COMMUNITY COMPOSITION AND PHYSIOLOGY OF ORAL BIOFILMS

拟议的研究的长期目标是开发简单的，定量的措施，口腔微生物（唾液和菌斑生物膜）的社区组成和生理，检查口腔社区的结构/功能关系的时间和空间变化，并表征社区组成和生理的营养和环境依赖性的变化。 这些目标旨在阐明菌斑生物膜，特别是牙周病病原体群落的进展的自然变化。本提案旨在定义一个物种组成的测量使用磷脂结合的脂肪酸（PLFA）配置文件的整个社区的生物量，并将验证与棋盘杂交和可培养的植物群的方法相关的测量。 将使用电子显微镜和激光共聚焦显微镜进行生物膜结构的分析，并将进行该数据与组成和生理数据的相关性。 将建立和应用于体外生物膜缩影和定义的物种财团的社区生理措施。 “功能块”将通过表征酶活性（蛋白酶、粘蛋白特异性外切糖苷酶、尿素酶）作为营养蛋白催化剂、粘蛋白催化剂和尿素水解的指标;葡萄糖发酵和酸产生的速率作为糖解发酵的指标和氨释放作为氨基酸代谢的指标来定义。 将使用BiologTM和API-ZymTM技术进行全群落代谢谱分析。 营养成分和环境pH值的变化的影响将通过监测社区生理，社区组成和架构进行检查。提出的工作的假设是：1）PLFA分析原则的应用证明是有效的，在社区分类的自然样品的口腔生物膜将产生快速和显着的信息的社区组成。 PLFA分析将允许简单，频繁和信息丰富的患者监测，从而提高我们对牙周病和龋齿进展的微生物群落组成变化的理解。 2)细菌群落组成和群落生理学以可理解的方式对营养成分和环境pH值的变化作出反应。组成和生理学是相关的，但不一定直接相关。牙菌斑诱导的变化被认为是牙周病进展的关键因素;对牙菌斑中正常和病原体群落的控制的了解将增强我们对牙周病和龋齿因果关系的理解，并支持新的干预策略的发展。

Long-term goals of the proposed research are development of simple, quantitative measures of oral microbial (saliva and plaque biofilm) community composition and physiology, examination of temporal and spatial variations in structure/function relationships in oral communities, and characterization of nutrient- and environment-dependent shifts in community composition and physiology. These goals seek to elucidate the shifts natural to the progression of plaque biofilms, especially pathogen communities of periodontal disease. The present proposal seeks to define a species-composition measurement using phospholipid-bound fatty-acid (PLFA) profiles of whole-community biomass, and will validate that measurement by correlation with checkerboard hybridization and cultivable flora methods. Analysis of biofilm architecture will be performed using electron microscopy and laser confocal microscopy, and correlation of that data with compositional and physiological data will be performed. Community physiology measures will be established and applied to in vitro biofilm microcosms and defined-species consortia. "Functional blocks" will be defined by characterizing enzymatic activities (proteases, mucin-specific exoglycosidases, urease) as indicators of nutrient-protein catabolism, mucin catabolism, and urea hydrolysis; the rate of glucose fermentation and of acid production as indicators of saccharolytic fermentation and ammonia release as an indicator of amino acid metabolism. Whole-community metabolic profiles will be performed using BiologTM and API-ZymTM technologies. The effects of shifts in nutrient composition and environmental pH will be examined by monitoring community physiology, community composition, and architecture. Hypotheses for the proposed work are: 1) that application of PLFA analysis principles proven effective in community taxonomy of natural samples to oral biofilms will yield rapid and significant information on community composition. PLFA analysis will permit simple, frequent, and information-rich patient monitoring and thus improve our understanding of microbial community-composition shifts that underlie the progression of periodontal disease and dental caries. 2) that bacterial community composition and community physiology respond in a understandable manner to shifts in nutrient composition and environmental pH. Composition and physiology are related though not necessarily directly linked. Ecologically induced shifts are presumed to be key factors in the progression of periodontal disease; knowledge of the controls over normal and pathogen communities in plaque will enhance our understanding of cause-and-effect in periodontal disease and caries as well as underpinning development of new intervention strategies.