Diversity and Dynamic Stability of the Ocular Surface Microbiome

眼表微生物组的多样性和动态稳定性

基本信息

批准号：
9123604
负责人：
VALERY I SHESTOPALOV
金额：
$ 63.06万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-01 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9123604
关键词：
Adrenal Cortex Hormones Age Antibiotic Therapy Antibiotics Bacteria Bacteriophages Bioinformatics Blindness Cataloging Catalogs Clinical Collaborations Communicable Diseases Communities Computer Simulation Conjunctivitis Contact Lenses Controlled Environment Corneal Ulcer Cross-Sectional Studies DNA Data Data Set Detection Development Disease Endophthalmitis Ethnic Origin Eye Eye Infections Eye diseases Gender Genomic DNA Genomics Geographic Locations Goals Health Human Hydrophilic Contact Lenses Indigenous Individual Infection Infectious Agent Karyotype determination procedure Keratitis Laboratories Locales Longitudinal Studies Lung Metagenomics Methods Microbe Molecular Molecular Diagnostic Testing Organism Outcome Parasites Pathologic Pharmaceutical Preparations Phylogeny Physiological Pilot Projects Principal Investigator Race Research Resistance Resources Ribosomal RNA Role Sampling Site Stress Structure Surface Techniques Technology Testing Time Tissues Topical Antibiotic Topical Corticosteroids Ursidae Family Virus Visual base clinically relevant conjunctiva deep sequencing disability experience fungus member microbial microbial community microbiome microbiota molecular diagnostics novel ocular surface pathogen pyrosequencing response

项目摘要

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.

描述（由适用提供）：眼科传染病，包括微生物角膜炎，结膜炎和内脑炎，仍然是引起潜在盲目疾病的重要原因。在许多情况下，传统的微生物培养方法无法识别出严重的生物；例如，角膜溃疡培养物的产量约为55％。引起眼部疾病的大多数传染性生物源于眼表。但是，迄今为止，该表面的成分未完全表征。现代分子生物学方法，包括最近可用的深层测序方法，可以对眼表微生物组进行空前的分析。两位主要研究人员利用16S核糖体测序的实验室的试点研究表明，1。）许多培养文化阴性角膜溃疡与新颖或不寻常的生物有关，并且2.）正常表面生物群体的多样性远大于传统，基于培养的方法。综上所述，这些发现为执行眼表（OS）微生物组的明确表征提供了动力。我们的总体假设是，与体内其他正常殖民地的位点类似，OS微生物组是一个通常受到有害感染保护的体内稳态控制环境。并且该微生物组中的扰动会易于病理状况。在研究的第一个目的中，将同时使用两种最先进的技术来研究OS微生物组。使用454个基于454的焦磷酸测序的第一个深16S/5.8S测序将用于生成100个健康受试者的结膜中发现的细菌和真菌属目录，这些受试者在地理位置（从迈阿密到西雅图），种族和性别多样化。我们还将在这些受试者的子集中查看跨时间的微生物组的稳定性。第二种技术，即硅核分型中的生物群体代表性，是一种发现以前未表征的物种的新方法，利用基于Illumina的深层测序对元基因组样品的定义基因组表示。使用这种技术，可以发现新型细菌，病毒，真菌，噬菌体或寄生虫，可以设计这些生物的分子诊断测试。在AIM 2和3中，可能会使用这些相同的技术测试可能破坏正常OS微生物组（特别是用局部抗生素，局部皮质类固醇和使用软接触透镜的治疗）的常见临床情况的影响。在这些研究的结论中，我们预计我们将得出对眼表面微生物组，并确定其在个体之间的可变性，其稳定性随着时间的流逝，一个人在眼睛之间，并了解该生物群落对药物治疗和软透镜磨损的反应。此信息对于旨在了解微生物组对眼部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方面具有多年的经验。通过协作，并包括生物信息学专家，可以在这两个问题上实现两种完整的生物群体表征的专业知识。此外，这两组的地理分离为理解环境对OS生物群落的影响提供了绝佳的机会。