|Year : 2017 | Volume
| Issue : 2 | Page : 151-155
Assessment of antimicrobial efficacy of bioceramic sealer, epiphany self-etch sealer, and AH-Plus sealer against Enterococcus faecalis: An in vitro study
Hegde Vibha, Ruchita Rathod
Department of Conservative Dentistry and Endodontics, YMT Dental College and Hospital, Navi Mumbai, Maharashtra, India
|Date of Web Publication||6-Nov-2017|
503, 56/A, Chintamani Chs Ltd, Near Pratiksha Nagar Bus Depot, Pratiksha Nagar, Sion - 400 022, Maharashtra
Source of Support: None, Conflict of Interest: None
Aim: The aim and objective of this in vitro study was to evaluate antimicrobial efficacy of root canal sealers (bioceramic [BC] sealer, Epiphany self-etch sealer, AH-Plus sealer) on Enterococcus faecalis.
Materials and Methods: An agar well diffusion assay method was used to determine the efficacy of the root canal sealer against E. faecalis (ATCC 29212). Root canal sealers were divided into three groups; BC sealer, Epiphany self-etch sealer, and AH-Plus sealer, and standard antibiotic disc of co-trimoxazole was kept as a control. The diameters of the growth inhibition zones against E. faecalis for each group were recorded and compared at 24 h and 48 h. The differences between groups were analyzed by one-way ANOVA and Tukey's post hoc tests for intergroup analysis.
Results: AH-Plus sealer exhibited larger zone of inhibition than BC sealer and Epiphany self-etch sealer against E. faecalis at 24 and 48 h. Antibiotic disc of co-trimoxazole, which was used as control exhibited the highest antimicrobial activity against E. faecalis at 24 and 48 h. Epiphany self-etch sealer showed least antimicrobial activity.
Conclusion: The AH-Plus root canal exhibits better antibacterial action against E. faecalis at 24 and 48 h.
Keywords: Agar diffusion test, brain-heart infusion broth, inhibition zone
|How to cite this article:|
Vibha H, Rathod R. Assessment of antimicrobial efficacy of bioceramic sealer, epiphany self-etch sealer, and AH-Plus sealer against Enterococcus faecalis: An in vitro study. Endodontology 2017;29:151-5
|How to cite this URL:|
Vibha H, Rathod R. Assessment of antimicrobial efficacy of bioceramic sealer, epiphany self-etch sealer, and AH-Plus sealer against Enterococcus faecalis: An in vitro study. Endodontology [serial online] 2017 [cited 2019 Nov 14];29:151-5. Available from: http://www.endodontologyonweb.org/text.asp?2017/29/2/151/217707
| Introduction|| |
Total canal disinfection and three-dimensional obturation are attained by proper root canal treatment. Microorganisms and their by-products have been responsible in dentinal, pulpal, and periapical pathology, which was observed by Miller in 1890. The typical polymicrobial flora of Gram-negative and Gram-positive bacteria is a endodontic microflora, which is controlled by obligate anaerobes. To seal all microchannels and dentinal tubules, various sealers have been applied along with gutta-percha obturating systems.
The most routinely microorganism perceived in endodontic failures is Enterococcus faecalis. E. faecalis is a Gram-positive, Group D streptococci, a facultative anaerobe. Due to their tendency to form biofilms, it can exist in extremely harsh environment. It has been shown to be highly invulnerable once demonstrated in the root canal system and is probably the species that can best adapt to and indulge the ecologically demanding conditions in the filled root canal. Adherence to host cells and extracellular matrix, tissue invasion, effect on immunomodulation, and toxin-mediated damage are caused by number of virulence factors of E. faecalis. Shaping and cleaning of the root canal space may abolish the majority of the bacteria found in the root canal system. Even after shaping and cleaning of the root canal system, microorganisms might still exist in these challenges due to the anatomical complexities such as dentinal tubules, ramification, deltas, and fins. E. faecalis is a recalcitrate candidate among the causative agents of failed endodontic treatment. According to Sundqvist et al. 1998, E. faecalis found in 38% of failed root canal system. These microorganism have tendency to remove the other microbes and also to grow in a low-nutrient environment. With chemomechanical preparation of root canal system, the eradication of E. faecalis from the root canal is challenging.
