Assessing Efficacy of Clinical Disinfectants for Pathogenic Fungi by Single-Cell Raman Microspectroscopy.

Fan Li,Pengfei Zhu,Xi Sun,Jian Xu,Lihui Ren,Fang Yang,Rongze Chen

doi:10.3389/fcimb.2022.772378

Abstract

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.

Highlights

Microbial infections in the pulp and periapical tissues could cause pulpitis, apical periodontitis, or even persistent inflammatory reaction
We started by selecting a proper concentration of D2O, via comparison among the growth curves of C. albicans under various D2O levels (Figure 1A)
Proper administration of intracanal disinfectants is vital for both long-term efficacy of antimicrobial root canal therapy (RCT) and reducing side effects that compromise patient experience

Summary

Introduction

Microbial infections in the pulp and periapical tissues could cause pulpitis, apical periodontitis, or even persistent inflammatory reaction In these endodontic infections, pathogenic fungi such as Candida albicans (C. albicans) are the most frequently isolated eukaryotes (Siqueira and Sen, 2004; Kumar et al, 2015). An ideal intracanal disinfectant should be effective in inhibiting the metabolism of all microbial members, capable of dissolving pulp tissue remnant and smear layer, and, nontoxic or nonallergic (Galler, 2016). Frequently, these desirable features are mutually exclusive: e.g., the more potent pathogeninhibitory effect can be linked to more severe side effects to host tissues (Gomes-Filho et al, 2008). There is an urgent need for method development to tackle this challenge

Methods

Results

Conclusion