Metronidazole resistance in Bacteroides fragilis: an integrated approach

描述(申请人提供)：意义：脆弱类杆菌(BF)是一种必须的厌氧菌，通常是肠道共生菌，可成为机会性病原体并导致严重感染。它是从人类厌氧感染中分离到的主要类杆菌(80%)。甲硝唑(MET)是治疗BF最常用的抗生素之一。不幸的是，MET耐药性在BF中正变得越来越普遍，MET耐药株(MET-R)已导致截肢、败血症和死亡。虽然在BF中MET抗性被典型地归因于Nim基因(编码NIM蛋白，防止有毒MET分子的形成)的存在，但我们的结果以及其他报告表明情况并非如此，提示MET抗性是多因素的。创新：该项目寻求使用一种结合经典遗传学、下一代测序技术和分子操纵的综合方法来识别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将这些技术用于BF研究，并拥有强大的全球同事网络，他们将为我们提供临床MET-R BF分离株。研究策略：在目标1A中，我们将研究自发的、顺序的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与MET抗性有关的基因(S)。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，并视情况删除或过度表达(AIM 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本进行比较。我们还将使用一种无偏见的方法，通过将临床MET-R毒株的基因组文库导入MET敏感的实验室菌株BF638R来寻找MET抗性相关基因。在目标2C中，我们将通过分子操作来展示这些基因的功能关联(与目标1C中一样)。在目标3中，我们将重点研究Nim介导的MET耐药性，并确定某些Nim等位基因是否更有可能产生临床耐药菌株的高MIC(Aim 3A)，以及Nim是否可能通过接合转座子移动元件在菌株之间转移(Aim 3B)。影响：拟议的工作将为类杆菌研究界提供重要的NGS数据，并建议一种新的综合方法来研究这种病原体。有关Nim基因活性的数据以及其他关键基因的参与将被用于开发新的Met抗性快速诊断试验。此外，识别MET耐药的多重因素将为药物开发人员设计治疗方案提供指导。

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.