Bioceramic (BC) sealers are known to possess biological activity. It is highly radiopaque and hydrophilic forms hydroxyapatite on setting and chemically bonds to both dentin and gutta-percha points. It is antibacterial during setting due to its highly alkaline pH. It exhibits absolutely zero shrinkage. Hence, when these sealers are used along with obturating systems, it may greatly affect the survival of bacteria adversely. The use of sealers with antibacterial properties may be advantageous, especially in clinical situations of persistent or current infection. The endodontic sealers have been shown to give the greatest antimicrobial effects immediately after spatulation, following which there is a gradual loss of antimicrobial effects over time.
Many studies have been performed to assess the antimicrobial efficacy of different root canal sealers. The agar diffusion test (ADT) was most commonly used technique. Antibacterial activity of the endodontic sealers is tested based on measuring the effect of close contact between test bacteria and tested material on kinetics of bacterial growth.
| Materials and Methods|| |
In the current study, the root canal sealers tested were as follows: Group 1 - Bioceramic Root Canal Sealer (Brasseler USA), Group 2 - Self-Etch Epiphany sealer (Real Seal, Sybronendo, Orange, California, USA), Group 3 - AH-Plus sealer (Dentsply, De-Trey, Konstanz, Germany). Standard antibiotic disc of co-trimoxazole disc was used as control group against E. faecalis.
Preparation of the medium for Enterococcus faecalis
The strains of microorganisms used for the study were standard strains of E. faecalis ATCC 29212 and were subcultured in blood agar plate and were incubated at 37°C for 24 h. A pure, single E. faecalis colony was isolated from the same cultured plate and Gram's staining was performed to confirm its growth, which was checked under oil immersion microscope and then inoculated with a brain-heart infusion (BHI) broth. The BHI-broth was incubated at 37°C for 24 h period and checked for bacterial growth by changes in turbidity.
A drop of BHI-broth containing E. faecalis was placed into saline solution and checked for correct bacterial concentration with a spectrophotometer. By analyzing the broth at a density associated to the barium sulfate standard of 0.5 McFarland units, which was equal in value to 1.5 × 108 CFU/ml, the density of the bacterial suspension is standardized.
Antimicrobial activity by agar diffusion test
Mueller-Hinton agar was used to prepare petri plates. The sterility of the plates was checked and the fresh inoculums of E. faecalis of 0.5 McFarland standard suspensions were formulated. A sterile nontoxic cotton swab was dipped on a wooden applicator into standardized inoculums and the soaked cotton swab was rotated firmly against the upper inside wall of the tube to express excess fluid. The plate was turned at 60° angle between each streaking. The inoculums were allowed to dry for 5–15 min with lid in place.
Four wells were created using 8 mm sterile cork borer. The desired amount of the root canal sealer to be tested was mixed and was placed in the well against E. faecalis. 10–15 min was allowed for diffusion of the medicament in agar and then was incubated immediately at 35 ± 2°C for 24 h. The whole experiment was carried out under aseptic conditions and was repeated twenty times to ensure reproducibility.
Measurement of inhibition zones
Zones of bacterial growth inhibition were measured at the end of 24 and 48 h using inhibition zone measuring scale.
