Role of extracellular DNA in Enterococcus faecalis biofilm formation and its susceptibility to sodium hypochlorite

Abstract Objective This study investigated the role of extracellular deoxyribonucleic acid (eDNA) on Enterococcus faecalis ( E. faecalis ) biofilm and the susceptibility of E. faecalis to sodium hypochlorite (NaOCl). Methodology E. faecalis biofilm was formed in bovine tooth specimens and the biofilm was cultured with or without deoxyribonuclease (DNase), an inhibitor of eDNA. Then, the role of eDNA in E. faecalis growth and biofilm formation was investigated using colony forming unit (CFUs) counting, eDNA level assay, crystal violet staining, confocal laser scanning microscopy, and scanning electron microscopy. The susceptibility of E. faecalis biofilm to low (0.5%) or high (5%) NaOCl concentrations was also analyzed by CFU counting. Results CFUs and biofilm formation decreased significantly with DNase treatment (p<0.05). The microstructure of DNase-treated biofilms exhibited less structured features when compared to the control. The volume of exopolysaccharides in the DNase-treated biofilm was significantly lower than that of control (p<0.05). Moreover, the CFUs, eDNA level, biofilm formation, and exopolysaccharides volume were lower when the biofilm was treated with DNase de novo when compared to when DNase was applied to matured biofilm (p<0.05). E. faecalis in the biofilm was more susceptible to NaOCl when it was cultured with DNase (p<0.05). Furthermore, 0.5% NaOCl combined with DNase treatment was as efficient as 5% NaOCl alone regarding susceptibility (p>0.05). Conclusions Inhibition of eDNA leads to decrease of E. faecalis biofilm formation and increase of susceptibility of E. faecalis to NaOCl even at low concentrations. Therefore, our results suggest that inhibition of eDNA would be beneficial in facilitating the efficacy of NaOCl and reducing its concentration.


Introduction
Enterococcus faecalis (E. faecalis) is a bacterium frequently recovered from infected root canal systems. 1,2 This bacteria is difficult to remove since it is able to form biofilms and survive under a wide range of acidic and basic conditions and prolonged periods of nutritional deprivation. 3,4 E. faecalis biofilms consist of exopolysaccharides, proteins, lipids, and extracellular deoxyribonucleic acid (eDNA). 5,6 The dense and protected environment of a biofilm may facilitate gene transfer and enhance biofilm stability. 7 As a major structural component of many different microbial biofilms, the importance of eDNA was first reported in Pseudomonas aeruginosa. 5 The eDNA is released via autolysis in a fratricidal or suicidal manner and/or active release through membrane vesicles and nanofibers in E. faecalis biofilms. 6,8 Previous reports have attributed a crucial role to eDNA in the formation, mechanical stability, and maturation of bacterial biofilms in general and E. faecalis biofilms in particular. [9][10][11] Irrigation is critical to remove microorganisms from root canal systems. NaOCl is the most frequently used material for endodontic treatment, it is an antiseptic and inexpensive lubricant that has been used at dilutions ranging from 0.5% to 5.25%. 12 Usually, it is assumed that a higher concentration of NaOCl increases the efficacy in removing bacteria within root canal systems; however, severe complications of NaOCl

Colony forming unit (CFU) counting and eDNA measurement
The specimens were washed with sterile water and then transferred into a 1.5 ml tube containing 1 ml of sterile water. Following, the specimens were sonicated to collect eDNA using a sonifier (10 s, two

NaOCl treatment and CFU counting
To study the E. faecalis susceptibility to NaOCl in biofilm treated with DNase, the specimens with E. faecalis (6×10 5 CFU/ml) and DNase (2 U/μl/ml) were cultured for 2 days at 37°C. After incubation, the biofilms formed on the specimens were treated with 0.5% or 5% NaOCl for 5 min. Next, the specimen was transferred to a 1.5 ml tube that contained 1 ml of sterile water and sonicated to break the biofilms.  NaOCl within the biofilms, 0.5% and 5% NaOCl were used to treat the biofilms with or without DNase for 2 days. As shown in Figure 4, the biofilms treated with NaOCl had significantly fewer CFUs when compared to the control that received no NaOCl treatment (p<0.05). Regardless of the concentration, NaOCl had significantly higher efficiency in eliminating E. faecalis when it was used with DNase (p<0.05). Furthermore, 5% NaOCl exhibited a higher bactericidal effect against E. faecalis than 0.5% with or without DNase. However, 0.5% NaOCl with DNase showed similar efficacy to 5% NaOCl without DNase (p>0.05). Lastly, we investigated the effect of eDNA removal from the biofilm on the susceptibility of E. faecalis to NaOCl. NaOCl is the most widely used material to irrigate in dental procedures due to its high antimicrobial activity and capacity to dissolve organic tissue. 35 However, NaOCl has drawbacks including an unpleasant odor and high toxicity that induces irritation when in contact with surrounding tissue. 36 Therefore, in an attempt to reduce the NaOCl concentration, we used DNase and investigated whether DNase increases the efficacy of NaOCl against E. faecalis Role of extracellular DNA in Enterococcus faecalis biofilm formation and its susceptibility to sodium hypochlorite J Appl Oral Sci. 2019;27:e20180699 7/8 in biofilms. According to the results of this study, 5% NaOCl exhibited a higher antibacterial effect against E. faecalis than 0.5% with or without DNase.

Discussion
However, the gap in elimination efficiency between 5% and 0.5% NaOCl became smaller when DNase was used, mainly for potentiation of the effect of the low NaOCl concentration. Notably, 0.5% NaOCl combined with DNase exhibited statistically similar values to 5% NaOCl without DNase, suggesting that DNase adjunctive treatment could represent a useful strategy for improving antimicrobial action while reducing NaOCl concentration.

Conclusions
The results suggest that inhibition of eDNA leads to decrease of E. faecalis biofilm formation and increase E. faecalis susceptibility to NaOCl especially at low concentration (0.5%). Therefore, our results suggest that inhibition of eDNA would be beneficial for removal of E. faecalis biofilm, especially by facilitating the efficacy of NaOCl and reducing its concentration for safer clinical use.