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率和发病率的主要原因，并且非常需要新的改善治疗的策略。限制细菌感染治疗的一个关键问题是无法快速确定抗体敏感性。常规的微生物诊断取决于从身体标本培养的，通常需要1-3天的孵化时间，这可以通过存在缓慢的或挑剔的病原体来进一步延迟。该建议的目的是制定一种流式细胞术策略，以快速确定抗生素的二种最常见的血液感染（BSI）引起的细菌。这种新的易感性策略旨在确定血液培养阳性4小时内的抗菌麦克风，为患者治疗结果改善了显着改善，并降低了由过量的抗生素使用引起的耐药率降低。此处提出的流式细胞仪方法依赖于统计，散射的光和选择性细菌（与哺乳动物细胞）的递送和富集技术最近在我们的实验室中开发的。该提议的核心假设是：“对于六个最常见的BSI引起的细菌，可以在4小时内产生抗菌易感性，使用流式细胞仪技术的组合，以测量抗生素诱导的ROS和散射细菌的变化，每种抗生素诱导的变化，每种均与严格的多维多维统计距离进行统计学距离，以实现量化量化量化。该提案中的实验将开发一组互补的流式细胞术技术，该技术在亚培养分离后或直接从阳性血液培养物中恢复后，迅速确定细菌的抗菌MIC。成功后，这些研究将使抗生素易感性确定的时间减少到〜4小时，即阳性血液培养。去除这种治疗瓶颈将使可操作的信息至少比目前实施的速度至少36小时，从而直接改善患者的预后并最大程度地减少抗生素耐药性。每个目标都利用高度创新的技术来为人类健康带来这些潜在的重大利益。在AIM 1中，我们的独特基于麦芽糊精的细菌靶向，30和我们高度敏感的荧光ROS Dosimeters31将合并并应用以量化所示的ROS产生42-44，近乎MIC抗生素暴露后。 AIM 2将表明，<1小时，近MIC抗生素暴露会引起特征性细菌形态的变化，这些变化可通过散射光检测到。开发了多维统计距离指标与配对对照，以量化这些变化并确定在存在生物可行性的情况下无标签的麦克风。 AIM 3将使用ROS探测AIM 1和2的创新，并使用3D统计距离指标散射，而对荧光团靶向细菌的门控迅速从血液培养中恢复为临床，将进一步降低到期时间的约束。拟议的实验涉及减少细菌感染的巨大人类和经济成本的巨大需求。