Modulation of virulence in Enterococcus faecalis cells surviving antimicrobial photodynamic inactivation with reduced graphene oxide-curcumin: An ex vivo biofilm model

Background: Enterococcus faecalis, as a major microorganism in persistent/secondary infections of endodontically treated teeth, is less likely to be eliminated during endodontic therapy. In this study, the effect of root canal disinfection and anti-virulence activities of photodynamic inactivation (PDI) of E. faecalis utilizing reduced graphene oxide-curcumin (rGO-Cur) as a photosensitizing agent following irradiation with light-emitting diode (LED), as well as intracellular ROS production were evaluated on ex vivo biofilms of E. faecalis in comparison with sodium hypochlorite (NaOCl) as the traditional endodontic irrigation solution. Materials and methods: After formulation and confirmation of synthesized rGO-Cur using scanning electron microscopy (SEM), Fourier transformation infrared (FT-IR), UV-Vis spectra, dynamic light scattering (DLS), and Zeta potential, the minimum biofilm inhibitory concentrations (MBICs) and in vitro anti-biofilm activity of rGO-Cur, light-emitting diode (LED) at the wavelength of 435 +/- 20 nm, and rGO-Cur-PDI were determined against 4-week-old pre-formed biofilms of E. faecalis. After preparation of ex vivo biofilm model in root canals, the ex-vivo anti-biofilm potential of rGO-Cur, LED, and rGO-Cur-PDI against E. faecalis were analyzed using the XTT assay and scanning electron microscopy (SEM) in comparison with NaOCl. The effects of sub-MBIC of rGO-Cur and NaOCl, sub-lethal dose of LED, and sub-significant inhibitory (SSI) potential of rGO-Cur-PDI for E. faecalis biofilms on virulence genes (efa, esp, gel, and fsr) expression of E. faecalis were analyzed using real-time polymerase chain reaction (qRT-PCR) assay. Intracellular reactive oxygen species (ROS) level was measured in rGO-Cur-PDI-treated bacterial cells compared to control cells with 2',7'-dichlorfluorescein-diacetate (DCFH-DA) fluorescent probe. Results: The FTIR, DLS, Zeta potential, SEM, and UV-Vis spectra analysis indicated the successful synthesis of rGO-Cur. The MBIC of rGO-Cur was 250 mu g/ml, which inhibited the growth ofE. faecalis. LED showed insignificant anti-biofilm activity against E. faecalis even after treating for a long irradiation time (300 s). According to checkerboard assay, the MBIC value of rGO-Cur-PDI was reduced noticeably compared to the individual MBIC values of rGO-Cur and LED for E. faecalis. The expression levels of efa, esp, gel, and fsr genes in pre-formed E. faecalis biofilms were markedly reduced after rGO-Cur, rGO-Cur-PDI, and NaOCl treatment in comparison with the control group. Conversely, LED revealed no significant change in the expression of the virulence genes. The intracellular ROS assay showed a significant increase (8.3-fold) in rGO-Cur-PDI when compared to the control. Conclusion: Our data support that rGO-Cur-PDI showed dual inhibitory effects on biofilm formation ability and virulence activity of E. faecalis with potential clinical applications for infection control in endodontics.