- Email: [email protected]
Disinfection of artificially contaminated Resilon cones with chlorhexidine and sodium hypochlorite at different time exposures
Disinfection of artificially contaminated Resilon cones with chlorhexidine and sodium hypochlorite at different time exposures Aysin Dumani, DDS,a Oguz Yoldas, DDS, PhD,b A. Sehnaz Isci, DDS,a Fatih Köksal, MD, PhD,c Begüm Kayar, MSc,d and Esra Polat, MD,d Adana, Turkey UNIVERSITY OF CUKUROVA
Objective. The aim of this study was to evaluate the efficiency of chlorhexidine (CHX) and sodium hypochlorite (NaOCl) solutions on Resilon cones that were artificially contaminated with microbial samples of Enterococcus faecalis or Candida albicans at various concentrations and time exposures. Study design. Resilon cones artificially contaminated with E faecalis or C albicans were left in contact with 1% NaOCl, 5% NaOCl, and 2% CHX disinfecting solutions for 1 and 5 minutes. The cones were then individually transferred to the test tubes, which contained 10 mL of thioglycollate media, and were incubated at 37°C for 7 days. The antimicrobial activities of tested agents were determined by microbial growth. Results. All of the Resilon cones contaminated with E faecalis or C albicans could be disinfected with 1% and 5% NaOCl for 1 and 5 minutes and with 2% CHX for 5 minutes. Three of 7 Resilon cones contaminated with E faecalis and 1 of 7 Resilon cones contaminated with C albicans could not be disinfected with 2% CHX at 1 minute of treatment. Conclusions. In conclusion, these results demonstrated that 1% and 5% NaOCl solutions are effective agents for disinfecting Resilon cones in 1- or 5-minute treatments. Two percent CHX was only effective after 5 minutes of treatment. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:e82-e85)
One of the major objectives of root canal treatment is the elimination of the microorganisms from the root canal system. Microorganisms play a fundamental role in the etiology of pulp and periapical diseases.1,2 Resilon (Resilon Research LLC, Madison, CT) is a thermoplastic synthetic polymer-based root canal filling material that performs like gutta-percha in handling properties and is similar to gutta-percha in sizes.3 Gutta-percha cones and Resilon cones cannot be sterilized by the conventional processes using moist or dry heat as this may alter their structures, therefore a rapid chemical disinfection is needed.4 Chemical disinfection of guttapercha has been described in previous studies.4-9 Sodium hypochlorite (NaOCl) is the most widely used disinfecting agent in endodontic therapy for irria
Research Assistant, Department of Conservative Dentistry and Endodontics, University of Cukurova, Faculty of Dentistry. b Associate Professor, Department of Conservative Dentistry and Endodontics, University of Cukurova, Faculty of Dentistry. c Professor, Department of Microbiology, University of Cukurova, Faculty of Medicine. d Research Assistant, Department of Microbiology, University of Cukurova, Faculty of Medicine. Received for publication May 4, 2006; returned for revision Jun 20, 2006; accepted for publication Oct 18, 2006. 1079-2104/$ - see front matter © 2007 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2006.10.013
e82
gation and cone disinfection. It is an effective antimicrobial agent, but its effectiveness is related to concentration and exposure time.7 Another irrigant and intracanal medicament is chlorhexidine (CHX).10,11 This agent has inhibitory effects on bacteria and yeasts that are commonly found in endodontic infections.12 The aim of this study was to evaluate the effectiveness of CHX and NaOCl on Resilon cones that were artificially contaminated with the microbial samples of Enterococcus faecalis and Candida albicans at various concentrations and time exposures. MATERIAL AND METHODS One hundred nineteen Resilon cones arranged in packages containing 7 units each were sterilized using ethylene oxide. The cones were divided into 12 experimental groups and 3 control groups. The experimental groups were arranged according to the type of solution (1% NaOCl, 5% NaOCl, 2% CHX), the time of exposure to each solution (1 and 5 minutes), and the type of the microorganisms (E faecalis and C albicans). Artificial contamination of Resilon cones E faecalis ATCC 29212 and C albicans ATCC 10231, as microbial suspensions containing approximately 108 CFU/mL in trypticase soy broth, were used. For artificial contamination, 56 Resilon cones were selected for each specie. The cones were immersed in
