Defining bacterial members of the ocular surface microbiome and assessing stability over time

项目摘要 最近的研究表明，有数据表明眼部微生物组存在并影响眼表 健康和疾病。尽管如此，眼表微生物组的“核心”组分的定义一直是不确定的。 困难主要是由于眼睛的低生物量性质，缺乏强大的数据集，仅依赖于下一个 代测序（NGS），以及无法显示微生物和宿主之间的因果关系 physiology.在此，我们概述了一个直接解决这些知识差距的建议， 未来研究的基础，旨在调查眼表微生物组如何影响健康， 疾病我们招募了一个多学科的研究团队，由临床和基础领域的专家组成， 眼科学研究、低生物量场所的微生物组和免疫学。在这个团队中，我们计划首先 在两个地理位置（匹兹堡和迈阿密）表征健康人眼部微生物组 使用分子技术（DNA和RNA测序）和培养组学纵向地（0、1周、3个月）观察。 分子技术将为我们提供最广泛的细菌群体，而培养组学将为我们提供 我们计划将这些细菌储存在坎贝尔实验室， 可以成为社区未来研究的资源。其次，由于免疫力通常产生于 微生物组的组成部分与宿主保持长时间的联系，我们计划使用 针对眼部细菌的人类免疫应答以区分定殖细菌和瞬时细菌 可能被冲走的细菌。为了进一步完善对眼部定植细菌的理解，我们计划 用人眼部细菌感染无菌（GF）和无特定病原体（SPF）小鼠。经过漫长的 一段时间后，我们计划从老鼠的眼睛中重新分离细菌。眼睛上仍然存在的细菌 因此，认为具有更高可能性的人的眼表面定殖物。的 开发这样的模型将允许其他人评估其他细菌的定殖能力， 被认为是眼表微生物组的核心组成部分。总之，我们提案中的数据将是一个 这是整个社区的资源，因为我们将：1）生成一组强大的眼部微生物组 测序数据将提供关于眼部微生物组特征的稳定性和一致性的信息， 2)创建了一个从健康人眼睛中获得的细菌库，3）定义了使用人类眼睛的协议， 免疫来测量细菌的定殖能力，4）开发了体内动物模型来评估如何 可以从眼睛中接种和再分离眼细菌。这些资源将免费用于 并将作为进一步研究眼部微生物组如何影响眼表的基础 健康和疾病。

PROJECT SUMMARY Recent studies have shown data suggesting that the ocular microbiome exists and influences ocular surface health and disease. Despite this defining the “core” components of the ocular surface microbiome has been difficult largely due to the low biomass nature of the eye, a lack of robust data sets, reliance only on next generation sequencing (NGS), and the inability to show causal relationships between microbes and host physiology. Herein, we have outlined a proposal that directly addresses these knowledge gaps and that will lay the foundation for future studies aimed at investigating how the ocular surface microbiome influences health and disease. We have recruited a multidisciplinary research team that consists of experts in clinical and basic ophthalmology research, microbiome of low biomass sites, and immunology. With this team, we plan to first characterize the healthy human ocular microbiome in two geographic locations (Pittsburgh and Miami) longitudinally (0, 1 week, 3 months) using molecular techniques (DNA and RNA sequencing) and culturomics. While molecular techniques will provide us with the broadest consortia of bacteria, culturomics will provide us with viable bacteria that we plan to bank in the Campbell Laboratory, so that bacteria from healthy human eyes can be a resource for the community in future studies. Next, because immunity is normally generated towards components of the microbiome that remain associated with the host for extended periods of time, we plan to use human immune responses directed against ocular bacteria to distinguish colonizing bacteria from transient bacteria that are likely washed away. To further refine the understanding of ocular colonizing bacteria, we plan to inoculate germ free (GF) and specific pathogen free (SPF) mice with human ocular bacteria. After an extended period of time, we plan to re-isolate bacteria from the eyes of mice. Bacteria that are still present on the ocular surface will be considered to have a higher likelihood of being an ocular surface colonizer in humans. The development of a model such as this will allow others to assess the colonizing ability of other bacteria that may be considered a core component of the ocular surface microbiome. In sum, data from our proposal will be a resource for the community at large because we will have: 1) generated a robust set of ocular microbiome sequencing data that will provide information on the stability and consistency of ocular microbiome signatures, 2) created a bank of bacteria that were acquired from healthy human eyes, 3) defined protocols to use human immunity to measure the colonizing ability of bacteria, 4) developed an in vivo animal model to assess how ocular bacteria can be inoculated and re-isolated from the eye. These resources will be free to use for the community and will act as a base to further investigate how the ocular microbiome influences ocular surface health and disease.