|Year : 2015 | Volume
| Issue : 3 | Page : 170-174
Antibacterial efficacy of three root canal sealers against Enterococcus faecalis and Candida albicans: An in vitro study
Rahul Bhargava1, K Shashikala2, Rangoli Bhargava3
1 Department of Conservative Dentistry and Endodontics, Darshan Dental College and Hospital, Udaipur, Rajasthan, India
2 Department of Conservative Dentistry and Endodontics, RV Dental College, Bengaluru, Karnataka, India
3 Department of Oral Medicine and Radiology, Darshan Dental College and Hospital, Udaipur, Rajasthan, India
|Date of Web Publication||4-Aug-2015|
Department of Conservative Dentistry and Endodontics, Darshan Dental College, Udaipur, Rajasthan
Aim: The aim of this in vitro study was to compare the antimicrobial efficacy of calcium hydroxide-based sealer (Apexit Plus), zinc oxide eugenol-based sealer (Endoflas FS) and resin-based sealer (EndoRez) root canal sealers against Enterococcus faecalis microbial type culture collection (MTCC) 439 and Candida albicans MTCC 239 using agar diffusion test. Materials and Methods: In the present study, 20 Mueller Hinton agar (MH agar) plates were employed. Three wells were made by removal of agar at equidistant points and filled with root canal sealers according to manufacturer's instructions. The strains of the bacteria and fungi used in this study were E. faecalis MTCC 439 and C. albicans MTCC 227. Both micro organisms were grown at 37°C for 24 h in MH Broth and seeded into MH agar to produce a turbidity of 0.5 on the McFarland scale, which corresponds to a concentration of 10 8 CFU/mL. This MH broth was used as a second layer. The seeded agar was then added over the plates immediately after the insertion of sealer cements. After incubation, the diameters of zones of inhibition around the plates were measured. Results: The results were statistically analyzed using two way ANOVA test. Against both the micro organisms used in this study, Endoflas FS showed the largest zones of inhibition followed by Apexit Plus and EndoRez. Conclusion: Within the limitations of this study, it can be concluded that: Root canal sealers showed different inhibitory effects depending on their types and microbial strains tested. Against Enterococcus faecalis and Candida albicans, zinc oxide based sealers showed the highest microbial zones of inhibition followed by the calcium hydroxide based sealer and resin based sealer respectively
Keywords: Antimicrobial activity, Apexit Plus, Candida albicans, EndoRez, Enterococcus faecalis
|How to cite this article:|
Bhargava R, Shashikala K, Bhargava R. Antibacterial efficacy of three root canal sealers against Enterococcus faecalis and Candida albicans: An in vitro study. SRM J Res Dent Sci 2015;6:170-4
|How to cite this URL:|
Bhargava R, Shashikala K, Bhargava R. Antibacterial efficacy of three root canal sealers against Enterococcus faecalis and Candida albicans: An in vitro study. SRM J Res Dent Sci [serial online] 2015 [cited 2020 Jul 13];6:170-4. Available from: http://www.srmjrds.in/text.asp?2015/6/3/170/162177
| Introduction|| |
The endodontic microflora is typically a polymicrobial flora consisting of Gram-positive and Gram-negative bacteria. Micro organisms and their products are the most frequent etiologic factors in causing pathologic conditions of pulpal and periapical origin. In the normal oral flora, there are about 300 different bacterial species, of which a great number of micro organisms can colonize in the root canal system. 
Failure, during and after endodontic treatment are linked to the presence of micro organisms in the root canal. Therefore, total elimination of micro organisms and their byproducts from the root canal system is the key goal of endodontic treatment. 
Enterococcus faecalis is a Gram-positive, Group D streptococci and a facultative anaerobe that occur singly, in pairs and in short chains and are capable of surviving in the harsh environment.  E. faecalis has been found to be the most predominant bacteria in tooth wherein root canal therapy fails, and it is often isolated from persistent cases, including retreatment cases of apical periodontitis.
The ability of E. faecalis to cause periapical disease and chronic failure of an endodontically treated tooth is due to its ability to bind to the collagen of the dentinal tubule and remain viable within the tubule. E. faecalis have been frequently found in obturated root canals exhibiting signs of chronic apical periodontitis, isolated in 23-70% of the positive cultures. ,,
It is also apparent from the dental literatures that E. faecalis is often difficult to eradicate from the root canal system with current intracanal medications.
