Capsular locus deep sequencing to study Klebsiella populations

荚膜位点深度测序研究克雷伯氏菌种群

• 批准号: 10679308
• 负责人: Michael Abbott Bachman
• 金额: $ 23.4万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-22 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679308
• 关键词: 16S ribosomal RNA sequencing; Acceleration; Admission activity; Affect; Alleles; Animal Model; Archives; Bacterial Antibiotic Resistance; Bacterial Infections; Bar Codes; Blood Circulation; Case/Control Studies; Clinical; Complex; Detection; Dideoxy Chain Termination DNA Sequencing; Feces; Genes; Genetic Variation; Genome; Goals; Healthcare; Hospitals; Human; Infection; Infection prevention; Intensive Care; Intensive Care Units; Intervention; Intestines; Klebsiella; Klebsiella Infections; Klebsiella oxytoca; Klebsiella pneumoniae; Liquid substance; Measures; Membrane; Methods; Modeling; Outcome Study; Pathogenesis; Pathogenicity; Patient Care; Patient-Focused Outcomes; Patients; Pneumonia; Population; Population Dynamics; Population Heterogeneity; Predisposition; Proteins; Rectum; Research; Risk; Sampling; Scheme; Serum; Site; Swab; Techniques; Testing; Therapeutic; Time; Urinary tract infection; Urine; biomarker identification; capsule; case control; cohort; comparative genomics; data integration; deep sequencing; efficacy evaluation; experimental study; fitness; gastrointestinal; genomic locus; gut colonization; healthcare-associated infections; high risk; infection risk; mutant; novel; novel therapeutics; pathogen; prevent; preventive intervention; rectal; tool; trait

Klebsiella species are a leading cause of healthcare-associated infections, and gastrointestinal colonization often precedes infection. Klebsiella colonize ~20% of intensive care patients, and 4% of these patients will develop pneumonia, bloodstream or urinary tract infections from this pathogen. In 80% of these cases, the infecting isolate is detectable by culture at the time of admission. These colonizing and infecting Klebsiella strains are highly diverse, as measured by typing of the capsule locus gene wzi and their gene content. In a case-control study of colonizing strains that progressed to infection or remained asymptomatic, we identified 147 wzi types across 245 isolates and found that some patients are colonized by more than one Klebsiella strain simultaneously. Over 22,000 genes varied between these isolates, suggesting that genetic diversity could lead to large differences in fitness and pathogenic potential. The diversity of colonizing strains between and within colonized patients raises fundamental questions about how the complex dynamics of colonization affect infection risk. This high diversity of wzi types also provides a novel tool to answer these questions: a barcoding scheme that can enable granular characterization of Klebsiella populations and large-scale competition experiments between non-isogenic strains. The long-term goal of our research is to identify markers of infection risk that can be used to prevent Klebsiella infections in colonized patients. The goal of this exploratory study is to develop deep sequencing of the wzi capsular locus as a tool to study Klebsiella population dynamics and enable rapid and large-scale competition experiments. Our hypothesis is that the wzi locus is a naturally-occurring barcode that, analogous to 16S sequencing, can be used to measure the relative abundance of Klebsiella strains in large, diverse populations. Our approach is to develop a wzi amplicon- sequencing method, apply it to measure population dynamics in patient samples and competitive fitness, and integrate these data with clinical modeling and comparative genomics. We will leverage an existing set of rectal swab samples from cases of infection and matched controls, and corresponding fully sequenced Klebsiella isolates, to complete the following Specific Aims: 1) Validate wzi deep sequencing and measure population dynamics during colonization. We will develop a wzi sequencing pipeline and validate it on contrived and archived rectal swab samples. Then we will pilot this technique on rectal swabs to measure diversity of Klebsiella during human colonization and its impact on infection risk. 2) Apply wzi deep sequencing to non- isogenic competition experiments in ex vivo colonization and infection models. We will measure relative fitness from >100 strains simultaneously in human urine, serum, bronchoalveolar fluid, and stool, and against a potential de-colonization intervention. We will correlate fitness with clinical case status, discover and validate novel fitness genes in each condition, and test them in animal models. The outcome of this study will be a powerful approach to accelerate progress in understanding how Klebsiella colonization progresses to infection.

克雷伯菌属是医疗保健相关感染和胃肠道定植的主要原因 通常在感染之前。克雷伯氏菌定植于约20%的重症监护患者，其中4%的患者将 由这种病原体引起的肺炎、血液或尿路感染。在80%的案例中， 在入院时通过培养可检测到感染分离物。这些定植和感染克雷伯氏菌 通过对荚膜基因座基因WZ 1和它们的基因含量进行分型来测量，菌株是高度多样化的。中 通过对进展为感染或保持无症状的定植菌株的病例对照研究，我们发现 在245个分离株中进行了147个wzi分型，并发现一些患者被一种以上的克雷伯菌定殖 同时应变。这些分离株之间有超过22，000个基因存在差异，这表明遗传多样性 可能导致适应性和致病潜力的巨大差异。不同地区定植菌株的多样性 而在殖民患者中，提出了一个基本问题，即殖民的复杂动力学 影响感染风险。WZI类型的高度多样性也为回答这些问题提供了一种新颖的工具： 条形码方案，可以实现克雷伯氏菌种群的颗粒表征和大规模 非等基因菌株之间的竞争实验。我们研究的长期目标是确定 感染风险的标志物，可用于预防定殖患者的克雷伯氏菌感染。这个目标 探索性研究是开发wzi荚膜位点的深度测序，作为研究克雷伯菌的工具 种群动态，并使快速和大规模的竞争实验。我们的假设是wzi 基因座是天然存在的条形码，其类似于16 S测序，可用于测量相对生物量。 克雷伯氏菌菌株在大的、不同的群体中的丰度。我们的方法是开发wzi扩增子- 测序方法，将其应用于测量患者样本中的群体动态和竞争适应度，以及 将这些数据与临床建模和比较基因组学相结合。我们将利用现有的一套直肠 来自感染病例和匹配对照的拭子样本，以及相应的完全测序的克雷伯氏菌 分离株，以完成以下特定目的：1）Escherwzi深度测序和测量群体 殖民时期的动态我们将开发一个wzi排序管道，并在人工和 存档的直肠拭子样本。然后，我们将在直肠拭子上试验这项技术，以测量 克雷伯氏菌在人体定植及其对感染风险的影响。2)将wzi深度测序应用于非 离体定殖和感染模型中的同基因竞争实验。我们将测量相对适合度 从人尿液、血清、支气管肺泡液和粪便中同时分离>100株菌株，并针对 潜在的非殖民化干预。我们将把健康与临床病例状态联系起来，发现并验证 在每种情况下都有新的适应基因，并在动物模型中进行测试。这项研究的结果将是一个 这是一种强有力的方法，可以加速了解克雷伯氏菌定植如何发展为感染。