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 (C. albicans) 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 on average by 0.4%, 93.9%, and 94.1% at 8 h, 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 h exposure. Moreover, combined use of NaClO+H₂O₂ (combination 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 pairwise combinations. MIC-MA is advantageous in critically assessing antifungal efficacy, and NaClO+H₂O₂ can potentially serve as a more efficient disinfectant formula for fungal pathogens.