|Year : 2014 | Volume
| Issue : 1 | Page : 1-6
Antimicrobial efficacy of chlorine dioxide against Candida albicans in stationary and starvation phases in human root canal: An in-vitro study
Shirur Krishnaraj Somayaji1, Shobha Karabylu Lakshminarayana2, Mohandas Rao Kappettu Gadahad3
1 Department of Conservative Dentistry and Endodontics, Manipal College of Dental Sciences, Manipal, Karnataka, India
2 Department of Microbiology, Manipal University, Manipal, Karnataka, India
3 Department of Anatomy, Melaka Manipal Medical College, Manipal University, Manipal, Karnataka, India
|Date of Web Publication||20-Mar-2014|
Shirur Krishnaraj Somayaji
Department of Conservative Dentistry and Endodontics, Manipal College of Dental Sciences, Manipal - 576 104, Karnataka
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
|How to cite this article:|
Somayaji SK, Lakshminarayana SK, Gadahad MK. Antimicrobial efficacy of chlorine dioxide against Candida albicans in stationary and starvation phases in human root canal: An in-vitro study. Sahel Med J 2014;17:1-6
|How to cite this URL:|
Somayaji SK, Lakshminarayana SK, Gadahad MK. Antimicrobial efficacy of chlorine dioxide against Candida albicans in stationary and starvation phases in human root canal: An in-vitro study. Sahel Med J [serial online] 2014 [cited 2019 Jan 16];17:1-6. Available from: http://www.smjonline.org/text.asp?2014/17/1/1/129144
| Introduction|| |
The most common fungal pathogen isolated from the oral cavity is C. albicans.  They exist in two major forms systemically; oval and pseudohyphal cells.  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.  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.  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. ,, C. albicans grows and survives by competing with other species of bacteria.  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. ,
Amphotericin- B has been the primary antifungal agent used to treat systemic invasive mycosis.  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. 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.  Chlorine dioxide oxidizes and kills the bacterium by altering the permeability of nutrients across the cell membrane.  Tissue dissolving capacity of chlorine dioxide has been shown to be equally effective to that of sodium hypochlorite.  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.  It has been used as endodontic irrigant because of its bacteriocidal effect and compatibility with living tissue.  The aim of this study was to check the efficacy of 13.8% chlorine dioxide against C. albicans in stationary and starvation phases.
| Materials and Methods|| |
Dentinal block preparation
Forty extracted single rooted human mandibular premolars were collected from the dental clinics of Manipal College of Dental Sciences. Teeth were cleaned ultrasonically and were then decoronated at cemento-enamel junction with the help of diamond disc. Two longitudinal grooves were made on the roots of teeth and split opened. Coronal portion of the roots were then prepared into blocks each of 4 × 4 × 1 mm size. The blocks were then irrigated with ethylene diamine tetra acetic acid (EDTA) for 1 min to remove the smear layer produced during the procedure. The blocks were autoclaved at 121°C for 15 min. Total of sixty such blocks were randomly selected and at least one block was taken from each tooth for the study.
Contamination with Candia albicans
0Twenty blocks were used for the study under stationary phase (10 blocks under 12 and 20 minutes each) using nutrient rich brain heart infusion (BHI) broth as medium. Each dentinal block was kept in the shaker along with 1 ml BHI broth suspended in eppendorf tube of 2cm size for twelve hours and incubated at 37°C for five days. The first twenty blocks grouped as stationary phase were kept in the shaker 100 rotations/min (MTS 2, IKA, Staufen, Germany) along with BHI broth in eppendorf tube. American type cell culture (ATCC) strain of C. albicans 10231was used in the present study. C. albicans was innoculated into the eppendorf tube containing the blocks. It was adjusted to contain 1 × 10 5 CFU/ml -1 (about 1,00,000 colonies) corresponding to 0.5 McFerlands tube.
