Metabolic Interaction Between Streptococcus Mutans And Candida Albicans Based On Raman Dual Labeling Technology

Objective:To evaluate cellular metabolism,metabolic activity and interactions of Streptococcus mutans and Candida albicans at the single cell level.Methods:（1）Streptococcus mutans,Candida albicans,and Enterococcus faecalis were cultured in each medium respectively until the stationary phase.Cells were sampled and treated as below:（i）Inoculated culture was monitored by the optical density at 600 nm to measure bacterial growth.（ii）Inoculated culture was sampled at logarithmic and stationary phase respectively to measure the Raman spectra,multivariate analysis was performed afterwards to differentiate between growth phases of a single species and strain types of different species.（2）Streptococcus mutans and Candida albicans were inoculated into culture medium with different concentrations of ¹³C-labeled glucose or D₂O until the stable period.We probed the sensitivity of bacterial growth to the¹³C or D₂O level in the medium.（3）Streptococcus mutans and Candida albicans were cultured in MM medium with 30%D₂O and 0.5%¹³C glucose for 12 hours.pure culture and co-culture cells were sampled at different hours for Raman measurement.We compared the dynamic changes of Raman spectra,glucose metabolic rate and cell activity between pure culture and mixed culture groups.Results:（1）The logarithmic phase and stationary phase of the three strains could be rapidly distinguished by the Raman technique.And the classification accuracy rates of random forest were 99.6%,99.86%,and 99.60%;（2）Streptococcus mutans could metabolize ¹³C glucose and be detected by the"red shift"of the phenylalanine peak;0.5%,1.0%of ¹³C glucose would promote the growth of Streptococcus mutans,and 2.5%of ¹³C would inhibit its growth and reduce its metabolic rate（p<0.01）;Candida albicans could metabolize ¹³C glucose and be detected by the peaks of protein,phenylalanine,and thymine.The growth of ¹³C glucose in each group of concentrations is stable and there were no significant differences between six groups（p>0.05）;（3）The growth of Streptococcus mutans and Candida albicans showed growth inhibition with the increase of heavy water concentration.Streptococcus mutans and Candida albicans did not show obvious growth inhibition when the heavy water concentration was lower than 40%;Besides,the Raman spectrum showed an obvious heavy water peak and the C-D Ratio was linearly positively correlated with the heavy water concentration（Streptococcus mutans,R²=0.9494;Candida albicans,R²=0.9612）,suggesting that the heavy water peak can quantify the ability of cells to metabolize heavy water to characterize cell activity.（4）The ¹³C glucose metabolism rate and cell activity of Streptococcus mutans which co-culture with Candida albicans were higher than those in the pure culture group at each time point（p<0.05）,while there was no statistical difference between the mixed and pure culture groups（p>0.05）;（5）The concentration of Candida albicans showed different promotion effects on the metabolism and cell activity of Streptococcus mutans and it showed best when Streptococcus mutans co-culture with Candida albicans at equal concentrations（p<0.001）.Conclusion:（1）Single-Cell Raman could be used to differentiate between growth phases of a single species and strain types of different species.（2）The growth of Streptococcus mutans is sensitive to ¹³C glucose concentration,while the growth and metabolism of Candida albicans is not affected by ¹³C glucose concentration.（3）Heavy water labeling can quantify the metabolic activity of Streptococcus mutans and Candida albicans.（4）Candida albicans enhances glucose metabolism and cell activity of Streptococcus mutans,and its cell concentration is a key factor affecting glucose metabolism and cell activity of Streptococcus mutans.（5）Streptococcus mutans has no effect on the glucose metabolism and metabolic activity of Candida albicans.