Studi di microbiologia endodontica: prevalenza di Candida spp. nei canali radicolari infetti ed azione antibatterica di due peptidi antimicrobici nei confronti di biofilm di Enterococcus faecalis

The research presented in this thesis aims to give further insight into the complexity of root canal infections and to investigate new root canal disinfection strategies. In the first part a systematic review the literature on the prevalence of Candida species in root canal infections was conducted. Extensive literature research was performed in the most important electronic biomedical databases, and additional studies have been identified from references from relevant articles. Studies were critically appraised using a modified version of the Joanna Briggs Institute Critical Appraisal Checklist. From 2225 unique records, 2118 were excluded on the basis of title and abstract. Of the remaining 107 studies, 50 were excluded after full-text review, and 57 were included for qualitative and quantitative analysis. The overall prevalence of Candida spp. in root canal infections was 8.20% (95% confidence interval, 5.56%–11.21%). Candida albicans was the most frequently isolated species. Significant heterogeneity among studies was observed (P < .001, I2 = 86.07%). Subgroup analyses revealed a higher prevalence of Candida spp. from African samples. All studies considered, a high or unclear risk of bias was prevalent regarding 6 out of the 8 items considered in the critical appraisal. According to our study, Candida spp. occurred in a small proportion of root canal infections and further research is needed to investigate the real contribution of Candida spp. to the microbial ecology in infected root canals. In the second part we assessed the antibacterial properties of two synthetic peptides, namely KP and L18R, against planktonic cells and biofilms of the endodontic pathogen Enterococcus faecalis and their cytotoxicity against fibroblast cells. The KP and L18R bactericidal activity against E. faecalis ATCC 29212 was evaluated by the colony forming unit assay and the half maximal effective concentration (EC50) was calculated. The peptide inhibition of E. faecalis biofilm mass and metabolic activity was assessed. Peptide effects on 2-day-old E. faecalis biofilms grown on hydroxyapatite discs were compared to a Ca(OH)2 saturated solution by confocal scanning laser microscopy (CLSM) and scanning electron microscopy (SEM). In order to test peptide cytotoxicity, L929 fibroblasts were exposed to KP and L18R for 24 hours and cell metabolism was evaluated by MTT assay. Both KP and L18R showed an effective bactericidal activity against planktonic E. faecalis and L18R proved to be 10-folds more effective compared to KP (EC50 value of 4.520 × 10‑6 M versus 3.624 × 10-7 M). The EC50 values, calculated with reference to the biomass and metabolic activity reduction, were 1.607 × 10-4 M and 1.235 × 10-4 M for KP and 1.411 × 10-5 M and 1.585 × 10-5 M for L18R, respectively. CLSM and SEM images showed that Ca(OH)2, KP and L18R remarkably impaired E. faecalis biofilms grown on hydroxyapatite discs and L18R was associated to a higher dead bacteria percentage compared to the other treatments. Toxicity studies demonstrated that 24-hour incubation with the two peptides at concentrations between 50 µg/ml and 400 µg/ml had no adverse effect on L929 fibroblast viability. KP and L18R effectively inhibited E. faecalis, both in planktonic and biofilm state and showed no cytotoxic effects in our experimental settings. L18R demonstrated a more potent antibacterial activity compared to KP. The obtained results show that antimicrobial peptides may represent a promising new strategy for endodontic infection control.