|Year : 2016 | Volume
| Issue : 1 | Page : 1-8
The synergistic effect of ultrasonic activation and irrigation on Enterococcus faecalis biofilm
Abeer A Al-Mahdi1, Hanan A Balto2
1 Postgraduate Student, College of Dentistry, King Saud University, Riyadh, Saudi Arabia
2 Department of Restorative Dental Science, Division of Endodontic, Dental Caries Research Chair, College of Dentistry, King Saud University, Riyadh, Saudi Arabia
|Date of Web Publication||16-Dec-2015|
Hanan A Balto
Department of Restorative Dental Science, Dental Caries Research Chair, College of Dentistry, King Saud University, Riyadh
Source of Support: None, Conflict of Interest: None
Aim: The aim of this investigation was to compare the efficacy of passive ultrasonic irrigation (PUI) with either 2.5% sodium hypochlorite (NaOCl) or saline, with that of conventional syringe irrigation on intraradicular Enterococcus faecalis biofilm. Materials and Methods: Biofilms of E. faecalis were established over 21 days in 80 single roots that had undergone biomechanical preparation followed by gamma radiation. Biofilms were treated for 1 min with 2.5% NaOCl/PUI (Group 1), 2.5% NaOCl (Group 2), sterile saline/PUI (Group 3), and sterile saline (Group 4). The positive control (n = 4) was used to confirm the presence of biofilm before various treatments. Additional four samples that served as a negative control were used to confirm the sterility of the samples. Biofilm eradication was evaluated by Colony Forming Unit (CFU) quantification and scanning electron microscopy (SEM). Results: The median of CFUs of S1 was significantly higher than that of S2 in all experimental groups. SEM examination showed a significant difference between the positive control and the experimental groups (P < 0.001), with the highest score of biofilm in the positive control group followed by Group 4 and both groups were not statistically significant from each other (P = 0.067). Following various treatments, the highest scores of biofilm were observed in the coronal third and the least were in the apical third. Conclusions: PUI did not increase the effectiveness of NaOCl irrigation on biofilm removal, however, PUI enhanced biofilm disturbance when used with saline. The least mean score of remaining biofilm was in the apical third of all treatment groups compared to other thirds.
Keywords: Biofilm, Enterococcus faecalis, irrigation, ultrasonic
|How to cite this article:|
Al-Mahdi AA, Balto HA. The synergistic effect of ultrasonic activation and irrigation on Enterococcus faecalis biofilm. Saudi Endod J 2016;6:1-8
|How to cite this URL:|
Al-Mahdi AA, Balto HA. The synergistic effect of ultrasonic activation and irrigation on Enterococcus faecalis biofilm. Saudi Endod J [serial online] 2016 [cited 2019 Nov 14];6:1-8. Available from: http://www.saudiendodj.com/text.asp?2016/6/1/1/171997
| Introduction|| |
The microbial flora present in infected root canals may exist as loose collection in mist canal lumen or as biofilms adhered to dentinal walls. The biofilm environment has been found to be advantageous for bacterial living; as it helps the bacteria to survive and multiply, inducing their metabolic products that will lead to the persistence of periapical infection. Several studies have shown that microorganisms in biofilms have stronger pathogenic potential than those in the planktonic state ,,, and its presence on the root canal walls, although sometimes nonviable, may prevent three-dimensional obturation of the root canal system. In addition, if bacteria or biofilm degrade after obturation, voids may result leading to potential therapeutic failure.
Many chemical irrigants have been used for the purpose of elimination of residual microbes in root canals accompanied with mechanical instrumentation. Sodium hypochlorite (NaOCl) is one of the most commonly used endodontic irrigants because of its ability to destroy a broad spectrum of microbes and dissolve organic materials. High concentrations of NaOCl have been shown to have a high degree of antimicrobial activity against both planktonic , and biofilm bacteria.,,,, However, full strength (5.25%) NaOCl has been found to cause a severe inflammatory reaction when placed in contact with vital tissues , and, therefore, low NaOCl concentrations were recommended.
