Metabolic Assessment of Anti-Microbial Susceptibility within One Cell Cycle

项目摘要 抗生素耐药细菌/真菌的日益出现已成为日益严重的全球威胁， 滥用和过度使用抗菌药物。为了对抗感染和减少抗微生物 因此，在早期检测和表征细菌/真菌对抗菌剂的敏感性是至关重要的 感染的各个阶段，以减少抗菌药物的不当使用和死亡率。为了解决这个 紧急医疗条件下，快速准确地确定抗菌药物敏感性至关重要， 细菌/真菌，以便可以在疾病过程的早期开出最佳治疗药物。常规 用于抗菌药物敏感性测试的方法，例如琼脂平板和肉汤稀释测定，检测表型 在存在所测试的抗微生物药物的情况下基于细菌/真菌生长的抗性。一个主要 这些方法的局限性在于它们是基于培养的，并且需要至少16至24小时来进行。到 针对这一未满足的需求，提出了一种微秒级受激拉曼光谱成像平台 为了能够在复杂环境中以亚微米分辨率原位检测单个细菌， 确定其对抗微生物药物的反应。一个跨学科小组将进行拟议的研究。 博士Ji-Xin Cheng（PI）是相干拉曼散射显微镜的发明者和领先专家。穆罕默德博士 Seleem（co-PI）是一名DVM科学家，在传染病和微生物学方面拥有广泛的专业知识。Dr. Ryan F. Relich（顾问），印第安纳州大学健康临床病毒学和血清学实验室的医学主任， 具有丰富的传染病临床诊断经验。该团队的核心假设是， 微秒尺度的相干拉曼光谱成像将使单个微生物细胞的原位分析成为可能 直接从临床样品（全血）富集。为了验证这一假设，研究小组将迅速证明 通过代谢产物的微秒级受激拉曼成像测定抗菌反应 活性在一个单一的活细菌（目标1），开发微秒级宽带受激拉曼 用于无标记鉴别细菌和测定抗微生物剂光谱显微镜 敏感性（目标2），并证明真菌早期检测和快速抗菌药物敏感性分析 感染（目标3）。所提出的快速AST方法适用于复杂环境中的细菌/真菌， 单细胞水平。因此，可以通过长时间的标本培养和传代培养来获得细菌/真菌分离株， 避免了这种方法的特点提供了一个显着的进步，目前的方法， 治疗细菌/真菌感染。

Project Summary The increasing emergence of antimicrobial-resistant bacteria/fungi has become a growing global threat due to the misuse and overuse of antimicrobial drugs. In order to combat infections and reduce anti-microbial resistance, it is essential to detect and characterize bacterial/fungal susceptibility to antimicrobial in the early stages of infections to reduce the inappropriate use of antimicrobial drugs and the death rate. To address this urgent medical condition, it is critical to rapidly and accurately determine the antimicrobial susceptibility of bacteria/fungi so that optimal therapy drugs can be prescribed early in the disease process. Conventional methods for antimicrobial susceptibility testing, such as agar plates and broth dilution assays, detect phenotypic resistance based on bacterial/fungal growth in the presence of antimicrobial drugs being tested. A major limitation of these methods is that they are based on culture and require at least 16 to 24 h to conduct. To address this unmet need, a microsecond-scale stimulated Raman spectroscopic imaging platform is proposed to enable in situ detection of a single bacterium in complex environment at sub-micron resolution and early determination of its response to an antimicrobial drug. An interdisciplinary team will conduct the proposed study. Dr. Ji-Xin Cheng (PI) is an inventor and leading expert in coherent Raman scattering microscopy. Dr. Mohamed Seleem (co-PI) is a DVM-scientist with broad expertise in infectious diseases and microbiology. Dr. Ryan F. Relich (consultant), Medical Director of the Indiana University Health Clinical Virology and Serology Laboratories, has extensive experience in clinical diagnosis of infectious diseases. The team’s central hypothesis that microsecond-scale coherent Raman spectroscopic imaging will enable in situ analysis of single microbial cells enriched directly from a clinical sample (whole blood). To test this hypothesis, the team will demonstrate fast determination of antimicrobial response through microsecond-scale stimulated Raman imaging of metabolic activity in a single living bacterium (aim 1), develop a microsecond-scale broadband stimulated Raman spectroscopic microscope for label-free discrimination of bacteria and determination of anti-microbial susceptibility (aim 2), and demonstrate early detection and fast antimicrobial susceptibility profiling of fungal infections (aim 3). The proposed rapid AST method works for bacteria/fungi in complex environment and at the single cell level. Therefore, long-time specimen culture and subculture to get bacterial/fungal isolate can be avoided. The characteristics of this approach offer a significant advancement over current approaches for treatment of bacterial/fungal infections.