Rapid antimicrobial susceptibility determination of bacterial pathogens

描述（由申请人提供）：细菌感染是世界上死亡率和发病率的主要原因，迫切需要新的策略来改善其治疗。限制细菌感染治疗的一个关键问题是无法快速确定抗生素的敏感性。传统的微生物诊断依赖于人体标本的培养，通常需要1-3天的孵育，可能会因存在挑剔的病原体而进一步延迟。本提案的目的是开发一种流式细胞术策略，用于快速确定抗生素对六种最常见的血流感染（BSI）的mic。这种新的药敏策略需要<1小时的抗生素孵育时间才能看到特征性反应，旨在确定4小时内血培养阳性的抗菌mic，预示着患者治疗结果的显着改善，并降低过度使用抗生素引起的耐药发生率。本文提出的流式细胞术方法依赖于我们实验室最近开发的统计、散射光和选择性细菌（相对于哺乳动物细胞）传递和富集技术。该提案的核心假设是“使用流式细胞术技术测量抗生素诱导的ROS和细菌中的分散变化，可以在阳性血培养后4小时内对六种最常见的bsi引起的细菌产生抗菌敏感性，每种技术都与严格的多维统计距离指标相结合进行量化。”本提案中的实验将开发一套互补的流式细胞术技术，用于快速检测培养物中生长的细菌或直接在阳性血培养物中标记的细菌的抗菌mic。如果成功，这些研究将减少到抗生素敏感性测定的时间少至4小时，阳性血培养后。消除这一治疗瓶颈将使收集可采取行动的信息比目前可能的至少快36小时，直接影响患者的门诊就诊，并最大限度地减少抗生素过度使用和耐药性问题。每个目标都利用高度创新的技术来实现这些对人类健康的潜在重大益处。在Aim 1中，我们独特的基于麦芽糖糊精的细菌靶向和我们高灵敏度的荧光ROS剂量计将结合起来，并应用于量化接近mic抗生素暴露时所演示的ROS产生。目的2将证明<1小时，接近mic抗生素暴露诱导特征性细菌形态变化，可通过散射光检测到。开发了多维统计距离度量与成对和非成对对照，以量化这些变化并确定存在生物变异的无标签mic。Aim 3将结合Aims 1和2的创新，使用ROS探测临床分离物，并使用3d统计距离度量进行分散，同时通过直接在血液培养中筛选荧光团靶向细菌，进一步缩短获得结果的时间。鉴于对新的细菌诊断方法的巨大需求以及细菌感染的巨大人力和经济成本，我们预计这里提出的实验将引起广泛的兴趣。

DESCRIPTION (provided by applicant): Bacterial infections are a major cause of mortality and morbidity in the world, and new strategies for improving their treatment are greatly needed. A key issue limiting treatment of bacterial infections is an inability to rapidly determine antibioti susceptibilities. Conventional microbiological diagnosis depends on culturing from bodily specimens, often requiring 1-3 day incubations that can be further delayed by the presence of fastidious pathogens. The objective of this proposal is to develop a flow cytometric strategy for rapidly determining MICs of antibiotics against the six most common blood stream infections (BSI). Requiring <1-hr antibiotic incubation times to see characteristic responses, this new susceptibility strategy is designed to determine antimicrobial MICs within 4 hours of blood culture positivity, portending significant improvements to patient treatment outcomes and lowered incidence of drug resistance caused by excessive antibiotic use. The flow cytometric methods proposed here rely on statistical, scattered light and selective bacterial (vs. mammalian cell) delivery and enrichment technologies recently developed in our laboratories. The central hypothesis of this proposal is that "Antimicrobial susceptibility can be generated within 4 hours of positive blood culture, for the six most common BSI-causing bacteria, using a combination of flow cytometry technologies that measure antibiotic-induced ROS and scatter changes in bacteria, each coupled with rigorous multidimensional statistical distance metrics for quantification." The experiments in this proposal will develop a complementary set of flow cytometric technologies that rapidly detect antibacterial MICs of bacteria either grown in culture or when tagged directly within positive blood cultures. When successful, these studies will reduce the time to antibiotic sensitivity determinations to as little as ~4 hours, post positive blood culture. Removing this treatment bottleneck will enable action- able information to be garnered at least 36 hours faster than currently possible, directly impacting patient out- comes and minimizing antibiotic overuse and resistance concerns. Each Aim utilizes highly innovative technologies to achieve these potential significant benefits to human health. In Aim 1, our unique maltodextrin-based bacterial targeting, and our highly sensitive fluorogenic ROS dosimeters will be combined and applied to quantify the demonstrated ROS production upon near-MIC antibiotic exposure. Aim 2 will demonstrate that <1-hr, near-MIC antibiotic exposure induces characteristic bacterial morphology changes are detectable with scattered light. Multidimensional statistical distance metrics vs. paired and unpaired controls are developed to quantify these changes and determine label-free MICs in the presence of biovariability. Aim 3 will combine the innovations of Aims 1 & 2 to probe clinical isolates using ROS and scatter with 3-D statistical distance metrics, while further decreasing time-to-result by gating on fluorophore-targeted bacteria directly within blood culture. We anticipate widespread interest in the experiments proposed here, given the great need for new bacterial diagnostics and the tremendous human and economic costs of bacterial infections.