Regulation of Bacteroides fragilis multidrug efflux pumps

脆弱拟杆菌多药外排泵的调节

• 批准号: 8242604
• 负责人: Hannah Wexler
• 金额: --
• 依托单位: VA GREATER LOS ANGELES HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-10-01 至 2014-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8242604
• 关键词: Accounting; Address; Aerobic; Aerobic Bacteria; Affect; Afghanistan; Amputation; Anaerobic Bacteria; Anti-Infective Agents; Antibiotic Resistance; Antibiotics; Antimicrobial Resistance; Area; Attention; Bacteremia; Bacteria; Bacterial Genome; Bacterial Infections; Bacteroides; Bacteroides fragilis; Bile fluid; Binding; Biochemical; Biological; Biology; Businesses; Cessation of life; Chronic; Clinical; Code; Collection; Colon; Communities; Complex; Consensus; Data; Development; Disease; Drug Efflux; Drug resistance; Elements; Environment; Escherichia coli; Explosion; Face; Family; Foundations; Frequencies; Funding; Funding Agency; Gastrointestinal tract structure; Gene Expression Profile; Genes; Genetic Transcription; Goals; Gram-Negative Bacteria; Health; Healthcare; Homologous Gene; Human; Human body; Individual; Industry; Infection; Knowledge; Laboratories; Lead; Liver diseases; Measures; Mediating; Medical center; Membrane; Metabolic; Metronidazole resistance; Microbial Biofilms; Molecular; Molecular Genetics; Morbidity - disease rate; Multi-Drug Resistance; Nosocomial Infections; One-Step dentin bonding system; Operon; Organ; Organism; Outcome; Outcome Study; Outcomes Research; Oxygen; Pathology; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Predisposition; Prevalence; Probability; Public Health; Publications; Published Comment; Pump; Race; Regimen; Regulation; Regulon; Reporting; Research; Research Personnel; Resistance; Resistance development; Resources; Rice; Risk; Risk Factors; Role; Scientist; Solid; Stress; Surface; System; Therapeutic; Time; Transcription factor genes; Up-Regulation; Virulence; Virulence Factors; Virulent; Work; abdominal wall; adverse outcome; antimicrobial; antimicrobial drug; bacterial resistance; biological research; capsule; clinical efficacy; combat; cost; design; drug development; drug discovery; efflux pump; functional genomics; genome sequencing; global environment; gut microbiota; information gathering; inhibitor/antagonist; member; methicillin resistant Staphylococcus aureus; microbiome; mortality; mutant; overexpression; pathogen; pathogenic bacteria; public health relevance; quorum sensing; resistance mechanism; resistant strain; response; salicylate; sensor; stressor; tool; transcription factor; translation factor; vector

DESCRIPTION (provided by applicant): Introduction: Despite an information explosion about antimicrobial resistance mechanisms in bacteria, patients are dying of untreatable infections for the first time since antibiotics were discovered. The main cause of multidrug resistance (MDR) in gram-negative bacteria is active efflux of drug by bacterial pumps. Knowing how the pumps are regulated is the key to understanding MDR development: bacteria can become MDR simply by increasing expression of these efflux pumps. Significance: Bacteroides fragilis are ubiquitous gut commensals that cause devastating disease when they escape the gut; their resistance to antimicrobials is rising significantly. In aerobes, MDR is caused by efflux pumps regulated by transcription factors (TFs). We hypothesize that MDR efflux pumps in B. fragilis are similarly regulated by TFs. These TFs, and the pumps they regulate, may also regulate other virulence factors. VA patients are particularly at risk for anaerobic infections. Mortality in Bacteroides bacteremia is high (25- 50%) and liver disease, common in VA patients, is a risk factor for increased mortality. We identified several MDR clinical isolates (including one from a GI in Afghanistan) with increased pump activity; these isolates were resistant to metronidazole and other agents and resulted in one death and one amputation, respectively. Preliminary Studies: In the previous funding period, we identified and characterized the B. fragilis bme efflux pump genes and showed that they were implicated in clinical MDR (described in 23 publications); we found that increases in pump levels and in resistance could be induced by a variety of agents and stressors including antimicrobials, salicylate, bile and quorum sensing molecules. We constructed new vectors and modified them to mimic the molecular tools available for use with aerobes. Research Plan: Our long term goal is to unravel the complex regulation of MDR in B. fragilis. Specifically, our goals now are to: AIM 1: Identify the TFs most important in efflux-mediated MDR (both activator and repressor TFs) using a functional genomic approach. AIM 2: Characterize TFs most important in efflux-mediated MDR. Determine the phenotype associated with the TF, measure MICs (minimal inhibitory concentrations) and determine the relevant bme genes. Demonstrate binding by physical and functional approaches. AIM 3: Determine the clinical prevalence of TF regulation in efflux mediated MDR. Measure TF transcription levels in clinical isolates, including the recently acquired MDR strains. Determine the effect of TF on efflux-mediated MDR and on ability to form biofilms. Assess the effect of bile exposure on TF transcription levels, MICs and biofilm forming ability. This study takes advantage of our unique resources: expertise with molecular and biochemical approaches, access to a massive collection of clinical isolates and associated MIC data, and excellent collaborative relationships with world-class experts. Once completed, this work, together with the studies already conducted, will reveal important new information about regulation of BF MDR pumps and will greatly contribute to our knowledge of regulation mechanisms in this bacterium that is subject to very different stresses than aerobic pathogens. B. fragilis is an important component of the gut microbiome (which has been called a separate "organ" within the human body) that is recognized as having its own impressive metabolic profile and profoundly affects almost every aspect of human health and disease. In this global environment, there is little time from the emergence of the first MDR strains to widespread resistance. Racing to develop effective drugs when untreatable infections emerge is not a viable public health strategy. Regulators of MDR efflux are important targets in drug discovery; identifying and understanding the TFs that are most important in these MDR organisms will lead to the development of new and effective interventional therapies that target the expression or translation of these factors. PUBLIC HEALTH RELEVANCE: Rising antimicrobial resistance costs 5-30 billion dollars and 90,000 lives in the US alone! Multi-drug efflux pumps are largely responsible for this resistance. Bacteroides fragilis is an important member of the gut microbiota that has critical roles in human health and disease. We have virtually no information about these pumps or their regulation in this important anaerobic pathogen.

