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 耐碳青霉烯类肠杆菌科 (CRE)，特别是肺炎克雷伯菌 (CRKp)，已成为 一种严重的医院获得性病原体，在美国和全世界造成相当大的发病率和死亡率。 目前，可用的“老一代”药物有限且效果较差。 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最后手段 但对碳青霉烯类耐药感染的患者剂量不佳，其可作为单药或联合用药。 使用效果较差的药物及其在牲畜中的使用，导致全球出现粘菌素抗药性 肺炎克雷伯菌。当地和全球分子监测研究表明，10%-20% CRKp 分离株。 与最近鉴定的碳青霉烯类敏感菌株相比，对粘菌素具有耐药性的人约为 2%。 质粒介导的 Mcr-1 粘菌素耐药性使对抗 CRKp 感染的斗争进一步复杂化。 肺炎克雷伯菌的耐药性主要是由于两成分系统（TCS）中的染色体基因突变造成的 及其负调节剂的结果是脂多糖（LPS）修饰；然而，直接因果效应。 特定的突变和耐药性仍不清楚，突变和耐药性水平之间的相关性 由于 TCS 之间串扰的性质仍然未知，因此复杂的监管强调网络。 在粘菌素耐药肺炎克雷伯菌中发现了粘菌素耐药性，并且表达途径发生了明显变化 值得注意的是，已知的粘菌素抗性基因没有发生突变。 在我们的初步研究中，约 25% 的抗性分离株中发现了这种基因，这表明还有其他基因参与 在这里，我们发现各种染色体突变导致了不同的粘菌素。 耐药结果和水平，可能涉及不同的监管途径，一些已披露，另一些尚未公布 在本提案中，我们将利用我们已建立的遗传和基因组平台来 1) 理解。 当地、区域和全球肺炎克雷伯菌粘菌素耐药性最常见的分子机制； 2）将不同的基因突变体与耐药水平相关联；3）揭示新的耐药机制。 捕获并表征大量来自当地（纽约州、新泽西州和 PA）、区域（美国各地）和全球来源，并选择全基因组的代表性分离株 测序揭示与粘菌素耐药性相关的最常见基因型（目标 1）。 尖端 CRISPR/Cas9 基因组编辑技术可为最常见的菌株生成同基因菌株 基因突变，并直接评估它们对粘菌素 MIC 的贡献，然后进行 RNAseq 转录组 分析揭示耐药性背后的差异基因表达途径（目标 2）。 结合WGS和RNAseq分析，将揭示耐药机制，并得到证实 通过 CRISPR/Cas9（目标 2）成功完成该提案将增加我们对粘菌素的了解。 抗性，并将在共同的遗传背景中产生一系列染色体突变体 明确的 MIC 和表达谱，这将极大地促进未来的药物发现、诊断计划、 以及菌株适应性和发病机制的研究。