Efficacy of relacin combined with sodium hypochlorite against Enterococcus faecalis biofilms

Abstract Objective Relacin is a synthetic molecule that targets RelA, an essential protein in a conserved bacterial stress response system. It was shown to inhibit bacterial growth. The aims of this study were to evaluate the antimicrobial effect of relacin combined with sodium hypochlorite (NaOCl) on Enterococcus faecalis biofilms and to evaluate the cytotoxicity of relacin. Material and Methods 48-h E. faecalis OG1RF biofilms were treated by various concentrations of relacin in order to determine its inhibitory concentration. Then, the 48-h biofilms were treated either with 1-min NaOCl (0.01%, 0.05%) alone, or in combination of relacin. As a means of comparison, the biofilms of ΔrelA were also treated by 1-min NaOCl (0.01%, 0.05%, 0.25%). The treatment efficacy was determined by agar plate count assays. The cytotoxicity of relacin was examined on human gingival epithelial cells Ca9-22 and murine fibroblasts NIH-3T3 by a methyl thiazolyltetrazolium (MTT) assay and a lactate dehydrogenase assay. Statistical analysis was performed by one-way or two-way analysis of variance (ANOVA) with Bonferroni’s post-hoc test and an independent Student’s t-test. A significance level of p<0.05 was used. Results Relacin inhibited the growth of OG1RF biofilms partially at 8 mM and fully at 14 mM. The relacin (14 mM) and NaOCl combined treatment resulted in significantly higher treatment efficacy than NaOCl treatment alone. At 0.05% NaOCl, the combined treatment resulted in 5.65 (±0.19) log reduction in biofilm viability. The ΔrelA biofilms were more susceptible to NaOCl treatment than the wild type biofilms at 0.25% NaOCl. Relacin at 14 mM was not toxic to host epithelial cells and fibroblasts. Conclusions The combination of relacin with a low concentration of NaOCl was effective and not cytotoxic.


Introduction
Root canal irrigation is a key step to successful root canal therapy. The biological role of irrigation is not only to reduce bacterial infection but also to remove debris in the infected root canal. Sodium hypochlorite (NaOCl) solution has become the most popular irrigant due to its broad antimicrobial spectrum, as well as its unique capacity to dissolve necrotic tissue remnants 29 . So far, there has been no consensus on the clinical concentration of NaOCl. The concentrations vary between 0.5% and 5.25%, depending on the routine of the individual clinic 9 . The irrigation efficacy of NaOCl increases with increasing concentration, but the risk of periapical tissue damage also increases due to the toxicity of NaOCl 5 . The extruding NaOCl into periradicular tissues can cause severe tissue reactions, such as pain, swelling, extensive bruise, and local necrosis 7 . Therefore, a combination of NaOCl with other antimicrobials or surfactants has been suggested to enhance the efficacy of irrigation while maintaining the concentration of NaOCl at a biocompatible level 14,23,29 .
Several agents have been applied together or in sequence with NaOCl. The combination of 2.5% NaOCl and 0.2% chlorhexidine (CHX), a broad-spectrum antimicrobial, has been shown to be more effective than NaOCl alone 14 . However, the interaction between NaOCl and CHX creates an orange-brown precipitate 4 , which is difficult to remove from the root canal system and may cause discoloration of the dental structures.
Alternatively, MTAD, a mixture of doxycycline, citric acid, and a detergent, was shown to inhibit the growth of Enterococcus faecalis effectively when applied as the final irrigant after 1.3% NaOCl 18 . However, the same combination was found unable to remove E. faecalis in another study 11 . Moreover, there was a concern that bacterial strains in an infected root canal might already be resistant to the doxycycline in MTAD 16 3 . This (p)ppGpp system is one of the essential systems in prokaryotic cells and is highly conserved among various bacterial species 3 .
Previous studies 1,27 demonstrated that the (p)ppGpp system was responsible for the resistance of E. faecalis to antibiotic treatment, starvation, and oxidative stress. Relacin is a novel compound designed to inhibit the synthetase function of the RelA enzyme, hence reducing the production of (p)ppGpp 25 . A recent study 25 showed that relacin could function as an antimicrobial agent by impairing the entry of bacterial cells into the stationary phase.
The aim of this study was to evaluate the antimicrobial effect of relacin combined with NaOCl on E. faecalis biofilms and to evaluate the cytotoxicity of relacin. Since E. faecalis is the predominant microbial species in persistent endodontic infections 30 and its resistance to stress has been linked to the (p)ppGpp system 1 , this bacterial species was used as a model microorganism in the evaluation. The null hypotheses were that pretreatment of relacin could allow for reduced concentration of NaOCl without comprising its antimicrobial efficacy, and that relacin had no significant cytotoxicity. In addition, the response of ΔrelA biofilms to NaOCl treatment was compared to that of the wild-type biofilms. To this end, the 48-h ΔrelA biofilms were treated with 0%, 0.01%, 0.05%, and 0.25% (wt/vol) NaOCl for 1 min as described previously.

