COMMUNITY COMPOSITION AND PHYSIOLOGY OF ORAL BIOFILMS

拟议研究的长期目标是开发简单、定量的口腔微生物(唾液和菌斑生物膜)群落组成和生理指标，检查口腔群落结构/功能关系的时间和空间变化，以及表征群落组成和生理依赖于营养和环境的变化。这些目标旨在阐明菌斑生物膜，特别是牙周病的病原体群落的自然变化。本提案试图定义一种利用整个群落生物量的磷脂结合脂肪酸(PLFA)谱来测量物种组成的方法，并将通过与棋盘杂交和可培养植物群方法的相关性来验证这一测量方法。将使用电子显微镜和激光共聚焦显微镜对生物膜结构进行分析，并将这些数据与成分和生理数据进行关联。将建立群落生理学措施，并将其应用于体外生物膜微观世界和确定的物种联合体。“功能块”的定义是：将酶活性(蛋白酶、粘蛋白特有的外糖苷酶、尿素酶)定义为营养蛋白分解代谢、粘蛋白分解代谢和尿素分解的指示物；将葡萄糖发酵和产酸的速率定义为糖解发酵的指示物；将氨释放作为氨基酸新陈代谢的指示物。整个社区的代谢概况将使用BiologTM和API-ZymTM技术进行。将通过监测群落生理、群落组成和建筑来检验营养成分和环境pH变化的影响。拟议工作的假设是：1)将PLFA分析原理应用于口腔生物膜的自然样品的群落分类被证明是有效的，将产生关于群落组成的快速和重要的信息。PLFA分析将允许简单、频繁和信息丰富的患者监测，从而提高我们对微生物群落组成变化的理解，这些变化是牙周病和龋齿进展的基础。2)细菌群落组成和群落生理以一种可以理解的方式对营养成分和环境pH的变化做出反应。成分和生理是相关的，但不一定是直接联系的。生态诱导的变化被认为是牙周病进展的关键因素；了解菌斑中正常和病原体群落的控制将增强我们对牙周病和龋病因果关系的理解，并为开发新的干预策略奠定基础。

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.