Antimicrobial efficacy of chlorine dioxide against Candida albicans in stationary and starvation phases in human root canal: An in-vitro study
| Abstract |
Introduction: Candida albicans (C. albicans) is the most commonly isolated fungal pathogen from dental root canal. C. albicans forms biofilm and develops resistance against root canal irrigants . This study determines the fungicidal efficacy of 13.8% chlorine dioxide in extracted human teeth at stationary and starvation phases of C. albicans. Materials and Methods: Teeth were decoronated and coronal portion of the roots were prepared into blocks, which were incubated at 37°C with C. albicans for five days. The samples were treated with chlorine dioxide for 12 and 20 minutes. Total of fifty blocks were taken in the study. Colony-forming units were counted in Sabourauds dextrose agar and scanning electron microscopic observation was done. Data were analyzed by one-way ANOVA and Bonferoni's post hoc test. Results: Teeth at stationary phase (12 min) showed mean colony count of 28,000 ± 1814 which is significantly (P < 0.001) less than control group. Teeth at starvation phase (12 min) showed colony count of 65,600 ± 1912 which is also significantly (P < 0.001) less than control group. Teeth irrigated at stationary phase (20 min) showed mean colony count of 23,400 ± 1776 (P < 0.001). Teeth irrigated at starvation phase (20 min) showed mean colony count of 48,100 ± 1663 which is also significantly (P < 0.001) less than that of control group. Conclusion: Treatment of chlorine dioxide reduces the C. albicans count in root canals of extracted human teeth at stationary and starvation phases. Efficacy of chlorine dioxide against C. albicans is relatively higher in stationary phase than that of starvation phase.
Keywords: Candida, chlorine dioxide activity, root canal
| Introduction |  |
The most common fungal pathogen isolated from the oral cavity is C. albicans. [1] They exist in two major forms systemically; oval and pseudohyphal cells. [2] Vacuolation of mother yeast cells and budding yeast has also been observed. Yeast cells pass through various phases of growth, undergo phenotypic changes and confer resistance to various agents. [3] Investigations have revealed that yeasts can be isolated from samples taken from apical periodontitis. Yeasts in the canal space are reported to represent a reservoir for the dissemination to the periphery via blood stream. [5] Candida binds to a variety of host cell receptors through lectin like or protein-protein interaction, including galactosyl or fucosyl receptors, also to fibronectin, laminin and collagen. [6],[7],[8] C. albicans grows and survives by competing with other species of bacteria. [9] Prevalence of C. albicans within the root canal space has been reported to be 55%, whereas another study reported Candida prevalence of 11.36% of teeth with pulpal lesions. [10],[11]
Amphotericin- B has been the primary antifungal agent used to treat systemic invasive mycosis. [12] Root canal irrigants are used in conjunction with mechanical preparation as an antimicrobial agent to flush out loose debris, to lubricate dentinal wall and to dissolve organic compounds. It was shown that 1% sodium hypochlorite was effective against C. albicans.[13] The physiological state of bacteria plays a major role in the outcome of antimicrobial treatment. It was shown that biofilms produced by E. faecalis in starvation phase is most resistant to sodium hypochlorite compared to that of exponential and stationary phases. [14] Chlorine dioxide oxidizes and kills the bacterium by altering the permeability of nutrients across the cell membrane. [15] Tissue dissolving capacity of chlorine dioxide has been shown to be equally effective to that of sodium hypochlorite. [16] Chlorine dioxide makes a likely substitute for sodium hypochlorite because it is less toxic. Chlorine dioxide does not hydrolyze to form HCl as chlorine does, but remains as true gas dissolved in solution. [16] It has been used as endodontic irrigant because of its bacteriocidal effect and compatibility with living tissue. [16] The aim of this study was to check the efficacy of 13.8% chlorine dioxide against C. albicans in stationary and starvation phases
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Treatment with chlorine dioxide
Following the contamination with candida, twenty blocks each from stationary and starvation phase groups were taken and subjected to treatment of chlorine dioxide for twelve (10 blocks) and twenty (10 blocks) minutes (Chlorine dioxide 13.8%, Frontier Pharmaceuticals, U.S.A). After the time duration, the blocks were washed with phosphate buffer saline and dentinal shavings were collected from the canal portion of teeth. The four blocks from each group (2 each from 12 and 20 minute subgroups) were prepared for scanning electron microscopic observation.
