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.

克雷伯氏菌是医疗保健相关感染和胃肠道定植的主要原因 通常发生在感染之前。克雷伯氏菌在约20%的重症监护患者中定植，其中4%的患者将 这种病原体会导致肺炎、血液或尿路感染。在80%的这种情况下， 感染分离株在入院时可通过培养检测到。这些定植和感染克雷伯氏菌 菌株高度多样化，通过对被膜基因WzI及其基因含量进行分型来衡量。在一个 对发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 245个分离株中的147种WzI类型，发现一些患者被一种以上的克雷伯氏菌定植 同时进行筛选。超过22,000个基因在这些分离株之间存在差异，这表明遗传多样性 可能会导致健康状况和致病潜力的巨大差异。两地定植菌株的多样性 在被殖民的患者体内，提出了关于殖民的复杂动态如何 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的工具： 条形码方案，可以实现克雷伯氏菌种群的细粒度表征和大规模 非同基因菌株间的竞争实验。我们研究的长期目标是确定 可用于预防克雷伯氏菌感染的感染风险标记物。这样做的目的是 探索性研究是对WzI囊膜基因进行深度测序，作为研究克雷伯氏菌的工具 并使快速和大规模的竞争实验成为可能。我们的假设是WZI 基因座是一种自然产生的条形码，类似于16S测序，可以用来测量亲属 克雷伯氏菌菌株在大而多样的种群中的丰富性。我们的方法是开发一种WZI扩增子- 测序法，应用它来测量患者样本中的种群动态和竞争适应度，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 感染病例和配对对照的拭子样本，以及相应的全序列克雷伯氏菌 分离，以完成以下具体目标：1)验证WZI深度测序和测量种群 殖民时期的动态。我们将开发一个WZI测序管道，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 克雷伯氏菌在人类定植期间及其对感染风险的影响。2)将WZI深度测序应用于非 体外定植和感染模型中的等基因竞争实验。我们将测量相对适合度 从人的尿液、血清、支气管肺泡液和粪便中同时分离出100个菌株，并对A 潜在的去殖民主义干预。我们将把健康状况与临床病例状态相关联，发现并验证 在每种情况下都有新的健康基因，并在动物模型中进行测试。这项研究的结果将是 加快了解克雷伯氏菌定植是如何进展到感染的有效方法。