Data_Sheet_1_Single-cell Raman spectroscopy identifies Escherichia coli persisters and reveals their enhanced metabolic activities.docx

Microbial persisters are the featured tiny sub-population of microorganisms that are highly tolerant to multiple antimicrobials. Currently, studies on persisters remain a considerable challenge owing to technical limitations. Here, we explored the application of single-cell Raman spectroscopy (SCRS) in the investigation of persisters. Escherichia coli (ATCC 25922) cells were treated with a lethal dosage of ampicillin (100 μg/mL, 32 × MIC, 4 h) for the formation of persisters. The biochemical characters of E. coli and its persisters were assessed by SCRS, and their metabolic activities were labeled and measured with D2O-based single-cell Raman spectroscopy (D2O-Ramanometry). Notable differences in the intensity of Raman bands related to major cellular components and metabolites were observed between E. coli and its ampicillin-treated persisters. Based on their distinct Raman spectra, E. coli and its persister cells were classified into different projective zones through the principal component analysis and t-distributed stochastic neighbor embedding. According to the D2O absorption rate, E. coli persisters exhibited higher metabolic activities than those of untreated E. coli. Importantly, after the termination of ampicillin exposure, these persister cells showed a temporal pattern of D2O intake that was distinct from non-persister cells. To our knowledge, this is the first report on identifying E. coli persisters and assessing their metabolic activities through the integrated SCRS and D2O-Ramanometry approach. These novel findings enhance our understanding of the phenotypes and functionalities of microbial persister cells. Further investigations could be extended to other pathogens by disclosing microbial pathogenicity mechanisms for developing novel therapeutic strategies and approaches.

微生物持留菌（Microbial persisters）是一类特征性的微小微生物亚群，对多种抗菌药物具有高度耐受性。当前，受限于技术瓶颈，针对持留菌的研究仍存在诸多挑战。本研究探索了单细胞拉曼光谱技术（single-cell Raman spectroscopy, SCRS）在持留菌研究中的应用：以致死剂量的氨苄青霉素（100 μg/mL，32倍最低抑菌浓度，处理4小时）诱导大肠杆菌（Escherichia coli，ATCC 25922）形成持留菌；通过单细胞拉曼光谱技术评估大肠杆菌及其持留菌的生化特性，并采用基于重水的单细胞拉曼光谱技术（D2O-based single-cell Raman spectroscopy, D2O-Ramanometry）标记并检测其代谢活性。研究观察到，大肠杆菌与氨苄青霉素诱导的持留菌之间，与主要细胞组分及代谢物相关的拉曼峰强度存在显著差异。基于二者独特的拉曼光谱，通过主成分分析与t分布随机邻域嵌入算法，可将大肠杆菌及其持留菌划分为不同的投影区域。基于重水吸收速率，大肠杆菌持留菌的代谢活性高于未处理的大肠杆菌。值得注意的是，在终止氨苄青霉素处理后，这些持留菌的重水摄入时序模式与非持留菌存在显著差异。据我们所知，本研究首次通过整合单细胞拉曼光谱技术与氘代拉曼光谱法，实现了大肠杆菌持留菌的鉴定及其代谢活性的评估。这些全新发现加深了我们对微生物持留菌表型与功能的认知。后续研究可通过揭示微生物致病机制，将该方法拓展至其他病原体，以开发新型治疗策略与手段。