Dynamics of mixed candida species biofilms in response to antifungal treatment

描述（由申请人提供）：念珠菌属的归因死亡率高达40- 50%，是美国医院获得性血流感染的第四大原因。这些真菌病原体通常在哺乳动物的粘膜小生境，特别是口腔中作为寄生虫被发现。在免疫功能低下的患者中，念珠菌过度生长导致生物膜的形成，这在感染的发展中起重要作用。生物膜是附着于表面并封闭在细胞外基质内的复杂微生物群落。许多研究表明，生物膜通常比单一的、无菌的细胞对抗菌剂更具抗性。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。即使C.白色念珠菌是鹅口疮中发现的主要微生物，由非白色念珠菌属（尤其是念珠菌）组成的鹅口疮分离株的发生率越来越高。glabrata、光叶隐翅虫C. dubliniensis和C. tropicalis）。这一发现是临床相关的，因为常规剂量的抗真菌药有时在清除这些口腔感染方面无效。虽然单种念珠菌生物膜已经得到了很好的研究，相当少的是已知的组成和相互作用的念珠菌物种在混合生物膜中的抗真菌治疗。我最近开发了一种高度准确的定量PCR为基础的方法，以确定混合念珠菌生物膜的精确物种组成。初步数据表明，C.混合生物膜中的glabrata在用氟康唑处理后显著增加。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。基于这些数据，我的假设是，在抗真菌治疗后，混合念珠菌属生物膜组成和空间结构的变化有利于固有耐药或快速产生耐药性的非白色念珠菌属。为了验证这一假设，我将进行实验，以解决以下具体目标：（1）确定在存在与不存在抗真菌治疗的情况下，由敏感和耐药菌株产生的混合念珠菌属物种生物膜的精确组成，以及（2）确定在存在和不存在抗真菌治疗的情况下产生的混合生物膜中念珠菌属物种的空间结构。总的来说，这些创新研究将产生重大影响，因为它们将提供有关混合念珠菌属生物膜如何与抗真菌药耐药性相关的新信息。这些研究也将测试我的 定量PCR测定用作混合念珠菌生物膜（即鹅口疮）的快速组成测定的诊断工具。由于混合念珠菌属生物膜更常见，这些研究将为开发更有效的抗真菌药物以改善患者结局铺平道路。此外，这个项目的成功完成将提供特殊的培训，促进我的职业发展，并准备我成为一名儿科牙科临床医生科学家。

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.