Operative Dentistry and Endodontics, Guanghua School of Stomatology, Affiliated Stomatological Hospital, Guangdong Province Key Laboratory of Stomatology, Sun Yat-Sen University , Guangzhou, Guangdong 510055, China.
Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences , Qingdao, Shandong 266101, China.
Anal Chem. 2017 Apr 4;89(7):4108-4115. doi: 10.1021/acs.analchem.6b05051. Epub 2017 Mar 24.
To combat the spread of antibiotic resistance, methods that quantitatively assess the metabolism-inhibiting effects of drugs in a rapid and culture-independent manner are urgently needed. Here using four oral bacteria as models, we show that heavy water (DO)-based single-cell Raman microspectroscopy (DO-Raman) can probe bacterial response to different drugs using the Raman shift at the C-D (carbon-deuterium vibration) band in 2040 to 2300 cm as a universal biomarker for metabolic activity at single-bacterial-cell resolution. The "minimum inhibitory concentration based on metabolic activity" (MIC-MA), defined as the minimal dose under which the median ΔC-D-ratio at 8 h of drug exposure is ≤0 and the standard deviation (SD) of the ΔC-D ratio among individual cells is ≤0.005, was proposed to evaluate the metabolism-inhibiting efficacy of drugs. In addition, heterogeneity index of MIC-MA (MIC-MA-HI), defined as SD of C-D ratio among individual cells, quantitatively assesses the among-cell heterogeneity of metabolic activity after drug regimens. When exposed to 1× MIC of sodium fluoride (NaF), 1× MIC of chlorhexidine (CHX), or 60× MIC of ampicillin, the cariogenic oral pathogen Streptococcus mutans UA159 ceased propagation yet remained metabolically highly active. This underscores the advantage of MIC-MA over the growth-based MIC in being able to detect the "nongrowing but metabolically active" (NGMA) cells that underlie many latent or recurring infections. Moreover, antibiotic susceptible and resistant S. mutans strains can be readily discriminated at as early as 0.5 h. Thus, DO-Raman can serve as a universal method for rapid and quantitative assessment of antimicrobial effects based on general metabolic activity at single-cell resolution.
为了遏制抗生素耐药性的传播，迫切需要开发能够快速且无需培养即可定量评估药物代谢抑制作用的方法。在这里，我们使用四种口腔细菌作为模型，展示了重水（DO）基单细胞拉曼微光谱（DO-Raman）可以通过在 2040 到 2300 cm 处的 C-D（碳-氘振动）带的喇曼位移来探测细菌对不同药物的反应，将其作为在单细胞分辨率下代谢活性的通用生物标志物。“基于代谢活性的最小抑制浓度”（MIC-MA），定义为药物暴露 8 小时时中位数 ΔC-D-比≤0 且个体细胞 ΔC-D 比标准差（SD）≤0.005 的最小剂量，被用来评估药物的代谢抑制效果。此外，MIC-MA 的异质性指数（MIC-MA-HI），定义为个体细胞间 C-D 比的 SD，定量评估了药物方案后代谢活性的细胞间异质性。当暴露于 1×MIC 的氟化钠（NaF）、1×MIC 的洗必泰（CHX）或 60×MIC 的氨苄西林时，致龋口腔病原体变形链球菌 UA159 停止繁殖但仍保持高度代谢活性。这凸显了 MIC-MA 优于基于生长的 MIC 的优势，因为它能够检测到许多潜伏或复发感染的“非生长但代谢活跃”（NGMA）细胞。此外，敏感和耐药的变形链球菌株可以在 0.5 小时时即可轻易区分。因此，DO-Raman 可以作为一种通用方法，用于快速和定量评估基于单细胞分辨率下的一般代谢活性的抗菌效果。
