Development of Protein-Based Beta-lactam Antibiotic Resistance Diagnostics

描述（由申请人提供）：？-内酰胺类抗生素，如青霉素和头孢菌素，是最常用的抗生素，占世界抗菌药物总消费量的60%以上。由于广泛？在使用-内酰胺抗菌素时，细菌耐药性一直在增加，现在对抗生素治疗的继续使用构成严重威胁。目前由耐抗生素革兰氏阴性杆状体引起的医院相关感染的情况至关重要，因为预计在不久的将来不会有新药来治疗这些感染。几种革兰氏阴性菌株的耐药率一直在上升，多药耐药是一个特殊问题，因为一些临床菌株对许多种类的抗生素具有耐药性；留给治疗的选择很少。细菌最常见的耐药机制是什么？-内酰胺类抗生素是如何合成的？水解药物产生无效产物的内酰胺酶。？-内酰胺酶根据氨基酸序列同源性可分为A、B、C、D四类。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谱，包括青霉素类、头孢菌素类和少数碳青霉烯类酶。A级TEM-1和SHV-1 ？-内酰胺酶是常见的质粒编码？革兰氏阴性细菌中的-内酰胺酶，是抗生素耐药性的广泛来源。近年来，A类KPC b-内酰胺酶在肺炎克雷伯菌和其他革兰氏阴性杆状菌中出现，由于其广泛的底物分布，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，难以诊断由KPC介导的碳青霉烯类耐药感染。的吗?-内酰胺酶抑制蛋白（BLIP）是由链霉菌clavuligerus产生的一种含有165个氨基酸的蛋白，它能结合和抑制几种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，该试剂可用于特异性鉴定KPC酶，同时不与其他a类酶结合。-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 -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，以创建能够唯一识别KPC ？-内酰胺酶，从而获得临床分离株抗生素耐药潜力的详细信息，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，使用拟议的方法开发针对其他新兴-内酰胺酶的类似检测。

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.