Unraveling colistin resistance in Klebsiella pneumoniae

解开肺炎克雷伯菌的粘菌素耐药性

• 批准号: 9919087
• 负责人: BARRY Neal KREISWIRTH
• 金额: $ 22.14万
• 依托单位: HACKENSACK UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-22 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9919087
• 关键词: Affinity; Alleles; CRISPR/Cas technology; Cancer Patient; Carbapenems; Catalogs; Cations; Cell Wall; Cells; Charge; Chemistry; Clinical; Colistin; Collection; Comparative Genomic Analysis; Cytolysis; Diagnostic; Dissection; Dose; Electrostatics; Elements; Epidemic; Ethanolamines; Event; Expression Profiling; Future; Gene Expression; Gene Mutation; Generations; Genes; Genetic; Genetic Transcription; Genome; Genomics; Genotype; Grant; Hospitals; Hot Spot; IS Elements; Infection; Klebsiella pneumonia bacterium; Laboratories; Lead; Life; Lipid A; Lipopolysaccharides; Livestock; Maps; Mediating; Membrane; Modification; Molecular; Morbidity - disease rate; Mutation; Nature; Outcome; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Plasmids; Polymyxin B; Polymyxins; Proteins; Public Domains; Regulatory Pathway; Resistance; Resolution; Resort; Sepsis; Sequence Analysis; Source; System; Technology; Therapeutic Agents; Time; Tissue-Specific Gene Expression; Transferase; Transplant Recipients; Up-Regulation; carbapenem resistance; carbapenem-resistant Enterobacteriaceae; colistin resistance; data mining; drug discovery; effective therapy; fitness; genome editing; genome sequencing; genomic platform; geographic population; inorganic phosphate; melting; mortality; mutant; novel; novel therapeutics; pathogen; patient population; phosphoethanolamine; programs; resistance gene; resistance mechanism; resistance mutation; resistant strain; sugar; surveillance study; transcriptome; transcriptome sequencing; transcriptomics; whole genome

Summary Carbapenem-resistant Enterobacteriaceae (CRE), particularly Klebsiella pneumoniae (CRKp), have emerged as a serious hospital-acquired pathogen causing considerable morbidity and mortality in the USA and worldwide. Currently, limited and less effective treatment options are available. “Old generation” agents, such as the polymyxins (polymyxin B and colistin) have been reintroduced as last-resort agents to treat life-threating carbapenem resistant infections. However, poor dosing of patients, its use as monotherapy or in combination with less effective agents, and its use in livestock, have resulted in the global emergence of colistin-resistant Klebsiella pneumoniae. Local and global molecular surveillance studies showed that 10%-20% CRKp isolates were resistant to colistin in comparison to ~2% in carbapenem susceptible isolates. Recent identification of plasmid mediated Mcr-1 colistin resistance further complicate the battle against CRKp infections. Colistin resistance in K. pneumoniae is mainly due to chromosomal gene mutations in two component systems (TCSs) and its negative regulators. The result is lipopolysaccharide (LPS) modification; however, direct causal effect of a given mutation and resistance remains unclear, and the correlation between mutations and resistance levels remains unknown. As is the nature of cross-talk among TCSs, complicated regulatory networks underscore colistin resistance, and distinct changes in expression pathways were found in colistin resistant K. pneumoniae using RNAseq transcriptome analysis. Significantly, no mutations in the known colistin resistance genes were identified in ~25% of resistant isolates in our preliminary study, suggesting that additional genes are involved in colistin resistance. Here we hypothesize that various chromosomal mutations contribute to diverse colistin resistance outcomes and levels, likely involving differential regulatory pathways, some revealed and others yet to be discovered. In this proposal we will utilize our established genetic and genomic platforms to 1) understand the most common molecular mechanisms of colistin resistance in K. pneumoniae locally, regional and globally; 2) correlate distinct gene mutants to the resistance levels; and 3) unravel novel resistance mechanisms. We will capture and characterize a large collection of colistin resistant K. pneumoniae isolates from local (NY, NJ and PA), and regional (across US) and global sources, and select representative isolates for whole genome sequencing to reveal the most common genotypes associated with colistin resistance (Aim 1). We will use the cutting-edging CRISPR/Cas9 genome editing technology to generate isogenic strains for those most common gene mutations, and directly evaluate their contribution to colistin MICs, followed by RNAseq transcriptome analysis to reveal differential gene expression pathways underling the resistance (Aim 2). In addition, novel resistance mechanisms will be unraveled using the combination of WGS and RNAseq analysis, and confirmed by CRISPR/Cas9 (Aim 2). Successful completion of this proposal will increase our understanding of colistin resistance and will produce a curated collection of chromosomal mutants in a common genetic background with defined MICs and expression profiles, which will greatly facilitate future drug discovery, diagnostic programs, and studies of strain fitness and pathogenesis.