The largest proportion of the fungal microbiota is made up of Candida species. The genus Candida is comprised of about 150 species, and Candida albicans has been the most commonly detected fungal species in endodontic infections. The incidence of C. albicans in the oral cavity has been reported to be 30-45% in healthy adults. The occurrence of fungi reported in infected root canals varies between 1% and 17%. The main objective of endodontic therapy is to eliminate micro organisms and their products from the root canal space and to prevent the regrowth of residual micro organisms. 
To date not much information is available on the antimicrobial efficacy of Apexit Plus and EndoRez against E. faecalis and C. albicans. This study was designed to compare the antimicrobial efficacy of three root canal sealers: Apexit Plus, Endoflas FS and Endorez against E. faecalis and C. albicans.
| Materials and Methods|| |
In the present study, three root canal sealers were used and they were: Apexit Plus (Ivoclar Vivadent), EndoRez (Ultradent) and Endoflas FS (SANLOR). Standard strains of Enterococcus faecalis MTCC 439 and Candida albicans MTCC 227, obtained from Institute of Microbial Technology (IMTECH), Chandigarh were used in the study.
Method of collection of data
Three root canal sealers were used in this study.
- Group A: Calcium hydroxide based sealer (Apexit Plus)
- Group B: Zinc oxide eugenol-based sealer (Endoflas FS)-control group
- Group C: Resin based sealer (EndoRez).
The study was conducted on 20 Mueller Hinton (MH) agar plates. The number of samples for all three groups were 10 each (n = 10). Three wells (10 mm) were made by removal of agar at equidistant points and filled with root canal sealers after being mixed according to manufacturer's instructions [Figure 1].
|Figure 1: (a) Mueller Hinton (MH) agar plates with three wells (b) Placement of sealers on MH agar plates (c) Group A-(e) Faecalis (d) Group B-(c) Albicans|
Click here to view
The standard strains of bacteria and fungi, E. faecalis MTCC 439 and C. albicans MTCC 227 were used in this study, respectively. Both micro organisms were grown at 37°C for 24 h in MH broth and seeded into MH agar to produce a turbidity of 0.5 on the McFarland scale, which corresponds to a concentration of 10 8 CFU/mL. This MH broth was used as 2 nd layer. The seeded agar was then added over the plates immediately after the insertion of sealer cements.
The plates were maintained at room temperature for 2 h for prediffusion of materials and then incubated at 37°C for 24 h. Aliquots of 10 mL triphenyl tetrazolium chloride gel were added, and the plated were incubated again at 37°C for 30 min. After incubation, the diameter of zones of inhibition around the plates was measured. Two-way ANOVA test was employed for statistical analysis.
| Results|| |
All sealers were tested using the agar diffusion test. After incubation, the diameter of zones of inhibition around the plates was measured, and the sealer, which exhibited the maximum zone of inhibition was considered as having the most efficient antimicrobial activity.
Mean inhibition produced by all the three sealers groups against E. faecalis and C. albicans were given in [Table 1]. Statistical analysis was conducted using the ANOVA test [Table 2] and test of difference were analyzed by Bonferroni method [Table 3] with a value of P < 0.05 being statistically significant at a 95% confidence interval.
The results from the ANOVA test demonstrated that there was a significant difference between all the three sealers tested against E. faecalis and C. albicans (P < 0.001).
Based on the statistical analysis, it was observed that Endoflas FS (control group) always yields a higher inhibition compared with Apexit plus (calcium hydroxide based sealer) and Endorez (resin-based sealer) against both E. faecalis and C. albicans [Table 1] and [Graph 1]. Multiple comparisons test using Bonferroni method were carried out to find the significant difference between all the three sealers and it was evident that that there was a significant difference between Apexit Plus and Endoflas FS (P < 0.001) as well as between Apexit Plus and Endorez (P < 0.001). It was also observed that there was a significant difference between Endoflas FS and Endorez (P < 0.001).
The results of this study showed that highest inhibition was recorded in Endoflas FS sealer followed by Apexit Plus and Endorez, respectively, and the difference between all the three sealers were statistically significant (P < 0.001).
| Discussion|| |
Micro organisms and their byproducts are the most frequent etiologic factors in pulpal and periapical pathology and the failure, during or after the endodontic treatment are linked to the presence of micro organisms in the root canal. Therefore, the main objective of endodontic treatment is the elimination of micro organisms and their byproducts from the root canal system and the prevention of subsequent reinfection. 
