Beta-lactamase fluorescent probes for bacterial detection

项目摘要 2014年9月，奥巴马总统签署了一项行政命令，指示联邦资源改善 检测和预防抗生素耐药性，目标是到2020年开发一种诊断测试，可以 在至少20分钟内区分细菌感染和病毒感染。这项研究的长期目标是 提供一种新的平台解决方案，用于快速、敏感和特异地检测病原性细菌感染 通过开发针对细菌中表达的单个β-内酰胺酶的荧光探针。 β-内酰胺酶是一类破坏β-内酰胺类抗生素和提供抗生素的细菌水解酶 细菌病原体的抗药性。今天，许多疑似感染的患者都接受了抗生素治疗。 (最常见的是β-内酰胺类)在经验上没有事先正确识别病原体，导致 抗生素的过度使用和耐药性的过度传播，每年全球有50万人死亡 可归因于这些感染。 这项提案将重点关注两种致命细菌：结核分枝杆菌(Mtb)和碳青霉烯耐药 肠杆菌科(Cre)。结核病是世界上最危险的呼吸道病原体之一 在人类历史上，每年有100多万人死亡，感染世界三分之一的人口。 结核杆菌表达一种Ambler A类β-内酰胺酶blc，因此结核分枝杆菌对β-内酰胺类具有固有的耐药性 抗生素。越来越多的结果，包括我们的结果，支持使用BARC作为生物标志物 结核杆菌检测。 广谱碳青霉烯类药物通常是治疗感染的最后选择 耐药细菌，但碳青霉烯耐药肠杆菌科(CRE)的出现超过 在过去的十年里，临床医生几乎没有治疗选择。CRE经常是由于产生了 碳青霉烯酶能有效水解碳青霉烯类抗生素和其他β-内酰胺类抗生素。 该项目将探索新的双猝灭、双靶向探针设计策略，以开发BLAC特异性 用于快速、高灵敏度检测结核分枝杆菌的底物探针(AIM#1)和碳青霉烯酶特异性底物 用于快速、高灵敏Cre检测的探头(目标2)。这些新的探头将在临床上进行评估 病人样本。 这项研究的预期结果是将发现一系列新型荧光探针作为一种 新的检测平台，能够快速、灵敏和特异地检测结核分枝杆菌和补体。我们设想这些 检测可以减少与细菌感染治疗相关的不必要的医疗费用，并防止 进一步扩大现有的耐药菌株。

Project Summary In September 2014, President Barack Obama signed an executive order directing federal resources to improve detection and prevention of antibiotic resistance, with a goal of developing a diagnostic test by 2020 that can distinguish between bacterial and viral infections in at least 20 min. The long-term goal of this research is to provide a novel platform solution for rapid, sensitive and specific detection of pathogenic bacterial infections through development of fluorescent probes specific to individual β-lactamases expressed in the bacteria. β-Lactamases are a class of bacterial hydrolases destroying β-lactam antibiotics and rendering antibiotics resistance in bacterial pathogens. Today many patients with suspected infections are administered antibiotics (most frequently β-lactams) empirically without prior proper identification of the causative agent, resulting in antibiotics overuse and overspread of antimicrobial resistance, with >500,000 deaths in the world annually attributable to these infections. This proposal will focus on two deadly bacteria: Mycobacterium tuberculosis (Mtb) and carbapenem-resistant Enterobacteriaceae (CRE). Tuberculosis represents one of the most dangerous respiratory pathogens in the history of mankind, killing over one million people each year and infecting one third of the world's population. Tubercle bacilli express BlaC, an Ambler class A β-lactamase, so Mtb has the intrinsic resistance to β-lactam antibiotics. Accumulating results including those from us have supported the use of BlaC as the biomarker for Mtb detection. Broad-spectrum carbapenem agents are frequently the last option for effective therapy of infections with antimicrobial resistant organisms, but the emergence of carbapenem-resistant Enterobacteriaceae (CRE) over the past decade has left clinicians with few treatment options. CRE is frequently due to the production of carbapenemase enzymes that efficiently hydrolyze carbapenems and other β-lactam antibiotics. This project will explore novel double-quenching, dual targeting probe design strategy to develop BlaC-specific substrate probes for rapid, highly sensitive Mtb detection (Aim #1) and carbapenemase-specific substrate probes for rapid, highly sensitive CRE detection (Aim #2). These new probes will be evaluated with clinical patient samples. The expected outcome of this research is that a series of novel fluorescent probes will be discovered as a novel assay platform to enable rapid, sensitive and specific detection of Mtb and CRE. We envision that these assays could reduce unnecessary healthcare costs associated with treatment of bacterial infection and prevent further expansion of existing drug-resistant strains.