Development of Protein-Based Beta-lactam Antibiotic Resistance Diagnostics

基于蛋白质的 β-内酰胺抗生素耐药性诊断的开发

• 批准号: 8112233
• 负责人: Timothy Palzkill
• 金额: $ 19.56万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-07 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8112233
• 关键词: Accounting; Address; Amino Acid Sequence Homology; Amino Acids; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Binding; Biological Assay; Carbapenems; Cephalosporins; Chimera organism; Clinical; Consumption; DNA; Detection; Development; Diagnosis; Diagnostic; Diagnostic Reagent; Drug resistance; Engineering; Enzyme-Linked Immunosorbent Assay; Enzymes; Escherichia coli; Exhibits; Future; Genetic Screening; Goals; Gram-Negative Bacteria; Hospitals; Hydrolysis; Infection; Infection Control; Klebsiella pneumonia bacterium; Laboratories; Lactamase; Lactams; Left; Libraries; Mediating; Methods; Monobactams; Multi-Drug Resistance; Mutation; Penicillins; Pharmaceutical Preparations; Plasmids; Property; Protein Binding; Proteins; Reagent; Research; Resistance; Sampling; Screening procedure; Sensitivity and Specificity; Source; Streptomyces; System; Testing; Validation; Variant; Work; antimicrobial; bacterial resistance; base; beta-Lactam Resistance; clinically relevant; drug resistant bacteria; efficacy testing; meetings; mutant; programs; research study; resistant strain; retinal rods; tool

DESCRIPTION (provided by applicant): ?-lactam antibiotics such as the penicillins and cephalosporins are the most often used antibiotics and account for more than 60% of total world consumption of antimicrobials. Due to widespread ?-lactam antimicrobial use, bacterial resistance has been increasing and now represents a serious threat to the continued use of antibiotic therapy. The current situation with hospital-associated infections resulting from antibiotic resistant gram-negative rods is critical in that no new drugs are expected in the near future to treat these infections. Resistance rates have been increasing for several gram-negative species and multidrug resistance is a particular problem in that some clinical strains are resistant to many classes of antibiotics; leaving few options for treatment. The most common mechanism of bacterial resistance to ?-lactam antibiotics is the synthesis of ?-lactamases that hydrolyze the drugs to generate ineffective products. ?-lactamases are classified into four groups A, B, C and D based on amino acid sequence homologies. Class A ?-lactamases are widespread in both gram-positive and gram-negative bacteria and exhibit broad substrate hydrolysis profiles which include penicillins, cephalosporins and, for a few enzymes, carbapenems. The class A TEM-1 and SHV-1 ?-lactamases are common plasmid-encoded ?-lactamases in gram-negative bacteria and are a widespread source of antibiotic resistance. The class A KPC b-lactamase has emerged in K. pneumoniae and other gram-negative rods in recent years and is a cause for concern due to its broad substrate profile that includes virtually all ?-lactam antibiotics including carbapenems. Adding to the concern is the difficulty in diagnosing infections with carbapenem resistance mediated by KPC. The ?-lactamase inhibitory protein (BLIP) is a 165 amino acid protein produced by Streptomyces clavuligerus which binds and inhibits several class A ?-lactamases. The goal of the project is to develop a BLIP-based protein reagent that can be used to specifically identify the KPC enzyme while not binding to other class A ?-lactamases such as the common TEM-1 and SHV-1 enzymes. In particular, the proposed experiments will utilize the class A ?-lactamase binding profile of BLIP in combination with a recently developed genetic screen to tailor the BLIP recognition properties to create variants that can uniquely recognize KPC ?-lactamase and thereby gain detailed information on the antibiotic resistance potential of clinical isolates that can be used to guide treatment and infection control strategies. In addition, the work will guide future studies using the proposed approaches for the development of similar assays targeting other emerging ?-lactamases. PUBLIC HEALTH RELEVANCE: This project addresses the need for identification of KPC ?-lactamase-mediated antibiotic resistance in gram-negative bacteria. ?-lactamases catalyze the destruction of b-lactam antibiotics and are the most common mechanism of resistance to these drugs. The proposed experiments will create an engineered version of the ?-lactamase inhibitory protein that is able to specifically recognize the clinically important KPC ?-lactamase and thereby can serve as an efficient diagnostic reagent to guide treatment and infection control strategies.

描述（由申请人提供）： β-内酰胺类抗生素，如青霉素类和头孢菌素类是最常用的抗生素，占世界抗菌药物总消费量的 60% 以上。由于β-内酰胺抗菌剂的广泛使用，细菌耐药性不断增加，现在对抗生素治疗的持续使用构成了严重威胁。目前由抗生素耐药性革兰氏阴性杆菌引起的医院相关感染的情况非常严峻，因为预计在不久的将来不会有新的药物来治疗这些感染。一些革兰氏阴性菌种的耐药率一直在增加，并且多重耐药性是一个特殊的问题，因为一些临床菌株对多种抗生素具有耐药性；留下的治疗选择很少。细菌对β-内酰胺抗生素产生耐药性的最常见机制是合成β-内酰胺酶，水解药物产生无效产物。根据氨基酸序列同源性，β-内酰胺酶分为A、B、C和D四组。 A 类 β-内酰胺酶广泛存在于革兰氏阳性和革兰氏阴性细菌中，并表现出广泛的底物水解特征，其中包括青霉素、头孢菌素以及少数酶的碳青霉烯类。 A 类 TEM-1 和 SHV-1 β-内酰胺酶是革兰氏阴性细菌中常见的质粒编码的 β-内酰胺酶，是抗生素耐药性的广泛来源。 A 类 KPC β-内酰胺酶近年来出现在肺炎克雷伯菌和其他革兰氏阴性杆菌中，由于其广泛的底物谱（包括碳青霉烯类在内的几乎所有 β-内酰胺抗生素）而引起人们的关注。更令人担忧的是诊断 KPC 介导的碳青霉烯类耐药性感染的难度。 β-内酰胺酶抑制蛋白 (BLIP) 是由棒状链霉菌 (Streptomyces clavuligerus) 产生的 165 个氨基酸的蛋白质，可结合并抑制多种 A 类 β-内酰胺酶。该项目的目标是开发一种基于 BLIP 的蛋白质试剂，可用于特异性识别 KPC 酶，同时不与其他 A 类 β-内酰胺酶（例如常见的 TEM-1 和 SHV-1 酶）结合。特别是，所提出的实验将利用 BLIP 的 A 类 β-内酰胺酶结合谱与最近开发的遗传筛选相结合，以定制 BLIP 识别特性，以创建能够独特识别 KPC β-内酰胺酶的变体，从而获得有关该酶的详细信息。临床分离株的抗生素耐药潜力可用于指导治疗和感染控制策略。此外，这项工作将指导未来的研究，使用所提出的方法来开发针对其他新兴β-内酰胺酶的类似测定方法。 公共卫生相关性：该项目解决了鉴定革兰氏阴性菌中 KPC β-内酰胺酶介导的抗生素耐药性的需求。 β-内酰胺酶催化β-内酰胺抗生素的破坏，是对这些药物产生耐药性的最常见机制。拟议的实验将创建β-内酰胺酶抑制蛋白的工程版本，该蛋白能够特异性识别临床上重要的KPC β-内酰胺酶，从而可以作为有效的诊断试剂来指导治疗和感染控制策略。