Delineating genetic determinants of polymyxin resistance in Serratia marcescens

描述粘质沙雷氏菌多粘菌素抗性的遗传决定因素

• 批准号: 10462801
• 负责人: Anne-Catrin Uhlemann
• 金额: $ 20.23万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-05 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10462801
• 关键词: Adjuvant; Alleles; Antibiotics; Arabinose; Automobile Driving; Biochemical; Biological Assay; Cations; Charge; Chemicals; Clinical; Collection; Complement; Data; Enzymes; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Determinism; Genetic Markers; Genome; Genomics; Geography; Glucans; Gram-Negative Bacteria; Hospitals; Imipenem; Impairment; Infection; Knowledge; Lactamase; Lactams; Lead; Lipid A; Mass Spectrum Analysis; Membrane; Membrane Proteins; Methods; Microbiology; Minimum Inhibitory Concentration measurement; Modeling; Modification; Molecular; Monitor; Multi-Drug Resistance; Mutation; New York City; Operon; Organism; Peptides; Pharmaceutical Preparations; Phenotype; Phylogenetic Analysis; Polymyxin Resistance; Polymyxins; Predisposition; Prevalence; Protein Biochemistry; Radiolabeled; Rapid screening; Recording of previous events; Regimen; Regulator Genes; Regulatory Pathway; Resistance; Resort; Resources; Role; Serratia marcescens; Structure; Techniques; Technology; Testing; Transferase; Urban Hospitals; Virulence; bacterial fitness; carbapenem resistance; carbapenem-resistant Enterobacteriaceae; design; effective therapy; experimental study; genome sequencing; improved; inhibitor; inorganic phosphate; molecular diagnostics; molecular marker; mutant; novel; pathogen; periplasm; reconstruction; resistance mechanism; response; screening; structural biology; whole genome

This proposal aims to define the molecular mechanisms of resistance to polymyxins in Serratia marcescens. The emergence of carbapenem resistance in this nosocomial pathogen poses a treatment dilemma as few treatment options exist. S. marcescens has long been considered intrinsically resistant to polymxyins, a mainstay for treatment of carbapenem-resistant Enterobacteriales. However, we unexpectedly noted that a substantial proportion of carbapenem-resistant S. marcescens (CR-SM) are fully susceptible to polymyxin. In addition, resistant isolates displayed two distinct phenotypes (low-level (R1) and high-level resistance (R2). Polymyxins are cationic peptides targeting negatively charged components of the bacterial outer membrane, notably Lipid A. Chemical modification of Lipid A through enzymatic transfer of L-Ara4N to Lipid A is the major contributor to polymyxin resistance and is catalyzed by the aminoarabinose transferase ArnT. Lipid A analysis of isolates representing the 3 phenotypic groups demonstrated a complete lack of L-Ara4N modification in susceptible isolates and distinct lipid A profiles between R1 and R2 phenotypes. Through whole genome sequencing we have begun to establish putative genetic determinants of polymyxin susceptibility. This includes a unique arnC-like gene only present in susceptible isolates and a flippase related to arnE only encoded in highly-resistant R2 isolates. In addition, R1 and R2 isolates harbored unique arnD and eptA alleles compared to susceptible isolates. Here, we aim to define the mechanisms underlying polymyxin susceptibility in detail to provide critical information on the potential suitability of polymyxins as a treatment option for CR-SM infections. We have assembled a highly skilled team with complementary expertise in microbiology, bacterial genomics and membrane protein biochemistry. In Aim 1, we will determine the prevalence of polymyxin susceptibility in a comprehensive collection of clinical multi-drug resistant (MDR) and non-MDR S. marcescens isolates. Through whole genome sequencing we will refine putative genetic markers of PR phenotypes and through phylogenetic reconstruction determine if the loss or PR occurred repeatedly or represents a stable sublineage. We will then validate candidate genetic markers on PR through a combination of complementation, gene editing and deletion experiments. In Aim 2, we will assess LPS structure and lipid A modifications of isogenic mutants generated in Aim 1 using radiolabeling techniques and mass spectrometry. We will evaluate gene expression of lipid A modifying enzymes and overall differences in transcriptional profiles across phenotypes. Lastly, we will examine the impact of PR mutants on bacterial fitness and virulence. Findings from our study will fill significant gaps in knowledge of the genomic and molecular determinants of intrinsic PR, provide information on the mechanisms of disruption of lipid A modifications, deliver molecular markers for rapid screening of polymyxin susceptibility phenotypes in CR-SM; and improve our understanding of the evolutionary trade-offs underlying the loss of the intrinsic PR phenotype.

该提案旨在确定粘质沙雷氏菌对多粘菌素耐药的分子机制。 这种医院病原体中碳青霉烯类耐药性的出现造成了治疗困境，因为很少有 存在治疗方案。长期以来，粘质沙门氏菌一直被认为对多粘菌素具有内在抗性，多粘菌素是一种 治疗耐碳青霉烯类肠杆菌的主要药物。然而，我们意外地注意到， 相当一部分耐碳青霉烯类粘质沙门氏菌 (CR-SM) 对多粘菌素完全敏感。在 此外，耐药菌株表现出两种不同的表型（低水平（R1）和高水平耐药（R2）。 多粘菌素是针对细菌外膜带负电成分的阳离子肽， 值得注意的是脂质 A。通过 L-Ara4N 酶促转移至脂质 A 对脂质 A 进行化学修饰是主要的 多粘菌素耐药性的贡献者，并由氨基阿拉伯糖转移酶 ArnT 催化。脂质A分析 代表 3 个表型组的分离株表明完全缺乏 L-Ara4N 修饰 R1 和 R2 表型之间的易感菌株和不同的脂质 A 谱。通过全基因组 通过测序，我们已经开始建立多粘菌素敏感性的推定遗传决定因素。这包括 一种独特的 arnC 样基因仅存在于易感菌株中，而与 arnE 相关的翻转酶仅在 高耐药性 R2 分离株。此外，相比之下，R1 和 R2 分离株具有独特的 arnD 和 eptA 等位基因 对易感分离株。在这里，我们的目标是详细定义多粘菌素敏感性的机制 提供有关多粘菌素作为 CR-SM 感染治疗选择的潜在适用性的关键信息。 我们组建了一支技术精湛的团队，在微生物学、细菌基因组学方面具有互补的专业知识 和膜蛋白生物化学。在目标 1 中，我们将确定多粘菌素敏感性的患病率 全面收集临床多重耐药 (MDR) 和非多重耐药粘质沙门氏菌分离株。通过 全基因组测序，我们将完善 PR 表型的假定遗传标记，并通过系统发育 重建确定丢失或 PR 是否重复发生或代表稳定的亚系。我们随后将 通过互补、基因编辑和 删除实验。在目标 2 中，我们将评估同基因突变体的 LPS 结构和脂质 A 修饰 使用放射性标记技术和质谱在目标 1 中生成。我们将评估基因表达 脂质 A 修饰酶的变化以及不同表型转录谱的总体差异。最后，我们 将检查 PR 突变体对细菌适应性和毒力的影响。我们的研究结果将填补 关于内在 PR 的基因组和分子决定因素的知识存在重大差距，请提供信息 研究脂质 A 修饰的破坏机制，提供分子标记以快速筛选 CR-SM 中的多粘菌素敏感性表型；并提高我们对进化权衡的理解 内在 PR 表型丧失的潜在原因。