Evaluation of the antimicrobial effectiveness of the combined use of irrigation solutions with different contents and various enzymes on enterococcus faecalis biofilm

In this study, it was aimed to investigate the antibacterial efficacy of 2.5% sodium hypochlorite (NaOCl), chlorhexidine (CHX), irritrol and silver chitosan nanoparticles (AgCNP) alone or in combination with deoxyribonuclease 1 (DNase1) and trypsin pre-enzyme applications in dentine blocks contaminated with Enterococcus faecalis (E. faecalis) using confocal laser scanning microscopy (CLSM). 152 semi-cylindrical dentin blocks of 4 mm x 4 mm x 0.7 mm obtained from 76 single root and single canal mandibular teeth were included in the study. All dentine blocks were sterilized in autovlave. It was confirmed by scanning electron microscopy (SEM) in 4 randomly selected samples that the smear layer was removed and the preexisting microbial colonization was eliminated after the sterilization process. E. faecalis inoculation was performed on 148 dentin blocks for 21 days. To confirm that E. faecalis was inoculated in dentin blocks and formed biofilm formation, 4 dentin blocks were divided into positive control group and examined by SEM. Following verification of inoculation, 144 dentine blocks were divided into 12 groups according to the final irrigation protocols to be tested (n= 12); NaOCl, CHX, Irritrol, AgCNP, Trypsin/ NaOCl, Trypsin/ CHX, Trypsin/ Irritrol, Trypsin/ AgCNP, DNase 1/ NaOCl, DNase 1/ CHX, DNase 1/ Irritrol and DNase 1/ AgCNP. After applying the final irrigation protocols, dentin blocks were stained using the Live/ Dead BacLight Bacterial Viability Kit in accordance with the manufacturer's instructions. After staining, dentin blocks were visualized with CLSM. Percentage of dead and viable bacteria on CLSM images was calculated using ImageJ software. The data obtained were statistically analyzed with the One-Way Anova and Post-Hoc Tukey tests, at the significance level of p<0.05, with SPSS software. A higher percentage of dead bacteria was obtained in CHX compared to NaOCl in the groups without pre-enzyme application and with DNase 1 as a pre-enzyme (p<0.05) and there was no statistically significant difference between the other final irrigation solutions (p>0.05). There was no statistically significant difference in the percentage of dead bacteria in the groups in which trypsin was applied as a pre-enzyme (p>0.05). While there is no statistical difference in the groups including NaOCl, CHX and irritrol, whether pre-enzyme is applied or not (p>0.05), AgCNP showed a higher percentage of dead bacteria with trypsin application as a pre-enzyme than other final irrigation solutions (p<0.05). Within the limitations of the current study, none of the final irrigation protocols tested could eliminate the E. faecalis biofilm 100%. CHX showed better antibacterial activity compared to NaOCl in groups where no pre-enzyme application was applied and DNase 1 was applied as a pre-enzyme. Application of DNase 1 as a pre-enzyme did not increase the antimicrobial activity of any irrigation solution. While the application of trypsin as a pre-enzyme improves the antimicrobial activity of AgCNP, it did not make any difference on other irrigation solutions. Keywords: DNase 1, E. faecalis, Irritrol, Silver chitosan nanoparticle, Trypsin.