To date not much information is available on the antimicrobial efficacy of Apexit Plus and EndoRez against E. faecalis and C. albicans. Therefore, the aim of this study was to evaluate the antimicrobial activity of Apexit Plus, EndoRez and Endoflas FS against E. faecalis and C. albicans using the agar diffusion test.
The agar diffusion test used in this study is one of the most frequently used methods to test the antimicrobial activity of dental materials. The advantages of this method is the creation of direct comparisons of the root canal sealers against test micro organisms and the visual indication of which sealer has the potential to eliminate micro organisms in the local microenvironment of the root canal system. ,, The main drawback of agar diffusion method is that it cannot differentiate bactericidal from bacteriostatic effect of the material and results of this method depend not only on the antimicrobial activity of the test material for the particular micro organisms but it is also highly influenced by the diffusibility of the material across the medium.  Therefore, the size of the inhibition zones does not indicate the absolute antimicrobial effect of sealer.
Enterococcus faecalis is a Gram-positive, Group D streptococci and a facultative anerobe that occur singly, in pairs and in short chains and is capable of surviving in the harsh environment. It is a commonly isolated species in persistent root canal infections. Although rare in primary root canal infections, it can be considered pathogenic, as it is often isolated in pure culture from persistent cases including retreatment cases of apical periodontitis. ,,,
Candida albicans is the fungal species most commonly isolated from infected root canals, and this species has been considered a dentinophilic microorganism because of its invasive affinity to dentin.  In the present study, standard strains E. faecalis MTCC 439 and C. albicans MTCC 227 were used.
Zinc oxide eugenol-based sealers have been traditionally the most commonly employed sealers during root canal treatment. They have served as the benchmark with which other sealers are compared, as it reasonably meets most of the Grossmann's requirements for sealers. The antimicrobial effect of zinc oxide eugenol cement was mainly attributed to the action of eugenol. Eugenol, a phenolic compound acts on micro organisms by protein denaturation whereby the protein becomes nonfunctional. , In order to improve the antimicrobial efficacy of zinc oxide eugenol sealers, known bactericidal agents such as iodoform have been incorporated resulting in modified zinc oxide eugenol-based sealers such as Endoflas FS and Medicated Canal Sealer (MCS). 
Apexit Plus is a recently introduced calcium hydroxide-based material. It contains calcium hydroxide 18.5%, disalicylate 23.8%, hydrated colophonium 27%, bismuth oxide 18.2%, fillers and auxiliary materials 12.5%. Apexit Plus is a two-component material, which sets by complex formation. For this complex formation the three components calcium hydroxide, salicylate, and water are needed. The antimicrobial effect of this sealer stems from the dissociative ability of Apexit Plus sealer into calcium and hydroxyl ions. 
Apexit Plus differs from Apexit in that it is supplied in a more convenient delivery form and has a more hydrophilic formulation.
EndoRez, is a hydrophilic, two-part chemical set material supplied in two dispense mixing and delivery syringe. It is basically a combination of resins, zinc oxide, barium sulfate and pigments in a matrix of urethane dimethacrylate resin.  This study was designed to evaluate the antimicrobial efficacy of EndoRez against E. faecalis and C. albicans, and the results demonstrated that it showed the minimal effect against both the tested micro organisms [Table 1]. The findings of the present study were in accordance with the earlier studies who also demonstrated that EndoRez is not as potent a microbial growth inhibitor.
The results of this study showed statistically significant difference between all the sealers tested and the antimicrobial effect of Endoflas FS was superior to Apexit Plus and EndoRez against both E. faecalis and C. albicans. However, it should be taken into considerations that the data presented here relate to in vitro conditions, and in vivo conditions such as the presence of dentin and serum might modify the antimicrobial efficacy of sealers. Hence, further in vivo studies are needed to evaluate the antimicrobial efficacy of sealers.
| Conclusion|| |
Within the limitations of this study, it can be concluded that:
Root canal sealers showed different inhibitory effects depending on their types, and microbial strains tested. Against E. faecalis and C. albicans, zinc oxide-based sealers showed the highest microbial zones of inhibition followed by the calcium hydroxide-based sealer and resin-based sealer, respectively. The antimicrobial efficacy of Calcium hydroxide-based sealer may be attributed to high pH and this sealer may provide additional benefits such as low toxicity, better biocompatibility, and superior osteogenic potential than that of zinc oxide-eugenol based and resin-based sealers. 