Mechanical activation such as passive ultrasonic irrigation (PUI) was advocated to improve the antibacterial effectiveness of low concentration of NaOCl. PUI showed its disinfection effect on both planktonic and biofilm bacteria, and when combined with NaOCl, it accelerates the chemical reactions, creates cavitational effects, and achieves superior cleansing action., However, there are limited studies that compared the synergistic effect of passive ultrasonic activation and irrigation on removing the biofilm from root canals.,, The aim of this investigation was to compare the efficacy of PUI with either 2.5% NaOCl or saline, with that of conventional syringe irrigation on intraradicular Enterococcus faecalis biofilm.
| Materials and Methods|| |
A total of 84 extracted human single-rooted teeth were used in this study. In order to exclude teeth with multiple root canals, all teeth were radiographed from both facial and proximal aspects. The selected teeth were then examined under magnifying dental loupes of ×2.5 magnification to ensure that they were free of any cracks, root resorption, and/or an open apex. Any tooth with calcified, and/or severely curved canals (>20° according to Schneider) was excluded. The anatomic crown of each tooth was resected perpendicular to the long axis of the root with a carborundum disk (Dentsply Maillefer, Tulsa, OK) under water spray, to standardize the root length of all samples at 16 mm. The working length was measured by deducting 1 mm from the lengths recorded when the tip of a #15 K-file was visible at the apical foramina. The canals were enlarged coronally with size 2 and 3 Gates-Glidden burs (Sendoline Perfect Endo, Sweden) and instrumented up to size 20 K-files to establish a glide path. The #15 K-file (Sendoline Perfect Endo, Sweden, Taby) was passed through the apical foramen of the canal before and after instrumentation to ensure patency. Standard root canal procedure was performed using K3Ni-Ti rotary instruments (SybronEndo, Glendora, CA, USA) until file #40/0.04 reached the working length. During instrumentation, Glyde File Prep (Dentsply Maillefer, Ballaigues Switzerland, USA) was used as a lubricant and the canals were irrigated with 2 ml of 2.5% NaOCl at each change of instrument. Finally, the canals were rinsed with 3 ml sterile saline solution to flush away residual irrigants. Each instrument was only used for the preparation of ten canals then discarded. The apical foramen of each root was sealed with Glass Ionomer Cement (Fuji II LC, GC Corporation, Tokyo, Japan) to avoid bacterial leakage through the apex or lateral canals during inoculation, the external root surface was coated with two layers of cyanoacrylate adhesive (SuperGlue, Alteco Chemical PTE, Ltd., Indonesia). All samples were sterilized using 25 KGy of gamma radiation, after which all procedures and microbiological manipulations were carried out in a Class II Biological Safety Cabinet (Baker Scientific Company, Maine, USA). After sterilization, the roots were embedded in modeling material that served as a base to stabilize the roots vertically during manipulation.
Enterococcus faecalis ATCC (29212) was taken from frozen stock culture of Dental Caries Research Chair, inoculated with brain heart infusion broth (BHIB) and grown overnight at 37°C. Cells were collected by centrifugation (900×g for 10 min), and the pellets were resuspended in BHIB. The culture was diluted (1:10) in fresh BHIB and used to inoculate the root canals every 2 days for 21 days. The teeth were maintained in a humid and anaerobic environment at 37°C throughout the experiment.
Roots were assigned randomly into four experimental groups (19 roots each) and two control groups (4 roots each). The pretreatment bacteriological sampling (S1) was taken for each root in the experimental and the positive control groups by inserting two sterilized paper points to the full length of the root canal for 4 s, then transferred to sterile tube containing 1-mL sterilized saline and vortexed for 30 s. Serial dilution of the resulting suspension were prepared and plated for quantification of Colony Forming Units (CFUs) with the aid of colony counter (Quebec Counter, California, USA). The teeth were then assigned to the following treatment protocols.
Group 1: Were irrigated with 3 ml of 2.5% NaOCl using a 27-G needle set 1 mm short of the working length, followed by ultrasonic activation with oscillating file size K 15 (IrriSafe, Satelec, Acteon, France) placed in the canal at a distance of 1 mm from the working length and moved passively using in and out movements for 1 min. The power setting on the ultrasonic unit (P-Max Newtron XS, Satelec, Aceton, France) was kept at 7. The irrigation was performed at a rate that ensured that the total irrigant volume (3 ml) was used within 1 min of activation. Final flush with 3 ml of 2.5% NaOCl was carried out to remove any dislodged remnants. To deactivate the NaOCl, the root canals were rinsed with 5% sodium thiosulfate for 1 min and then underwent a final flush with 2 ml of sterile saline.
