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.

-内酰胺类抗生素，如青霉素和头孢菌素是最常用的抗生素，占全球抗菌药总消费量的60%以上。由于β-内酰胺类抗菌药物的广泛使用，细菌耐药性一直在增加，现在严重威胁着抗生素治疗的继续使用。目前由抗生素耐药革兰氏阴性杆菌引起的医院相关感染的情况很关键，因为预计在不久的将来不会有新的药物来治疗这些感染。几种革兰氏阴性菌的耐药率一直在上升，多药耐药是一个特别的问题，因为一些临床菌株对多种抗生素具有耐药性；几乎没有治疗选择。细菌对β-内酰胺类抗生素耐药最常见的机制是合成β-内酰胺酶，这种酶能使药物产生无效产物。根据氨基酸序列同源性，β-内酰胺酶可分为A、B、C和D四类。A类β-内酰胺酶广泛存在于革兰氏阳性菌和革兰氏阴性菌中，具有广泛的底物水解谱，包括青霉素类、头孢菌素类和碳青霉烯类。A类TEM1和SHV-1β-内酰胺酶是革兰氏阴性菌中常见的质粒编码β-内酰胺酶，是抗生素耐药性的广泛来源。近年来，肺炎克雷伯菌和其他革兰氏阴性杆菌中出现了A类KPCβ-内酰胺酶，由于其广泛的底物谱几乎包括所有的β-内酰胺类抗生素，包括碳青霉烯类抗生素，因此引起了人们的关注。更令人担忧的是，在诊断由KPC介导的碳青霉烯耐药感染方面存在困难。β-内酰胺酶抑制蛋白(BLIP)是棒状链霉菌产生的一种由165个氨基酸组成的蛋白质，可结合和抑制多种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，该试剂可用于特异性鉴定KPC酶，而不与其他A-内酰胺酶结合，如常见的TEM-1和SHV-1酶。特别是，拟议的实验将利用BLIP的A类β-内酰胺酶结合谱结合最近开发的遗传筛选来定制BLIP识别属性，以创建能够唯一识别KPC-内酰胺酶的变体，从而获得关于临床分离株的抗生素耐药性潜力的详细信息，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，使用建议的方法来开发针对其他新兴β-内酰胺酶的类似检测方法。 公共卫生相关性：该项目解决了鉴定革兰氏阴性细菌中KpC？-内酰胺酶介导的抗生素耐药性的需要。β-内酰胺酶催化β-内酰胺类抗生素的破坏，是这些药物最常见的耐药机制。拟议的实验将创造一种工程版本的β-内酰胺酶抑制蛋白，能够特异性地识别临床上重要的KPC-内酰胺酶，从而可以作为一种有效的诊断试剂来指导治疗和感染控制策略。