Susceptibility and resistance of multidrug-resistant gram-negative bacteria to novel beta-lactam/beta-lactamase inhibitor combinations

项目摘要/摘要 β-内酰胺/b-内酰胺酶抑制剂(BLBLI)类抗生素是抗菌药物的支柱之一 革兰氏阴性菌的治疗，如大肠杆菌和铜绿假单胞菌。直到最近，所有的b- 这些组合中的内酰胺酶抑制剂本身就是缺乏直接抗菌剂的β-内酰胺类化合物。 活动。然而，细菌对目前所有可用的BLBLI都产生了越来越多的抗药性。致信地址 对于这个问题，正在开发新的组合，其中包括新的二氮杂双环辛烷(DBO)b- 内酰胺酶抑制剂，是具有内在直接抗菌活性的非β-内酰胺类化合物 通过与青霉素结合蛋白2(PBP2)结合而介导。虽然这些新的扩展频谱 组合是有希望的，它们将被用来治疗的高度耐多药细菌容易 其他耐药机制的发展，特别是PBP2介导的抗菌活性 已知在治疗过程中容易出现耐药性。这个项目的总体目标是 表征对新型含DBO的BLBLIs的抗性发展，以发现如何最好地 在利用它们的同时，防止出现耐药性。在目标1中，对DBO的抵抗率- 将在一大批不同的革兰氏阴性细菌中对含有BLBLI进行评估，包括 大肠埃希菌、肺炎克雷伯氏菌、阴沟肠杆菌复合体和铜绿假单胞菌。在目标2中，机制 对这些药物的耐药性将使用两种不同的组学方法进行调查。第一，全基因组 测序将用于识别已经产生抗药性的菌株的突变。第二，基因 使用RNA-Seq的表达谱将被用来研究两者的转录反应 对含有DBO的BLBLI处理的敏感和抗药性细菌。目标3的目标是了解如何 在更好地模拟体内治疗条件的模型中防止对含有DBO的BLBLI的耐药性。一个 时间杀死试验，一种中空纤维感染模型，允许模拟变化的抗生素浓度 随着时间的推移，小鼠大腿感染模型将被用来识别防止耐药性的组合 在更长的药物暴露时间内，耐药菌株的全基因组测序将用于 将这些模型中发生的导致耐药性的突变与在体外观察到的标准突变进行比较 化验。拟议的项目完成后，将为利用小说的最有效方式提供指南 含DBO的BLBLIs在有效治疗MDR感染患者的同时防止其出现 抵抗的力量。

Project Summary/Abstract Antibiotics in the b-lactam/b-lactamase inhibitor (BLBLI) class are among the mainstays of antimicrobial treatment for gram-negative bacteria such as E. coli and Pseudomonas aeruginosa. Until recently, all b- lactamase inhibitors in these combinations were themselves b-lactam compounds that lacked direct antimicrobial activity. However, bacteria are increasingly developing resistance to all currently available BLBLIs. To address this problem, new combinations are being developed that incorporate novel diazabicyclooctane (DBO) b- lactamase inhibitors, which are non-b-lactam compounds that possess intrinsic direct antimicrobial activity mediated by binding to penicillin-binding protein 2 (PBP2). While the expanded spectrum of these new combinations is promising, the highly multidrug-resistant bacteria they will be used to treat are prone to the development of additional resistance mechanisms, and PBP2-mediated antibacterial activity in particular is known to be vulnerable to the emergence of resistance during treatment. The overall goal of this project is to characterize the development of resistance to novel DBO-containing BLBLIs in order to discover how best to make use of them while preventing the emergence of resistance. In Aim #1, rates of resistance to DBO- containing BLBLIs will be assessed among a large, diverse collection of gram-negative bacterial strains including E. coli, Klebsiella pneumoniae, Enterobacter cloacae complex, and P. aeruginosa. In Aim #2, mechanisms of resistance to these agents will be investigated using two different ‘omics approaches. First, whole genome sequencing will be used to identify mutations in strains in which resistance has developed. Second, gene expression profiling using RNA-Seq will be employed to investigate the transcriptomic response of both susceptible and resistant bacteria to DBO-containing BLBLI treatment. The goal of Aim #3 is to understand how to prevent resistance to DBO-containing BLBLIs in models that better simulate in vivo treatment conditions. A time-kill assay, a hollow-fiber infection model, which allows for simulation of changing antibiotic concentrations over time, and a mouse thigh infection model will be employed to identify combinations that prevent resistance over longer periods of drug exposure, and whole genome sequencing of resistant isolates will be used to compare resistance-conferring mutations that occur in these models to those observed in standard in vitro assays. The proposed project, when completed, will provide a guide to the most effective ways to utilize novel DBO-containing BLBLIs in order to effectively treat patients with MDR infections while preventing the emergence of resistance.