Metronidazole resistance in Bacteroides fragilis: an integrated approach

描述（由申请方提供）：意义：脆弱拟杆菌（BF）是一种专性厌氧菌，通常是肠道寄生菌，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的主要拟杆菌属（80%）。甲硝唑（MET）是最常用的抗生素之一。不幸的是，BF中的MET耐药性变得越来越普遍，并且MET耐药（MET-R）菌株已导致截肢、败血症和死亡。虽然BF中的MET耐药性已被规范地归因于nim基因（编码NIM蛋白，其防止毒性MET分子的形成）的存在，但我们的结果以及其他报告表明并非如此，并表明MET耐药性是多因素的。创新：该项目旨在通过结合经典遗传学、下一代测序技术和分子操作的综合方法，将功能与特定基因联系起来，确定BF中赋予MET抗性的机制。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文库中鉴定负责MET抗性的基因。鉴定为参与MET抗性的可能候选基因将被靶向用于分子操作，并酌情删除或过表达（目标1C）。在Aim 2A中，我们将通过基因组测序和基于RNA-SEQ的转录组分析研究临床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比较。我们还将使用无偏倚的方法，通过将临床MET-R菌株制成的基因组文库引入MET敏感实验室菌株BF 638 R中，来寻找MET耐药相关基因。在Aim 2C中，我们将通过分子操作（如Aim 1C）证明这些基因的功能关联。在目标3中，我们将关注nim介导的MET抗性，并确定某些nim等位基因是否更可能赋予临床抗性菌株中观察到的高MIC（目标3A），以及nim是否可能通过接合转座子移动的元件在菌株之间转移（目标3B）。影响力：拟议的工作将提供重要的NGS数据为拟杆菌研究界，并提出了一个新的综合方法来研究这种病原体。关于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耐药的多种因素将为药物开发人员设计治疗提供指导。

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.