Antimicrobial Resistance and Horizontal Gene Transfer in the Human Gut Microbiome in Response to an Antibiotic

项目总结 抗菌素耐药性(AMR)是一个日益普遍和严重的世界性问题。大多数情况下， AMR由水平基因转移(HGT)产生，涉及可移动的遗传元件(MGE)，如质粒 还有噬菌体。人类的肠道是AMR进化和传播的热点；共生体是一种 通过HGT治疗病原体的AMR来源。减缓AMR的演变和传播既是可能的，也是必要的。 了解AMR在体内发展和传播的进化史是必要的，以促进 有效的管理。然而，这方面的知识仍然有限。AMR进化的重要方面变得显而易见 只有在复杂的环境和不同社区的背景下才能这样做。我们将研究抗生素的反应 体内人体肠道微生物区系的暴露，以及复杂的粪便衍生特定受试者社区的暴露 在体外，在高时间分辨率下并使用创新的方法。我们将把AMR和其他MGE- 利用高通量染色体构象捕获(Hi-C)技术将相关基因与宿主核心基因组相关联 并在接触抗生素之前、期间和之后监测细菌宿主中的这些基因和元素。短小的 拟议工作的长期目标是描述和评估从头突变和 HGT对抗生素暴露期间AMR在人体肠道微生物区系中的传播和发展的影响。长的- 学期目标是通过识别进化中的关键事件或转变来改进抗生素管理 AMR在体内的传播和传播，以及使这些事件发生的可能性较小的因素和条件。 目的1.确定健康人群肠道微生物区系中耐药基因的分布 人类。我们将使用Hi-C和元基因组测序从粪便中分离菌株水平的微生物基因组 在接触环丙沙星之前，在8周的无抗生素间隔内收集了60名健康成年人的样本。 我们将识别潜在的AMR基因，并确定它们在核心和辅助基因组中的分布，以及 与可移动的遗传元素相关。 目的2.研究环丙沙星对人乳腺癌细胞AMR基因丰度和动员的影响 活体中的人体肠道微生物区系。我们将使用Hi-C和元基因组测序来检测收集的粪便样本 在环丙沙星暴露期间和之后的相同的60名受试者中，并表征其组成和 影响AMR出现的选择性扫掠的动力学。 目的3.表征环丙沙星对人工合成的人AMR基因动员的影响 肠道来源的微生物群落在体外。我们将从体外传播暴露前的粪便群落 所有60名受试者，以及从暴露前的5名受试者样本中产生复杂的合成群落， 然后在多种环丙沙星制度下厌氧通过原生和合成群落。我们 将在体外通过HGT和从头突变来确定影响AMR出现的因素和条件。

PROJECT SUMMARY Antimicrobial resistance (AMR) is an increasingly prevalent and serious problem worldwide. Most often, AMR arises from horizontal gene transfer (HGT), involving mobile genetic elements (MGE) such as plasmids and phages. The human gut is a hotspot for both the evolution and spread of AMR; commensals serve as a source of AMR for pathogens via HGT. Slowing the evolution and spread of AMR is both possible and necessary. Knowledge about the evolutionary history of AMR development and dissemination in vivo is essential to facilitate effective stewardship. Yet, this knowledge remains limited. Important aspects of AMR evolution become evident only in complex environments and in the setting of diverse communities. We will study responses to antibiotic exposure in the human gut microbiota in vivo, as well as in complex stool-derived subject-specific communities in vitro, at high temporal resolution and using innovative approaches. We will link AMR and other MGE- associated genes to their host core genomes using high throughput chromosome conformation capture (Hi-C) and monitor these genes and elements in bacterial hosts before, during and after antibiotic exposure. The short term-objectives of the proposed work are to characterize and assess the contributions of de novo mutations and HGT to the spread and development of AMR in the human gut microbiota during antibiotic exposure. The long- term objectives are to improve antibiotic stewardship by identifying critical events or transitions in the evolution and dissemination of AMR in vivo, and the factors and conditions that make those events less likely. Aim 1. Determine the distribution of antimicrobial resistance genes in the gut microbiota of healthy humans. We will use Hi-C and metagenomic sequencing to resolve strain-level microbial genomes from stool samples of 60 healthy adults collected over an 8-week antibiotic-free interval, prior to a ciprofloxacin exposure. We will identify potential AMR genes and determine their distribution within core and accessory genomes, and in association with mobile genetic elements. Aim 2. Characterize the effects of ciprofloxacin on the abundance and mobilization of AMR genes in the human gut microbiota in vivo. We will use Hi-C and metagenomic sequencing to assay stool samples collected from the same 60 subjects during and after the ciprofloxacin exposure, and to characterize the composition and dynamics of selective sweeps that affect the emergence of AMR. Aim 3. Characterize the effects of ciprofloxacin on the mobilization of AMR genes in synthetic, human gut-derived microbial communities in vitro. We will propagate pre-exposure fecal communities ex vivo from all 60 subjects, as well as generate complex, synthetic communities from pre-exposure samples of 5 subjects, and then passage both bulk and synthetic communities anaerobically under multiple ciprofloxacin regimes. We will identify factors and conditions that affect emergence of AMR through HGT and de novo mutations in vitro.