Heteroresistance Interdisciplinary Research Unit (Project 3)

摘要 异源耐药性（HR）是一种现象，其中抗生素耐药细菌的少数群体维持在细菌中。 由对抗生素敏感的细胞控制的种群。我们已经证明，HR通常无法通过临床检查检测到。 然而，这些结果表明，HR中存在的耐药细胞亚群可导致体内治疗失败。至关重要 深入了解HR，以便设计更有效和更灵敏的诊断方法，并 指导临床治疗。一个主要的未解决的调查领域是哪些参数控制的动态 项目3的目标是阐明抗性亚群的动态， 实验测试，由药理学，种群和进化动力学的定量建模支持， 异源抗性细菌重要的是，这将包括对抗生素治疗后的动力学以及 在去除药物后群体向易感性的逆转。为了实现这一目标，我们将开发和 分析了HR的数学模型的性质，其基础是从 项目1和2中的分子、遗传和单细胞微流控研究。用于数值分析的参数 这些模型的特性的1/4将用HR肠杆菌科的临床分离株（肠杆菌， 大肠杆菌、克雷伯氏菌）和鲍曼不动杆菌从核心B获得，并在项目1和2中进行了深入研究。为 我们将估计每个亚群：（i）综合药效学功能的参数，（ii） 敏感和抗性状态之间的过渡，以及（iii）这些抗性状态的适应性成本。利用中空纤维 生物反应器，分批培养和微流体，我们将评估模型的效果，并对其进行独立估计。 参数，适合HR-细菌面对抗生素的药效学，并且使用连续培养装置， 这些模型如何解释异质耐药感染的药物治疗动力学。连续转移培养 我们将估计在去除抗生素后向基线敏感状态转变的速率。基于 这些实验的结果，并在迭代过程中，模型将被修改，使它们更准确， HR药效学的预测类似物。对于研究的每个HR分离株，我们还将进行实验， 确定抗性亚群的频率是否由于抗生素介导的选择而改变（即，如果 耐药细胞的基线频率增加），并阐明是否存在HR将 取而代之的是持久的抵抗。这项研究的结果将首次提供一个广泛而详细的 了解HR的动态，有助于了解控制频率的参数 抵抗的亚群。这些研究将对诊断程序的发展产生重大影响， 检测HR和设计用于治疗对治疗抗生素表现出HR的细菌感染的方案。

ABSTRACT Heteroresistance (HR) is a phenomenon in which minority populations of antibiotic resistant bacteria are maintained in populations dominated by cells susceptible to that antibiotic. We have shown that HR is often undetected by clinical diagnostics, but that the subpopulations of resistant cells present in HR can lead to in vivo treatment failure. It is critical to gain a thorough understanding of HR in order to design more effective and sensitive diagnostics for its detection, and to guide clinical treatment. One major unaddressed area of investigation is which parameters control the dynamics of the resistant subpopulations in HR. The goal of Project 3 is to elucidate the dynamics of resistant subpopulations through experimental testing, supported by quantitative modeling of the pharmaco-, population- and evolutionary dynamics of heteroresistant bacteria. Importantly, this will include studies of both their dynamics upon antibiotic treatment as well as the reversion of the population toward susceptibility upon removal of the drug. Toward this end we will develop and analyze the properties of mathematical models of HR based on the mechanisms of heteroresistance derived from molecular, genetic and single-cell microfluidic studies in Projects 1 and 2. The parameters used for the numerical analyses of the properties of these models will be estimated with clinical isolates of HR Enterobacteriaceae (Enterobacter, Escherichia, Klebsiella) and Acinetobacter baumannii obtained from Core B and studied in depth in Projects 1 and 2. For each subpopulation we will estimate: (i) parameters of comprehensive pharmacodynamic functions, (ii) the rates of transition between susceptible and resistant states, and (iii) the fitness costs of these resistant states. Using Hollow Fiber Bioreactors, batch culture, and microfluidics, we will evaluate how well the models, with independent estimates of their parameters, fit the pharmacodynamic of HR-bacteria confronted with antibiotics and, with continuous culture devices, how well these models account for the dynamics of drug treatment of heteroresistant infections. In serial transfer culture we will estimate the rates of transition to baseline susceptible states following the removal of the antibiotics. Based on the results of these experiments, and in an iterative process, the models will be modified to make them more accurate and predictive analogs of the pharmacodynamics of HR. For each HR isolate studied, we will also perform experiments to determine if the frequency of the resistant subpopulations change as a consequence of antibiotic-mediated selection (i.e. if the baseline frequency of the resistant cells increases), and elucidate if there are conditions under which HR will be replaced by permanent resistance. The results from this research will for the first time, provide a broad and detailed understanding of the dynamics of HR, facilitating an understanding of the parameters that control the frequency of the resistant subpopulations. These studies will have a major impact on the development of diagnostic procedures to detect HR and the design of protocols for treating infections with bacteria that exhibit HR to the treating antibiotic.