The next twenty blocks were used for the study of starvation phase (10 blocks under 12 and 20 minutes each) using nutrient deprived peptone water. C. albicans was innoculated into the eppendorf tube containing the blocks. It was adjusted to contain 1 × 10 5 CFU/ml -1 (about 1,00,000 colonies) corresponding to 0.5 McFerlands tube. For inducing starvation phase, the cells were initially grown in BHI medium for 24 hours and later were washed with phosphate buffered saline, then resuspended in peptone water for five days.
The remaining 20 blocks were kept as control in BHI medium containing C. albicans. They were treated with normal saline (10 blocks for 12 minutes and 10 blocks for 20 minutes).
Optical density measurement
Optical density is the optical thickness which measures the total light blocking power of a certain medium with certain thickness. The effect of drugs against bacterial multiplication can be measured by optical density. If the optical density increases, the drug is not effective. The optical density of chlorine dioxide against candida at stationary and starvation phase was measured at 620nm using photospectometer (Bio-Rad laboratories India). The chlorine dioxide was in direct contact with C. albicans in different media like BHI, peptone water and normal saline. Growth of C. albicans in different media was recorded.
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.
Statistical analyses were made using statistical software Graph Pad Prism version 3.00, Graph Pad Software, Inc. San Diego CA. One-way ANOVA was applied to analyze the data and Bonferoni's post hoc test was used to see the significance between different groups.
The study was approved by Institutional Ethical Committee of Manipal University.
| 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|
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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].
|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|
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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|
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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].
|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|
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|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|
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| 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.  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.  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. , Masuoka and Hazen in their study showed that C. albicans enters stationary phase in twenty four to forty eight hours.  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.  In addition, in stationary phase, C. albicans has the ability to generate true hyphal cells which are clinically important. 
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.  It has been shown that C albicans produces biofilms of lesser amount in starvation phase when compared to stationary phase.  However, biofilms which are formed in starvation phase are relatively more resistant to the irrigants than the stationary phase.  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. 
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. , Other reason for this could be the formation of biofilms. , Adherence, hyphal formation, thigmotropism, protease secretion and phenotypic switching were the most reported virulence factors of C. albicans. C albicans in its yeast form in apical periodontitis has been shown to be the cause of failure of root canal treated teeth.  However, Ning et al., have reported that the biofilm formation in starvation phase is less when compared to that of stationary phase. 
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.
| References|| |
|1.||Sen BH, Safavi KE, Spanberg LS. Growth patterns of C. albicans in relation to radicular dentin. Oral Surg Oral Med Oral Pathol 1997;84:68-73. |
|2.||Odds FC. Pathogenesis of candida infections. J Am Acad Dermatol 1994;31:2-5. |
|3.||Beggs WH. Growth phase in relation to ketaconazole and miconazole susceptibilities of Candida albicans. Antimicrob Agents Chemother 1984;25:316-8. |
