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，非常需要改进其治疗的新策略。限制细菌感染治疗的关键问题是不能快速确定抗生素敏感性。常规的微生物诊断依赖于从身体样本培养，通常需要1-3天的孵育时间，这可能由于缓慢生长或挑剔的病原体的存在而进一步延迟。该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）致病细菌的MIC。需要<1小时的抗生素孵育时间才能看到特征性反应，这种新的敏感性策略旨在确定血培养阳性4小时内的抗菌药物MIC，预示着患者治疗结果的显著改善和过度使用抗生素引起的耐药性发生率的降低。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（与哺乳动物细胞）递送和富集技术。该提案的中心假设是，“对于六种最常见的导致BSI的细菌，可以在血液培养阳性的4小时内产生抗菌药物敏感性，使用流式细胞术技术的组合，测量细菌中的细菌诱导的ROS和散射变化，每种技术都结合了严格的多维统计距离度量进行量化。“本提案中的实验将开发一套互补的流式细胞术技术，该技术可以在传代分离后或直接从阳性血培养物中回收后快速确定细菌的抗菌MIC。如果成功，这些研究将在血培养阳性后将抗生素敏感性测定时间缩短至约4小时。消除这一治疗瓶颈将使可采取行动的信息能够比目前实施的时间快至少36小时，直接改善患者结局并最大限度地减少抗生素耐药性选择。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利益。在目标1中，我们独特的基于麦芽糖糊精的细菌靶向30和我们的高灵敏度荧光ROS剂量计31将被组合并应用于量化在接近MIC抗生素暴露时所证明的ROS产生42 -44。目标2将证明<1小时，接近MIC的抗生素暴露诱导特征性细菌形态学变化，这些变化可以用散射光检测到。开发了多维统计距离度量与配对对照，以量化这些变化并确定存在生物变异性时的无标记MIC。目标3将联合收割机与目标1和2的创新相结合，使用ROS和具有3-D统计距离度量的散射来探测临床分离株，而对从血液培养中快速回收的荧光团靶向细菌进行门控将进一步减少获得结果的时间限制。所提出的实验解决了减少细菌感染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.