Combinatorial Imaging of the Oral Microbiome

描述(申请人提供)：人类口腔被一个极其复杂的微生物群落定居，该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栖息的生物膜，口腔微生物群落参与了龋病、牙周病、牙槽骨炎、扁桃体炎、链球菌性咽炎和中耳炎等多种感染性疾病的发生发展。关于口腔中存在哪些生物的问题已经被广泛研究，并已鉴定出600多个物种或系统型，其中只有大约一半目前可以培养。最近利用16S rRNA克隆进行的调查表明，分类群的相对丰度是不均匀的，约有250个类群占所观察到的克隆的90%。目前用于确定存在哪些分类群的DNA微阵列或标签测序方法需要将样本均质化以提取DNA用于进一步分析。虽然可以保留厘米级的信息(例如，牙菌斑是从哪颗牙齿采集的，或者样本是从舌侧还是舌尖上取的)，但微米级别的空间信息--即微生物群落组织的级别--会丢失。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本中的许多微生物分类群，以获得关于口腔微生物组空间组织的微观信息。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，但通常只允许同时分化两到三个类群。我们建议将FISH的能力至少扩展一个数量级，使用荧光报告组的组合来创建“光谱特征”，允许同时编码和成像数十到可能数百个不同的微生物系统类型。在该项目的整个过程中，将在培养细胞、体外生物膜和从志愿者那里获得的斑块上设计和测试单独的鱼探针，以及标记了不同荧光团组合的15个或更多组鱼探针。该项目的重点是翻译技术的开发，扩大可用于检测口腔微生物分类群的探针组，获得关于分类群之间和相对于宿主(人体)组织的位置的信息，并推动这项技术应用于小簇和生物膜中的牙科微生物的实际限度。这项技术有可能被用于开发高通量的转化科学分析方法，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。 与公共卫生相关：数百种细菌生活在人类口腔中；牙菌斑是由微生物构建和栖息的生物膜；口腔微生物群落参与了许多口腔疾病的发展，包括龋齿和牙周炎。这项提议是要开发创新的“组合成像”技术，以研究不同种类细菌的精确空间组织，这些细菌包括在口腔中发现的微生物群落。该技术引入了“光谱特征”，使用针对完整细菌的荧光标记探针的二元组合，并使用光谱成像来同时区分大量标记的探针。这种方法将使我们能够确定分类群相对于彼此和相对于宿主(人类)组织的位置，这将为新的快速诊断分析和研究口腔微生物群落如何工作以及致病物种如何入侵宿主组织和导致疾病奠定基础。

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.