|4.||Sen BH, Piskin B, Demirci T. Observation of bacteria and fungi in infected root canals and dentinal tubules by SEM. Endod Dent Traumatol 1995;11:6-9. |
|5.||Debelian GJ, Olsen I, Tronstad L. Bacteremia in conjunction with endodontic therapy. Endod. Dent Traumatol 1995;11:142-9. |
|6.||Cannon RD, Chaffin WL. Oral colonization by Candida albicans. Crit Rev Oral Biol Med 1999;10:359-83. |
|7.||Tosh FD, Douglas LJ. Characterization of a fucoside-binding adhesin of Candida albicans. Infect Immun 1992;60:4734-9. |
|8.||Jimenez-Lucho V, Ginsburg V, Krivan HC. Cryptococcus neoformans, Candida albicans, and other fungi bind specifically to the glycosphingolipidlactosylceramide (Gal βl-4Glc β1-1Cer), a possible adhesion receptor for yeasts. Infect Immun 1990;58:2085-90. |
|9.||Cannon RD, Chaffin WL. Oral colonization by Candida albicans. Crit Rev Oral Biol Med 1999;10:359-83. |
|10.||Nair RG, Samaranayake LP. The effect of oral commensal bacteria on Candida adhesion to human buccal epithelial cells in vitro. J Med Microbiol 1996;45:179-85. |
|11.||Najzar-FlegerD, Filipovic D, Prpic G, Kobler D. Candida in root canal in accordance with oral ecology (Abstract). Int Endod J 1992;25:40. |
|12.||Kubo CH, Gomes AP, Jorge AO. Isolating candida from root canal and assessing its sensitivity to medication used in endodontic therapy. Rev Odontol UNICID 1997;9:119-30. |
|13.||Gallis HA, Drew RH, Pickard WW. Amphotericin B: 30 years of clinical experience. Rev Infect Dis 1990;12:308-28. |
|14.||Fidalgo TK, Barcelos R, Petrópolis DB, Azevedo BR, Primo LG, Silva FC. Inhibitory activity of root canal irrigantsagainst Candida albicans, Enterococcus faecalis and Staphylococcus aureus. Braz Oral Res 2010;24:406-12. |
|15.||Portneir I, Walmtimo T, Ostavik D, Haapasalo M. The susceptibility of starved, stationaryphase and growing cells of enterococcus faecalis to endodontic medicaments. J Endod 2005;31:380-6. |
|16.||Cobankara FK, Ozkan B, Terlemez A. Comparision of organic tissue dissolution capacities of sodium hypochlorite and chlorine dioxide. J Endod 2010;36:272-4. |
|17.||Wei MK, Wu QP, Huang Q, Wu JL, Zhang JM. Plasma membrane damage to Candida albicans caused by chlorine dioxide. Lett Appl microbiol 2008;47:67-73. |
|18.||Brändle N, Zehnde M, Weiger R, Waltimo T. Impact of Growth Conditions on Susceptibility of Five Microbial Species to Alkaline Stress. J Endod 2008;34:579-82. |
|19.||Cassone A, Kerridge D, Gale EF. Ultrastructural changes in the cell wall of Candida albicans following cessation of growth and their possible relation to their development of polyene resistance. J Gen Microbiol 1979;110:339-49. |
|20.||Dudani AK, Prasad R. Differencesin amino acid transport and phospholipid contents during the cell cycle of Candida albicans. Folia Microbiol (Praha) 1985;30:493-500. |
|21.||Masuok J, Hazen KC. Differences in the acid-labile component of Candida albicans mannan from hydrophobic and hydrophilic yeast cells. Glycobiolog 1999;9:1281-6. |
|22.||King RD, Lee JC, Morris AL. Adherence of Candida albicans and other Candida species to mucosal epithelial cells. Infect Immun 1980;27:667-74. |
|23.||Ning Y, Hu X, Ling J, Du Y, Liu J, Liu H, et al. Candida albicans survival and biofilm formation under starvation condition. Int Endod J 2013;46:62-70. |
|24.||Liu H, Wei X, Ling J, Wang W, Huang X. Biofilm formation capacity of Enterococcus faecalis cells in starvation phase and its susceptibility to sodium hypochlorite. J Endod 2010;36:650-5. |
|25.||Uppuluri P, Chaffin WL. Defining Candida albicans stationary phase by cellular and DNA replication, gene expression and regulation. Mol Microbiol 2007;64:1572-86. |
|26.||Kinirons MJ. Candidal invasion of dentine complicating hypodontia. Br Dent J 1983;154:400-1. |
|27.||Sena NT, Gomes BP, Vianna ME, Berber VB, Zia AA, Ferraz CC, et al. In vitro antimicrobial activity of sodium hypochlorite and Chlorhexidine against selected single species biofilms. Int Endod J 2006;39:878-85. |
|28.||Turk BT, Ates M, Sen BH. The effect of treatment of radicular dentin of colonization pattern of Candida albicans. Oral Surg Oral Med Oral Pathol Endod 2008;106:457-62. |
|29.||Waltimo TM, Sen BH, Meurman JH, Ørstavik D, Haapasalo MP. Yeasts in apical periodontitis. Crit Rev Oral Biol Med 2003;14:128-37. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]