Population genomic analysis of gut microbial colonization in premature infants

描述(申请人提供)：与人体相关的微生物群落，特别是胃肠道，在健康和疾病中起着至关重要的作用。这项建议的目的是了解肠道微生物演替的特定模式如何与健康和疾病有关，特别是与新生儿坏死性小肠结肠炎(NEC)有关。尽管现有证据表明肠道微生物参与了NEC的发病机制，但这种关系的细节仍然知之甚少。目前，对早产儿肠道定植过程以及使用和不使用NEC的早产儿肠道定植过程的差异知之甚少。我们建议补充高通量的系统发育和元基因组分析来研究早产儿肠道在出生后的前三周的微生物定植。我们拟议的工作将以高分辨率阐明早产儿肠道定植期间发展的微生物群落的种群结构，并研究早期殖民者、胃肠道介导的选择、迁徙、移动元件对基因组变异和微生物生存的影响，并研究这些过程与NEC发病的关系。我们将使用“下一代”测序来解决在肠道定植的关键初始阶段的物种和种群水平的群落继承模式，这些婴儿已经和不会继续发展为NEC。我们将使用高通量的16S rRNA标签对出生后三周内每天收集的粪便样本进行测序来描述社区发展。然后，我们将对这些粪便时间序列样本中的一半进行微生物DNA的深度元基因组测序，以重建共存的细菌、古生菌、噬菌体和质粒群的基因组。这将使我们能够跟踪物种成员、群落结构、代谢潜力和种群水平的遗传异质性。我们将使用这些数据来测试初始联合体预测后续群落多样性的程度[目标1]，由噬菌体、插入元件和质粒介导的原位多样性与迁徙在确定种群结构和代谢潜力方面的重要性[目标2]，并确定与健康和疾病相关的生态轨迹[目标3]。我们的初步元基因组数据最终表明，所提出的方法可以用于从具有足够种群深度的早产儿肠道样本中重建共存生物的近完整基因组，以分析种群的异质性和动态。提高对早产儿肠道定植过程的了解，可以通过提出更有效的疾病预防和治疗战略来改善早产儿的结局。更广泛地说，这项研究将揭示生态系统殖民动态的一些方面，这些方面对人类健康和环境的其他方面有影响。好了！ 公共卫生相关性：肠道定植对于建立正常运作的消化系统至关重要，并与整个婴儿发育密切相关。正常肠道定植的紊乱可能会引发坏死性小肠结肠炎(NEC)等疾病的发生，NEC是早产儿最常见的胃肠道急症。使用在细菌菌株水平上解析的元基因组方法，我们将检查正常肠道定植的特征，并测试病毒与宿主的相互作用是否在疾病的发展中发挥作用。好了！

DESCRIPTION (provided by applicant): Microbial communities associated with the human body, in particular the gastrointestinal tract, play crucial roles in health and disease. The objective of this proposal is to understand how specific patterns in gut microbial succession are related to health and disease, and specifically to neonatal necrotizing enterocolitis (NEC). Although available evidence suggests that intestinal microbes contribute to the pathogenesis of NEC, the details of this relationship remain poorly understood. At present, relatively little is known about the gut colonization process in premature newborns, and about differences between this process in premature infants with and without NEC. We propose complementary high-throughput phylogenetic and metagenomic analyses to study microbial colonization of the premature infant gut during the first three weeks after birth. Our proposed work will elucidate, at high resolution, the population structure of microbial communities that develop during colonization of the premature infant gut and examine the roles of early colonists, gastrointestinal tract-mediated selection, immigration, the effects of mobile elements on genomic variation and microbial survival, and examine how these processes relate to onset of NEC. We will use "next-generation" sequencing to resolve species- and population-level community succession patterns during the critical initial period of gut colonization in babies that do and do not go on to develop NEC. We will profile community development using high-throughput 16S rRNA tag sequencing of stool samples collected daily during the first three weeks after birth. We will then carry out deep metagenomic sequencing of microbial DNA from half of these fecal time series samples to reconstruct genomes for coexisting bacterial, archaeal, phage, and plasmid populations. This will allow us to track species membership, community structure, metabolic potential, and population-level genetic heterogeneity. We will use these data to test the extent to which initial consortia predict succeeding community diversity [Aim 1], the importance of in situ diversification mediated by phage, insertion elements, and plasmids vs. immigration in determining population structure and metabolic potential [Aim 2], and to define ecological trajectories that correlate with health and disease [Aim 3]. Our preliminary metagenomic data conclusively demonstrate that the proposed approach can be used to reconstruct near-complete genomes of coexisting organisms from premature infant gut fecal samples with sufficient population depth to analyze population heterogeneity and dynamics. Improved understanding of the colonization process in the premature infant gut could translate to improved outcomes for premature babies by suggesting more effective strategies for disease prevention and treatment. More broadly, this research will uncover aspects of ecosystem colonization dynamics that have implications for other aspects of human health and the environment. ! PUBLIC HEALTH RELEVANCE: Gut colonization is critical in establishing a functioning digestive system and is intimately connected to overall infant development. Perturbation of normal gut colonization may trigger onset of diseases such as necrotizing enterocolitis (NEC), the most common gastrointestinal emergency of premature babies. Using a metagenomic approach with resolution at the level of bacterial strains, we will examine features of normal gut colonization and test whether virus-host interactions might play a role in the development of disease. !