The minimum inhibitory concentration (MIC) and MIC based on metabolic activity (MIC-MA) of NaF against S. mutans were measured through the broth dilution test and D₂O-labeled single-cell Raman microspectroscopy, respectively, to explore the inhibitory effect of NaF on the growth and metabolism of S. mutans. The effects of different concentrations of C. albicans supernatant on the metabolic activity of S. mutans were evaluated by using D₂O-labeled single-cell Raman microspectroscopy. The correlation between C. albicans and S. mutans was explored throughassessing the drug resistance of S. mutans against NaF in a mixed system containing C. albicans supernatant by using D₂O-labeled single-cell Raman microspectroscopy.

The MIC and MIC-MA of NaF against S. mutans were 0.4 g·L^-1 and 1.2 g·L^-1, respectively. At the MIC level, S. mutans showed completely inhibited growth but retained high metabolic activity. Therefore, S. mutans may regain its virulence. At the MIC-MA level, the metabolic activity of S. mutans was inhibited only when the NaF concentration reached 3×MIC. At the concentration of OD₆₀₀≥0.5, the C. albicans supernatant significantly promoted the metabolic activity of S. mutans at different time points (P<0.05). In the mixed system, the susceptibility of S. mutans to NaF decreased, and statistical differences at different time points were observed (P<0.05). The metabolic activity of S. mutans was inhibited completely only at the concentration of 4×MIC (1.6 g·L^-1).

D₂O-labeled single-cell Raman microspectroscopy is suitable for evaluating the effects of drugs on bacterial metabolic activity. In the mixed system, the metabolites in the C. albicans supernatant at the concentration of OD₆₀₀≥0.5 significantly promoted the metabolic activity of S. mutans and reducedthe susceptibility of S. mutans to NaF.