Additive effect of photoactivated disinfection on the antibacterial activity of QMix 2in1 against 6-week Enterococcus faecalis biofilms: An in vitro study

Abstract

Background:

Evaluation of the additive effect of photodynamic therapy (PDT) on the antibacterial activity of 2.5% sodium hypochlorite (NaOCl) and QMix against 6-week Enterococcus faecalis biofilms contaminated root canals.

Aims:

To establish the most suitable irrigant for eradication of 6-week E. faecalis biofilms.

Settings and Design:

In vitro study.

Materials and Methods:

A 6-week E. faecalis (ATCC 29212) biofilm was formed in 190 extracted teeth that were subsequently subjected to irrigation protocols as follows. Group A₁: normal saline, Group A₂: 2.5% NaOCl, Group A₃: QMix, Group B₁: normal saline and photoactivated disinfection (PAD), Group B₂: 2.5% NaOCl and PAD, Group B₃: QMix and PAD, Group C: no irrigation. For PAD, irradiation was done three times for 5 s each with 10 s interval on continuous mode with a 980 nm diode laser. Samples from the root canals were collected and plated onto brain heart infusion agar plates to determine the colony-forming unit/ml.

Statistical Analysis Used:

One-way ANOVA, post hoc Tukey's honest significant difference test.

Results:

Maximum percentage of disinfection (99%) was seen in Group B₂ (NaOCl with PDT), which was similar to Groups A₂ (97.6%) and B₃ (98.8%) (P < 0.0001).

Conclusions:

NaOCl with PDT gave maximum disinfection.

INTRODUCTION

The microbiota associated with the secondary root canal infections markedly differs from that of untreated teeth. Enterococcus faecalis is the most common microorganism isolated from root-filled teeth. It plays a major role in the etiology of persistent periradicular lesions after root canal treatment due to its several virulence factors and ability to form calcified biofilms at 6-weeks. Thus, its eradication is of utmost importance for successful root canal treatment of reinfected cases.[1]

Several powerful irrigating solutions such as sodium hypochlorite (NaOCl) and chlorhexidine (CHX), along with chelating agents and surfactants have been employed against E. faecalis.[2]

QMix 2in1 (Dentsply, Tulsa, USA) is a new irrigating solution that contains 2% CHX, ethylenediaminetetraacetic acid (EDTA), and a surfactant. It is recommended as a one-step final rinse for 60–90 s after irrigation with NaOCl.[3]

In spite of the potent antimicrobial activity of individual irrigants and their combinations, no irrigation protocol can render the root canals 100% bacteria-free. Hence, newer methods need to be employed as an adjunct with conventional irrigation protocol for the additional disinfection of root canals.

Photoactivated disinfection (PAD) or photodynamic therapy (PDT) is an antimicrobial strategy consisting of two components: A nontoxic photosensitizer and a laser. The photosensitizer first binds to the bacterial membrane and enters the cytoplasm of the target cells. It is excited by a laser light of specific wavelength producing singlet oxygen species and free radicals which are cytotoxic to the DNA and cell membrane of the target cells.[4]

With new irrigant combinations and strategies being introduced for root canal disinfection, it was necessary to check the efficacy of PAD as an adjunct to these new irrigants in eradicating E. faecalis biofilms. In addition, limited literature is available on the antimicrobial properties of various disinfecting solutions on 6-week E. faecalis biofilms. Thus, the aim of this study was to compare the additive effect of PDT on the antibacterial activity of 2.5% NaOCl, and QMix used as a final rinse against 6 weeks E. faecalis biofilms contaminating root canals.

MATERIALS AND METHODS

In the present study, the sample size was determined to be 180 with 99% confidence interval, 90% power, 4.0 standard deviation (SD), and 1.67 differences between two groups. To this, ten samples were added to include the negative control group.

After obtaining ethical clearance, 190 extracted human single-rooted anteriors were collected and kept in NaOCl solution (Prime Dental Products Pvt. Ltd., Kalher, Thane, India) for 2 h to remove organic debris followed by storage in 10% formalin (Narsipur Chemicals Pvt. Ltd., Navi Mumbai, India) for 7 days. After disinfection, the outer surfaces were cleaned with an ultrasonic scaler.

