Combinatorial Imaging of the Oral Microbiome

描述（由申请人提供）：人类口腔中定植着一个极其复杂的微生物群落，在人类健康和疾病中起着关键作用。牙菌斑是由微生物构建和居住的生物膜，口腔微生物群落参与了多种感染性疾病的发展，如龋齿、牙周病、牙槽骨炎、扁桃体炎、链球菌性咽喉炎和中耳炎。关于口腔中存在哪些生物体的问题已经得到了广泛的研究，已经确定了600多个物种或种类，其中只有大约一半目前可以培养。最近使用16 S rRNA克隆的调查表明，类群的相对丰度是不均匀的，约250个类群占观察到的克隆的90%。目前用于鉴定存在哪些分类群的DNA微阵列或标签测序方法需要将样品均质化以提取DNA用于进一步分析。虽然厘米级的信息可以保留（例如，牙菌斑是从哪颗牙齿上取的，或者样本是来自舌头的侧面还是顶部），但微米级的空间信息-即微生物群落组织的水平-却丢失了。该项目的目标是开发一种新的“组合成像”方法，用于同时成像和识别人类口腔样品中的许多微生物类群，从而获得有关口腔微生物组空间组织的微米级信息。荧光原位杂交（FISH）技术靶向核糖体RNA被广泛用于识别微生物，可以是特异性和敏感性，但作为常用的，它只允许同时区分两个或三个类群。我们建议将FISH的能力扩展至少一个数量级，采用荧光报告基团的组合来创建“光谱特征”，从而允许同时编码和成像数十种至可能数百种不同的微生物微生物群类型。在该项目的过程中，将设计单个FISH探针，以及用不同荧光团组合标记的15个或更多FISH探针组，并在培养细胞，体外生物膜和从志愿者获得的斑块上进行测试。该项目的重点是转化技术开发，扩大可用于检测口腔微生物类群的探针组，获得有关类群相对于彼此和相对于宿主（人类）组织的位置的信息，并推动该技术应用于小簇和生物膜中的牙科微生物的实际限制。该技术有可能被用于开发高通量的翻译科学检测，用于快速和具有成本效益的口腔社区监测。它将为未来研究正常微生物植物群在健康和患病口腔中的作用奠定基础。 公共卫生相关性：数百种细菌生活在人类口腔中;牙菌斑是由微生物构建和居住的生物膜;口腔微生物群落参与许多口腔疾病的发展，包括龋齿和牙周炎。该提案旨在开发创新的“组合成像”技术，以研究口腔中发现的不同种类细菌（包括微生物群落）的精确空间组织。该技术引入了“光谱特征”，使用荧光标记探针的二元组合靶向完整的细菌，并使用光谱成像来区分大量的标记探针同时。这种方法将使我们能够确定分类群相对于彼此以及相对于宿主（人类）组织的位置，这将为新的快速诊断检测奠定基础，并为研究口腔微生物群落如何工作以及致病物种如何侵入宿主组织并导致疾病奠定基础。

DESCRIPTION (provided by applicant): The human mouth is colonized by a microbial community of enormous complexity that plays a key role in human health and disease. Dental plaque is a biofilm constructed and inhabited by microbes, and the oral microbial community is involved in the development of several infectious diseases such as dental caries, periodontal disease, alveolar osteitis, tonsillitis, strep throat and otitis media. The question of which organisms are present in the oral cavity has been extensively studied and over 600 species or phylotypes have been identified, of which only about half can be cultivated at the present time. Recent surveys using 16S rRNA clones indicate that the relative abundance of taxa is uneven, with about 250 taxa accounting for 90% of the clones observed. The DNA microarray or tag sequencing methods currently used to identify which taxa are present require that a sample be homogenized to extract the DNA for further analysis. Although centimeterscale information can be retained (for example, which tooth the plaque was taken from, or whether a sample was from the side or the top of the tongue), spatial information at the micron level--namely the level of microbial community organization--is lost. The objective of this project is to develop a novel, "combinatorial imaging" method for simultaneous imaging and identification of many microbial taxa in samples from the human mouth, so as to obtain micronscale information about the spatial organization of the oral microbiome. The technique of fluorescence in situ hybridization (FISH) targeting ribosomal RNA is widely used to identify microbes and can be specific and sensitive, but as commonly employed it allows the differentiation of only two or three taxa simultaneously. We propose to extend the capabilities of FISH by at least an order of magnitude, employing a combination of fluorescent reporter groups to create "spectral signatures" that allow simultaneous encoding and imaging of tens to potentially hundreds of different microbial phylotypes. Over the course of the project, individual FISH probes, and sets of fifteen or more FISH probes labeled with different combinations of fluorophores, will be designed and tested on cultured cells, in vitro biofilms and on plaque obtained from volunteers. The focus of the project is translational technology development, expanding the set of probes that can be employed to detect oral microbial taxa, obtaining information about where taxa are located relative to each other and relative to host (human) tissues, and pushing the practical limits of this technique as applied to dental microbes in small clusters and in biofilms. The technology has the potential to be used for developing high throughput translational science assays for the rapid and cost-effective monitoring of oral communities. It will lay the foundation for future studies examining the role of the normal microbial flora in both healthy and diseased mouths. PUBLIC HEALTH RELEVANCE: Hundreds of species of bacteria live in the human mouth; dental plaque is a biofilm constructed and inhabited by microbes; and the oral microbial community is involved in the development of many oral diseases including dental caries and periodontitis. This proposal is to develop innovative, "combinatorial imaging" technology to study the precise spatial organization of different kinds of bacteria comprising microbial communities found in the mouth. The technology introduces "spectral signatures" using binary combinations of fluorescently labeled probes targeting intact bacteria and using spectral imaging to differentiate large numbers of labeled probes simultaneously. This method will allow us to determine where taxa are located relative to each other and relative to the host (human) tissues, which will lay the foundation for new, rapid diagnostic assays and for studies of how oral microbial communities work and how pathogenic species invade host tissues and cause disease.