Programmable benchtop bioreactors for scalable eco-evolutionary dynamics of the human microbiome

项目摘要/摘要 抗药性微生物病原体对公众健康构成严重而紧迫的威胁。随着吸毒率的上升- 耐药感染和新抗生素治疗方法的武器库不断减少，迫切需要 更好地了解、预测和预防抗菌素耐药性(AMR)出现的方法。至 为此，实验进化方法，其中微生物在实验室中进化 用户定义的条件提供了一个强大的范例来定义AMR的进化路径。这 方法阐明了进化抗性的遗传途径，并可以定义可以利用的因素 引导药物敏感状态并指导新的临床策略。然而，这一潜力 理解AMR演进的方法从根本上受到以下技术壁垒的限制 进行持续的培养和进化实验，这需要以下关键能力：1)规模 在不同的微生物、实验条件和抗生素之间进化；2)频繁的自动化 在长实验时间尺度上的扰动和反馈；3)控制以重现 哺乳动物的肠道环境，是AMR在体内进化的主要场所。所有现有工具都会在一次或多次故障中失败 更多这样的能力。更重要的是，实验室进化研究未能解释 细菌群落影响AMR的进化轨迹、动态和结果。我们建议填补 这一技术和实验空白是通过开发一流的桌面技术实现的， 自动化和受控的微生物进化研究，并将其应用于AMR中的两个紧迫问题。因为 肠道环境会耗尽氧气(厌氧)，目前的技术缺乏完全的氧气控制， 我们将首先开发一个通过微型生物反应器单独控制大气条件的系统。 (空气调节器)。我们将在Evolver平台上实现这一点，Evolver平台是一个开源微生物培养系统，用于 对生长条件的自动控制，易于适应新的控制功能，并且非常 可扩展。Evolver-atmostat的初步结果显示，连续 严格厌氧肠道微生物在台面上的培养和进化。第一项研究将确定 氧分压对大肠埃希菌AMR突变适合度的影响我们将实施一个 结合氧气梯度的大气恒定控制的自动抗生素选择制度，并使用 在AMR中元基因组测序以绘制氧、抗生素和菌株背景的相互作用图。这个 第二项研究将通过进化E. 具有跨越多种抗生素的肠道群落的大肠杆菌菌株。应用最先进的丰度量化方法 随着时间的推移和种群遗传学的方法，我们将定义生态和进化的图景 肠道中的大肠杆菌。总而言之，这项工作将产生一种变革性的技术，供 世界各地的研究人员，并开始揭示病原体如何在人类肠道生态系统中进化AMR。

PROJECT SUMMARY/ABSTRACT Antibiotic-resistant microbial pathogens are a grave and urgent threat to public health. With rising rates of drug- resistant infections and a diminishing arsenal of new antibiotic treatments, there is pressing need for approaches to better understand, predict, and prevent the emergence of antimicrobial resistance (AMR). To this end, experimental evolution approaches, in which microbial organisms are evolved in the laboratory in user-defined conditions, provide a powerful paradigm to define the evolutionary paths toward AMR. This approach has illuminated genetic pathways to evolving resistance, and can define factors that can be exploited to steer toward drug-susceptible states and guide new clinical strategies. However, the potential of this approach for understanding AMR evolution is fundamentally constrained by technological barriers in conducting continuous culture and evolution experiments, which requires the following key capacities: 1) Scale to evolve across a diversity of microbes, experimental conditions, and antibiotics; 2) Automation for frequent perturbations and feedback over long experimental time scales; 3) Control to reproduce key features of the mammalian gut environment, a primary site for the evolution of AMR in vivo. All existing tools fail in one or more of these capacities. And critically, laboratory evolution studies fail to account for how interactions within bacterial communities impact the evolutionary trajectory, dynamics, and outcomes of AMR. We propose to fill this technological and experimental void by developing a first-in-class, benchtop technology for scalable, automated, and controlled microbial evolution studies, and apply it to two pressing problems in AMR. Because the gut environment is depleted of oxygen (anaerobic), and current technology lacks complete oxygen control, we will first develop a system for individual control of atmospheric conditions across mini-bioreactors (atmostat). We will achieve this in the eVOLVER platform, an open-source microbial culture system for automated control of growth conditions that is easily adapted to new control features, and is exceedingly scalable. Preliminary results of eVOLVER-atmostat demonstrate unprecedented scale for continuous culture and evolution of strict anaerobic gut microbes on the benchtop. The first study will determine the effects of oxygen tension on the mutational fitness landscapes of AMR in E. coli strains. We will implement an automated antibiotic selection regime in combination with atmostat control of oxygen gradients, and employ metagenomic sequencing to map the interactions of oxygen, antibiotics, and strains backgrounds in AMR. The second study will determine how AMR emerges in the ecological context of the gut microbiome, by evolving E. coli strains with a gut community across multiple antibiotics. Applying state-of-the-art abundance quantification over time and population genetics approaches, we will define both the ecological and evolutionary landscape of E. coli in the gut community. Collectively, this work will produce a transformative technology to be used by researchers worldwide, and begin to reveal how pathogens evolve AMR in the human gut ecosystem.