Access preparation was made, and apical patency confirmed with #15 K-file (K-files, Mani Inc., Utsunomiya, Japan). Working length was established 1 mm short of the apical foramen. Coronal flaring was done using sequential Gates Glidden drills (K-Files, Mani Inc., Utsunomiya, Japan). Initial apical file (IAF) was selected as the first snugly fitting file at the apex, and apical preparation was done to three sizes larger than the IAF. Root canals were prepared using rotary instruments (ProTaper, Maillefer-Dentsply, Baillagues, Switzerland) by crown-down technique to an apical size corresponding to the last file used at the apex and were irrigated with 2 ml of 2.5% NaOCl between each instrumentation.

After preparation, the teeth were immersed in an ultrasonic bath of EDTA (Safe Plus, Neelkanth Health Care Pvt. Ltd., Jodhpur, India) for 10 min to remove smear layer, followed by NaOCl bath for 5 min, and normal saline ultrasonic bath for 10 min. Then, the apical foramina were sealed using light cure restorative glass ionomer cement (Fusion i-Seal, Prevest DenPro Ltd. Jammu, India), and the roots were coated with two layers of nail varnish.

Each sample was transferred to a sterile glass test tube containing sterile brain heart infusion (BHI) broth (EOS Laboratories, Thane, Mumbai, India), and all samples were autoclaved under 15 psi at 121°C for 40 min. Samples were incubated in their sealed tubes for 48 h at 37°C. Daily inspection was carried out to reveal any signs of turbidity, and the samples showing turbidity were excluded from the study. Now, all infection-free samples were taken for further evaluation.

An overnight pure culture of E. faecalis (ATCC 29212, KWIK-STIK Microbiologics, France) in BHI broth was prepared for inoculation. The bacterial suspension was adjusted to match the turbidity of a McFarland 0.5 scale. A 0.01 ml aliquot of the suspension was used to inoculate each canal with a sterile micropipette. The samples were incubated for 6 weeks at 37°C in aerobic conditions for biofilm formation. The inoculums inside the canal were replaced with 0.01 ml of fresh bacterial suspension every 3 days in an ultraviolet chamber within 6 inches of a gas burner. Random sampling with Gram stain kit (Biolab Diagnostics India Pvt. Ltd., Maharashtra, India) was carried out every 7 days to confirm E. faecalis cultures.

After 6 week biofilm formation, the samples were randomly divided into the following groups, two main groups (n = 90) with three subgroups each (n = 30) and one negative control group (n = 10) and were subjected to irrigation protocol as follows:

• Group A (irrigation alone):

  • Group A₁: Irrigation with 15 ml of 10% saline for 3 min
  • Group A₂(positive control): Irrigation with 15 ml of 2.5% NaOCl for 3 min
  • Group A₃: Irrigation with 5 ml of 2.5% NaOCl solution, followed by 5 ml normal saline, and finally, 5 ml QMix 2in1 (Dentsply Tulsa Dental Specialities, USA) for a total of 3 min as per the manufacturer's instructions.

• Group B (irrigation with PDT):

  • Group B₁: 15 ml normal saline for 3 min and then irradiated with PAD
  • Group B₂: Irrigation with 15 ml of 2.5% NaOCl solution for 3 min and irradiated with PAD
  • Group B₃: Irrigation with 5 ml of 2.5% NaOCl solution, followed by 5 ml of normal saline, and then, 5 ml QMix 2in1 for 3 min as per the manufacturer's instructions and irradiation with PAD.

• Group C (negative control): No irrigation.

For PAD, a methylene blue dye (SDFCL Industries, Mumbai, India) was prepared by dissolution in BHI broth and was filter-sterilized immediately before use. The final concentration used was 25 μg/ml. The dye was then injected into the canals of each sample. The irradiation source was a diode laser (Sunny Optoelectronic Technology Co., Ltd., Shanghai, China, Model Number: MDL50) with an output power of 1.5W and a wavelength of 980 nm. A 200 μm diameter optical fiber was used. The laser handpiece was held at an angle of 10° between the fiber and root canal wall. Laser irradiation was performed three times for 5 s each with an interval of 10 s between irradiations on continuous mode. The laser irradiation was delivered into the canal up to 1 mm short of the working length while moving coronally without any water spray or air cooling.

Now, for microbiological evaluation, the canals of all the samples were dried with sterile paper points and refilled with normal saline. Using a sterile #30 H- file (H-files, Mani Inc., Utsunomiya, Japan), circumferential filing was performed for 20 s to collect dentin chips. Samples from inside the canal were collected using two sterile paper points. The sampling paper points and the sampling H-file were placed in a test tube containing 5 ml of sterile saline and vortexed for 20 s. Fifty microliters of the vortexed saline were introduced to culture plates (EOS Laboratories, Thane, Mumbai, India) and incubated at 37° C for 48 h. After 48 h, colony-forming unit [CFU]/ml was calculated for each sample by a single-blinded investigator. All data were collected, tabulated, and statistical analysis was done using one-way ANOVA and post hoc and Tukey's honest significant difference tests.

