Rapid antimicrobial susceptibility determination of bacterial pathogens

描述(申请人提供)：细菌感染是世界上死亡和发病的主要原因，迫切需要新的战略来改进他们的治疗。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。传统的微生物学诊断依赖于人体标本的培养，通常需要1-3天的孵化时间，如果存在生长缓慢或挑剔的病原体，可能会进一步推迟。这项建议的目的是开发一种流式细胞术策略，用于快速测定抗生素对六种最常见的血液感染(BSI)细菌的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典型的反应，旨在确定血培养阳性后4小时内的抗菌素MIC，预示着患者治疗结果的显著改善，并降低因过度使用抗生素而导致的耐药率。这里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(与哺乳动物细胞)传递和浓缩技术。这项提议的中心假设是，“使用流式细胞术技术的组合，测量抗生素诱导的ROS和细菌中的分散变化，可以在血培养阳性的4小时内产生对六种最常见的BSI细菌的抗菌素敏感性，每种技术都结合严格的多维统计距离度量进行量化。”这项计划中的实验将开发一套补充的流式细胞仪技术，在继代培养分离后或直接从阳性血培养中回收后，快速测定细菌的抗菌最低抑菌浓度。如果成功，这些研究将把抗生素敏感性测定的时间减少到血培养阳性后的最少4小时。消除这一治疗瓶颈将使可操作信息的获取速度比目前实施的时间至少快36小时，直接改善患者的预后，并将抗生素耐药性选择降至最低。每个目标都利用高度创新的技术来实现对人类健康的这些潜在的重大好处。在目标1中，我们独特的基于麦芽糊精的细菌靶向30和我们的高灵敏度荧光ROS剂量计31将结合在一起，并应用于量化在接近MIC的抗生素暴露下展示的ROS产量42-44。目的2将证明-lt；1小时，近MIC抗生素暴露诱导特征的细菌形态变化，这是散射光可以检测到的。与配对对照相比，多维统计距离度量被开发来量化这些变化，并在存在生物可变性的情况下确定无标记MIC。AIM 3将结合AIMS 1和AIMS 2的创新，使用ROS和散布技术检测临床分离株，并使用3-D统计距离度量，而对从血培养中快速回收的荧光团靶标细菌进行门控将进一步减少结果的时间限制。拟议的实验解决了减少细菌感染带来的巨大人力和经济成本的巨大需求。

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 incubation times that can be further delayed by the presence of slow- growing or finicky 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 infection (BSI)-causing bacteria. 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 determine antibacterial MICs of bacteria either after subculture isolation or after being recovered directly from positive blood cultures. When successful, these studies will reduce the time to antibiotic susceptibility determinations to as little as ~4 hours, post positive blood culture. Removing this treatment bottleneck will enable actionable information to be garnered at least 36 hours faster than currently implemented, directly improving patient outcomes and minimizing antibiotic resistance se- lection. Each Aim utilizes highly innovative technologies to achieve these potential significant benefits to human health. In Aim 1,our unique maltodextrin-based bacterial targeting,30 and our highly sensitive fluorogenic ROS dosimeters31 will be combined and applied to quantify the demonstrated ROS production42-44 upon near- MIC antibiotic exposure. Aim 2 will demonstrate that <1-hr, near-MIC antibiotic exposure induces characteristic bacterial morphology changes that are detectable with scattered light. Multidimensional statistical distance metrics vs. paired 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 gating on fluorophore-targeted bacteria rapidly recovered from blood culture will further decrease time-to-result constraints. The proposed experiments address the great need to reduce the tremendous human and economic costs of bacterial infections.