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（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，这可能因难养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）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。“这项提案中的实验将开发一套互补的流式细胞技术，快速检测细菌的抗菌MIC，无论是在培养物中生长还是直接在阳性血培养物中标记。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能够采取行动的信息比目前可能的速度快至少36小时，直接影响患者的预后，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利益。在目标1中，我们独特的基于麦芽糊精的细菌靶向和我们高度灵敏的荧光活性氧剂量计将结合起来，并应用于量化接近MIC抗生素暴露后所证明的活性氧产生。目标2将证明<1小时，接近MIC的抗生素暴露诱导的特征性细菌形态学变化可用散射光检测。开发了多维统计距离度量与配对和未配对对照，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将联合收割机结合目标1和2的创新，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，同时通过直接在血液培养物中对荧光团靶向细菌进行门控来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，我们预计本文提出的实验会引起广泛的兴趣。