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.

项目总结 耐碳青霉烯类肠杆菌科细菌(CRE)是最紧迫的耐药菌之一。 公共卫生威胁。Cre大致分为产碳青霉烯酶和非碳青霉烯酶。 生产(即CP-Cre和非Cp-Cre)。虽然CP-CRE已经被深入研究，但系统地 来自美国和其他地方的研究表明，非CP-Cre约占Cre总量的50%。 最近公布的数据显示，感染非CP-CRE患者的预后与那些 感染产CP菌株。因此，缓解非CP-CRE发展和传播的战略是 急需之物。在这一应用的初步数据中，我们表明非CP-CRE可以从一个 染色体上转座子单位产生超广谱β-内酰胺酶的背景 (Tu)介导的β-内酰胺酶编码基因的扩增。此外，我们已经确定这些TU可以 在孔蛋白编码基因内插入和扩增。鉴于孔蛋白是碳青霉烯类抗生素的关键机制 进入细菌细胞，TU插入和扩增都增加了β-内酰胺酶的产量和 减少碳青霉烯类进入，从而导致非CP-CRE的出现。这就是我们的目标 建议确定β-内酰胺酶基因扩增在非慢性前列腺炎中的患病率和机制 Cre包括含有AMR元件的TU中断孔蛋白编码基因的频率。另外， 我们将使用一个实验性的进化系统来观察临床ESBL肠杆菌科细菌是如何分离出来的 非CP-Cre的进展和β-内酰胺酶基因扩增带来的适应成本。以特定的目标 1，我们将应用我们最近开发的组合长读/短读全基因组测序(WGS) 大群大肠埃希菌和肺炎克雷伯菌非CP-Cre分离株检测方法的探讨 编码β-内酰胺酶基因扩增的发生率及其机制。此外，我们将确定 编码β-内酰胺酶基因的增强表达对非Cp-Cre耐药性的影响 使用可诱导表达系统。在具体目标2中，我们将评估临床ESBL大肠杆菌和K。 肺炎杆菌对多种β-内酰胺类抗生素的反应进展为非CP-CRE 微流控系统，允许对实验进化进行纵向评估。通过应用我们的 结合WGS方法对系列分离株，我们将能够通过以下方式最终评估进化轨迹 哪些临床分离的超广谱β-内酰胺酶菌株在不产生碳青霉烯酶的情况下对碳青霉烯类产生耐药性。 最后，我们将通过以下方式确定在临床分离株中开发非CP-Cre的潜在适合性成本 在没有抗菌药物选择压力的情况下传代菌株。建议完成的研究 这将为更深入地探索基因的作用和机制奠定基础 广泛的临床重要AMR病原体的扩增。