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 碳青霉烯类耐药肠杆菌科（CRE）是最紧迫的抗生素耐药（AMR）之一 公共健康威胁。CRE大致分为产碳青霉烯酶和不产碳青霉烯酶 生产（即CP-CRE和非CP-CRE）。虽然CP-CRE已被深入研究，系统 美国及其他地方的研究显示，非职业退休计划的综合招聘考试约占综合招聘考试总数的50%。 最近发表的数据显示，感染非CP-CRE的患者的结局与那些 感染CP产生菌株。因此，缓解非CP-CRE发展和传播的策略是 迫切需要。在本申请的初步数据中，我们表明，非CP-CRE可以从一个 染色体定位转座子单元产生超广谱β-内酰胺酶（ESBL）背景 (TU)介导的β-内酰胺酶编码基因扩增。此外，我们已经确定，这些TU可以 插入并扩增孔蛋白编码基因。鉴于孔蛋白是碳青霉烯类抗生素 进入细菌细胞后，TU插入和扩增都增加了β-内酰胺酶的产生， 减少碳青霉烯类进入，从而导致非CP-CRE的出现。这是我们的目标 建议确定非CP患者中β-内酰胺酶基因扩增的患病率和潜在机制， CRE包括孔蛋白编码基因被含有AMR元件的TU中断的频率。此外，本发明还 我们将使用实验进化系统来观察临床ESBL肠杆菌科菌株如何 非CP-CRE的进展以及β-内酰胺酶基因扩增产生的适应性成本。具体目标 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） 一个大队列的大肠埃希菌和肺炎克雷伯菌非CP-CRE分离株的方法，以确定 β-内酰胺酶编码基因扩增的发生率及机制。此外，我们将确定 β-内酰胺酶编码基因表达增强对非CP-CRE抗菌药物敏感性的影响 使用诱导表达系统。在具体目标2中，我们将评估临床ESBL E。埃希菌和 使用一种新的β-内酰胺类抗生素治疗肺炎进展为非CP-CRE 微流体系统，允许对实验进化进行纵向评估。通过应用我们 结合WGS方法对系列分离株，我们将能够明确评估进化轨迹， 临床ESBL分离株在不产生碳青霉烯酶的情况下产生碳青霉烯耐药性。 最后，我们将通过以下方式确定临床分离株中非CP-CRE开发的潜在适应性成本： 在不存在抗微生物选择压力的情况下传代菌株。完成拟议的研究 本文将为更深入地探索基因的作用和机制奠定基础 在广泛的临床上重要的AMR病原体中的扩增。