Therefore, in endodontic therapy, root canal sealers should be selected which are characterized by adequate antimicrobial activity, good sealability, and an acceptable biocompatibility. However, the data presented here relate to in vitro conditions, and in vivo conditions such as the presence of dentin, root canal complexities might modify the antimicrobial efficacy of sealers. Hence, further in vivo studies are needed to evaluate the antimicrobial efficacy of sealers.
| References|| |
Grossman LI, Oliet S, Rio CE. Endodontic Practice. 11 th
ed. India: Varghese Publishing House; 1991. p. 234-41.
Smadi L, Mahafzah A, Khraisat A. An in vitro
evaluation of the antimicrobial activity of nine root canal sealers. J Contemp Dent Pract 2008;9:60-7.
Gupte S. Medical Microbiology. 8 th
ed. Jaypee Brothers 4838/24, Ansari Road, Daryaganj, New Delhi, India 110002, 2002; p. 166-72.
Portenier I, Waltimo TMT, Haapsalo M. Enterococcus faecalis
-the rootcanal survivor and star in post treatment disease. Endod Topics 2003;6:135-59.
Evans M, Davies JK, Sundqvist G, Figdor D. Mechanisms involved in the resistance of Enterococcus faecalis
to calcium hydroxide. Int Endod J 2002;35:221-8.
Murray BE. The life and times of the Enterococcus
. Clin Microbiol Rev 1990;3:46-65.
Siqueira JF Jr, Sen BH. Fungi in endodontic infections. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:632-41.
Ribaric SP, Brekalo I, Maza A, Miletic I, Ivica A, Ferreri S. Antibacterial effects of various root filling materials. Acta Stomatol Croat 2000;34:55-7.
Mirzana V, Branca R. Antimicrobial activity of root canal sealers. Stom Glass 2006;53:104-12.
Pinheiro CR, Guinesi AS, Pizzolitto AC, Bonetti-Filho I. In vitro
antimicrobial activity of Acroseal, Polifil and Epiphany against Enterococcus faecalis
. Braz Dent J 2009;20:107-11.
Saleh IM, Ruyter IE, Haapasalo M, Orstavik 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.
Sipert CR, Hussne RP, Nishiyama CK, Torres SA. In vitro antimicrobial activity of Fill Canal, Sealapex, Mineral trioxide aggregate, Portland cement and Endorez. Int Endod J 2005;38:539-43.
Leonardo MR, da Silva LA, Tanomaru Filho M, Bonifácio KC, Ito IY. In vitro
evaluation of antimicrobial activity of sealers and pastes used in endodontics. J Endod 2000;26:391-4.
Portenier I, Waltimo T, Orstavik D, Haapasalo M. The susceptibility of starved, stationary phase, and growing cells of Enterococcus faecalis
to endodontic medicaments. J Endod 2005;31:380-6.
Pirani C, Bertacci A, Cavrini F, Foschi F, Acquaviva GL, Prati C, et al.
Recovery of Enterococcus faecalis
in root canal lumen of patients with primary and secondary endodontic lesions. New Microbiol 2008;31:235-40.
Waltimo TMT, Haapsalo M, Zehnder M, Meyer J. Clinical aspects related toendodontic yeast infections. Endod Topics 2004;9:66-78.
Gopikrishna AV, Kandaswamy D, Rajan KJ. Comparative evaluation of theantimicrobial efficacy of five endodontic root canal sealers against Enterococcus faecalis
and Candida albicans
. J Conserv Dent 2006;9:2-11.
Markowitz K, Moynihan M, Liu M, Kim S. Biologic properties of eugenol and zinc oxide-eugenol. A clinically oriented review. Oral Surg Oral Med Oral Pathol 1992;73:729-37.
Kayaoglu G, Erten H, Alaçam T, Orstavik D. Short-term antibacterial activity of root canal sealers towards Enterococcus faecalis
. Int Endod J 2005;38:483-8.
Mickel AK, Nguyen TH, Chogle S. Antimicrobial activity of endodontic sealers on Enterococcus faecalis
. J Endod 2003;29: 257-8.
[Table 1], [Table 2], [Table 3]