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.

总结 碳青霉烯类耐药肠杆菌科（CRE），特别是肺炎克雷伯氏菌（CRKp），已经出现， 一种严重的医院获得性病原体，在美国和全世界引起相当大的发病率和死亡率。 目前，可用的治疗选择有限且效果较差。“老一代”特工，如 多粘菌素（多粘菌素B和粘菌素）已被重新引入作为治疗危及生命的 碳青霉烯耐药感染。然而，患者的剂量差，其作为单药治疗或联合使用 使用不太有效的药剂，以及其在牲畜中的使用，导致了全球出现了粘菌素耐药 肺炎克雷伯氏菌。当地和全球分子监测研究表明，10%-20%的CRKp分离株 对粘菌素耐药，而碳青霉烯敏感菌株中约为2%。最近的研究发现 质粒介导的Mcr-1粘菌素抗性进一步使对抗CRKp感染的战斗复杂化。粘菌素 电阻K。肺炎主要是由于双组分系统（TCS）中的染色体基因突变 和它的负调节器。结果是脂多糖（LPS）修饰;然而， 特定的突变和耐药性尚不清楚，突变和耐药性水平之间的相关性 仍然未知。由于TCS之间相互影响的性质，复杂的监管网络强调， 结果表明，在对黏菌素产生抗性的K.肺炎 使用RNAseq转录组分析。值得注意的是，在已知的粘菌素耐药基因中没有突变， 在我们的初步研究中，在约25%的耐药菌株中鉴定出了这种基因，这表明另外的基因参与了 粘菌素耐药在这里，我们假设各种染色体突变有助于产生不同的粘菌素 耐药结果和水平，可能涉及不同的调控途径，一些已经揭示，另一些尚未 等待被发现在这项提案中，我们将利用我们建立的遗传和基因组平台，1）了解 K. pneumoniae本地，区域和全球; 2)将不同的基因突变体与抗性水平相关联;以及3）揭示新的抗性机制。我们将 捕获和表征大量的粘菌素抗性K.来自当地（NY，NJ和 PA）、区域（美国）和全球来源，并选择全基因组的代表性分离株 测序以揭示与粘菌素耐药性相关的最常见基因型（Aim 1）。我们将使用 最前沿的CRISPR/Cas9基因组编辑技术，为那些最常见的 基因突变，并直接评估其对粘菌素MIC的贡献，然后是RNAseq转录组 分析，以揭示差异基因表达途径下的电阻（目的2）。此外，小说 将使用WGS和RNAseq分析的组合来解开抗性机制，并证实 CRISPR/Cas9（Aim 2）成功完成本提案将增加我们对粘菌素的了解 耐药性，并将产生一个共同的遗传背景下的染色体突变体的策划收集， 确定的MIC和表达谱，这将极大地促进未来的药物发现，诊断程序， 以及菌株适应性和发病机制的研究。