RESULTS

The mean and standard deviation of CFU/ml of each group was calculated and subjected to statistical analysis [Tables 1 and 2]. The results showed Groups A₂, B₂, and B₃ to perform significantly better than other groups in reducing the total bacterial count with over 97% disinfection (P < 0.001). Group A₁ performed the worst of all six experimental groups with the least percentage of disinfection of 50% while B₂ performed the best of all with the maximum percentage of disinfection of 99% [Figure 1].

Table 1.

Mean, standard deviation, minimum, and maximum values and P value of colony-forming unit/ mL of all groups

Table 2.

Comparison of colony-forming unit/ml between all groups using post hoc and Tukey's honestly significant difference tests

Figure 1.

A bar graph showing the mean percentage of disinfection of all groups after irrigation protocols

DISCUSSION

In the present study, E. faecalis was selected as it is known to form calcified monoculture biofilms at 3 weeks and can be easily cultivated in vitro. In our study, the samples were inoculated for 6 weeks as E. faecalis biofilm shows signs of mineralization at 6 weeks making it further difficult to eradicate.[5]

For PAD, a variety of diode lasers with a wide range of wavelengths have been used. Limited literature is available on the effect of diode laser of 980 nm on root canal disinfection. Hence, in the present study, a high-power diode laser of 980 nm with 25 μg/ml methylene blue dye was utilized.[6,7]

In the present study, the total volume of irrigants and the time of irrigation were kept constant for standardization. A total volume of 15 ml of irrigants was used for the disinfection of root canal of each sample in all experimental groups while irrigating for a total of 3 min.

Our results demonstrated Group B₁: normal saline with PAD to significantly reduce bacterial counts when compared to saline used alone. This is in accordance to the study by Mehrvarzfar et al., Souza et al., and Soukos et al. and can be attributed to the cytotoxic effect of the photosensitizer dye or the cavitational effects and heat generated by high-power diode laser in methylene blue aqueous medium. Thus, PAD alone can be considered to have some antimicrobial action, but it is significantly less than that of NaOCl or the combination of antimicrobials with PAD.[6,8,9]

In the present study, 2.5% NaOCl (Group A₂) was found to eliminate 97.6% of E. faecalis biofilms owing to its tissue dissolving properties.[9,10,11] In addition, an increase in the antimicrobial activity with the combination of 2.5% NaOCl, and PAD was seen which can be due to the increased depth of penetration of laser into dentinal tubules. Gutknecht et al. found 980 nm-diode laser to disinfect dentinal tubules at a greater depth of 500 μm as compared to 100 μm depth achieved by standard irrigants.[7,12,13,14]

QMix (Group A₃), in the present study, showed a kill rate of 89.7% against E. faecalis biofilms, which was significantly less than that of 2.5% NaOCl in accordance to the results of studies by Del Carpio-Perochena et al. and Dunavant et al.[11,15] In addition, Wang et al. found QMix to be significantly less effective than NaOCl in eradicating 3-week E. faecalis biofilms while they were equally effective in eliminating 1 day old E. faecalis biofilm. Thus, it proves that QMix is less effective than NaOCl in eradicating mature biofims.[5,16] However, when combined with PAD, QMix showed an increase in the percentage disinfection of E. faecalis biofilm to 98.8%, which was similar to Group B₂.[17]

In contrast to the results of this study, Mehrvarzfar et al. found that PAD did not show significantly better results when combined with 2% CHX. In their study, they found 2% CHX and 2% CHX in combination with PAD to be equally effective in eradicating 2 week biofilm of E. faecalis. This may be due to the shorter period of incubation. QMix is known to be less effective in eradicating mature E. faecalis biofilms. In the present study, a 6-week biofilm was used which is more resistant to disinfection procedures.[9,18]

Thus, within the limitations of the present study, it can be concluded that in lieu of the safe therapeutic window, the application of PAD might be a useful adjunct to conventional endodontic irrigation in eradicating E. faecalis biofilms. However, PAD must be used only as a supplement and not as an alternative to conventional irrigation protocols.

CONCLUSION

PAD with 2.5% NaOCl proved to be the most effective in disinfecting root canals containing mature E. faecalis biofilms.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.