Group 2: Were treated in the same manner as Group 1 but without ultrasonic activation.
Group 3: Were irrigated with 3 ml of sterile saline followed by ultrasonic activation for 1 min as in Group 1, flushed with 3 ml of saline to remove any dislodged remnants and finally underwent a flush with 2 ml of sterile saline.
Group 4: Were treated in the same manner as Group 3 but without ultrasonic activation.
The negative control group was placed individually in sterile centrifuge tubes containing 5 ml of BHIB and incubated overnight at 37°C and checked for turbidity. The positive control group did not receive any treatment after bacterial inoculation and were used to confirm the presence of biofilm.
Posttreatment samples (S2) were obtained and counted for all experimental groups in the same manner as pretreatment samples (S1). After bacterial sampling, all the samples including the control groups were immersed individually in 1.5 ml plastic tubes containing 1% formaldehyde and left for 24 h for fixation.
Scanning electron microscopy evaluation
Roots were grooved longitudinally on the external surface with a carborundum diamond disc (Dentsply Maillefer, Tulsa, OK) and split into two halves with a chisel. The roots were marked at three levels 5, 10, and 15 mm from the root apices using a diamond bur and then washed with normal saline, labeled, assigned to its designated group, and processed for scanning electron microscopy (SEM) examination.
Each half was examined individually under SEM (JEOL JSM-636OLV, Tokyo, Japan) at an accelerating voltage of 25 kV. Digital photomicrographs at ×200, ×1000, ×2000, and ×3000 were taken. Biofilm coverage was evaluated using the three-point scoring system: Score (1) indicates 0–30% root canal wall coverage, score of (2) indicates 30–60% root canal wall; and score of (3) indicates >60% root canal wall coverage. Two examiners performed the scoring procedure blindly and independently. In case of disagreement, the two examiners discussed the case to reach a consensus.
The reduction in CFU was compared using Wilcoxon signed-rank test followed by Kruskal-Wallis test. The intra-examiner's reliability was assessed by Pearson correlation coefficient test. One-way ANOVA and Bonferroni tests were used for comparison between each third in each group with significance level set at P < 0.05.
| Results|| |
Wilcoxon sign rank test showed a significant difference in median of CFUs between S1 and S2 in all experimental groups. The median of CFUs of S1 was significantly (P< 0.05) higher than that of S2 in experimental groups [Table 1]. Meanwhile, Kruskal–Wallis test showed no significant difference in biofilm eradication (P = 0.274) between S1 and S2 among the four experimental groups using CFU counts [Table 2].
|Table 1: Comparison of median CFUs counts between S1 and S2 among the experimental groups|
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|Table 2: Kruskal-Wallis equality of populations rank test comparing biofilm eradication between S1 and S2 among the four experimental groups|
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An excellent inter-examiner reproducibility for both observers with value of 0.96 was observed. SEM examination revealed a statistically significant difference between the positive control group and the experimental groups (P< 0.001), with the highest score of biofilm in the positive control group followed by Group 4 and both groups were not statistically significant from each other (P = 0.067) [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5] and [Table 3]. A significant difference in the biofilm eradication among the three levels of the root was observed in the control and the experimental groups, except between the apical and middle third of the control group, Groups 2 and 4. However, in general, the highest scores of biofilm were observed in the coronal third and the least were in the apical third following various treatments [Table 4].
|Figure 1: Scanning electron microscopy photograph of the positive control group showing biofilm colonization around the dentinal tubules (×1000)|
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|Figure 2: Scanning electron microscopy photograph of the (a) coronal, (b) middle, and (c) apical third at ×200 after final flushing with 2.5% sodium hypochlorite and passive ultrasonic irrigation, showing disrupted biofilm around dentinal tubules with few debris|
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|Figure 3: Scanning electron microscopy photograph of the (a) coronal, (b) middle, and (c) apical third at ×200 after final flushing with 2.5% sodium hypochlorite, showing disrupted biofilm around dentinal tubules with few debris|
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|Figure 4: Scanning electron microscopy photograph of the (a) coronal, (b) middle, and (c) apical third at ×200 after final flushing with saline/passive ultrasonic irrigation, showing heavy biofilm around dentinal tubules with few debris|
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|Figure 5: Scanning electron microscopy photograph of the (a) coronal, (b) middle, and (c) apical third at ×200 after final flushing with Saline, showing heavy biofilm around dentinal tubules with few debris|
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|Table 3: One-way ANOVA and Bonferroni tests comparing biofilm eradication among the control and experimental groups using SEM|
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|Table 4: One-way ANOVA and Bonferroni tests comparing biofilm eradication at each third of the root canal in each group|
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| Discussion|| |
The biofilm model used in this study was adopted from Estrela et al. where the inoculation was made every other alternate day for period of 21 days. Although, single bacterial species is not representative of the polymicrobial infection in the root canal, this could simplify the biofilm formation as the multispecies biofilms have different modes of growth, which is difficult to control and represents a problem in disruption assessment.
