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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

基本信息

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
项目类别：
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

项目摘要

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