Assessing Efficacy Of Root Canal Irrigants For Candida Albicans By D₂o-labeled Single-cell Raman Microspectroscopy

Objectives:The universality of heavy water（D₂O）-labeled Single-cell Raman Microspectroscopy（D₂O-Ramanometry）for evaluating fungal metabolic activity was explored and the antifungal efficacy of clinical root canal irrigants（CHX,Na Cl O,H₂O₂）on C.a respectively.These will help to guide the rational use of root canal irrigants,reduce the chance of recurring infections,refractory periapical periodontitis and treatment failure.Methods:（1）The universality of D₂O-Ramanometry was investigated by exploring the growth of C.a under various concentrations of D₂O and the regularity of D₂O intake by C.a on the basis of the temporal change in OD₆₀₀and C-D ratio,respectively.（2）We determined the effects of inhibiting the growth and metabolism of CHX,Na Cl O and H₂O₂on C.a according to minimum inhibitory concentration（MIC）and minimum inhibitory concentration based on metabolic activity（MIC-MA）values,using broth dilution test and D₂O-Ramanometry respectively.（3）We measured the effects of inhibiting metabolism of multiple combinations of different root canal irrigants.Results:（1）The growth of C.a was not significantly affected at concentrations below or equal to 30%D₂O（p>0.05）,and C.a can actively metabolize D₂O.Moreover,the C-D ratio of C.a at the stationary phase was positively correlated with D₂O concentration（R²=0.9894,p<0.01）.The C-D peak can be used to quantify the ability of C.a to metabolize D₂O to characterize the metabolism activity of cells.And the 30%D₂O was selected as the default D₂O concentration in subsequent experiments.（2）The MIC of CHX,Na Cl O and H₂O₂were 4,168,60μg/ml respectively,and the MIC-MA of them were 8,336,120μg/ml respectively.Under the MIC of CHX,Na Cl O and H₂O₂,despite the completely halted growth,metabolic activity of C.a cells were inhibited by merely 0.4%,93.9%and 94.1%.Metabolic activity can be inhibited only when the concentration of MIC-MA was reached.（3）The metabolism of C.a can be completely inhibited by the combination of Na Cl O and H₂O₂at their each lower concentration.And no apparent synergistic effect in metabolic inhibition was observed when combining CHX and either Na Cl O or H₂O₂.Conclusions:D₂O-Ramanometry is suitable for accurately and quantitatively evaluating the effect of root canal irrigants on fungal metabolic activity at the single-cell resolution.At their respective MIC level of CHX,Na Cl O and H₂O₂,the growth of C.a was completely inhibited,but metabolic activity was active,suggesting the presence of“nongrowing but metabolically active”（NGMA）cells that may underlie refractory infections for each of the treatments（particularly for CHX）.Hence,MIC-MA would be an advantageous parameter than MIC in assessing intracanal irrigants-pathogen interaction.The combination of Na Cl O and H₂O₂was the better group to inhibit the metabolic activity of C.a cells.This study lays a foundation for the rapid screening of commonly used clinical drugs,the precise direction of drug use and the improvement of treatment success rates.