A Novel Technology to Image Human Oral Microbial Communities

描述（由申请人提供）：本提案的目标是开发一种新的组合标记和检测技术，用于成像和区分大多数（如果不是全部）人类口腔微生物群落的成员。16S rRNA序列的荧光原位杂交（FISH）将直观地识别微生物群落的单个成员，无论是收集还是原位。FISH的功能将通过采用荧光报告组的组合来扩展，以创建“光谱签名”，允许同时编码和成像数十到数百种不同的微生物类群。增强的、最先进的光谱成像技术将记录光谱特征。将开发新的算法来解构标记微生物的光谱特征。这种新的成像能力将允许在人类口腔微生物群落的种型的全面普查，以及该群落的空间组织的确定。原则上，可以扩展组合标记策略，同时使用针对16S rRNA的探针标记微生物进行系统发育鉴定，同时使用针对mRNA或蛋白质的探针标记微生物以获取代谢或结构信息。评估特定微生物的相对丰度、组织和功能的能力将为口腔微生物群落生态学的全面和详细研究开辟新的途径。口腔微生物与龋齿、牙龈炎和牙周炎等疾病有关，这些疾病是人类最常见的细菌感染，也与全身疾病有关，包括心血管疾病、肺部感染、低出生体重和早产结局。没有单一的微生物物种被认为是这些疾病的病原，相反，假设社区中多种微生物类群的复杂关联负责维持健康和引起疾病。这里提出的技术的发展将使我们能够全面了解正常的人类口腔菌群，并促进开发新的有效策略来研究、监测和控制感染。

DESCRIPTION (provided by applicant): The objective of this proposal is to develop a novel combinatorial labeling and detection technology for imaging and distinguishing between most if not all of the members of the human oral microbiological community. Fluorescence In Situ Hybridization (FISH) for 16S rRNA sequences will visually identify individual members of the microbial community either as collected or in situ. The capabilities of FISH will be extended by employing a combination of fluorescent reporter groups to create "spectral signatures" that allow simultaneous encoding and imaging of tens to hundreds of different microbial phylotypes. Enhanced, state-of-the-art spectral imaging techniques will record the spectral signatures. Novel algorithms will be developed to deconstruct spectral signatures of labeled microbes. This new imaging capability will allow a comprehensive census of phylotypes in the human oral microbial community as well as determination of the spatial organization of this community. In principle, the combinatorial labeling strategy could be extended by simultaneously labeling microbes with probes against 16S rRNA for phylogenetic identification and probes against mRNA or proteins for metabolic or structural information. The capability to assess the relative abundance, organization and function of particular microbes will open new avenues for comprehensive and detailed study of the ecology of the oral microbial community. Oral microbes are implicated in diseases such as caries, gingivitis, and periodontitis, which are among the most common bacterial infections in humans, as well as systemic diseases including cardiovascular disease, pulmonary infections, and low birth-weight and pre-term pregnancy outcome. No single microbial species is implicated as the etiological agent of these diseases, rather, it is hypothesized that the complex association of multiple microbial phylotypes in communities are responsible for maintaining health and causing disease. Development of the technology proposed here will allow a comprehensive understanding of the normal human oral flora and foster the development of new and effective strategies to study, monitor, and control infection.