Rapid antimicrobial susceptibility determination of bacterial pathogens

细菌病原体的快速抗菌药敏测定

• 批准号: 8711615
• 负责人: ROBERT M DICKSON
• 金额: $ 37.91万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-15 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8711615
• 关键词: 3-Dimensional; Accounting; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotic susceptibility; Antibiotics; Antimicrobial susceptibility; Bacteria; Bacterial Infections; Blood; Characteristics; Clinic; Clinical; Coupled; Data; Detection; Diagnosis; Diagnostic; Diagnostic Procedure; Dimensions; Drug resistance; Dyes; Ensure; Epiphysial cartilage; Flow Cytometry; Fluorescence; Health; Hospitals; Hour; Human; Incidence; Infection; Kinetics; Label; Laboratories; Mammalian Cell; Measurement; Measures; Medical; Metabolism; Methodology; Methods; Metric; Molecular; Morbidity - disease rate; Morphology; Multi-Drug Resistance; Natural regeneration; Nature; Optics; Outcome; Patients; Pharmaceutical Preparations; Population; Predisposition; Procedures; Production; Public Health; Reactive Oxygen Species; Reporting; Resistance; Resolution; Risk; Sampling; Sepsis; Side; Specimen; Speed; Statistical Methods; Stream; Technology; Testing; Time; Translations; Treatment outcome; antimicrobial; base; clinically relevant; cyanine; design; economic cost; effective therapy; fluorophore; improved; innovation; innovative technologies; interest; 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 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.

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身体标本的培养，通常需要1-3天的孵化时间，如果存在挑剔的病原体，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，快速检测在培养中生长的细菌或直接标记在阳性血培养中的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采取行动的信息比目前可能的速度至少快36小时，直接影响患者的出院，并将抗生素过度使用和耐药性问题降至最低。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和我们的高灵敏度荧光ROS剂量计将结合起来，并应用于量化在接近MIC的抗生素暴露时展示的ROS产生。目的2将证明1小时，近MIC抗生素暴露引起的特征细菌形态变化是可以用散射光检测到的。多维统计距离度量与配对和非配对对照的对比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，同时通过直接在血液培养中对荧光团靶标细菌进行选通，进一步缩短结果时间。考虑到对新的细菌诊断的巨大需求以及细菌感染带来的巨大的人力和经济成本，我们预计会对这里提出的实验产生广泛的兴趣。