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Table of Contents   
ORIGINAL ARTICLE  
Year : 2014  |  Volume : 17  |  Issue : 3  |  Page : 276-279
Evaluation of antifungal activity of white-colored mineral trioxide aggregate on different strains of Candida albicans in vitro


1 Department of Conservative Dentistry and Endodontics, Vananchal Dental College and Hospital, Garhwa, Jharkhand, India
2 Department of Oral and Maxillofacial Surgery, Vananchal Dental College and Hospital, Garhwa, Jharkhand, India
3 Department of Conservative Dentistry and Endodontics, Shri Dharmasthala Manjunatheshwara College of Dental Sciences and Hospital, Dharwad, Karnataka, India

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Date of Submission21-Dec-2013
Date of Decision28-Feb-2014
Date of Acceptance01-Feb-2014
Date of Web Publication2-May-2014
 

   Abstract 

Aim: The purpose of this study was to evaluate the antifungal action of various concentrations of white mineral trioxide aggregate (MTA) against seven different strains of Candida albicans using the tube dilution test.
Materials and Methods: Fresh mix of MTA was prepared at concentrations of 100, 50, 25, and 12.5 mg/ml and added to a broth tube containing Sabouraud's liquid medium. A total of 1287 broth tubes were prepared and divided into experimental and control groups. Stock cultures of seven strains of C. albicans were obtained. Fresh inoculate of the microorganism was prepared by growing overnight cultures. Aliquots of the test C. albicans were taken and added to the test tubes. All tubes were incubated at 37°C for 1-, 24-, 72-, and 168-h time periods. At each time period, the presence of C. albicans colonies was assessed.
Statistical analysis used: Differences among the groups were statistically analyzed using Kruskal-Wallis and Mann-Whitney U tests.
Results: Results showed that one strain showed resistance even after 3 days at the lower MTA concentrations of 12.5 and 25 mg/ml. Growth reoccurred with three strains at MTA concentration of 12.5 mg/ml after 7 days. A significant difference was found between strain 3 and other strains at MTA concentrations of 12.5 and 25 mg/ml at the 3-days time period and between tubes containing 12.5 mg/ml and tubes containing higher concentrations of MTA at the 7-days time period.
Conclusion: White MTA in concentrations of 100 and 50 mg/ml is effective in inhibiting the seven tested strains of C. albicans for periods up to 1 week.

Keywords: Candida albicans; mineral trioxide aggregate; tube dilution

How to cite this article:
Bhardwaj A, Bhardwaj A, Rao N. Evaluation of antifungal activity of white-colored mineral trioxide aggregate on different strains of Candida albicans in vitro. J Conserv Dent 2014;17:276-9

How to cite this URL:
Bhardwaj A, Bhardwaj A, Rao N. Evaluation of antifungal activity of white-colored mineral trioxide aggregate on different strains of Candida albicans in vitro. J Conserv Dent [serial online] 2014 [cited 2019 Jul 22];17:276-9. Available from: http://www.jcd.org.in/text.asp?2014/17/3/276/131799

   Introduction Top


Microorganisms play a key role in the development of pulpal and periapical disease. [1],[2] The role of fungi in endodontic disease is also well documented. [3],[4] Persistence of virulent microorganisms and their byproducts in the root canal system, or the surrounding tissues, following initial endodontic treatment, is the main cause for treatment failure. [5]

It has been reported that failing root canal treatments associated with periradicular pathosis frequently demonstrated fungi colonization. [4],[6] The most commonly recovered fungi were Candida albicans. These microorganisms showed an ability to colonize canal walls and invade dentinal tubules. [4],[7]

The exact factors affecting the colonization of the root canal by fungi are not completely understood. It seems that the most common predisposing factors are certain intracanal medicaments, local and systemic antibiotics, [8] and previous unsuccessful endodontic treatment. [5],[9] It has been hypothesized that the reduction of specific strains of bacteria in the root canal during endodontic treatment may allow fungi overgrowth in the low nutrient environment. [5],[9] In addition, it is possible that fungi, such as C. albicans, may gain access to the root canal from the oral cavity as a result of poor asepsis during endodontic treatment procedures or because of coronal leakage. In fact, the presence of C. albicans in root canals was found to be directly associated with its presence in the oral cavity of the same patients. [3] Multiple unrelated strains of C. albicans may be harbored in the same individual. [3],[7]

Failure of initial endodontic treatment can often be treated successfully by retreatment or endodontic surgery. Elimination of the microbial flora and infected tissues as well as a complete seal of the root canal system, to prevent future recontamination, will enhance treatment success. In this regard, mineral trioxide aggregate (MTA) has become a popular material to seal off communications between the root canal system and the external surface of the root. It has been mostly used as a retrograde filling material during apical surgery and as a sealant of root perforations. [10],[11] However, MTA is difficult to use because of its longer setting time and poor handling properties. [10],[12]

