Rapid AST through Metabolic Imaging at Single Cell Level

项目摘要 为了对抗感染和减少抗菌素耐药性，检测和表征 感染早期阶段细菌/真菌对抗菌药物的敏感性，以减少抗菌药物的不当使用 抗生素和死亡率。根据R 01 AI 141439（2018年至2022年），我们一直在解决这一紧迫问题。 需要通过开发一个快速AST通过高速受激拉曼散射（SRS）成像的单细胞 新陈代谢.通过葡萄糖-d 7代谢的SRS成像，我们确定了抗生素敏感性 1个细胞周期内的细菌和真菌谱。最近，通过D2 O掺入的SRS成像，我们 在培养菌落不到2.5小时内获得单细胞代谢灭活浓度（SC-MIC） 取得成果。尽管取得了初步成功，但在将这项技术转化为临床方面仍存在差距。 首先，单色SRS信号取决于细菌的大小，因此在预测单光子散射时不是定量的。 细胞代谢活性。其次，我们过去研究中使用的激光器体积太大，无法用于临床。第三，SRS 仅仅成像不能分辨病原体的身份。第四，单井成像室不允许 高通量测量由一组不同浓度的抗生素单独处理的样品。 在R 01 AI 141439的竞争性更新中，我们建议通过三项技术来克服这些障碍。 创新。为了解决第一个和第二个差距，我们将建立一个快速调谐SRS显微镜的基础上， 紧凑型光纤OPO激光器我们已经证明了这种方法在多窗口SRS成像中的可行性 单细胞代谢的一部分。通过在C-D和C-H振动之间快速调谐，我们将能够使用CD/（CD+CH） SRS信号比率来量化单个细胞的代谢活性。为了解决第三个差距，我们将建立多- 彩色荧光原位杂交（FISH）到紧凑的SRS显微镜，使快速识别 和快速AST。我们已经证明了FISH-SRS成像的可行性 肠道微生物组中细菌的代谢活性。为了解决第四个差距，我们将展示高通量 AST通过液体标本的机器人处理和多孔盒设计。一个具有互补性的团队 专家将进行拟议的研究。虽然我们使用尿路感染作为一个重点试验台， 建议，我们的技术广泛适用于其他重要的敏感性的快速测定 感染（例如，血液感染、脑膜炎、肺炎），从而对全球 公共卫生

Project Summary In order to combat infections and reduce antimicrobial resistance, it is essential to detect and characterize bacterial/fungal susceptibility to antimicrobials in the early stages of infections to reduce the inappropriate use of antimicrobials and the rate of death. Under R01AI141439 (2018 to 2022), we have been addressing this urgent need by developing a rapid AST through high-speed stimulated Raman scattering (SRS) imaging of single-cell metabolism. Through SRS imaging of glucose-d7 metabolism, we determined the antimicrobial susceptibility profile of bacteria and fungi within 1 cell cycle. More recently, through SRS imaging of D2O incorporation, we obtained a single-cell metabolism inactivation concentration (SC-MIC) in less than 2.5 h from culturing a colony to obtaining results. Despite this initial success, there are remaining gaps in translating this technology into clinic. First, the single-color SRS signal depends on the bacterial size and thus is not quantitative in predicting single- cell metabolic activity. Second, the laser used in our past studies is too bulky to be used in clinic. Third, SRS imaging alone does not tell the identity of the pathogens. Fourth, the single-well imaging chamber does not allow high-throughput measurement of samples separately treated by a panel of antibiotics at various concentrations. In this competitive renewal of R01AI141439, we propose to overcome these barriers through three technical innovations. To address the first and second gaps, we will build a fast-tuning SRS microscope based on a compact fiber-OPO laser. We have demonstrated the feasibility of this approach in multi-window SRS imaging of single cell metabolism. By fast tuning between C-D and C-H vibration, we will be able to use CD/(CD+CH) SRS signal ratio to quantify the metabolic activity of a single cell. To address the third gap, we will build multi- color fluorescence in situ hybridization (FISH) into the compact SRS microscope to enable rapid identification and fast AST on a single microscope platform. We have demonstrated the feasibility of FISH-SRS for imaging metabolic activity of bacteria in a gut microbiome. To address the fourth gap, we will demonstrate high-throughput AST through robotic handling of liquid specimens and a multi-well cartridge design. A team with complementary expertise will carry out the proposed research. While we use urinary tract infection as a focused testbed in this proposal, our technology is broadly applicable to rapid determination of susceptibility for other important infections (e.g., bloodstream infections, meningitis, pneumonia), thus having a broad, positive impact on global public health.