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

项目概要/摘要 b-内酰胺/b-内酰胺酶抑制剂（BLBLI）类中的抗生素是抗微生物药物的支柱之一。 革兰氏阴性菌如大肠杆菌的治疗。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是缺乏直接抗微生物活性的β-内酰胺化合物， 活动然而，细菌对目前所有可用的BLBLI越来越多地产生耐药性。解决 为了解决这个问题，正在开发新的组合，其包含新的二氮杂双环辛烷（DBO）B- 内酰胺酶抑制剂，其为具有内在直接抗微生物活性的非β-内酰胺化合物 通过结合青霉素结合蛋白2（PBP 2）介导。虽然这些新的扩展频谱 联合用药是有希望的，他们将被用来治疗的高度多重耐药的细菌容易发生耐药。 其他耐药机制的发展，特别是PBP 2介导的抗菌活性， 已知在治疗过程中易出现耐药性。该项目的总体目标是 表征对含DBO的新型BLBLI的抗性的发展，以发现如何最好地 利用它们，同时防止出现耐药性。在目标#1中，对DBO的抗性率- 将在大量不同的革兰氏阴性细菌菌株中评估含有BLBLI的菌株，包括 E.大肠杆菌、肺炎克雷伯氏菌、阴沟肠杆菌复合菌和铜绿假单胞菌。在目标#2中， 将使用两种不同的组学方法研究对这些试剂的抗性。第一，全基因组 测序将用于鉴定已产生耐药性的菌株中的突变。第二，基因 使用RNA-Seq的表达谱分析将用于研究两种基因的转录组学应答。 对含DBO的BLBLI处理敏感和耐药的细菌。目标3的目标是了解如何 以防止在更好地模拟体内治疗条件的模型中对含DBO的BBLLI产生耐药性。一 时间杀灭试验，一种中空纤维感染模型，允许模拟不断变化的抗生素浓度 随着时间的推移，将采用小鼠大腿感染模型来识别防止耐药性的组合 耐药菌株的全基因组测序将用于 将这些模型中发生的耐药突变与标准体外试验中观察到的耐药突变进行比较 测定。拟议的项目完成后，将提供一个最有效的方式来利用新的指南， 含DBO的BLBLI，以有效治疗MDR感染患者，同时预防 抗战

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.