Combinatorial Imaging of the Oral Microbiome

口腔微生物组的组合成像

• 批准号: 7830369
• 负责人: Gary G Borisy
• 金额: $ 47.58万
• 依托单位: MARINE BIOLOGICAL LABORATORY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-22 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7830369
• 关键词: Accounting; Algorithms; Alveolar periostitis; Arts; Bacteria; Biological Assay; Cells; Censuses; Communicable Diseases; Communities; Cultured Cells; DNA; DNA Microarray Chip; Dental; Dental Plaque; Dental caries; Development; Diagnostic; Disease; Family; Fluorescence Microscopy; Fluorescent in Situ Hybridization; Foundations; Future; Goals; Growth and Development function; Harvest; Health; Human; Image; Image Analysis; Imagery; Imaging Techniques; Imaging technology; In Vitro; Individual; Institutes; Invaded; Label; Life; Metabolic; Methods; Microbe; Microbial Biofilms; Molecular; Monitor; Mouth Diseases; Oligonucleotide Probes; Oral; Oral cavity; Organism; Otitis Media; Periodontal Diseases; Periodontitis; Pharyngeal structure; Play; Positioning Attribute; Protocols documentation; Relative (related person); Reporter; Ribosomal RNA; Role; Sampling; Side; Surveys; Taxon; Techniques; Technology; Testing; Time; Tissues; Tongue; Tonsillitis; Tooth structure; Translational Research; Work; combinatorial; community organizations; cost; design; feeding; fluorophore; human tissue; imaging modality; imaging probe; innovation; member; microbial; microbial community; novel; oral biofilm; oral microbiome; public health relevance; research study; technology development; tool; volunteer

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.

描述（由申请人提供）：人口是由一个巨大复杂性的微生物社区殖民的，在人类健康和疾病中起着关键作用。牙菌斑是一种由微生物建造和居住的生物膜，口腔微生物群落参与了几种传染病的发展，例如龋齿，牙周疾病，牙槽骨骨炎，扁桃体炎，链球菌炎，链球菌或情疼痛和耳炎。已经对口腔中存在哪些生物存在的问题进行了广泛的研究，并且已经确定了600多种或系统型，目前只能培养其中约一半。最近使用16S rRNA克隆的调查表明，分类单元的相对丰度不平衡，约有250个分类单元占观察到的克隆的90％。目前用于识别存在哪个分类单元的DNA微阵列或TAG测序方法要求将样品均质化以提取DNA以进行进一步分析。尽管可以保留厘米尺度的信息（例如，从牙菌斑中取出哪种牙齿，或样品是从舌头还是舌头上的样本），但在微米层的空间信息（即微生物社区组织的水平）丢失了。该项目的目的是开发一种新颖的“组合成像”方法，用于同时成像和鉴定人口样品中许多微生物分类群，以获取有关口腔微生物组空间组织的微尺度信息。靶向核糖体RNA的原位原位杂交（FISH）的荧光技术被广泛用于识别微生物，并且可以是特定和敏感的，但是通常使用的是，它可以同时区分两个或三个分类单元。我们建议将鱼类的能力扩展到至少一个数量级，并采用荧光报告基团的组合来创建“光谱特征”，从而可以同时编码和成像，以使数百种不同的微生物系统型。在整个项目过程中，将在培养的细胞，体外生物膜和从志愿者获得的牙菌斑上设计和测试，在培养细胞，体外生物膜上设计和测试，将单个鱼类探针和15个或更多的鱼类探针组进行设计和测试。该项目的重点是转化技术开发，扩展了可用于检测口腔微生物分类单元的一组探针，获取了有关彼此相对的何处以及相对于宿主（人）组织的何处，并在小物物和生物膜中推动了该技术的实际限制。该技术有可能用于开发高通量转化科学测定法，以快速且具有成本效益的口腔社区监测。它将为未来的研究奠定基础，以研究正常微生物菌群在健康和患病的口腔中的作用。 公共卫生相关性：数百种细菌生活在人口中；牙菌斑是一种由微生物建造和居住的生物膜。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。该建议是开发创新的“组合成像”技术，以研究各种细菌的精确空间组织，其中包括在口腔中发现的微生物群落。该技术使用针对完整细菌的荧光标记的探针的二元组合引入“光谱特征”，并使用光谱成像同时区分了大量标记的探针。这种方法将使我们能够确定类群相对于彼此的位置以及相对于宿主（人）组织的位置，这将为新的快速诊断测定奠定基础，并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并引起疾病。