概括 抗碳青霉烯型肠杆菌科（CRE），尤其是克雷伯氏菌（Klebsiella）肺炎（CRKP），已经出现为 严重的医院可获得的病原体，在美国和全球引起了可考虑的发病率和死亡率。 当前，有限且效率较低的治疗选择可用。 “旧一代”代理商，例如 多霉菌素（多粘蛋白B和colistin）已被重新引入，以治疗威胁生命 碳青霉烯抗性感染。但是，患者的剂量不佳，用作单一疗法或组合使用 由于效率较低的代理及其在牲畜中的使用，已导致全球抗菌素的出现 克雷伯氏菌肺炎。局部和全球分子监测研究表明，10％-20％CRKP分离株 与碳青霉烯易感分离株相比，对大肠菌素的抗性能力。最近的识别 质粒介导的MCR-1结肠癌抗性进一步使与CRKP感染的斗争变得复杂。 colistin K.肺炎的耐药性主要是由于两个组分系统（TCSS）中的染色体基因突变引起的 及其负面监管机构。结果是脂多糖（LPS）修饰；但是，直接因果关系 给定的突变和电阻尚不清楚，突变与电阻水平之间的相关性 仍然未知。与TCSS之间的串扰性质一样，复杂的监管网络强调 在肺炎菌群中发现了结肠蛋白抗性和表达途径的明显变化 使用RNASEQ转录组分析。值得注意的是，已知的colistin耐药基因中没有突变是 在我们的初步研究中，在约25％的抗性分离株中鉴定出来，这表明其他基因参与 结肠蛋白抗性。在这里，我们假设各种染色体突变有助于潜水大肠杆菌素 阻力结果和水平，可能涉及差异调节途径，有些揭示了 被发现。在此提案中，我们将利用我们既定的遗传和基因组平台来1）了解 在本地，区域和全球范围内，肺炎链球菌抗性的最常见分子机制； 2）将不同的基因突变体与抗性水平相关联； 3）揭开新颖的抗性机制。我们将 捕获并表征了大量抗体蛋白耐药的K.肺炎。 PA），区域（整个美国）和全球来源，并选择整个基因组的代表性分离株 测序以揭示与大肠菌蛋白抗性相关的最常见基因型（AIM 1）。我们将使用 加工CRISPR/CAS9基因组编辑技术，为最常见的人产生同基因菌株 基因突变，并直接评估其对Colistin MIC的贡献，其次是RNASEQ转录组 分析以揭示抗电阻的差异基因表达途径（AIM 2）。此外，新颖 使用WGS和RNASEQ分析的组合将揭示电阻机制，并确认 由CRISPR/CAS9（AIM 2）。成功完成这项建议将增加我们对colistin的理解 抗性，并将在共同的遗传背景中产生策划的染色体突变体的集合 定义的麦克风和表达概况将极大地促进未来的药物发现，诊断计划， 以及应变适应性和发病机理的研究。