Effects Of Candida Albicans On The Metabolic Activity And Drug Resistance Of Streptococcus Mutans Based On D₂O-labeled Single-cell Raman Micro-spectroscopy

Chapter One:Construction of Research System of Metabolism Interaction Between Streptococcus mutans and Candida albicansObjective:With S.mutans UA159 and C.albicans ATCC10231 as the target strains,find the most suitable D₂O concentration that does not affect the growth of the experimental strains.Analyze whether the D₂O-labeled single-cell Raman micro-spectroscopy can be used to detect the cell metabolic activity of the experimental strains,so as to construct a research system for the metabolic interaction between S.mutans UA159 and C.albicans ATCC10231.Methods:Cultivate S.mutans UA159 and C.albicans ATCC10231 respectively to the stationary phase.Detect the change of absorbance value OD₆₀₀ of S.mutans UA159and C.albicans ATCC10231 in different concentrations of D₂O medium（0%,10%,20%,30%,40%,50%）with time,and draw a growth curve to evaluate the growth status of the experimental strains;The Single-cell Raman Spectra（SCRS）of S.mutans UA159 and C.albicans ATCC10231 in different concentrations of D₂O medium were randomly selected from different fields of view,analyze the law of D₂O absorption by experimental strains;According to the results of previous experiments,use 30%D₂O medium to cultivate S.mutans UA159 and C.albicans ATCC10231,and samples were taken on a time gradient to compare the changes in the time dimension of the experimental strain C-D ratio and OD₆₀₀.Results:The growth of S.mutans UA159 and C.albicans ATCC10231 will be inhibited when they are cultured in a medium with a concentration of more than 30%D₂O（p<0.05）,and the growth will not be inhibited when cultured in a medium with a concentration of 10%～30%D₂O（p>0.05）;The Single-cell Raman Spectra of S.mutans UA159 and C.albicans ATCC10231 in different concentrations of D₂O medium showed C-D signature peaks.Moreover,the C-D ratio of S.mutans UA159 and C.albicans ATCC10231 at the stationary phase was positively correlated with D₂O concentration（S.mutans UA159:R²=0.9973,p<0.001;C.albicans ATCC10231:R²=0.9985,p<0.001）.It shows that the C-D signature peaks can be used to characterize the metabolic level of cells.Conclusions:S.mutans UA159 and C.albicans ATCC10231 can well intake D₂O and can be detected by SCRS.Using 30%D₂O for this research will not affect the growth of the experimental strains,and it can also show the intake of D₂O by the experimental strains.Therefore,D₂O-labeled single-cell Raman micro-spectroscopy can be used as a real-time,accurate,quantitative and non-destructive technical method to study cell metabolism.Chapter Two:Effects of Candida albicans on the Metabolic Activity and Drug Resistance of Streptococcus mutansObjective:Using D₂O-labeled single-cell Raman micro-spectroscopy,with S.mutans UA159 and C.albicans ATCC10231 as the target strains,the effect of C.albicans ATCC10231 on the metabolic activity and drug resistance of S.mutans UA159 was systematically studied on a single-cell scale.A deep understanding of the interaction between different microorganisms will lay the foundation for the prevention and treatment of microbial infections.Methods:We explore the effective drug concentration of Ampicillin against S.mutans UA159 according to minimum inhibitory concentration（MIC）and minimum inhibitory concentration based on metabolic activity（MIC-MA）values,using broth dilution test and D₂O-labeled single-cell Raman micro-spectroscopy,respectively;The Single-cell Raman Spectra of S.mutans UA159 under different concentrations of C.albicans ATCC10231 supernatant was selected from different fields of view to explore the changes of C.albicans ATCC10231 on the metabolic activity of S.mutans UA159;Under the action of different concentrations of Ampicillin,the Single-cell Raman Spectra of S.mutans UA159 in different fields were randomly selected to analyze the effect of C.albicans ATCC10231 on the resistance of S.mutans UA159.Results:The MIC of Ampicillin is 0.8mg/L,and under the stimulation of up to50×MIC drug concentration,S.mutans UA159 can still maintain high metabolic activity,so there is no MIC-MA.Compared with the control group（without C.albicans ATCC10231 supernatant）,when the concentration of C.albicans ATCC10231supernatant was OD₆₀₀≥0.5,the metabolic activity of S.mutans UA159 increased,and there were statistical differences at different time points（p<0.05）,when the concentration of C.albicans ATCC10231 supernatant was OD₆₀₀<0.5,the metabolic activity of S.mutans UA159 did not change significantly（p>0.05）;Under the action of different concentrations of Ampicillin,the metabolic activity of S.mutans UA159 cultured with the supernatant of C.albicans ATCC10231 was higher than that of S.mutans UA159cultured without the supernatant of C.albicans ATCC10231,and there were statistical differences at different time points（p<0.05）.Conclusions:D₂O-labeled single-cell Raman micro-spectroscopy can quickly and accurately evaluate the effect of drugs on the metabolic activity of experimental strains at the single-cell level.A higher concentration of C.albicans ATCC10231 supernatant can not only promote the metabolic activity of S.mutans UA159,but also enhance the resistance of S.mutans UA159 to Ampicillin.