Diversity and Dynamic Stability of the Ocular Surface Microbiome

描述(申请人提供)：眼部感染性疾病，包括微生物角膜炎、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，角膜溃疡的培养得率约为55%。大多数引起眼部疾病的感染性生物都起源于眼表。然而，到目前为止，这种表面的成分还没有完全被表征。现代分子生物学方法，包括最近可用的�深度测序�方法，使前所未有的眼表微生物组分析成为可能。最近，两个首席研究员�S的实验室利用16S核糖体测序进行的初步研究表明：1)许多培养阴性的角膜溃疡与新的或不寻常的生物有关。�Normal�眼表生物群的多样性远远超过了传统的、基于培养的方法。综上所述，这些发现为确定眼表(OS)微生物群的特征提供了动力。我们的主要假设是，与身体上其他正常定居的部位类似，OS微生物群是一个动态平衡控制的环境，通常对有害感染具有保护作用；并且这个微生物群中的扰动将易于发生病理情况。在这些研究的第一个目标中，将并行使用两种最先进的技术来研究OS微生物组。第一次，使用基于454的焦磷酸测序的深度16S/5.8S测序将被用于生成在100名健康受试者的结膜中发现的细菌和真菌属的目录，根据地理位置(从迈阿密到西雅图)、种族和性别而多样化。我们还将在这些受试者的子集中观察微生物组随时间的稳定性。这个 第二种技术，Silico核型鉴定中的生物组代表性，是一种发现以前未描述的物种的新方法，利用基于Illumina的对已定义的元基因组样本的基因组表示进行深度测序。利用这项技术，可以发现新的细菌、病毒、真菌、噬菌体或寄生虫，并可以设计出对这些生物的分子诊断测试。在目标2和目标3中，将使用这些相同的技术测试可能扰乱正常OS微生物群的常见临床情景的效果(具体而言，使用局部抗生素、局部皮质类固醇治疗和使用软性隐形眼镜)。根据这些研究的结论，我们预计我们将得出关于 眼表面微生物群，并测定其在个体间的变异性，其在 一个人在眼睛之间和随着时间的推移，并了解这个生物群对药物治疗和软性隐形眼镜佩戴的反应。这些信息将对旨在了解微生物组对眼睛健康的影响的后续研究至关重要。这项研究的主要研究人员在分子诊断学方面有多年的经验。通过合作，并包括一名生物信息学专家，可以利用两种互补的生物群表征方法的专业知识来解决这一问题。此外，这两个群体在地理上的分离为了解地区对操作系统生物群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.