Enterococcus faecalis was selected in this study for many reasons; it can survive very harsh environment, being the dominant microorganism in posttreatment apical periodontitis, has often been isolated from the root canal in pure culture,, exhibited a high level of resistance to wide range of antimicrobial agents, has intrinsic resistance to hypochlorite, has high capability to form biofilm, and it is easy to culture. Although, E. faecalis is a facultative anaerobic bacteria, in this study; it was cultured under anaerobic environment to simulate conditions in an infected root canal where a very low oxygen tension would be expected.
In this study, 1-min ultrasonic file activation was used based on previous finding, which showed that 1-min PUI when combined with 1% NaOCl was significantly effective in reducing CFU in canal walls and dentinal tubules. Variations in the canal configuration (oval or round) could affect the surface area colonized with bacteria, therefore, every attempt was made to distribute equal number of round and oval canals among the groups.
The results of the present study showed no significant difference in CFU counts among the four treatment protocols. However, all treatment protocols significantly reduced intracanal E. faecalis counts (S2) compared to the baseline (S1). A possible explanation for the lack of sensitivity of CFU method to detect the enhancing effect of PUI, if any, could be due to the fact that a CFU could be formed by a single cell or a clump of cells; therefore, using PUI might have dispersed the biofilm bacterial cells allowing their CFU numbers to be higher comparable to a similar amount of biofilm that was not subjected to PUI.
In addition, studies using infected root segments or tooth models have reported less promising results when NaOCl was used as the test agent., The differences could be attributed to the inactivation of NaOCl by the organic component in root dentine and the possible inability of NaOCl to penetrate into accessory anatomy or dentinal tubules in these models.
Colony Forming Unit quantification is the simplest and most commonly used method to assess the antimicrobial effectiveness of endodontic treatment measures.,, However, it has many limitations including that only the planktonic or free floating bacteria within the root canal system can be evaluated while those located in inaccessible areas such as fins, accessory canals, and isthmuses cannot be assessed., Therefore, SEM was used in this study as an adjunct to describe the details of biofilm structure and estimate the proportion of the surface colonized.
The findings of SEM demonstrated that the least mean score was in Group 1, yet it was not statistically significant from Groups 2 and 3. Whereas, the highest score of biofilm was observed in the positive control group followed by Groups 4 and both groups were not statistically significant from each other. This means that the use of passive ultrasonic activation with NaOCl did not improve the potential for decontamination in root canals infected with E. faecalis. Similar results were found in previous studies, with no significant difference between groups receiving endodontic irrigants with or without ultrasonic agitation.,, Lack of significant difference between NaOCl and saline when used with PUI indicate that the ultrasonic activation was able to partially eradicate the biofilm when combined with saline. This was consistent with Spoleti et al. where they have shown that ultrasonic activation of saline significantly reduced the surviving bacterial colonies, although, they have evaluated the tested irrigants against planktonic bacteria.
The least scores of remaining biofilm was in the apical third of the experimental groups, this could be explained by the fact that in Groups 1 and 3, where PUI was used, the vibration of the small file resulted in concentration of hydrodynamic stresses apically, and with the tapering shape of the canal and smaller surface area; greater acoustic streaming could be generated and improved the effectiveness of ultrasonic debridement. Less score means were found in the apical third of Groups 2 and 4, this could be due to the deep insertion of the irrigating needle tip which resulted in higher efficacy of irrigation procedure.
| Conclusions|| |
Within the limitations of this study, our finding showed that PUI did not increase the effectiveness of NaOCl irrigation on biofilm removal, however, PUI enhanced biofilm disturbance when used with saline.
This study was registered in the College of Dentistry Research Center, King Saud University (NF# 2177), and conducted in collaboration with Dental Caries Research Chair.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4]