After data collection, data were entered into Microsoft Excel and analyzed using the SPSS software v20 (IBM Corp., Armonk, NY, USA). Descriptive statistics were shown as mean and standard deviation. To evaluate the differences between the antimicrobial efficacy Endosequence BC sealer, Epiphany self-etch sealer, and AH-Plus sealer, one-way ANOVA test [Table 1] was used and for comparison within the groups, Tukey's multiple comparison test [Table 2] was used. P < 0.05 was considered statistically significant.
|Table 1: Statistical analysis ANOVA against Enterococcus faecalis at 24 and 48 h|
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|Table 2: Statistical analysis Tukey's post hoc multiple comparisons against Enterococcus faecalis|
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| Results|| |
The means of the diameters of the growth inhibition zones for each group of root canal sealers and control group are presented in [Figure 1] and [Graph 1]. The range of inhibitory values between experimental groups varied broadly. One-way ANOVA was used to calculate P value and showed significant differences (P< 0.0001) [Table 1]. Overall, AH-Plus sealer (Group 3) had the largest zones of growth inhibition in the well diffusion assay [Figure 1] and [Graph 1] than the BC sealer. Epiphany self-etch sealer showed least amount of inhibitory effect against E. faecalis. Standard antibiotic disk of co-trimoxazole which serves as control exhibited highest zone of growth inhibition against E. faecalis.
|Figure 1: Zone of inhibition exhibited by AH-Plus sealer was higher than other two sealers. Whereas standard antibiotic disk of co-trimoxazole showed highest zone of inhibition among all the groups|
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| Discussion|| |
Kirby-Bauer method is the choice of method study chosen. Agar diffusion method is the most commonly used method to evaluate the antimicrobial activity of different endodontic sealers. Using this method, we can compare of root canal sealers against test microorganisms and also the visual indication of sealer potential to eliminate microorganisms in the local microenvironment of the root canal system.
E. faecalis is used as a target microorganism for the determination of result of antibacterial agents on it.,,E. faecalis is more susceptible to infected failed endodontic treated teeth than with a primary infected teeth. There are many reasons for the isolation of E. faecalis in failed root canal treated teeth and could be as follows: (a) a small amount of enteric bacteria is already present in the infected canal at the initiation of the treatment and their relative proportion increases during the treatment as other bacteria are susceptible to therapy or (b) enteric bacteria could be introduced the root canal during the treatment due to (i) inadequate isolation, (ii) a leaking due to temporary filling, or (iii) the root canal has been left open for drainage. Therefore, it is important to keep in mind that the objective of the root canal treatment is complete elimination of infection and also prevention of reinfection of the treated root canal system.
Since E. faecalis is the preferred microorganism in this study. Three endodontic sealers were used to study antimicrobial property (BC sealer, Epiphany self-etch sealer, and AH-Plus sealer). These sealers were used against E. faecalis at 24 and 48 h using the agar diffusion method.
According to the Performance Standards for Antimicrobial Disk Susceptibility Test (Clinical and Laboratory Standards Institute, formerly NCCLS) guidelines, in the present study, standard antibiotic disc of co-trimoxazole was used as a control against E. faecalis (ATCC 29212) strain. Results demonstrated that control group (standard antibiotic disc of co-trimoxazole) showed the highest zone of inhibition followed by AH-Plus and Endosequence BC sealer. This bactericidal action is due to the double inhibition of bacterial folic acid synthesis.
BC sealer is newly introduced endodontic sealer (Endosequence BC Sealer, Brasseler USA, Savannah, GA, USA). It has an alkaline pH, high calcium ions release, and suitable radiopacity and flow capacity. It also exhibits antibacterial activity and biocompatibility. BC sealers are highly hydrophilic which allows them to spread easily over the root canal walls and fill the lateral microcanals too. During setting, these sealers expand and form chemical bond with the canal walls.
The present study found significant antimicrobial activity of BC sealer against the test microorganism. The antibacterial effect of the BC sealer may be due to the combination of high pH and active calcium hydroxide diffusion. AH-Plus sealer had significantly greater ability to eliminate E. faecalis than Endosequence BC sealer. Studies by Candeiro et al. have similar findings as that of our study and they have shown that AH-Plus sealer had significantly greater ability to eliminate E. faecalis than Endosequence BC sealer when assessed by the ADT.
