Quantification of bacterial strain count in the human gut microbiome in health and disease

项目摘要 在炎症性肠病（IBD）中，肠道微生物组表现出物种多样性减少， 然后在FMT之后被逆转。然而，没有研究确定微生物组改变的共同模式 与IBD有因果关系，也没有任何研究阐明FMT在IBD中成功的机制。 有反应的IBD个体的子集。在FMT中，物种和品系多样性都在增加， 目前的研究只跟踪物种多样性的变化，导致微生物组的基本特征 这是变化的，但没有在试验中或在与健康和IBD比较的横断面研究中测量 微生物群落因此，目前还不清楚是否是物种或菌株多样性的增加是关键的FMT 成功或IBD微生物组的菌株水平结构是否存在独特差异。由于微生物 在菌株水平上发现了功能变异，这是对IBD中微生物组组成的功能性理解 可能处于应变水平。然而，到目前为止，还没有研究系统地表征应变水平 健康或疾病中的微生物组结构。该建议旨在定义应变计数-唯一的 菌株每种细菌物种稳定地保持在个体中-在健康和IBD微生物组中，并且进一步 研究疾病中菌株计数的功能影响。我们将通过使用高- 通量培养技术，以分离和测序数千种肠道细菌分离株，用于所有可培养的 物种的宽度为重点的方式，并验证我们的宽度为重点的量化使用深度- 集中的方法，从十种最常见的肠道物种中对更多的基因组进行密集测序。为我们 在深度测序中，我们将使用喂食不同饮食的gnotobiotic小鼠来富集低丰度菌株。这 实验方法使我们能够提高效率，通过这种方法，我们可以捕获特定的菌株， 物种，并实现更准确的菌株计数定量。目标1-根据我们的初步调查结果， 我们预计每个物种在肠道微生物群中可以维持的菌株数量有限， 我们的方法对足够数量的基因组进行测序以量化菌株计数。我们的目标是定义应变 健康和IBD微生物组计数，这将允许更高分辨率的功能研究 微生物组的组成。在目标2-我们将研究增加应变的影响 计算结肠炎的严重程度。我们将联合收割机将从IBD分离的菌株和健康 图4显示了已知菌T细胞转移结肠炎模型中的微生物群。定义的社区将允许我们具体地 增加菌株数量，同时保持物种多样性稳定。通过研究应变计数的功能作用， 可以揭示微生物组菌株水平结构对疾病过程的潜在贡献， 确定优化FMT治疗IBD的适当靶点。

PROJECT SUMMARY In Inflammatory Bowel Disease (IBD), the gut microbiome demonstrates reduced species diversity that can then be reversed following FMT. However, no study has identified a common pattern of microbiome alteration that is causally related to IBD nor has any study elucidated the mechanisms underlying FMT success in the subset of individuals with IBD that respond. In FMT, both species and strain diversity are increasing but currently studies only track changes in species diversity, resulting in a fundamental feature of the microbiome that is changing but not measured in trials or in cross sectional studies of comparing with healthy and IBD microbiomes. Thus, it is unclear whether it is the increase in species or strain diversity that is critical to FMT success or if there are unique differences in the strain-level structure of the IBD microbiome. Since microbial functional variation is found at the strain level, a functional understanding of microbiome composition in IBD may lie at the strain level. However, no studies so far have systematically characterized the strain-level microbiome structure in health or disease. This proposal aims to define strain count—the number of unique strains each bacterial species stably maintains in an individual—in healthy and IBD microbiomes and to further investigate the functional impact of strain count in disease. We will quantify strain count by employing a high- throughput culturing technique to isolate and sequence thousands of gut bacterial isolates for all culturable species in a breadth-focused manner and validate our breadth-focused quantifications by using a depth- focused approach to intensely sequence more genomes from ten of the most common gut species. For our depth-sequencing, we will use gnotobiotic mice fed different diets to enrich for low abundance strains. This experimental approach allows us to increase the efficiency by which we capture the strains of a particular species and achieve a more accurate quantification of strain count. Aim 1—Based on our preliminary findings, we anticipate that there is a limited number of strains each species can maintain in the gut microbiota and that our method sequences a sufficient number of genomes to quantify strain count. We aim to define strain count in healthy and IBD microbiomes which will allow for higher resolution, functional investigations into microbiome composition in disease. In Aim 2—we will investigate the effect of increasing strain count on colitis severity. We will combine defined communities of strains isolated from an IBD and healthy microbiota in a gnotobiotic T cell transfer colitis model. The defined communities will allow us to specifically increase strain count while holding species diversity stable. By studying the functional role of strain count, we can uncover the potential contributions of microbiome strain-level architecture to the disease process and identify appropriate targets for optimizing FMT for the treatment of IBD.