Diversity and Dynamic Stability of the Ocular Surface Microbiome

描述（由申请方提供）：眼部感染性疾病，包括微生物性角膜炎、结膜炎和眼内炎，仍然是潜在致盲疾病的重要原因。传统的微生物培养方法在许多情况下无法识别致病微生物;例如，角膜溃疡培养的产率约为55%。引起眼部疾病的大多数传染性生物体起源于眼表。然而，该表面的成分至今尚未完全表征。现代分子生物学方法，包括最近可用的“深度测序”方法，允许对眼表微生物组进行前所未有的分析。最近两个主要研究者的实验室利用16 S核糖体测序的试点研究表明，1。许多培养阴性的角膜溃疡与新的或不常见的微生物有关，以及2.）“正常”眼表生物群系的多样性远远大于传统的基于培养的方法所认识到的。总之，这些发现为进行眼表（OS）微生物组的明确表征提供了动力。我们的总体假设是，与身体上的其他正常定殖部位类似，OS微生物组是一种稳态控制的环境，通常可以保护有害感染;并且该微生物组的扰动将易患病理条件。在这些研究的第一个目标中，将并行采用两种最先进的技术来研究OS微生物组。第一个，使用基于454焦磷酸测序的深度16 S/5.8S测序将用于生成在100名健康受试者的结膜中发现的细菌和真菌属的目录，这些受试者因地理位置（从迈阿密到西雅图）、种族和性别而多样化。我们还将研究这些受试者的一个子集中微生物组随时间的稳定性。的 第二种技术Biome Representational in Silico Karyotyping是一种用于发现先前未表征的物种的新方法，其利用基于Illumina的宏基因组样品的定义的基因组表示的深度测序。使用这种技术，可以发现新的细菌，病毒，真菌，噬菌体或寄生虫，并可以设计这些生物体的分子诊断测试。在目标2和3中，将使用这些相同的技术测试可能破坏正常OS微生物组的常见临床情况（具体而言，局部抗生素治疗、局部皮质类固醇治疗和软性角膜接触镜的使用）的影响。在这些研究的结论中，我们预计我们将得出对 眼表微生物组，并确定其在个体之间的变异性， 一个人之间的眼睛和随着时间的推移，并了解该生物群落的反应，以治疗药物和软接触透镜磨损。这些信息对于旨在了解微生物组对眼部健康影响的后续研究至关重要。本研究的主要研究者具有多年的分子诊断经验。通过合作，并包括一名生物信息学专家，在两个互补的方法来生物群系表征的专业知识可以承担这个问题。此外，这两个群体的地理分离为理解地点对OS生物群系的影响提供了极好的机会。

DESCRIPTION (provided by applicant): Ocular infectious diseases, including microbial keratitis, conjunctivitis, and endophthalmitis, remain a significant cause of potentially blinding disease. Traditional microbial culture methods are unable to identify causative organisms in many cases; yields for cultures of corneal ulcers, for example, are around 55%. Most infectious organisms causing ocular disease originate in the ocular surface. However, the constituents of this surface have been incompletely characterized to date. Modern molecular biologic methods including recently available �deep sequencing� methods allow unprecedented analysis of the ocular surface microbiome. Recent pilot studies from both principal investigator�s laboratories utilizing 16S ribosomal sequencing have demonstrated that 1.) many culture-negative corneal ulcers are associated with novel or unusual organisms, and 2.) the diversity of the �normal� ocular surface biome is far greater than has been appreciated by traditional, culture-based methods. Taken together, these findings provide an impetus for performing a definitive characterization of the ocular surface (OS) microbiome. Our overarching hypothesis is that, similar to other normally-colonized sites on the body, the OS microbiome is a homeostatically controlled environment which is normally protective of deleterious infection; and that perturbations in this microbiome will predispose to pathologic conditions. In the first Aim of thes studies, two state-of-the-art techniques will be employed in parallel to study the OS microbiome. The first, deep 16S/5.8S sequencing using 454-based pyrosequencing will be used to generate a catalog of bacterial and fungal genera found in the conjunctiva of 100 healthy subjects, diversified for geographic location (from Miami to Seattle), ethnicity, and gender. We will additionally look at the stability of the microbiome across time in a subset of these subjects. The second technique, Biome Representational in Silico Karyotyping, is a novel method for discovery of previously uncharacterized species, utilizing Illumina-based deep sequencing of a defined genomic representation of a metagenomic sample. Using this technique, novel bacteria, viruses, fungi, phage, or parasites can be discovered and molecular diagnostic tests for these organisms can be devised. In Aims 2 and 3, the effect of common clinical scenarios that may disrupt the normal OS microbiome (specifically, treatment with topical antibiotics, topical corticosteroids, and use of soft contact lenses) will be tested using these same techniques. At the conclusions of these studies we anticipate we will have derived a definitive description of the ocular surface microbiome, and determined its variability amongst individuals, its stability within an individual between eyes and over time, and understood the response of this biome to treatment with medications and soft contact lens wear. This information will be essential to subsequent studies aimed at understanding the influence of the microbiome on ocular health. The principal investigators of this study have many years experience in molecular diagnostics. By collaboration, and with inclusion of a bioinformatics expert, expertise in the two complementary approaches to biome characterization can be brought to bear on this problem. Additionally, the geographic separation of the two groups provides excellent opportunity for understanding the effect of locale on the OS biome.