描述（由申请人提供）： 简介：尽管有关细菌抗菌素耐药机制的信息激增，但自抗生素被发现以来，患者首次死于无法治愈的感染。革兰氏阴性菌产生多药耐药性（MDR）的主要原因是细菌泵主动流出药物。了解泵的调节方式是了解 MDR 发展的关键：细菌只需增加这些外排泵的表达即可成为 MDR。意义：脆弱拟杆菌是普遍存在的肠道共生菌，当它们逃离肠道时会引起毁灭性的疾病。他们对抗菌药物的耐药性正在显着上升。在需氧菌中，MDR 是由转录因子 (TF) 调节的外排泵引起的。我们假设脆弱拟杆菌中的 MDR 外排泵同样受到 TF 的调节。这些转录因子及其调节的泵也可能调节其他毒力因子。 VA 患者尤其面临厌氧菌感染的风险。拟杆菌菌血症的死亡率很高（25-50%），而 VA 患者常见的肝病是死亡率增加的危险因素。我们发现了几种具有增加泵活性的 MDR 临床分离株（包括来自阿富汗胃肠道的一种）；这些分离株对甲硝唑和其他药物具有耐药性，分别导致 1 例死亡和 1 例截肢。初步研究：在之前的资助期间，我们鉴定并表征了脆弱拟杆菌 bme 外排泵基因，并表明它们与临床 MDR 有关（在 23 篇出版物中进行了描述）；我们发现泵水平和阻力的增加可以由多种药物和应激源引起，包括抗菌剂、水杨酸盐、胆汁和群体感应分子。我们构建了新的载体并对其进行了修改以模仿可用于需氧菌的分子工具。研究计划：我们的长期目标是阐明脆弱拟杆菌中 MDR 的复杂调控。具体来说，我们现在的目标是： 目标 1：使用功能基因组方法识别外排介导的 MDR 中最重要的 TF（激活子和阻遏子 TF）。目标 2：表征外排介导的 MDR 中最重要的 TF。确定与 TF 相关的表型，测量 MIC（最低抑制浓度）并确定相关的 bme 基因。通过物理和功能方法展示结合力。目标 3：确定外排介导的 MDR 中 TF 调节的临床患病率。测量临床分离株（包括最近获得的 MDR 菌株）中的 TF 转录水平。确定 TF 对外排介导的 MDR 和形成生物膜的能力的影响。评估胆汁暴露对 TF 转录水平、MIC 和生物膜形成能力的影响。这项研究利用了我们独特的资源：分子和生化方法的专业知识、大量临床分离株和相关 MIC 数据的获取，以及与世界一流专家的良好合作关系。一旦完成，这项工作连同已经进行的研究将揭示有关 BF MDR 泵调节的重要新信息，并将极大地有助于我们了解这种细菌的调节机制，这种细菌受到与需氧病原体截然不同的压力。脆弱拟杆菌是肠道微生物组（被称为人体内的一个独立“器官”）的重要组成部分，它被认为具有自己令人印象深刻的代谢特征，并深刻影响人类健康和疾病的几乎各个方面。在这种全球环境下，从第一个耐多药菌株的出现到广泛耐药性的时间很短。当出现无法治疗的感染时竞相开发有效药物并不是可行的公共卫生策略。 MDR 流出的调节因子是药物发现的重要目标；识别和理解这些 MDR 生物体中最重要的转录因子将导致针对这些因子的表达或翻译的新的有效介入疗法的开发。 公共卫生相关性： 仅在美国，不断上升的抗菌素耐药性就造成了 5-300 亿美元的损失和 90,000 人的生命！多药外排泵是造成这种阻力的主要原因。脆弱拟杆菌是肠道微生物群的重要成员，在人类健康和疾病中发挥着关键作用。我们几乎没有关于这些泵或其在这种重要厌氧病原体中的调节的信息。