Biofilm Architecture of Subgingival Plaque

描述（由申请人提供）：全世界有相当数量的成年人受到牙周病的影响。这些牙齿支持组织的慢性生物膜感染是牙齿脱落的主要原因，也与全身性疾病有关。尽管口腔菌群的大量清查和口腔生物膜的一般结构信息是可用的，但对引起牙周病的生物膜中的物种分布及其相互作用的详细分析在很大程度上是缺失的。本项目的长期目标是建立荧光原位杂交（FISH）技术，以详细检查牙龈下重要的生物膜特征，包括物种的存在和丰度、定位模式、生物膜特异性形态、尚未培养的生物的作用以及鉴定在生物膜结构形成中起关键作用的细菌。FISH被选为一种工具，因为这种多功能技术可以在所有分类水平上独立检测微生物，并在未受干扰的样品中可视化其分布。此外，特定微生物在建筑生物膜方面的作用以及它们的相互作用将在定量水平上进行研究。根据国家牙周病研究中心的使命，这将为开发治疗牙周病的新诊断和医疗目标提供重要信息，牙周病由于感染的持久性而仍然难以治愈。在这个特定的应用中，我们建议使用FISH来确定生物膜群落中选定的假定牙周病原体的比例、空间分布和共定位模式。该方法将与基于载体的体内模型系统结合应用，该系统允许对自然生长的牙周生物膜进行采样。将检查这些生物膜是否存在可疑的口腔病原体（如口腔密螺旋体、梭杆菌等）。，牙龈卟啉单胞菌，连翘Tannerella， alocis Filifactor, TM7 complex)。对整个和切片载体进行显微镜检查将能够对它们各自的丰度、空间分布和共定位（指示微生物之间的相互作用）进行定性评估。为了能够对所获得的数据进行定量分析，将采用针对FISH标记样品进行优化的新型数字图像分析程序。

DESCRIPTION (provided by applicant): A significant number of the adult population world-wide is affected by periodontal diseases. These chronic biofilm infections of the tooth-supporting tissues that are the major cause of tooth loss have also been associated with systemic diseases. Even though extensive inventory of the oral flora and general structural information about oral biofilms are available, detailed analysis of the species distribution and their interactions in biofilms causing periodontal disease is largely missing. The long-term objective of this project is to establish fluorescence in situ hybridisation (FISH) for the detailed examination of important subgingival biofilm features, including species presence and abundance, localization pattern, biofilm-specific morphotype, the role of as-yet-uncultured organisms and indentification of bacteria with pivotal role in biofilm structure formation. FISH is chosen as a tool since this versatile technique enables culture independent detection of microorganisms on all taxonomic levels and visualisation of their distribution in undisturbed samples. Furthermore, the role of specific microorganisms in architectoral biofilm aspects as well as their interaction will be examined on a quantitative level. In compliance with the mission of the NIDCR, this will provide important information for the development of new diagnostic and medical targets in the treatment of periodontal diseases which are still difficult to cure due to the persistent nature of the infection. In this specific application we propose to employ FISH to determine proportion, spatial distribution and co- localisation pattern of selected putative periodontal pathogens within the biofilm community. This method will be applied in combination with a carrier-based in vivo model system that allows sampling of naturally grown periodontal biofilms. These biofilms will be examined for the presence of suspected oral pathogens (e.g. oral treponemes, Fusobacterium ssp., Porphyromonas gingivalis, Tannerella forsythia, Filifactor alocis, TM7 complex) using specific FISH probes. Microscopic examination of whole and sectioned carriers will enable qualitative evaluation of their respective abundance, spatial distribution and co-localisation (indicative for interactions between microorganisms). To enable quanitative analysis of the data obtained, the novel digital image analysis program daime that was optimized for FISH labeled samples will be employed. This study will validate current ideas of periodontal disease development and provide detailed information about the bacteria involved including those that cannot be studied in laboratory test tube cultures. Knowing which bacteria play central roles in the build-up of the biofilm communities in periodontitis could identify new marker organisms for diagnostic tests. This could lead to treatments in which only key harmful organisms will be eliminated compared to the "kill all" approaches of existing therapies in which beneficial bacteria are not spared. Furthermore, failed treatments and treatments outcomes could be easily monitored. Project Narrative: Chronic bacterial infections of the tooth-supporting tissue (gum and bone), the periodontal diseases affect a significant portion of adults. It is the major cause for tooth loss and may be related to so-called systemic conditions such as heart disease. Periodontal diseases are difficult to cure permanently. Repetitive treatments are necessary to keep these infections in check since their cause, highly structured bacterial communities and yet unknown bacteria that reside within the periodontal pockets are difficult to remove completely. Our research will help to understand which type of bacteria are relevant for formation of these anchoring structures that keep other bacteria in place, who is most abundant and which types of bacteria may interact with each other to promote disease. If successful, this study will provide useful information for the development of new diagnostic and therapeutic tools.