Material and methods
Each treatment was tested in triplicate. All experiments were repeated three times.

Biofilm viability assay
The viability of the biofilm before and after 24 h relacin treatments and of those after NaOCl treatments were examined with the biofilm viability assay.  Response of E. faecalis ΔrelA biofilms to NaOCl As shown in Figure 3, NaOCl treatments resulted in a dose-dependent reduction in biofilm viability, irrespective of the strains tested. Significantly higher reduction was observed in ΔrelA biofilms than in the wild type biofilms, when NaOCl concentration was 0.25%.

Cytotoxicity of relacin
The MTT readings showed that the viability of epithelial cells Ca9-22 was not affected by the 24-h treatment with 14 mM relacin. In addition, the viability of fibroblasts NIH-3T3 was hardly reduced (approximately 10% reduction) ( Figure 4A). Similar findings could be found using the LDH release assay ( Figure 4B). After the relacin treatment, a low percentage of cell death was observed for Ca9-22 (2.08 % ± 0.65) and NIH-3T3 (7.24% ± 0.90), respectively.

Discussion
This study showed that the 1-min application of 0.05% NaOCl after 14 mM relacin pretreatment resulted in approximately 6-log reduction in the viability of E. faecalis biofilms. Relacin at 14 mM did not exhibit obvious cytotoxicity to host cells. Therefore, the combined treatment can reduce the potential cytotoxicity of NaOCl by lowering its concentration and application time 24 , without compromising the antimicrobial efficacy of NaOCl.
It was hypothesized that relacin is not harmful for human tissue because relacin is designed to bind specifically to the synthetase site of a microbial RelA protein, which is not present in the eukaryotic cells 21 .
The data of this study supports this hypothesis. In this study, the cytotoxicity of relacin was evaluated by using two types of host cells, gingival epithelial cells and fibroblasts, which represent the major cell types in oral mucosa and periradicular tissue. The cytotoxicity was examined with two types of assays, MTT and LDH. Both assays were used to assess the biosafety of novel dental materials or treatments. MTT reduction is a marker reflecting viable cell metabolism, while the detection of LDH activity in the extracellular medium indicates irreversible cell death due to cell membrane damage 6 . This study showed that the results from these two assays were in line with each other and that the overall cytotoxicity of relacin was low. Collectively, relacin itself is harmless to the host cells and it can improve the safety of NaOCl by reducing its treatment concentration. Relacin is a promising candidate to be used together with NaOCl.
Our data showed that the pre-treatment of relacin reduced the effective concentration of NaOCl to 0.05%. This result is comparable to what was reported for the combination of CHX/calcium hydroxide and NaOCl.
After the pre-treatment of chlorhexidine or calcium hydroxide, a minimum of 1% NaOCl was needed for 3 to 6-log reduction in bacterial cell counts 20,23 .
The enhanced killing efficacy of NaOCl after relacin treatment may be explained by inhibition of the (p) ppGpp system. It is likely that relacin inactivated RelA by binding to the synthetase site of the enzyme, hence inhibiting the production of (p)ppGpp 25  faecalis was able to form matured biofilms after incubation for 48 h on polystyrene blocks. Accordingly, in this study, a 48-h E. faecalis biofilms can be applied for the antimicrobial assays. However, dentin may be much closer to the clinical situation 12 . Therefore, further study could be performed on the dentin with different ages of biofilms. Despite the limitations of the biofilm model, the data of this study can support that relacin has the potential to allow reduced concentration of NaOCl without comprising its antimicrobial efficacy.

Conclusions
The data of this study demonstrated that the application of relacin allowed reduced concentrations of NaOCl without comprising its antimicrobial efficacy.
The combination of relacin with a low concentration of NaOCl was effective and not cytotoxic.