Colony-forming units (CFU) of viable cells were determined on sabouraud dextrose agar plates in triplicates of 1:10 dilution (phosphate buffer saline). Sabouraud dextrose agar plates were incubated in 5% CO 2 at 37°C in carbon dioxide incubator and the number of fungal colony-forming units was counted at 24hrs. Plates containing 30 to 300 fungal colonies were used preferentially for data analysis.
Tooth preparation for scanning electron microscope
Dentinal blocks were washed with phosphate buffer saline. They were then fixed with 4% glutaraldehyde for 8hrs at 4 to 6°C. Following this, super fixation was done with 1% osmium tetroxide for 4hrs at 5°C. Blocks were then dehydrated with ascending concentrations of ethanol (30%, 50%, 70%, 90%, 100%). Critical point drying was done by keeping the preparations in dryer. Preparations were processed for gold sputtering. Blocks were examined in SEM (Cambridge, England) at 20Kv. Different images of the entire dentinal blocks were captured and recorded. Qualitative analysis and comparison of areas of biofilm formed by different groups were done.
Following the contamination with candida, twenty blocks each from stationary and starvation phase groups were taken and subjected to treatment of chlorine dioxide for twelve (10 blocks) and twenty (10 blocks) minutes (Chlorine dioxide 13.8%, Frontier Pharmaceuticals, U.S.A). After the time duration, the blocks were washed with phosphate buffer saline and dentinal shavings were collected from the canal portion of teeth. The four blocks from each group (2 each from 12 and 20 minute subgroups) were prepared for scanning electron microscopic observation.
Colony-forming units (CFU) of viable cells were determined on sabouraud dextrose agar plates in triplicates of 1:10 dilution (phosphate buffer saline). Sabouraud dextrose agar plates were incubated in 5% CO 2 at 37°C in carbon dioxide incubator and the number of fungal colony-forming units was counted at 24hrs. Plates containing 30 to 300 fungal colonies were used preferentially for data analysis.
Tooth preparation for scanning electron microscope
Dentinal blocks were washed with phosphate buffer saline. They were then fixed with 4% glutaraldehyde for 8hrs at 4 to 6°C. Following this, super fixation was done with 1% osmium tetroxide for 4hrs at 5°C. Blocks were then dehydrated with ascending concentrations of ethanol (30%, 50%, 70%, 90%, 100%). Critical point drying was done by keeping the preparations in dryer. Preparations were processed for gold sputtering. Blocks were examined in SEM (Cambridge, England) at 20Kv. Different images of the entire dentinal blocks were captured and recorded. Qualitative analysis and comparison of areas of biofilm formed by different groups were done.
| Results | |
Optical density measurement
Optical density reading (OD620) of C. albicans grown in different media showed increase in fungal growth on day one and remained constant for four days and started declining during eighth day. Maximum growth was observed in BHI medium followed by peptone water and normal saline [Figure 1].
 | Figure 1: Optical density reading (OD620) of C. albicans grown in different media (NS-Normal saline, BHI: Brain heart infusion, PW: Peptone water). Graph representing increase in bacterial growth on day one remained constant for four days and declined during eighth day. Maximum growth is observed in BHI medium followed by PW and NW Click here to view |
Colony forming units
In the present study, teeth of all the groups were inoculated with about 10 5 (1,00,123) of C. albicans initially and is taken as initial count of C albicans.
After the treatment, the number of C albicans colony-forming units recovered was represented in the form of colony counts (numerical form) in all test samples.
