens form polymicrobial biofilms. We will determine how extracellular matrix secreted by C. albicans and bacteria drives polymicrobial biofilm formation, which ECM components are important for this, and the impact of fungal and bacterial matrix on antimicrobial susceptibility of fungi and bacteria in mixed biofilms. We will study the mechanisms leading to high antimicrobial resistance of polymicrobial biofilms. This sort of fundamental knowledge is essential for guiding development of more efficient antibiofilm therapies. We will screen the combinations of antifungal drugs and antibacterial drugs for optimal activities against fungal-bacterial polymicrobial biofilms. We will further optimise these antibiofilm regimens for improved activities against mixed biofilms, by selecting the "right" concentration for each individual agent. We will also examine new strategies that dissemble fungal matrix from the polymicrobial biofilms and combine it with the previously established antibiofilm regimems, for the best anti-polymicrobial-biofilm activities. We hypothesized that disrupting C. albicans biofilm ECM will disable polymicrobial biofilm formation and drastically improve the antimicrobial susceptibility of mixed biofilms. Collectively, this work will provide new knowledge of how fungal and bacterial pathogens form polymicrobial biofilms and lay a foundation for the treatment of deadly polymicrobial biofilm infections, as well as high impact publications in influential journal through the new break-through knowledge that we will generate.

白色念珠菌是引起住院患者严重的系统性感染的最主要真菌病原体，常与医院源性的细菌共存而形成混合生物膜。目前关于白色念珠菌与细菌混合生物膜的形成机制尚不明确，临床治疗手段对此类生物膜感染效果甚微，且此类感染死亡率极高，因此急需开发有效的治疗策略来应对此类感染。我们前期研究发现白色念珠菌生物膜细胞外基质（ECM）具有促进其与金黄色葡萄球菌混合生物膜形成的重要作用。本研究将结合微生物学、分子生物学、生物化学以及动物模型等方法综合分析白色念珠菌与细菌各自分泌的ECM在混合生物膜形成和产生高度耐药性中的作用，探索混合生物膜高度耐药的机制。在这基础上我们还将研究治疗白色念珠菌与细菌混合生物膜感染的有效的抗真菌-细菌药物组合，并在这组合中加入以ECM为靶点的抗生物膜药物，作为最优化的混合生物膜治疗方案。本研究将阐明白色念珠菌与细菌混合生物膜的形成和耐药机制，并为有效治疗致死性混合生物膜感染提供理论基础。

白色念珠菌和细菌混合生长的生物膜是一个急需解决的医学难题。在临床上白色念珠菌和细菌的混合生物膜比单纯白色念珠菌生物膜感染或者单纯的细菌生物膜更难治疗。 我们收集了白色念珠菌实验室标准株和基因敲除株，以及一些常见的细菌，包括金黄色葡萄球菌、表皮葡萄球菌、 头状葡萄球菌、乳酸杆菌和阴道加德纳菌，用传统的实验室生物膜培养、动态点滴-流动生物膜反应器、创新型的导管管道生物膜模型， 以及动物阴道白色念珠菌生物膜模型，对真菌和细菌在生长为真菌细菌混合生物膜过程中的相互影响做了分析。我们还进一步研究了真菌和细菌的生物膜细胞外基质体（EPS）的主要成分，以及其在混合生物膜形成中的作用。我们发现白色念珠菌的EPS在混合生物膜的生长中能有效的促使细菌的粘附。而单个EPS的组成成分并不起明显的作用。细菌的EPS对真菌的生物膜形成的促进作用有限。我们进一步研究了生物膜的耐药机制，发现其高密度的密集生长是生物膜耐药的主要机制，而EPS对药物的阻碍作用和水解作用、群体感应系统对耐药性的产生并没有显著的影响。高密度的密集生长导致微生物的总体代谢率下降而产生耐药。另外，我们的动物模型实验提示持留细胞的产生也是生物膜耐药的重要机理之一。根据混合生物膜的耐药机理，我们进行了混合生物膜的有效治疗的研究。我们发现对混合生物膜EPS进行水解，并不能有效的提高生物膜的药物敏感度。我们采用葡萄菌素和一线抗菌药物的联合使用，发现其对杀灭混合生物膜一定的有效性。我们也引入了对混合生物膜可能有效的鸟苷酸化的聚甲基丙烯酸脂，并在活体动物上验证了其对白色念珠菌生物膜混合阴道加德纳菌的感染的有效性。