Assessing The Antimicrobial Effects Of Root Canal Irrigants For Enterococcus Faecalis Via D₂O-labeled Single-Cell Raman Microspectroscopy

Objective:Enterococcus faecalis ATCC29212 was used as the targeted strain and D₂O-labeled Single-Cell Raman Microspectroscopy was employed as a non-invasive and quantitative technology to evaluate the antimicrobial effects of root canal irrigants for Enterococcus faecalis based on metabolic activity at the single-cell level.That would be beneficial to guide the rational use of root canal irrigants in clinic to reduce the rate of the endodontic treatment failures,the occurrence of refractory periapical periodontitis and bacterial resistance.Methods:E.faecalis ATCC29212 was cultured until stationary phase,then the growths of E.faecalis under different concentrations of D₂O were measured via the Bioscreen C.E.faecalis cells were cultivated at different doses of D₂O and sampled at 12h for the acquisition of Single-cell Raman Spectra?SCRS?.Moreover,samples of E.faecalis cells were obtained at a series of timepoints at the level of 30%D₂O for acquisition of SCRS,for the quantification of C-D ratio change in SCRS and for OD₆₀₀ using a Synergy HT microplate reader.The measurement of MIC in different doses of hydrogen peroxide?H₂O₂?,sodium hypochlorite?NaClO?and chlorhexidine gluconate?CHX?was performed using the broth dilution method.Moreover,E.faecalis cells were further incubated at brain heart infusion broth?BHI?containing 30%D₂O with different root canal irrigants and sampled at a series of timepoints for the acquisition of SCRS to measure MIC-MA.C-D ratio represented the metabolic activity at the single-cell level.MIC-MA is defined as the minimal dose under which the median?C–D ratio at 8 h of drug exposure is?0 and the standard deviation?SD?of the?C–D ratio among individual cells is?0.005.The chemometrics analyses were performed using R?version 3.6.1?.Results:First,a broad C-D Raman band(between 2,040 to 2,300 cm^-1 region)emerged in SCRS for E.faecalis ATCC29212 texted at different D₂O levels except D₂O-free conditions,and the intensity of the C-D Raman band was proportional to the concentration of D₂O in the medium?R²=0.9585,p<0.001?.Moreover,the concentration of D₂O in the medium with 30%did not inhibit the growth of E.faecalis significantly?p>0.05?,and the dynamic range of the C-D band at the level of 30%D₂O was allowed the quantification of C-D ratio change in SCRS.Second,the measurement of MIC for E.faecalis demonstrated that the MIC values of each root canal irrigants were 0.45 g/L for NaClO,110 mg/L for H₂O₂ and6 mg/L for CHX.Third,the temporal dynamics of the C-D ratio showed distinct dose-dependency to each root canal irrigants for E.faecalis,which C-D ratio decreased gradually with the increase of root canal irrigants concentration.Finally,the MIC-MA test for E.faecalis demonstrated that the MIC-MA values of each root canal irrigants were 0.9 g/L for NaClO,220 mg/L for H₂O₂ and 12 mg/L for CHX,which were higher than the MIC values of each root canal irrigants.And most E.faecalis cells?about 2/3?still had metabolic activity under the MIC concentration of each root canal irrigants.Conclusions:All of those three irrigants including NaClO,H₂O₂ and CHX showed antibacterial efficacy on E.faecalis.The MIC-MA values are higher than the corresponding MIC values,which indicated that the concentration of bacterial growth suppression could not necessarily lead to completely inhibit cellular metabolic activity.These results suggested that although E.faecalis could not grow,they still maintain metabolic activity,which might contribute to the root canal reinfection,bacterial drug resistance and refractory periapical periodontitis.The D₂O-labeled Single-Cell Raman Microspectroscopy as a new method could be used to accurately and quantitatively evaluate the antimicrobial effects of H₂O₂,NaClO and CHX for E.faecalis ATCC29212 based on metabolic activity at the single-cell level,which lays a foundation for precision medicine with MIC-MA as the quantitative index.