Rapid antimicrobial susceptibility determination of bacterial pathogens

细菌病原体的快速抗菌药敏测定

• 批准号: 8811098
• 负责人: ROBERT M DICKSON
• 金额: $ 37.77万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8811098
• 关键词: 3-Dimensional; Accounting; Address; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotic susceptibility; Antibiotics; Antimicrobial susceptibility; Bacteria; Bacterial Infections; Blood; Characteristics; Clinic; Clinical; Coupled; Detection; Diagnosis; Diagnostic Procedure; Dimensions; Drug resistance; Ensure; Epiphysial cartilage; Excision; Flow Cytometry; Fluorescence; Growth; Health; Hospitals; Hour; Human; Incidence; Infection; Kinetics; Label; Laboratories; Mammalian Cell; Measurement; Measures; Medical; Metabolism; Methodology; Methods; Minimum Inhibitory Concentration measurement; Molecular; Morbidity - disease rate; Morphology; Natural regeneration; Optics; Outcome; Patients; Population; Predisposition; Procedures; Production; Public Health; Reactive Oxygen Species; Recovery; Resolution; Sampling; Sepsis; Side; Specimen; Speed; Statistical Methods; Stream; Technology; Testing; Time; Translations; Treatment outcome; antimicrobial; base; clinically relevant; cost; cyanine; design; economic cost; effective therapy; fluorophore; improved; innovation; innovative technologies; light scattering; maltodextrin; mortality; new technology; pathogen; research study; response; uptake

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.

描述（由申请人提供）：细菌感染是世界上死亡和发病的主要原因，非常需要改善其治疗的新策略。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敏感性。传统的微生物学诊断依赖于身体样本的培养，通常需要 1-3 天的培养时间，如果存在生长缓慢或挑剔的病原体，则可能会进一步延迟。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针对六种最常见的引起血流感染 (BSI) 的细菌的 MIC。这种新的药敏策略需要不到 1 小时的抗生素孵育时间才能看到特征反应，旨在在血培养呈阳性后 4 小时内确定抗菌药物 MIC，这预示着患者治疗结果的显着改善，并降低了因过度使用抗生素而导致的耐药性发生率。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（相对于哺乳动物细胞）递送和富集技术。该提案的中心假设是，“通过结合流式细胞技术测量抗生素诱导的 ROS 和细菌中的散射变化，可在血培养呈阳性的 4 小时内对六种最常见的引起 BSI 的细菌产生抗菌敏感性” ，每个都加上严格的多维统计距离度量以进行量化。”该提案中的实验将开发一套互补的流式细胞术技术，可在传代培养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呈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获得可操作信息的速度比目前实施的速度至少快 36 小时，从而直接改善患者的治疗效果并最大限度地减少抗生素耐药性的选择。每个目标都利用高度创新的技术来实现这些对人类健康潜在的重大益处。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 将结合起来，用于量化近 MIC 抗生素暴露后所证实的 ROS 产生 42-44。目标 2 将证明，<1 小时、接近 MIC 的抗生素暴露会引起特征性细菌形态变化，这些变化可通过散射光检测到。开发多维统计距离指标与配对对照的比较是为了量化这些变化并确定存在生物变异性的情况下的无标记 MIC。目标 3 将结合目标 1 和 2 的创新，使用 ROS 探测临床分离株，并通过 3D 统计距离度量进行散射，同时对从血培养中快速恢复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。拟议的实验解决了减少细菌感染造成的巨大人力和经济成本的巨大需求。