Metronidazole resistance in Bacteroides fragilis: an integrated approach

脆弱拟杆菌中的甲硝唑耐药性：综合方法

• 批准号: 8624211
• 负责人: Hannah Wexler
• 金额: $ 18.45万
• 依托单位: BRENTWOOD BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-19 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8624211
• 关键词: Alleles; Amputation; Anaerobic Bacteria; Antibiotics; Antimicrobial Resistance; Back; Bacteria; Bacteroides; Bacteroides fragilis; Benign; Cessation of life; Clinical; Code; Collection; Communities; Data; Databases; Diagnostic Procedure; Diagnostic tests; Elements; Escherichia coli; Expression Library; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Genome; Genomic Library; Genomics; Goals; Human; Infection; Knock-out; Lead; Libraries; Maps; Measures; Mediating; Metronidazole; Metronidazole resistance; Microbe; Molecular; Molecular Target; Mutagenesis; Mutation; Parents; Pharmaceutical Preparations; Phenotype; Plasmids; Proteins; RNA; RNA Sequences; Reading; Regulon; Reporting; Research; Research Design; Resistance; Resistance development; Scientist; Sepsis; Sequence Analysis; Structure; Techniques; Technology; Testing; Therapeutic; Time; Tinidazole; Virulent; Work; base; comparative; gene function; innovation; interest; mutant; next generation sequencing; overexpression; pathogen; pressure; prevent; public health relevance; resistance factors; resistance mechanism; resistant strain; therapy design; trait; vector

DESCRIPTION (provided by applicant): Significance: Bacteroides fragilis (BF), an obligatory anaerobic bacterium that is normally a gut commensal, can become an opportunistic pathogen and cause serious infections. It is the major Bacteroides species isolated from human anaerobic infections (80%). Metronidazole (MET) is among the most commonly used antibiotics against BF. Unfortunately, MET resistance among BF is becoming more prevalent and MET resistant (MET-R) strains have resulted in amputations, sepsis and death. While MET resistance in BF has been canonically ascribed to the presence of the nim gene (coding for the NIM protein which prevents the formation of the toxic MET molecule), our results as well as other reports indicate otherwise and suggest that MET resistance is multifactorial. Innovation: This project seeks to identify mechanisms that confer MET resistance in BF using an integrated approach combining classical genetics, Next Generation Sequencing technology and molecular manipulation to relate function to specific genes. Our lab has become skilled in using these techniques for BF research and has a strong global network of colleagues who will send us clinical MET-R BF isolates. Research strategy: In Aim 1A we will study transcriptome changes among spontaneous, sequential MET-R mutants. In Aim 1B, we will identify gene(s) responsible for MET resistance in a saturated transposon mutant library. The genes identified as likely candidates for involvement in MET resistance will be targeted for molecular manipulation and either deleted or overexpressed, as appropriate (Aim 1C). In Aim 2A, we will study the clinical MET-R isolates with genomic sequencing and RNA-SEQ based transcriptome analysis with subsequent comparisons with genomes and transcriptomes from other clinical and lab isolates. We will also use an unbiased approach to find MET resistance-associated genes by introducing genomic libraries made from clinical MET-R strains into the MET susceptible lab strain, BF638R. In Aim 2C, we will demonstrate functional associations of these genes by molecular manipulation (as in Aim 1C). In Aim 3 we will focus on nim-mediated MET resistance and determine whether certain nim alleles are more likely to confer the high MICs seen in clinically resistant strains (Aim 3A) and whether nim might be transferred between strains via a conjugative transposon mobile element (Aim 3B). Impact: The proposed work will provide important NGS data for the Bacteroides research community and suggest a new integrated approach with which to study this pathogen. The data regarding nim gene activity as well as involvement of other key genes will be used to develop new rapid diagnostic tests for MET resistance. In addition, identifying the multiple factors in MET resistance will provide guidance t drug developers in designing therapy.

描述（由申请人提供）： 意义：脆弱拟杆菌 (BF) 是一种必需厌氧细菌，通常是肠道共生菌，可成为机会致病菌并引起严重感染。它是从人类厌氧菌感染中分离出的主要拟杆菌属物种（80%）。甲硝唑 (MET) 是最常用的 BF 抗生素之一。不幸的是，BF 中的 MET 耐药性变得越来越普遍，MET 耐药性 (MET-R) 菌株已导致截肢、败血症和死亡。虽然 BF 中的 MET 抗性通常归因于 nim 基因（编码 NIM 蛋白，可防止有毒 MET 分子形成）的存在，但我们的结果以及其他报告表明情况并非如此，并表明 MET 抗性是多因素的。创新：该项目旨在利用结合经典遗传学、下一代测序技术和分子操作的综合方法来确定 BF 中 MET 抗性的机制，以将功能与特定基因联系起来。我们的实验室已经熟练地使用这些技术进行 BF 研究，并拥有强大的全球同事网络，他们将向我们发送临床 MET-R BF 分离株。研究策略：在目标 1A 中，我们将研究自发的连续 MET-R 突变体之间的转录组变化。在目标 1B 中，我们将在饱和转座子突变体库中鉴定负责 MET 抗性的基因。被确定为可能参与 MET 抗性的候选基因将被作为分子操作的目标，并酌情删除或过度表达（目标 1C）。在目标 2A 中，我们将通过基因组测序和基于 RNA-SEQ 的转录组分析来研究临床 MET-R 分离株，并随后与其他临床和实验室分离株的基因组和转录组进行比较。我们还将采用公正的方法，通过将临床 MET-R 菌株制成的基因组文库引入 MET 易感实验室菌株 BF638R 中，来寻找 MET 抗性相关基因。在目标 2C 中，我们将通过分子操作演示这些基因的功能关联（如目标 1C 中所示）。在目标 3 中，我们将重点关注 nim 介导的 MET 耐药性，并确定某些 nim 等位基因是否更有可能赋予临床耐药菌株中所见的高 MIC（目标 3A），以及 nim 是否可以通过接合转座子移动元件在菌株之间转移（目标 3B）。影响：拟议的工作将为拟杆菌研究界提供重要的 NGS 数据，并提出一种新的综合方法来研究这种病原体。有关 nim 基因活性以及其他关键基因参与的数据将用于开发新的 MET 抗性快速诊断测试。此外，确定 MET 耐药性的多种因素将为药物开发人员设计治疗方法提供指导。