Comparative resistomics of Gram-negative bacterial pathogens

摘要 增加抗生素耐药性需要扩大对细菌感染机制的研究 病原体获得并维持抗药性。尽管快速扩大的基因组图谱 临床分离株和实验室研究中导致耐药性的突变，我们对动力学和 抗菌素耐药性演变的潜在机制仍然不充分。为了填补这一空白，作者们 将连续培养装置中的实验进化与时间相结合-- 对进化中的细菌培养物进行超深基因组测序。已开发的产品的实用性 基于抗菌素的工作流程得到已发表和正在进行的已建立的抗菌药研究的支持 和实验药物候选人。范围内比较耐药组学研究的初步结果 革兰氏阴性细菌的耐药性的进化提供了初步的支持 抗病毒药通过一组有限的轨迹进行，这些轨迹是由阻力和适应性的组合定义的 限制了近似的临床进化，这有利于低频/高适应度的选择，而不是高频率 频率/低适应度突变体。一种比较耐药组学方法使普遍的和 在最近的一项关于实验进化的概念验证研究中展示了应变特有的机制 3株革兰氏阴性杆菌对环丙沙星的耐药性拟议中的5年项目将考验中央 通过追求以下具体目标，提出和扩展抗菌素耐药性的假设和探索：(I)在 目标1，将使用已建立的基于稳定期的工作流程来确定主要的驱动机制 对广谱临床抗生素环丙沙星、粘菌素、替格环素和美罗培南耐药 难治性革兰阴性杆菌鲍曼不动杆菌ATCC17978、铜绿假单胞菌 ATCC27853、大肠杆菌ATCC25922和肺炎克雷伯菌ATCC13883；(Ii)在AIM 2中， 选定的克隆将被系统地表征，以评估单个突变和 它们的组合对获得性抵抗力和适合性的影响；(3)目标3将利用基于moribiostat的 以鲍曼不动杆菌为重点的多重耐药实验进化的第一步工作流程 并从单一药物进化研究中选择的克隆开始。将在所有方面取得的结果 计划中的研究将作为深入生物信息学分析的主题(包括与公共数据的比较 临床分离株)、预测性建模、集成和与广泛研究社区共享 关于抗菌素耐药性进化的综合基因组学(IGEAR)的专门网站。建议数 研究有望在推进支持合理优化的方法论方面产生翻译影响 抗生素治疗方案和将耐药性降至最低的新药的开发。

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.