12 minute treatment
Teeth treated with normal saline for 12 minutes showed mean C. albicans colony count of 99.7 × 10 3 , which is considered as control. Teeth irrigated with chlorine dioxide during stationary phase showed mean C. albicans colony counts of 28 × 10 3 which is significantly (71.91%, P < 0.001) less when compared to that of control group. Teeth irrigated with chlorine dioxide during starvation phase showed mean C. albicans colony count of 65.6 × 10 3 which is also significantly (34.2%, P < 0.001) less when compared to the control group. When mean colony count of C. albicans at stationary phase was compared with that of starvation phases, the stationary phase showed significantly (P < 0.001) lesser amount of C. albicans colonies [Figure 2].
20 minute treatment
In the present study, teeth of all the groups were inoculated with about 10 5 (1,00,123) of C. albicans initially and is taken as initial count of C albicans.
After the treatment, the number of C albicans colony-forming units recovered was represented in the form of colony counts (numerical form) in all test samples.
12 minute treatment
Teeth treated with normal saline for 12 minutes showed mean C. albicans colony count of 99.7 × 10 3 , which is considered as control. Teeth irrigated with chlorine dioxide during stationary phase showed mean C. albicans colony counts of 28 × 10 3 which is significantly (71.91%, P < 0.001) less when compared to that of control group. Teeth irrigated with chlorine dioxide during starvation phase showed mean C. albicans colony count of 65.6 × 10 3 which is also significantly (34.2%, P < 0.001) less when compared to the control group. When mean colony count of C. albicans at stationary phase was compared with that of starvation phases, the stationary phase showed significantly (P < 0.001) lesser amount of C. albicans colonies [Figure 2].
 | Figure 2: C albicans count after irrigating with normal saline (Control) and chlorine dioxide for 12 minutes at stationary and starvation phases. Colony count at stationary phase is significantly (***P<0.001) less than that of control, starvation phase. Similarly, there is significant (###P<0.001) decrease in C albicans colony count at starvation phase when compared to that of control Click here to view |
20 minute treatment
Teeth treated with normal saline for 20 minutes showed mean C albicans colony count of 99.68 × 10 3 which is considered as control. Teeth irrigated with chlorine dioxide during stationary phase showed mean C. albicans colony count of 23.4 × 10 3 which is significantly (76.5%, P < 0.001) less when compared to that of control group. Teeth irrigated with chlorine dioxide during starvation phase showed mean C. albicans colony count of 48.1 × 10 3 which is also significantly (51.7%, P < 0.001) less when compared to the control group. When mean colony count of C. albicans at stationary phase was compared with that of starvation phase, the stationary phase showed significantly (P < 0.001) lesser amount of C. albicans colonies [Figure 3].
 | Figure 3: C albicans count after irrigating with normal saline (Control) and chlorine dioxide for 20 minutes at stationary and starvation phases. Colony count at stationary phase is significantly (***P<0.001) less when compared to that of control, starvation phase. Similarly, there is significant (###P<0.001) decrease in C albicans colony count at starvation phase when compared to that of control Click here to view |
Scanning electron microscopic observation
Scanning electron microscopic photomicrographs of two samples from each group were taken. An untreated sample was also taken and observed for C. albicans biofilm formation. In the samples irrigated with the chlorine dioxide at stationary and starvation phases, a qualitative assessment of areas of biofilm left behind after the irrigation was carried out. Areas of biofilm were compared between different groups. Chlorine dioxide treated samples showed minimal areas of biofilm in both stationary and starvation phases at the end of 12 minutes and 20 minutes [Figure 4] and [Figure 5].
Scanning electron microscopic photomicrographs of two samples from each group were taken. An untreated sample was also taken and observed for C. albicans biofilm formation. In the samples irrigated with the chlorine dioxide at stationary and starvation phases, a qualitative assessment of areas of biofilm left behind after the irrigation was carried out. Areas of biofilm were compared between different groups. Chlorine dioxide treated samples showed minimal areas of biofilm in both stationary and starvation phases at the end of 12 minutes and 20 minutes [Figure 4] and [Figure 5].
