Drug resistance enablers and their role in antibiotic treatment failure

项目1：概要 抗生素耐药性是全球最大的健康挑战之一，缺乏明确的解决途径。 一个解决方案最主要的问题是医院内的疾病，这些疾病不适合抗菌治疗， 与传染病无关的可治疗疾病转化为生命终结事件。这种抗生素的来源 治疗失败（ATF）各不相同，但失败本身是非常常见的，与医疗保健相关的肺炎 导致高达70%的案件都是由ATF造成的虽然ATF通常与耐药性的传播有关， 细菌，很大一部分病例不能如此容易地解释。此外，收购 抗菌素耐药性（AMR）本身似乎比最初设想的更复杂， 突变似乎存在于微生物种群中，这些微生物种群可能是发展的垫脚石。 的AMR。这些前体突变是促成因素，导致增加药物耐受性，持久性， 异源抗性，其结果是亚群可以在存在异源抗性的情况下保持活力。 随着抗生素浓度的涨落，抗生素允许耐药群体的生长。的 这项工作的中心假设是双重的：这些使能突变增加了微生物的进化能力， 获得抗菌素耐药性，并且这些具有使能突变的菌株可以被证明引起 ATF，即使在没有获得临床定义的AMR的情况下。为了验证这一假设， 建议分析革兰氏阴性菌鲍曼不动杆菌，医院感染的病原体， 由于获得多药耐药性，肺炎变得越来越难以治疗。 研究A.鲍曼不动杆菌启动子变异体、ATF和药物副产物 抗性突变体，提出了一系列的实验，系统地确定了一个大的光谱， 使能突变，包括必需基因的部分功能损伤。这些使能因素的特点是， 确定为AMR的发展提供前体库的亚群的相对大小， 包括与项目3和科学核心的合作。这将有助于评估以下可能性： 一种变体最终会导致AMR。一旦确定了促成因素库，将根据 它们在培养条件下引起ATF的能力以及它们产生ATF的能力， 抗性突变体类似地，ATF和AMR的临床发展将在小鼠肺炎中建模。 模型这将通过首先确定在不存在使能突变的情况下， AMR的已知决定因素，可以导致ATF，然后确定使能突变是否增加 在疾病过程中对耐药性的进化。

PROJECT 1: SUMMARY Antimicrobial resistance is one of the greatest worldwide health challenges lacking a clear path toward a solution. Chief among the concerns are nosocomial diseases recalcitrant to antimicrobial treatment, turning treatable illnesses unrelated to infectious diseases into end-of-life events. The source of this antibiotic treatment failure (ATF) varies, but failure itself is remarkably common, with healthcare-associated pneumonia resulting ATF in up to 70% of the cases. Although ATF is often tied to the spread of antimicrobial resistant bacteria, a large proportion of cases cannot be so easily explained. Furthermore, the acquisition of antimicrobial resistance (AMR) itself appears to be more complex than originally envisioned, as precursor mutations appear to exist in microbial populations that may be required as stepping stones for the development of AMR. These precursor mutations are enablers, resulting in increasing drug tolerance, persistence, or heteroresistance, with the consequence that a subpopulation can remain viable in the presence of antimicrobials allowing the outgrowth of resistant populations as antibiotic concentrations ebb and flow. The central hypothesis of this work is twofold: these enabler mutations increase the evolvability of the microbe to acquire antimicrobial resistance, and these strains having enabler mutations can be demonstrated to cause ATF even in the absence of acquisition of clinically-defined AMR. To test this hypothesis, experiments are proposed to analyze the Gram-negative bacterium Acinetobacter baumannii, a causative agent of nosocomial pneumonia that has become increasingly difficult to treat due to the acquisition of multidrug resistance. To study the connection between A. baumannii enabler variants, ATF and the outgrowth of drug resistant mutants, a series of experiments are proposed that systematically identifies a large spectrum of enabler mutations, including partial function lesions in essential genes. The enablers will be characterized to determine the relative size of subpopulations that provide precursor pools for the development of AMR, in work involving collaboration with Project 3 and the Scientific Core. This will allow an evaluation of the likelihood that a variant will eventually give rise to AMR. Once the pool of enablers is identified, they will be tested under multiple growth conditions for their ability to cause ATF in culture conditions as well as their ability to generate resistant mutants. Similarly, the clinical development of ATF and AMR will be modeled in a murine pneumonia model. This will be accomplished by first determining if acquisition of an enabler mutation, in the absence of known determinants of AMR, can result in ATF, and then determining if enabler mutations increase the evolvability to drug resistance during the course of disease.