COMMUNITY COMPOSITION AND PHYSIOLOGY OF ORAL BIOFILMS

该研究的长期目标是开发口腔微生物（唾液和牙菌斑生物膜）群落组成和生理的简单定量测量方法，检查口腔群落结构/功能关系的时空变化，以及表征群落组成和生理的营养和环境依赖变化。这些目标旨在阐明菌斑生物膜进展的自然变化，特别是牙周病的病原体群落。本提案旨在利用整个群落生物量的磷脂结合脂肪酸（PLFA）谱来定义一种物种组成测量方法，并将通过与棋盘杂交和可培养菌群方法的相关性来验证该测量方法。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。将建立群落生理学测量方法，并将其应用于体外生物膜微观环境和物种群落。“功能块”将通过酶活性（蛋白酶、粘蛋白特异性外糖苷酶、脲酶）作为营养蛋白分解代谢、粘蛋白分解代谢和尿素水解的指标来定义；葡萄糖发酵速率和产酸速率是糖解发酵的指标，氨释放是氨基酸代谢的指标。将使用BiologTM和api - zyntm技术进行全群落代谢谱分析。营养成分和环境pH值变化的影响将通过监测群落生理学、群落组成和结构来检验。本研究的假设是：1)将PLFA分析原理应用于口腔生物膜的天然样品群落分类中，将获得快速而有意义的群落组成信息。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理解的方式响应营养成分和环境ph值的变化。组成和生理是相关的，但不一定直接相关。生态诱导的转移被认为是牙周病进展的关键因素；对菌斑中正常菌群和病原菌群控制的了解将增强我们对牙周病和龋齿的因果关系的理解，并为新的干预策略的发展奠定基础。

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.