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

项目摘要 抗生素耐药性（AMR）是全球范围内日益普遍和严重的问题。大多数时候， AMR由水平基因转移（HGT）引起，涉及移动的遗传元件（MGE），如质粒 和。人类肠道是AMR进化和传播的热点;肠道是AMR的主要传播途径。 通过HGT检测病原体的AMR来源。减缓抗生素耐药性的发展和蔓延既是可能的，也是必要的。 了解体内AMR发展和传播的进化史对于促进 有效的管理。然而，这方面的知识仍然有限。AMR演变的重要方面变得明显 只有在复杂的环境和不同社区的背景下。我们将研究对抗生素 体内人体肠道微生物群以及复杂粪便来源的受试者特异性群落中的暴露 在体外，在高时间分辨率和使用创新的方法。我们将把AMR和其他MGE- 使用高通量染色体构象捕获（Hi-C）将相关基因与其宿主核心基因组关联 并在抗生素暴露之前、期间和之后监测细菌宿主中的这些基因和元件。短 术语-拟议工作的目标是表征和评估从头突变的贡献， HGT对抗生素暴露期间人类肠道微生物群中AMR的传播和发展。很长的- 长期目标是通过识别进化中的关键事件或转变来改善抗生素管理 和体内AMR的传播，以及使这些事件不太可能发生的因素和条件。 目标1。确定健康人肠道微生物群中抗菌素耐药基因的分布 人类我们将使用Hi-C和宏基因组测序来解析来自粪便的菌株水平的微生物基因组 在暴露于环丙沙星之前，在8周的无抗生素间隔期间收集60名健康成人的样品。 我们将识别潜在的AMR基因，并确定其在核心和辅助基因组中的分布， 与移动的遗传因子有关。 目标2.表征环丙沙星对大肠杆菌中AMR基因的丰度和动员的影响。 人体肠道微生物群我们将使用Hi-C和宏基因组测序来分析收集的粪便样本 来自相同的60名受试者在环丙沙星暴露期间和之后，并表征该组合物， 影响AMR出现的选择性扫描的动态。 目标3。表征环丙沙星对合成的人巨噬细胞中AMR基因动员的影响， 肠道衍生的微生物群落体外。我们将在体外繁殖暴露前的粪便群落， 所有60名受试者，以及从5名受试者的暴露前样本中生成复杂的合成社区， 然后在多个环丙沙星方案下厌氧传代本体和合成群落。我们 将通过体外HGT和从头突变确定影响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.