 | Figure 4: (a) Representative scanning electron photomicrograph showing candidal colonization prior to treatment with chlorine dioxide. (b and c) are representative scanning electron photomicrographs of candidal biofilm remaining after treatment with chlorine dioxide for 12minutes in stationary and starvation phases respectively. Encircled area shows candidal cells and arrow indicates the eroded biofilm area after treatment in stationary phase Click here to view |
 | Figure 5: (a) Representative scanning electron photomicrograph showing candidal colonization prior to treatment with chlorine dioxide. b and c are representative scanning electron photomicrographs of biofilm remaining after treatment with chlorine dioxide for 20 minutes in stationary and starvation phases respectively. Arrows indicate candidal cells (CC) and psuedohyphal cells (PH) in stationary phase. Encircled area shows eroded surface of biofilm after treatment with chlorine dioxide in stationary phase. In picture C, arrow indicates irregular surface of biofilm after treatment with chlorine dioxide for 20 minutes at starvation phase Click here to view |
| Discussion | |
The main purpose of endodontic treatment is to reduce microorganisms and other species in root canal system. Calcium hydroxide is one of the commonly used intra-canal medicaments. However, Brandle et al., have reported that C. albicans is resistant to calcium hydroxide. [17] Another commonly used irrigant is sodium hypochlorite. Though it is proved to be effective in eradicating biofilms, it is reported to produce carcinogens like trihalo-methanes. [16] Hence, there is a need of an alternative irrigant which is effective as well as less toxic. In the present study, candidal cells in stationary and starvation phases were significantly reduced though they were not completely eliminated by treatment with chlorine dioxide.
Regarding the growth phases of C. albicans, there are differences among the authors in determining its stationary and starvation phases. Some studies have shown that stationary phase of C. albicans starts between 3 rd and 8 th day. [18],[19] Masuoka and Hazen in their study showed that C. albicans enters stationary phase in twenty four to forty eight hours. [20] In our study, the teeth along with C. albicans were incubated for five days to allow the candida to enter into the stationary phase. The stationary and starvation are the two vital physiological phases during the growth of the C albicans. It is reported that C. albicans in these phases harbour properties like better adherence, virulence and elevated drug resistance. [21] In addition, in stationary phase, C. albicans has the ability to generate true hyphal cells which are clinically important. [22]
The present study revealed that treatment with chlorine dioxide has more effectively reduced number of candidal cells in stationary phase when compared to that of starvation phase. This is in agreement with the previous studies and may be due to release of catabolite repression in starvation phase. [14] It has been shown that C albicans produces biofilms of lesser amount in starvation phase when compared to stationary phase. [23] However, biofilms which are formed in starvation phase are relatively more resistant to the irrigants than the stationary phase. [24] Efficacy of chlorine dioxide to reduce the C albicans count significantly is due to its ability to alter the cell membrane integrity of C. albicans and to cause leakage of potassium and adenosine triphosphate, thereby damaging the candidal cells. [15]
Fungi within the root canal system have been shown to be the cause of failure in root canal treated teeth with periapical lesion. The failure of chlorine dioxide to eradicate entire cell population of C. albicans may be due to their adhesion to dentin and penetration deep inside dentinal tubules. [1],[25] Other reason for this could be the formation of biofilms. [26],[27] Adherence, hyphal formation, thigmotropism, protease secretion and phenotypic switching were the most reported virulence factors of C. albicans.[28] C albicans in its yeast form in apical periodontitis has been shown to be the cause of failure of root canal treated teeth. [29] However, Ning et al., have reported that the biofilm formation in starvation phase is less when compared to that of stationary phase. [23]
Prolonged irrigation with chlorine dioxide combined with intracanal medication might help to eliminate candidal cells in stationary and starvation phases in root canal environment. Further studies have to be conducted to study the effect of chlorine dioxide for longer duration and to determine whether it can more effectively be used synergistically with other irrigants.
Prolonged irrigation with chlorine dioxide combined with intracanal medication might help to eliminate candidal cells in stationary and starvation phases in root canal environment. Further studies have to be conducted to study the effect of chlorine dioxide for longer duration and to determine whether it can more effectively be used synergistically with other irrigants.
| Conclusion | |
Treatment of chlorine dioxide reduces the C. albicans count in root canals of extracted human teeth at stationary and starvation phases. Efficacy of chlorine dioxide against C. albicans is relatively higher in stationary phase than that of starvation phase.
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