Role of β-lactamase encoding gene amplification in the development of non-carbapenemase producing, carbapenem-resistant Enterobacteriaceae

β-内酰胺酶编码基因扩增在产生非碳青霉烯酶、耐碳青霉烯肠杆菌科细菌中的作用

• 批准号: 10373951
• 负责人: SAMUEL A SHELBURNE
• 金额: $ 19.96万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-17 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10373951
• 关键词: Accounting; Antibiotic Resistance; Antimicrobial Resistance; Antimicrobial susceptibility; Carbapenems; Cells; Chromosomal Duplication; Clinical; Data; Development; Elements; Enterobacteriaceae; Enzymes; Escherichia coli; Evolution; Exposure to; Extended-spectrum β-lactamase; Frequencies; Gene Amplification; Generations; Genes; Genomics; Goals; Individual; Interruption; Investigation; Klebsiella pneumoniae; Laboratories; Laboratory Study; Measurement; Mediating; Membrane; Methods; Microfluidics; Mutation; Outcome; Patient-Focused Outcomes; Pharmaceutical Preparations; Phenotype; Predisposition; Prevalence; Prevention strategy; Production; Public Health; Publishing; Research; Role; System; Technology; Testing; United States; VDAC1 gene; antimicrobial; antimicrobial resistant infection; antimicrobial resistant pathogen; bacterial resistance; base; beta-Lactam Resistance; beta-Lactamase; beta-Lactams; carbapenem resistance; carbapenem-resistant Enterobacteriaceae; carbapenemase; cohort; cost; fitness; genome sequencing; inducible gene expression; innovation; insight; nanopore; novel; pressure; promoter; response; treatment strategy; whole genome

PROJECT SUMMARY Carbapenem resistant Enterobacteriaceae (CRE) are among the most urgent antimicrobial resistant (AMR) public health threats. CRE are broadly divided into carbapenemase producing and non-carbapenemase producing (i.e. CP-CRE and non-CP-CRE). Although CP-CRE has been intensely investigated, systematic studies from the United States and elsewhere show that non-CP-CRE make up some 50% of total CRE. Recently published data show that the outcomes for patients infected with non-CP-CRE are as poor as those infected with CP producing strains. Thus, strategies to mitigate non-CP-CRE development and spread are urgently needed. In the preliminary data to this application, we show that non-CP-CRE can emerge from an extended spectrum β-lactamase (ESBL) producing background by a chromosomally located transposon unit (TU) mediated amplification of β-lactamase encoding genes. Moreover, we have identified that these TUs can insert and amplify within porin encoding genes. Given that porins are key mechanisms by which carbapenems enter the bacterial cells, the TU insertion and amplification both increases β-lactamase production and decreases carbapenem entry, thereby engendering the emergence of non-CP-CRE. It is the goal of this proposal to determine the prevalence and mechanisms underlying β-lactamase gene amplification in non-CP- CRE including how often porin encoding genes are interrupted by TUs containing AMR elements. Additionally, we will use an experimental evolution system to observe how clinical ESBL Enterobacteriaceae isolates progress to non-CP-CRE and the fitness costs engendered by β-lactamase gene amplification. In specific aim 1, we will apply our recently developed combined long-read/short-read whole genome sequencing (WGS) approach to a large cohort of Escherichia coli and Klebsiella pneumoniae non-CP-CRE isolates to determine the prevalence and mechanisms of β-lactamase encoding gene amplification. Additionally, we will determine the impact of augmented expression of β-lactamase encoding on non-CP-CRE antimicrobial susceptibility using an inducible expression system. In specific aim 2, we will assess how clinical ESBL E. coli and K. pneumoniae progress to non-CP-CRE in response to various β-lactam antimicrobials using a novel microfluidics system which allows for longitudinal assessments of experimental evolution. By applying our combined WGS approach to serial isolates, we will be able to definitively assess the evolutionary trajectory by which clinical ESBL isolates develop carbapenem resistance in the absence of producing a carbapenemase. Finally, we will determine the potential fitness costs of non-CP-CRE development in clinical isolates by passaging strains in the absence of antimicrobial selective pressure. Completion of the research proposed herein will set the stage for more in-depth exploration of the role and mechanisms underlying gene amplification in a wide range of clinically important AMR pathogens.

项目摘要 抗碳青霉型肠杆菌科（CRE）是最紧急的抗菌抗药性（AMR）之一 公共卫生威胁。 CRE大致分为碳青霉酶产生和非核糖烯酶 生产（即CP-CRE和非CP-CRE）。尽管已经积极研究了CP-CRE 美国和其他地方的研究表明，非CP-CRE占总CRE的50％。 最近发布的数据显示，感染非CP-CRE的患者的结果与 感染CP产生菌株。那是减轻非CP-CRE开发和传播的策略 迫切需要。在此应用程序的初步数据中，我们表明非CP-CRE可以从一个 扩展光谱β-内酰胺酶（ESBL）由染色体位于转座子单元产生背景 （TU）介导的编码基因的β-内酰胺酶的扩增。而且，我们已经确定这些tus可以 插入和放大孔蛋白编码基因。鉴于孔素是碳青霉烯的关键机制 进入细菌细胞，TU插入和扩增都会增加β-内酰胺酶的产生，并 减少碳青霉烯的进入，从而导致非CP-CRE的出现。这是目标的目标 提议确定非CP-中β-内酰胺酶基因扩增的患病率和机制 包括孔蛋白编码基因的频率被包含AMR元素的TU中断。此外， 我们将使用实验进化系统来观察临床ESBL肠杆菌科的分离株如何 进展到非CP-CRE以及由β-内酰胺酶基因扩增产生的适应性成本。在特定目标中 1，我们将应用我们最近开发的长阅读/短阅读的整个基因组测序（WGS） 大量大肠杆菌和肺炎肺炎非CP-CR-CRE分离株的方法 编码基因扩增的β-内酰胺酶的患病率和机制。此外，我们将确定 编码β-内酰胺酶增强表达对非CP-CR抗菌易感性的影响 使用诱导表达系统。在特定的目标2中，我们将评估临床ESBL E. Coli和K. 肺炎使用新颖的 微流体系统允许对实验进化进行纵向评估。通过应用我们的 联合WGS的连续分离株方法，我们将能够通过 在没有产生碳青霉酶的情况下，临床ESBL分离出会产生碳青霉烯耐药性。 最后，我们将通过 在没有抗菌选择性压力的情况下，通过菌株。拟议的研究完成 此处将为对基因的作用和机制进行更深入的探索奠定基础 在各种临床重要的AMR病原体中放大。