title: Metabolic-Activity-Based Assessment of Antimicrobial Effects by D2O-Labeled Single-Cell Raman Microspectroscopy.
abstract: 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 (D2O)-based single-cell Raman microspectroscopy (D2O-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-1as 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, D2O-Raman can serve as a universal method for rapid and quantitative assessment of antimicrobial effects based on general metabolic activity at single-cell resolution.
authors.affiliation: 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.
authors.forename: Yifan
authors.initials: Y
authors.lastname: Tao
authors.affiliation: 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.|University of Chinese Academy of Sciences , Beijing, 100049, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Wang
authors.affiliation: 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.|University of Chinese Academy of Sciences , Beijing, 100049, China.
authors.forename: Shi
authors.initials: S
authors.lastname: Huang
authors.affiliation: 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.|University of Chinese Academy of Sciences , Beijing, 100049, China.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Zhu
authors.affiliation: Department of Engineering Science, University of Oxford , Parks Road, Oxford OX1 3PJ, United Kingdom.
authors.forename: Wei E
authors.initials: WE
authors.lastname: Huang
authors.affiliation: 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.
authors.forename: Junqi
authors.initials: J
authors.lastname: Ling
authors.affiliation: 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.|University of Chinese Academy of Sciences , Beijing, 100049, China.
authors.forename: Jian
authors.identifier: 0000-0002-0548-8477
authors.initials: J
authors.lastname: Xu
mesh_terms: D000667:Ampicillin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D017666:Deuterium Oxide / Q000737:chemistry* / Q000378:metabolism; D052197:Limosilactobacillus fermentum / Q000166:cytology / Q000187:drug effects* / Q000254:growth & development; D059010:Single-Cell Analysis; D013059:Spectrum Analysis, Raman; D013291:Streptococcus / Q000166:cytology / Q000187:drug effects* / Q000254:growth & development
chemical_list: D000900:Anti-Bacterial Agents; D000667:Ampicillin; D017666:Deuterium Oxide