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 通常先于感染。克雷伯菌定植于约 20% 的重症监护患者中，其中 4% 将 由这种病原体引起肺炎、血液或尿路感染。在 80% 的此类案例中， 入院时可通过培养检测到感染分离株。这些定植并感染克雷伯氏菌 通过荚膜基因座基因 wzi 的分型及其基因含量来测量，菌株具有高度多样性。在一个 对进展为感染或保持无症状的定植菌株进行病例对照研究，我们发现 对 245 个分离株中的 147 个 WZ 类型进行了研究，发现一些患者体内定植了不止一种克雷伯氏菌 同时应变。这些分离株之间有超过 22,000 个基因存在差异，表明遗传多样性 可能导致适应性和致病潜力的巨大差异。定植菌株之间的多样性 在被殖民的患者体内，提出了关于殖民的复杂动态如何发生的基本问题 影响感染风险。 Wzi 类型的高度多样性也提供了一个新颖的工具来回答这些问题： 条形码方案，可以实现克雷伯氏菌种群的精细表征和大规模 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 感染风险标记，可用于预防定植患者的克雷伯氏菌感染。此举的目标 探索性研究是开发 wzi 荚膜基因座的深度测序作为研究克雷伯氏菌的工具 种群动态并实现快速和大规模的竞争实验。我们的假设是 wzi 位点是一种天然存在的条形码，类似于 16S 测序，可用于测量相对 大量不同种群中存在丰富的克雷伯氏菌菌株。我们的方法是开发一个 wzi 扩增子 测序方法，应用它来测量患者样本中的群体动态和竞争适应性，以及 将这些数据与临床模型和比较基因组学相结合。我们将利用现有的一组直肠 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，以完成以下具体目标：1) 验证 wzi 深度测序并测量群体 殖民时期的动态。我们将开发一个 wzi 测序流程并在人为和验证上验证它 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来测量 人类定植期间的克雷伯氏菌及其对感染风险的影响。 2）将wzi深度测序应用于非 离体定植和感染模型中的同基因竞争实验。我们将测量相对适合度 同时从人尿液、血清、支气管肺泡液和粪便中检测到超过 100 种菌株，并针对 潜在的非殖民化干预。我们将把健康状况与临床病例状态联系起来，发现并验证 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 加速了解克雷伯氏菌定植如何进展为感染的有力方法。