Comparative resistomics of Gram-negative bacterial pathogens

摘要 抗生素耐药性的增加需要扩大对细菌耐药机制的研究。 病原体获得并保持耐药性。尽管快速扩大基因组图谱， 临床分离株和实验室研究中的耐药突变，我们对动力学和 抗菌素耐药性演变的潜在机制仍然不足。为了填补这一空白，作者 该建议的联合收割机在连续培养装置中的实验进化，morbidostat，与时间- 解决了进化细菌培养物的超深度基因组测序。发达国家的效用 基于莫比司他的工作流程得到已发表和正在进行的已确定抗菌剂研究的支持 和实验性候选药物在一个范围内比较阻力学研究的初步结果 革兰氏阴性菌种的耐药性的进化提供了初步的支持， morbidostat通过由抵抗力和适应性的组合定义的一组有限的轨迹进行 限制近似临床进化，这有利于选择低频/高适应性而不是高适应性。 频率/低适应性突变体。一种比较阻力学的方法，使映射的普遍和 应变的具体机制，如在最近的概念验证研究的实验演变， 三种革兰阴性菌对环丙沙星耐药。五年计划将测试中央 通过追求以下具体目标，对抗菌剂耐药组的假说和扩展探索进行了研究：（i）在 目的1、建立基于莫比司他的工作流程，确定主要驱动机制 对广谱临床抗生素，环丙沙星，粘菌素，替加环素和美罗培南的耐药性， 难治性革兰氏阴性细菌病原体，鲍曼不动杆菌ATCC 17978，铜绿假单胞菌 ATCC27853，E. coli ATCC 25922和K.肺炎链球菌ATCC 13883;（ii）在Aim 2中，一组代表性的 选择的克隆将被系统地表征以评估单个突变的影响， （iii）目标3将利用基于moribidostat的 工作流程，使第一步的多药耐药性的实验演变集中在A。鲍曼不 并从单一药物进化研究中选择的克隆开始。将在所有项目中获得的结果 计划中的研究将是深入生物信息学分析的主题（包括与公开数据的比较 用于临床分离株）、预测建模、通过 抗生素耐药性进化的综合基因组学（iGEAR）。拟议 研究预计将在推进方法学方面产生转化影响，以支持合理优化 抗生素治疗方案和开发具有最小耐受性的新药。

ABSTRACT Increasing antibiotic resistance necessitates expanding research into the mechanisms by which bacterial pathogens acquire and perpetuate drug resistance. Despite rapidly expanding genomic mapping of resistance-conferring mutations in clinical isolates and laboratory studies, our knowledge of dynamics and mechanisms underlying evolution of antimicrobial resistance is still insufficient. To fill-in this gap, the authors of this proposal combine experimental evolution in a continuous culturing device, morbidostat, with time- resolved ultradeep genomic sequencing of evolving bacterial cultures. The utility of the developed morbidostat-based workflow is supported by published and ongoing studies with established antimicrobials and experimental drug candidates. The preliminary results of comparative resistomics studies over a range of Gram-negative bacterial species provided initial support to a premise that evolution of drug resistance in morbidostat proceeds via a limited set of trajectories defined by a combination of resistance and fitness constrains approximating clinical evolution, which favors selection of low-frequency/high-fitness over high- frequency/low-fitness mutants. A comparative resistomics approach enables mapping of both universal and strain-specific mechanisms as demonstrated in a recent proof-of-concept study on experimental evolution of ciprofloxacin resistance in three Gram-negative bacteria. The proposed 5-year project will test the central hypothesis and extend exploration of antimicrobial resistome by pursuing the following specific aims: (i) in Aim 1, the established morbidostat-based workflow will be used to determine major mechanisms driving resistance to broad-spectrum clinical antibiotics, ciprofloxacin, colistin, tigecycline and meropenem, in four difficult-to-treat Gram-negative bacterial pathogens, Acinetobacter baumannii ATCC17978, P. aeruginosa ATCC27853, E. coli ATCC25922, and K. pneumoniae ATCC13883; (ii) in Aim 2, a representative panel of selected clones will be systematically characterized to assess the effects of individual mutations and combinations thereof on acquired resistance and fitness; (iii) Aim 3 will leverage a moribidostat-based workflow to make first steps toward experimental evolution of multidrug resistance focusing on A. baumannii and starting from clones selected in single-drug evolution studies. The results that will be obtained in all planned studies will be a subject of in-depth bioinformatics analysis (including comparison with public data for clinical isolates), predictive modeling, integration and sharing with broad research community via a specialized web-site on integrative Genomics of Evolution of Antimicrobial Resistance (iGEAR). The proposed study is expected to have translational impacts in advancing methodology to support rational optimization of antibiotic treatment regimens and development of new drugs with minimized resistibility.