According to Epiphany's manufacturer (Pentron Clinical Technologies, LLC Wallingford, Connecticut), this dual-cure, resin root canal sealer is nonmutagenic, noncytotoxic, biocompatible, and less irritating when compared to epoxy resin or zinc oxide-eugenol-based sealers. According to studies, Epiphany has a great sealing capacity to the root canal walls, but this is based on limited information.,
In the present study, Epiphany sealer showed a little antimicrobial activity against E. faecalis. This result partly agrees with the previous studies of Pinheiro et al. and Bodrumlu and Semiz that investigated the antibacterial efficacy of Endomethasone, Sultan, Sealapex, Diaket, Epiphany, and AH 26 against E. faecalis using the agar diffusion method. They found that Epiphany had a little effect against the tested microorganism., Slutzyk Goldberg et al. reported that epiphany had no antibacterial effect against E. faecalis. It showed that the least antimicrobial effect of this material may result from its hydrophilic resin form.
AH-Plus has highest antimicrobial activity in evaluation periods 24 and 48 h. It also showed antimicrobial activity on Candida albicans, Staphylococcus aureus, and Escherichia More Details coli. Besides, Yasuda showed that AH-Plus has higher antimicrobial activity against all the tested microorganisms (S. aureus, E. faecalis, C. albicans, Staphylococcus mutans, Streptococcus saguinis). The action of AH-Plus is due to the presence of bisphenol-A-diglycidyl ether. Components of paste A (containing epoxy resin) and paste B (containing amines) are mixed together, whereby the sealer reduced the cell viability. Furthermore, this sealer has a good flow, thereby diffusing into the dentinal tubules and creating microbial inhibition by means of entombment. It has been also reported that material released formaldehyde in the polymerization process. This results in the sealers antibacterial property. Pizzo et al. reported that in direct contact test, only fresh AH-Plus showed antibacterial activity and is found inactive in 24 h and 7-day-old samples did not show antibacterial effect against E. faecalis. Previous studies also demonstrated the high antibacterial effectiveness of AH-Plus sealer against E. faecalis.
In the present study, AH-Plus sealer exhibited the highest zone of inhibition compared to Endosequence BC sealer and Epiphany sealer. This result could imply that these sealers contain more potent antibacterial inhibitors than Endosequence BC sealer and Epiphany sealer. An alternative explanation is that the antibacterial components of these sealers have better diffusion properties.
The diameters of the zones of microbial growth inhibition of all three root canal sealers at 48 h were the same as those measured at 24 h, only the substances continued to diffuse, and only the 24 h values were considered for the study.
However, there are certain limitations associated with this study. The results of agar diffusion method could be influenced by diffusion and affinity of the material to the culture medium since a material that diffuses easily usually results in larger zone of inhibition of bacterial growth. Furthermore, agar diffusion method is not completely reliable as it has its own limitations such as intensity of agar, condition of plate storage, time of incubation, size and number of specimen or plate, quantity of culture medium, inability to distinguish between bacteriostatic and bacteriocidal properties and can be tested only in water-soluble agents. There are contemporary and more reliable methods available to check antibacterial efficacy which can also be tried in the future to test the same.
| Conclusion|| |
Within the experimental conditions of the present study, it can be concluded that
- BC sealer showed moderate antimicrobial activity against E. faecalis though it was lesser than AH-Plus sealer
- Epiphany self-etch sealer showed least antimicrobial activity
- AH-Plus sealer exhibited larger zone of inhibition than BC sealer and Epiphany self-etch sealer against E. faecalis
- Antibiotic disc of co-trimoxazole, which was used as control exhibited highest antimicrobial activity against E. faecalis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Oh S, Perinpanayagam H, Kum D, Lim S, Yoo Y, Chang S, et al
. Evaluation of three obturation techniques in the apical third of mandibular first molar mesial root canals using micro-computed tomography. J Dent Sci 2016;11:95-102.
Seelan RG, Kumar AA, Emil Sam RJ, Maheswari SU. Antimicrobial efficacy of different root canal sealers by using real-time polymerase chain reaction: An ex vivo
study. J Conserv Dent 2015;18:474-8.
] [Full text]
Portenier I, Waltimo T, Haapasalo M. Enterococcus faecalis-
The root canal survivor and 'star' in post-treatment disease. Endod Topics 2003;6:135-59.
