Single-cell Raman microspectroscopy-based assessment of three intracanal disinfectants' effect on Enterococcus faecalis

Enterococcus faecalis (E. faecalis) is frequently encountered in asymptomatic, persistent endodontic infections; thus, its control and eradication via disinfectants are important. To explore a disinfectant formulation that is effective yet with minimal side effects, here, we evaluated the susceptibility of E. faecalis to three intracanal disinfectants. We quantitatively assessed and compared the growth- or metabolism-inhibiting effects of intracanal disinfectants via minimum inhibitory concentration (MIC) and minimum inhibitory concentration based on metabolic activity (MIC-MA), based on the broth dilution test and D2O-probed single-cell Raman spectra (SCRS), respectively. For sodium hypochlorite (NaClO), hydrogen peroxide (H2O2), and chlorhexidine gluconate (CHX), the corresponding MIC was 0.45 g/L, 110 mg/L, and 6 mg/L, respectively. Under their respective MIC doses, metabolic activity of bacterial cells was reduced to 2%, 4%, and 2% remained at 8 h, yet recovered to a retention level of 90%, 97%, and 2% at 24 h. Despite the halting of growth, the remained metabolic activity suggests a nongrowing but metabolically active (NGMA) state that may lead to potential recurring infections. In contrast, at their respective MIC-MA doses of 0.9 g/L, 220 mg/L, and 12 mg/L, metabolic activities of all cells were completely inhibited throughout 24-h exposure. Furthermore, lower combined concentration of above three intracanal disinfectants can elicit equivalent metabolism-inhibiting effect with that of solo use of each one at the MIC-MA dose. Thus, binary combined use of disinfectants can outperform their solo use in controlling infection of E. faecalis and reducing side effects. In conclusion, the MIC-MA derived from D2O-probed single-cell Raman microspectroscopy is a promising approach in quantitatively assessing disinfectants' antimicrobial efficacy via metabolic activity.

Automated summary

For Enterococcus faecalis, sodium hypochlorite, hydrogen peroxide, and chlorhexidine gluconate have different minimum inhibitory concentrations, but at their respective minimum inhibitory concentrations, the bacteria may still be in a non-growing but metabolically active state, which can lead to recurring infections. In contrast, at their respective minimum inhibitory concentration based on metabolic activity, all three disinfectants completely inhibit the metabolic activity of the bacteria. The combination of these disinfectants at lower concentrations can achieve an equivalent metabolism-inhibiting effect as using each disinfectant alone at the minimum inhibitory concentration, thereby controlling the infection of E. faecalis and reducing side effects.