Dynamics of mixed candida species biofilms in response to antifungal treatment

描述(由申请人提供)：假丝酵母菌具有40-50%的高可归因性死亡率，是美国医院获得性血液感染的第四大原因。这些真菌病原体通常作为共生菌存在于哺乳动物的粘膜壁龛中，尤其是口腔。在免疫功能低下的患者中，念珠菌的过度生长导致生物膜的形成，而生物膜在感染的发展中起着重要的作用。生物膜是附着在表面并包裹在细胞外基质中的复杂微生物群落。许多研究表明，生物膜通常比单一的浮游细胞对抗菌剂更具抵抗力。这些生物膜与 机会性酵母菌感染，口腔念珠菌病(口腔鹅口疮)，发现于新生儿和许多艾滋病、糖尿病和癌症患者。尽管口腔鹅口疮中以白色念珠菌为主，但由非白色念珠菌(特别是光滑念珠菌、都柏林念珠菌和热带念珠菌)组成的口腔鹅口疮分离物的发病率也在增加。这一发现具有临床意义，因为常规剂量的抗真菌药物有时在清除这些口腔感染方面无效。虽然单一菌种的念珠菌生物膜已经得到了很好的研究，但对混合生物膜中假丝酵母菌的组成和相互作用对抗真菌治疗的影响却知之甚少。我最近开发了一种基于高精度定量-聚合酶链式反应的方法来确定混合念珠菌生物膜的精确物种组成。初步数据显示，经氟康唑处理后，光滑葡萄球菌在混合生物膜中的比例显著增加。我的初步数据还表明，当这四种念珠菌在不同的营养环境下混合时，生物膜的代谢活性发生了变化，这表明生物膜的组成和动力学也发生了变化。根据这一数据，我的假设是，在抗真菌治疗后，混合念珠菌生物膜组成和空间结构的变化有利于具有内在耐药性或迅速产生耐药性的非白色念珠菌。为了验证这一假设，我将进行实验以解决以下具体目标：(1)确定敏感和耐药菌株在存在和不存在抗真菌处理的情况下产生的混合念珠菌生物膜的准确组成，以及(2)确定在存在和不存在抗真菌处理的混合生物膜中念珠菌物种的空间结构。总的来说，这些创新研究将产生重大影响，因为它们将提供关于混合念珠菌生物膜如何与抗真菌耐药性相关的新信息。这些研究还将测试我的 定量-聚合酶链式反应作为一种快速检测混合念珠菌生物被膜(如口腔鹅口疮)成分的诊断工具。由于混合念珠菌生物被膜更频繁，这些研究将为开发更有效的抗真菌药物以改善患者预后铺平道路。此外，这个项目的成功完成将为我提供特殊的培训，促进我的职业发展，并为我成为一名儿科临床医生科学家做好准备。

DESCRIPTION (provided by applicant): With a high attributable mortality rate of 40-50%, Candida species represent the fourth leading cause of hospital-acquired bloodstream infections in the United States. These fungal pathogens are normally found as commensals in mammalian mucosal niches, especially the oral cavity. In immunocompromised patients, Candida overgrowth leads to the formation of biofilms, which play an important role in the development of infection. Biofilms are complex microbial communities that adhere to a surface and are enclosed within an extracellular matrix. Many studies have shown that biofilms are generally more resistant to antimicrobial agents than single, planktonic cells. These biofilms are associated with the opportunistic yeast infection, oral candidiasis (oral thrush), found in newborns and many patients with AIDS, diabetes and cancer. Even though C. albicans is the predominant organism found in oral thrush, there is an increasing incidence of oral thrush isolates consisting of non-albicans Candida species (especially C. glabrata, C. dubliniensis and C. tropicalis). This finding is clinically relevant since conventional doses of antifungals are sometimes ineffective at clearing these oral infections. While single-species Candida biofilms have been well-studied, considerably less is known about the composition and interaction of Candida species in mixed biofilms in response to antifungal treatment. I have recently developed a highly accurate quantitative-PCR-based approach to determine the precise species composition of mixed Candida biofilms. Preliminary data shows that the proportion of C. glabrata in the mixed biofilm increases significantly upon treatment with fluconazole. My preliminary data also indicates that the metabolic activity of biofilms is altered when these four Candida species are mixed under different nutritional environments, suggesting that biofilm composition and dynamics have changed as well. Based on this data, my hypothesis is that the shift in mixed Candida species biofilm composition and spatial architecture following antifungal treatment favors non-albicans Candida species that are intrinsically resistant or rapidly develop drug resistance. In order to test this hypothesis, I will perform experiments to address the following specific aims: (1) To determine the precise composition of mixed Candida species biofilms generated from susceptible and drug-resistant strains in the presence vs. absence of antifungal treatment and (2) To determine the spatial architecture of Candida species in mixed biofilms generated in the presence and absence of antifungal treatment. Collectively, these innovative studies will have significant impact since they will provide new information about how mixed Candida species biofilms are associated with antifungal resistance. These studies will also test the efficacy of my quantitative-PCR assay to serve as a diagnostic tool for rapid composition determination of mixed Candida biofilms (i.e. oral thrush). Since mixed Candida species biofilms are encountered more frequently, these studies will pave the way for the development of more effective antifungals to improve patient outcomes. Moreover, successful completion of this project will provide exceptional training, advance my career development and prepare me to become a pediatric dental clinician scientist.