Heteroresistance Interdisciplinary Research Unit (Project 3)

摘要 异耐药(HR)是一种少数群体的抗生素耐药细菌维持在 由对这种抗生素敏感的细胞主导的种群。我们已经证明，HR通常是临床上没有发现的 诊断，但HR中存在的耐药细胞亚群可能导致体内治疗失败。这是至关重要的 对人力资源有透彻的了解，以便为其检测设计更有效和更灵敏的诊断方法，并 指导临床救治。一个尚未解决的主要研究领域是哪些参数控制着 HR中的抗性亚群。项目3的目标是阐明抗性亚群的动态，通过 实验测试，由药物、种群和进化动力学的定量建模支持 异源耐药细菌。重要的是，这将包括对它们在抗生素治疗中的动态以及对 在停药后，人群对易感性的回归。为此，我们将制定和实施 基于异质电阻机理的HR数学模型的特性分析 项目1和2中的分子、遗传和单细胞微流体研究。用于数值分析的参数 这些模型的特性将用HR肠杆菌科临床分离株(肠杆菌， Escherichia，Klebsiella)和鲍曼不动杆菌，在项目1和2中进行了深入研究。 我们将估计每个亚群：(I)综合药效学函数的参数，(Ii) 敏感状态和抗性状态之间的转换，以及(Iii)这些抗性状态的适应成本。使用中空光纤 生物反应器、间歇培养和微流控技术，我们将评估这些模型的效果，并对其进行独立评估 参数，符合面对抗生素的HR-细菌的药效学，并在连续培养装置中， 这些模型很好地解释了异耐药感染的药物治疗动态。在连续转移培养中 我们将估计在撤除抗生素后过渡到基线敏感状态的比率。基于 这些实验的结果，在迭代过程中，将对模型进行修改，使其更加准确和 HR药效学的预测性类似物。对于所研究的每个HR分离株，我们还将进行实验以 确定耐药亚群的频率是否由于抗生素介导的选择而改变(即 耐药细胞的基线频率增加)，并阐明是否存在HR将 取而代之的是永久抵抗。这项研究的结果将第一次提供广泛和详细的 了解人力资源的动态，有助于了解控制频率的参数 抗性亚群的数量。这些研究将对制定诊断程序产生重大影响 检测HR和治疗细菌感染的方案设计，这些细菌对治疗抗生素表现出HR。

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.