MTA (ProRoot MTA, Dentsply/Tulsa Dental, Tulsa, OK) is marketed in gray-colored and white-colored preparations; both are 75% Portland cement, 20% bismuth oxide, and 5% gypsum by weight. Recently, the use of the white-colored preparation became more popular because of esthetic considerations. MTA is a powder that consists of fine hydrophilic particles that in the presence of water, or moisture, forms a colloidal gel that solidifies to form hard cement within approximately 4 h. The principal constituents of the gray-colored formula are tricalcium oxide, tricalcium silicate, bismuth oxide, dicalcium silicate, tricalcium aluminate, tetracalcium aluminoferrite, and calcium sulfate dihydrate. The more esthetic white-colored preparation lacks the tetracalcium aluminoferrite. [10],[12]

It has been reported that a fixed concentration of white-colored MTA was effective against C. albicans in vitro for periods of up to 3 days. [13] It has also been shown that white-colored MTA in concentration of 50 mg/ml was effective in killing C. albicans for periods of up to 3 days, whereas lower concentrations were not as effective. [14] In another study, fungal growth occurred during 1-h incubation in both freshly mixed and 24-h set gray and white MTA cements, whereas by increasing the incubation time, no fungal growth was observed in 24 and 72 h. [15] It was also reported that both gray-colored and white-colored MTA in concentrations of 50 mg/ml and 25 mg/ml were effective in killing C. albicans for periods up to 1 week. [16] Lower concentrations of gray-colored MTA might still be effective, whereas lower concentrations of white-colored MTA might not. [16] However, the antifungal effect of MTA on multiple strains of C. albicans over longer periods of time is less investigated.

The purpose of this study was to evaluate the antifungal action of various concentrations of white-colored MTA against seven different C. albicans strains for various exposure periods using the tube dilution test.


   Materials and methods Top


Fresh mix of white-colored MTA (Pro-Root MTA, batch # 05002015, Dentsply/Tulsa Dental, Tulsa, OK) was prepared at concentrations of 100, 50, 25, and 12.5 mg/ml by dilution with distilled water. Each freshly prepared mix was added to a broth tube containing Sabouraud's liquid medium (Hi-Media Laboratories, Mumbai, India). A total of 1287 broth tubes were prepared and divided into seven sets with experimental groups of 11 tubes each and control groups. Broth tubes without MTA served as positive control and tubes without C. albicans served as negative control.

The antifungal action of MTA against C. albicans was evaluated by using the tube dilution test. [17] Stock cultures of six strains of C. albicans clinically isolated from the oral cavity (strains 1-6: C-89, E-175, C-304, E-259, C-176, and E-170) and a standard strain (strain 7: Microbial type culture collection (MTCC) 3017) were obtained. Fresh inoculate of the microorganism was prepared by growing overnight cultures until a complete suspension of growth was achieved. The cultures were diluted in a pre-warmed broth to achieve a final density of 10 4 colony-forming unit (CFU)/spot.

One-milliliter aliquots of the test C. albicans were taken from the broth culture and added to sterile capped test tubes of each experimental and positive control group. All experimental and control groups were then incubated at 37°C and evaluated at 1-, 24-, 72-, and 168-h (7 days) time periods. At each time period, 0.1 ml of samples from each tube was subcultured on Sabouraud's dextrose agar plates (Hi-Media Laboratories, Mumbai, India). Any growth appearing was smeared and stained to confirm presence of C. albicans.

Differences among the groups were statistically analyzed using Kruskal-Wallis and Mann-Whitney U tests (Statistical Package for the Social Sciences (SPSS) 7 software). If the calculated critical values were at least as large as the respective critical values at the 0.05 level, then calculations were considered statistically significant in this study.


   Results Top


Generally, a direct correlation was found between MTA concentrations and its inhibition effect on C. albicans growth. Differing behavior patterns of various strains of C. albicans were also seen [Table 1]. Strains 1 and 2 were inhibited at all concentrations during all the time periods. Strains 5, 6, and 7 displayed intermediate resistance to MTA, whereas strains 3 and 4 appeared to be most resistant. The negative controls showed no fungal growth at any time period, whereas the positive controls always showed fungal growth.
Table 1: Effect of various MTA concentrations on growth of C. albicans strains

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Statistically, a significant difference was found between tubes containing either strain 3 or 4 and tubes containing other strains at all MTA concentrations at the 1-day time period (P < 0.001). A significant difference was found between tubes containing strain 3 and tubes containing other strains at MTA concentrations of 12.5 and 25 mg/ml at the 3-days time period (P < 0.001). A significant difference was also found between tubes containing 12.5 mg/ml and tubes containing higher concentrations of MTA at the 7-days time period (P < 0.001).


