Assessing efficacy of clinical disinfectants for pathogenic fungi by Single-cell Raman microspectroscopy

Disinfectants are crucial for root canal therapy (RCT), as metabolism of canal-inhabiting microbes can cause refractory infections. To develop effective yet patient- and environment-friendly disinfectant formulations, we quantitatively assessed the metabolism-inhibiting effects of intracanal disinfectants via D₂O-probed Single-Cell Raman Spectra (SCRS), using Candida albicans (Ca) as a pathogen model. For chlorhexidine gluconate (CHX), sodium hypochlorite (NaClO) and hydrogen peroxide (H₂O₂), at their MIC of 4, 168 and 60 µg/mL respectively, despite the complete growth halt, metabolic activity of individual fungal cells was reduced averagely for 0.4%, 93.9% and 94.1% at 8 hours, revealing a "nongrowing but metabolically active" (NGMA) state that may underlie potential refractory infections, particularly for CHX. In contrast, at their Metabolic Activity-based Minimum Inhibitory Concentrations (MIC-MA) of 8, 336 and 120 µg/mL respectively, metabolic activity of all cells was completely halted throughout 8 hours′exposure. Moreover, combined use of NaClO+H₂O₂ (mixture at 0.5× MIC-MA each) outperforms solo uses of CHX, NaClO, H₂O₂ or other binary combinations. Furthermore, dynamics of SCRS revealed distinct fungicidal mechanisms of CHX, NaClO, H₂O₂ and their pair-wise mixtures. MIC-MA is advantageous in critically assessing antifungal efficacy, and NaClO+H₂O can potentially serve as a more efficient disinfectant formula for fungal pathogens.