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：菌丝（BF）是一种强制性的厌氧细菌，通常是肠道的，可以成为机会性病原体并引起严重的感染。它是从人类厌氧感染中分离出的主要杀菌物质（80％）。甲硝唑（MET）是针对BF的最常用的抗生素之一。不幸的是，BF之间的MET耐药性变得越来越普遍，并且耐MET（MET-R）菌株导致截肢，败血症和死亡。虽然BF中的MET耐药性已归因于NIM基因的存在（编码防止有毒MET分子形成的NIM蛋白的编码），但我们的结果以及其他报告表明，表明MET抗性是多因素的。创新：该项目旨在使用结合经典遗传学，下一代测序技术和分子操作以将功能与特定基因相关联的综合方法来确定在BF中赋予抗药性的机制。我们的实验室已经熟练使用这些技术进行BF研究，并且拥有强大的全球同事网络，这些网络将向我们发送临床MET-R BF隔离株。研究策略：在AIM 1A中，我们将研究自发的，顺序MET-R突变体之间的转录组变化。在AIM 1B中，我们将确定在饱和的转座子突变库中负责MET抗性的基因。被识别为参与MET耐药性的候选者的基因将用于分子操作，并在适当的情况下删除或过表达（AIM 1C）。在AIM 2A中，我们将研究基于基因组测序和基于RNA-SEQ的转录组分析的临床MET-R分离株，随后与其他临床和实验室分离株的基因组和转录组进行了比较。我们还将使用一种无​​偏的方法通过将临床MET-R菌株制成的基因组文库引入MET易感实验室菌株BF638R来找到与抗性相关的基因。在AIM 2C中，我们将通过分子操作（如AIM 1C）来证明这些基因的功能关联。在AIM 3中，我们将重点放在NIM介导的MET耐药性上，并确定某些NIM等位基因是否更有可能赋予在临床上抗性菌株中看到的高麦克风（AIM 3A），以及是否可以通过结合型转孔元件在菌株之间转移NIM（AIM 3B）。影响：拟议的工作将为细菌研究界提供重要的NGS数据，并提出一种研究这种病原体的新综合方法。有关NIM基因活性以及其他关键基因的参与的数据将用于开发新的快速诊断测试以进行MET抗性。此外，确定MET抗性中的多个因素将为指导药物开发人员设计治疗。