Antimicrobial peptide GH12 as root canal irrigant inhibits biofilm and virulence of Enterococcus faecalis

Abstract

Aim The objectives of this laboratory‐based study were to investigate the effects of GH12 on Enterococcus faecalis biofilm and virulence. Methodology Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of GH12 against E. faecalis were first determined. A time‐kill assay was further conducted. The effects of GH12 on the expression of virulence and stress genes in E. faecalis were evaluated by RT‐qPCR. Crystal violet stain was used to investigate the effects of GH12 on E. faecalis biofilm formation and 1‐day‐old biofilm. Finally, an ex vivo tooth model contaminated with E. faecalis was used to evaluate the antimicrobial activity of GH12 as an irrigant by CFU counting, SEM and CLSM. One‐way anova and Tukey’s multiple comparisons test were used to compare the differences amongst groups (α = 0.05). Results The MICs and MBCs of GH12 against E. faecalis were 8.0 ± 0.0 and 16.0 ± 0.0 mg L−1, respectively, and GH12 at 32.0 mg L−1 reduced the bacterial numbers by more than 99.9% within 1 min. Various virulence genes (efaA, esp and gelE) and stress genes (dnaK, groEL, ctsR and clpPBCEX) in E. faecalis were significantly downregulated by GH12 at sub‐MIC levels (P < 0.05). Additionally, both E. faecalis biofilm formation and the biomass of 1‐day‐old E. faecalis biofilm were significantly reduced by GH12 (P < 0.05). Elimination of E. faecalis in biofilms from root canal walls was achieved through irrigation with 64.0 mg L−1 GH12 for 30 min. CLSM analysis revealed that GH12 at 64.0 mg L−1 was most effective in eliminating bacteria within dentinal tubules (P < 0.05). Conclusion In a laboratory setting, and when used as an irrigant, GH12 suppressed E. faecalis, downregulated specific virulence and stress‐associated genes, eliminated intracanal E. faecalis protected by biofilms and killed bacteria in dentinal tubules. These results emphasize the need for preclinical and clinical studies to explore the potential of GH12 as an antimicrobial agent during root canal treatment.

Microbiological Aspects of Root Canal Infections and Disinfection Strategies: An Update Review on the Current Knowledge and Challenges

“…Nisin used as an irrigant was found to reduce E. faecalis growth and disrupt its biofilm structure [ 244 ]. Peptide GH12 exhibited multimode mechanisms against E. faecalis by influencing its genetic expression and virulence whilst also proving to be bactericidal in vitro and ex vivo [ 245 ]. Peptide DJK-5, with or without chlorhexidine was shown to have efficacy against multispecies biofilms [ 246 ].…”

A critical analysis of research methods and experimental models to study irrigants and irrigation systems

“…The crystal violet assay is a colorimetric assay that has been used for rapid approximate quantification of the biofilm mass after exposure to irrigants (Christensen et al, 1985; Li et al, 2020; Mohmmed et al, 2016). This assay has been applied to biofilms grown on microtiter plates (Alves et al, 2013; Li et al, 2020; Mohmmed et al, 2016; Wilson et al, 2015) and also inside artificial root canals created in acrylic blocks (Layton et al, 2015; Townsend & Maki, 2009). It is easy to perform, inexpensive, and it can be applied to different bacterial species.…”

“…Planktonic cultures have been widely used to determine the antimicrobial effect of root canal irrigants (Generali et al, 2020; Li et al, 2020; Nudera et al, 2007; Tong et al, 2011; Torabinejad et al, 2003). These simple tests on bacteria in a liquid phase measure the minimal inhibitory concentration and minimal bactericidal concentration of an irrigant (Andrews, 2011).…”

Cationic Antimicrobial Peptides Are Leading the Way to Combat Oropathogenic Infections

“…96 Additionally, by acting as an irrigant, GH12 has been effective in reducing both the virulence and biofilm formations of E. faecalis. 126 As Enterococcus species can contribute to apical periodontitis after root canal procedures, it has further emphasized GH12's protective role in oral surgery. 127 Given its ability to disrupt biofilms, GH12 can suppress the cariogenic properties of these pathogenic species, thus lowering the risk of caries by influencing the composition of the oral microbiome.…”