Development of Protein-Based Beta-lactam Antibiotic Resistance Diagnostics

描述（由申请人提供）：内酰胺抗生素如青霉素和头孢菌素是最常用的抗生素，占世界抗微生物剂总消耗量的60%以上。由于广泛？随着内酰胺类抗生素的使用，细菌耐药性一直在增加，现在对抗生素治疗的持续使用构成了严重威胁。目前由耐抗生素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药治疗这些感染。几种革兰氏阴性菌的耐药率一直在增加，多药耐药性是一个特别的问题，因为一些临床菌株对许多类别的抗生素都有耐药性;几乎没有治疗选择。最常见的细菌耐药机制是？-内酰胺类抗生素是合成？-水解药物产生无效产物的内酰胺酶。?-根据氨基酸序列同源性，内酰胺酶分为A、B、C和D四组。A级？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性，包括青霉素、头孢菌素和碳青霉烯类（对于少数酶）。A类TEM-1和SHV-1？内酰胺酶是常见的质粒编码的？革兰氏阴性菌中的内酰胺酶，并且是抗生素耐药性的广泛来源。A类KPC β-内酰胺酶已在克雷伯氏菌中出现。肺炎和其他革兰氏阴性杆菌，是一个令人关注的原因，由于其广泛的底物概况，包括几乎所有？内酰胺类抗生素，包括碳青霉烯类。更令人担忧的是，难以诊断KPC介导的碳青霉烯耐药感染。什么？-内酰胺酶抑制蛋白（BLIP）是由棒状链霉菌产生的165个氨基酸的蛋白质，其结合并抑制几种A？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？内酰胺酶，如常见的TEM-1和SHV-1酶。特别是，拟议的实验将利用A类？- BLIP的内酰胺酶结合特征与最近开发的基因筛选相结合，以定制BLIP识别特性，从而产生能够唯一识别KPC？的变体。通过对内酰胺酶的分析，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，这些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，使用提出的方法开发类似的检测方法，针对其他新兴的？内酰胺酶。 公共卫生相关性：该项目解决了查明科索沃保护团的需要。内酰胺酶介导的革兰氏阴性菌抗生素耐药性。?-内酰胺酶催化β-内酰胺抗生素的破坏，并且是对这些药物的抗性的最常见机制。拟议中的实验将创造一个工程版本的？-内酰胺酶抑制蛋白，能够特异性识别临床重要的KPC？-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。

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.