   Discussion Top


The method used in the present study was the tube dilution test, which is an effective method to evaluate the antifungal and antibacterial properties of any filling material or solution. [18] This method allows direct contact between fungal cells and MTA. It was considered appropriate for evaluating the antifungal activity of MTA, which has a low solubility and diffusibility. [19]

C. albicans was the test organism in this study. It is a frequent colonizer of the oral cavity, and multiple unrelated strains showing varying pathogenicity may be present in the same individual. [3],[7] Mechanisms of pathogenicity of different strains may include the power of adaptability to a variety of environmental conditions attributable to the switching of gene expression dictated by environmental changes and evasion and immunomodulation of the host defenses by different mechanisms. [4] C. albicans is frequently associated with failing root canal treatments and has the ability to form biofilms on different surfaces, which makes it more pathogenic than species that are less able to form biofilms. [4] As it may be involved in cases of persistent and secondary infections, usually present during apical surgery and perforation repair procedures, the antifungal action of the repair material may further assist in the management of such infections and enhance tissue healing.

In this study, growth of seven strains of C. albicans in four concentrations of MTA was assessed for periods of up to 7 days. A direct correlation was found between the MTA concentration and antifungal activity. However, variations were seen between different strains in terms of susceptibility to MTA. The results showed that 3-days exposure was sufficient to eradicate most organisms. Only one strain showed relative resistance even after 3 days at the lower MTA concentration of 12.5 and 25 mg/ml. Recurrence of fungal growth was seen with three strains at MTA concentration of 12.5 mg/ml after 7 days. It might indicate that MTA only exhibits a fungistatic action at lower concentrations, whereas a fungicidal action may be expected at higher concentrations. These results also point toward the strain variations within a single species. This highlights the significance of using seven strains in the present study.

In a previous study, Al-Nazhan and Al-Judai [13] reported white-colored MTA to be effective in killing C. albicans in vitro for a period of up to 3 days. However, in that study a higher concentration of MTA was used. In another in vitro study, Al-Hezaimi et al., [14] assessed the antifungal action of different concentrations of white-colored MTA against C. albicans in vitro. White-colored MTA in concentration of 50 mg/ml was effective in inhibiting C. albicans for periods of up to 3 days, whereas lower concentrations were not effective. Mohammadi et al., [15] observed fungal growth during 1-h incubation in both freshly mixed and 24-h set gray and white MTA cements, whereas by increasing the incubation time, no fungal growth was observed in 24 and 72 h. Another study also showed that both white and gray MTA in concentrations of 50 and 25 mg/ml were effective in inhibiting C. albicans for periods of up to 7 days. [16] Kangarlou et al., [20] assessed the antifungal activity of Proroot MTA and MTA-Angelus and concluded that MTA-angelus was a more effective antifungal agent compared with ProRoot MTA at concentrations of 50 and 100 mg/ml.

Regarding the antifungal action of MTA, it has been shown that the mechanism of action of MTA involves the dissolution of calcium oxide, which increases pH by releasing Ca +2 and OH . [21],[22] The high pH increases the permeability of cell membranes with leakage of intracellular components. [13] Therefore, MTA and calcium hydroxide seem to possess similar antimicrobial action. Several studies have evaluated the susceptibility of C. albicans to calcium hydroxide. Waltimo et al., [23] reported that C. albicans survived incubation in calcium hydroxide solution for 1 and 6 h and was killed after 6 h of incubation. Ferguson et al., [24] found that an aqueous solution of calcium hydroxide had no activity against C. albicans. However, when maintained in direct contact with C. albicans cells, calcium hydroxide paste was very effective in killing this fungus. [24] Therefore, it can be assumed that MTA is more effective in killing C. albicans when placed in direct contact with the fungus. In addition, Samiei et al., reported that adding silver nanoparticles to MTA improved its antimicrobial efficacy. [25]

In conclusion, it appears that under the conditions of this study, white-colored MTA in concentrations of 100 and 50 mg/ml is effective in inhibiting the seven tested strains of C. albicans for periods up to 1 week. Lower concentrations may not be effective. However, the antifungal activity of white-colored and gray-colored MTA over longer periods of time on more number of strains merits further investigation.

 
   References Top

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Correspondence Address:
Archana Bhardwaj
Abhirachna Dental Trauma and Implant Center, GA-4, City Center, Sector 4, Bokaro Steel City - 827 004, Jharkhand
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-0707.131799

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