Combinatorial Imaging of the Oral Microbiome

描述（由申请人提供）：人类口腔是一个极其复杂的微生物群落的定植，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传染病的发展，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。口腔中存在哪些生物的问题已经得到了广泛的研究，已经确定了600多个物种或种型，其中目前只有大约一半可以培养。最近对16S rRNA克隆的调查表明，类群的相对丰度是不均匀的，大约250个类群占观察到的克隆的90%。目前用于识别分类群的DNA微阵列或标签测序方法需要将样品均匀化以提取DNA以供进一步分析。虽然可以保留厘米尺度的信息（例如，牙菌斑是从哪颗牙齿上取下来的，或者样本是来自舌头的侧面还是顶部），但微米级别的空间信息——即微生物群落组织水平——却丢失了。本项目的目标是开发一种新的“组合成像”方法，用于同时成像和鉴定人类口腔样品中的许多微生物分类群，从而获得有关口腔微生物组空间组织的微观尺度信息。针对核糖体RNA的荧光原位杂交（FISH）技术被广泛用于微生物鉴定，具有特异性和敏感性，但作为常用的技术，它只能同时分化两个或三个分类群。我们建议将FISH的功能扩展至少一个数量级，采用荧光报告组的组合来创建“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种型。在整个项目过程中，将在培养细胞、体外生物膜和志愿者获得的斑块上设计和测试单个FISH探针，以及15个或更多的标记有不同荧光团组合的FISH探针。该项目的重点是转化技术的发展，扩大可用于检测口腔微生物分类群的探针集，获得分类群相对于彼此以及相对于宿主（人类）组织的位置信息，并推动该技术应用于小集群和生物膜中的牙齿微生物的实际限制。该技术有潜力用于开发高通量转化科学分析，以快速和经济有效地监测口腔社区。这将为未来研究正常微生物菌群在健康和患病口腔中的作用奠定基础。

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.