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