Anumula L, Kumar S, Kumar VS, Sekhar C, Krishna M, Pathapati RM, et al.
An assessment of antibacterial activity of four endodontic sealers on Enterococcus faecalis
by a direct contact test: An in vitro
study. ISRN Dent 2012;2012:989781.
Sundqvist G, Figdor D, Persson S, Sjögren U. Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86-93.
Geetha RV, Veeraraghavan VP. Evaluation of antibacterial activity of five root canal sealants against Enterococcus faecalis
– Anin vitro
study. Int J Pharm Sci Rev Res 2016;40:221-3.
Asgary S, Kamrani FA. Antibacterial effects of five different root canal sealing materials. J Oral Sci 2008;50:469-74.
Arora D, Rawat D, Bhayya D. A comparative evaluation of antimicrobial efficacy of three endodontic sealers: Endoflas FS, AH plus and sealapex against Enterococcus faecalis - An in vitro
study. IOSR J Dent Med Sci 2014;13:90-3.
McHugh CP, Zhang P, Michalek S, Eleazer PD. pH required to kill Enterococcus faecalis in vitro
. J Endod 2004;30:218-9.
Cobankara FK, Altinöz HC, Ergani O, Kav K, Belli S.In vitro
antibacterial activities of root-canal sealers by using two different methods. J Endod 2004;30:57-60.
Saleh IM, Ruyter IE, Haapasalo M, Østavik D. Survival of Enterococcus faecalis
in infected dentinal tubules after root canal filling with different root Canal sealers in vitro
. Int Endod J 2004;37:193-8.
Kayaoglu G, Ørstavik D. Virulence factors of Enterococcus faecalis
: Relationship to endodontic disease. Crit Rev Oral Biol Med 2004;15:308-20.
Aravind S, Gopikrishna V, Jeyavel RK, Kandaswamy D. Comparative evaluation of the antimicrobial efficacy of five endodontic root canal sealers against Enterococcus faecalis
and Candida albicans
. J Conserv Dent 2006;9:2-12.
Al-Haddad A, Che Ab Aziz ZA. Bioceramic-based root canal sealers: A review. Int J Biomater 2016;2016:9753210.
Tyagi S, Tyagi P, Mishra P. Evolution of root canal sealers: An insight story. Eur J Gen Dent 2013;2:199-218. [Full text]
Zhang H, Shen Y, Ruse ND, Haapasalo M. Antibacterial activity of endodontic sealers by modified direct contact test against Enterococcus faecalis
. J Endod 2009;35:1051-5.
Shipper G, Teixeira FB, Arnold RR, Trope M. Periapical inflammation after coronal microbial inoculation of dog roots filled with gutta-percha or resilon. J Endod 2005;31:91-6.
Shipper G, Ørstavik D, Teixeira FB, Trope M. An evaluation of microbial leakage in roots filled with a thermoplastic synthetic polymer-based root canal filling material (Resilon) J Endod 2004;30:342-7.
Pinheiro C, Guinesi A, Pizzolitto, Bonetti-Filho I. In Vitro
Antimicrobial Activity of Acroseal, Polifil and Epiphany against Enterococcus faecalis. Braz Dent J 2009;20:107-111.
Bodrumlu E, Semiz M. Antibacterial activity of a new endodontic sealer against Enterococcus faecalis
. J Can Dent Assoc 2006;72:637-640.
Slutzky-Goldberg I, Slutzky H, Solomonov M, et al
. Antibacterial properties of four endodontic sealers. J Endod 2008;34:735-8.
Miyagak DC, de Carvalho EM, Robazza CR, Chavasco JK, Levorato GL.In vitro
evaluation of the antimicrobial activity of endodontic sealers. Braz Oral Res 2006;20:303-6.
Pizzo G, Giammanco GM, Cumbo E, Nicolosi G, Gallina G. In vitro
antibacterial activity of endodontic sealers. J Dent 2006;34:35-40.
[Table 1], [Table 2]