OOOOE Volume 103, Number 3
Dumani et al. e83
Table I. Microbial growth of Enterococcus faecalis and Candida albicans after disinfection procedures E faecalis Solutions
Groups
1% 1% 5% 1% 2% 2%
Group Group Group Group Group Group
NaOCl, 1 min NaOCl, 5 min NaOCl, 1 min NaOCl, 5 min CHX, 1 min CHX, 5 min
1 2 3 4 5 6
C albicans
Number of samples
Microbial growth
7 7 7 7 7 7
0 0 0 0 3 0
20 mL of each microbial suspension (108 CFU/mL) for 30 minutes. The cones were then transferred to sterile pads in petri dishes and allowed to air-dry for 10 minutes at room temperature under a laminar flow hood. Then, for each group, 7 contaminated cones were immersed in separate glass tubes that contained 20 mL of the chemical agents at determined time intervals. The groups of Resilon cones that were artificially contaminated with E faecalis immersed in different solutions at different time periods are as follows: group 1: 1% NaOCl, 1-minute treatment; group 2: 1% NaOCl, 5-minute treatment; group 3: 5% NaOCl, 1-minute treatment; group 4: 5% NaOCl, 5-minute treatment; group 5: 2% CHX, 1-minute treatment; and group 6: 2% CHX, 5-minute treatment. The groups of Resilon cones that were artificially contaminated with C albicans immersed in different solutions at different time periods are as follows: group 7: 1% NaOCl, 1-minute treatment; group 8: 1% NaOCl, 5-minute treatment; group 9: 5% NaOCl, 1-minute treatment; group 10: 5% NaOCl, 5-minute treatment; group 11: 2% CHX, 1-minute treatment; and group 12: 2% CHX, 5-minute treatment. After immersing in chemical agents, the cones were washed for 5 minutes in 10 mL of detergent solution (3% Tween 80 and 5% sodium thiosulfate). The cones were then rinsed with 10 mL of sterile distilled water to neutralize any residual effects of the test agents. Subsequently, the cones were individually transferred to the test tubes containing 10 mL of thioglycollate media and incubated at 37°C for 7 days. All test tubes were observed at 24-hour intervals. The antimicrobial activities of the tested agents were determined by the microbial growth. After 7 days, if microbial growth occurred, 10 mL of thioglycollate media was inoculated to blood agars with 7% horse blood for E faecalis or Sabouraud dextrose agars for C albicans. The plates were incubated for 24 to 48 hours aerobically at 37°C. Microbial growth was also confirmed with Gram staining, colony morphology, and with a microbial growth identification kit (API 20 Strep, bioMeuriex SA, Marcy-l’ Etoile, France).
Groups Group Group Group Group Group Group
7 8 9 10 11 12
Number of samples
Microbial growth
7 7 7 7 7 7
0 0 0 0 1 0
Control groups Seven Resilon cones were individually cultivated in thioglycollate media to confirm sterility with ethylene oxide (control group 1, negative control). Seven Resilon cones contaminated with E faecalis and 7 Resilon cones contaminated with C albicans were individually cultivated in thioglycollate media to control the artificial contamination procedures (control group 2, positive control). For washing controls, 7 Resilon cones contaminated with E faecalis and 7 Resilon cones contaminated with C albicans were washed for 5 minutes with 10 mL of detergent solution (3% Tween 80 and 5% sodium thiosulfate), then rinsed with 10 mL of sterile distilled water and cultivated in thioglycollate media (control group 3, washing control). RESULTS Data obtained from experimental groups are summarized in Table I. In group 5, 3 of 7 Resilon cones contaminated with E faecalis could not be disinfected with 2% CHX at 1 minute of treatment. In addition, in group 11, one of 7 Resilon cones contaminated with C albicans could not be disinfected with 2% CHX at 1 minute of treatment. All the other Resilon cone groups contaminated with C albicans and E faecalis were totally disinfected. All positive controls and washing controls had microbial growth within the first 24 hours. Negative controls did not have any microbial growth for 1 week. DISCUSSION The disinfection of gutta-percha cones has been considered unnecessary because they are produced under aseptic conditions and have antimicrobial properties.13 However, there is a risk of gutta-percha contamination by aerosols and physical sources during the storage process.5,7 Gutta-percha cones cannot be sterilized by heat. Therefore, a chemical agent should be used in routine endodontic practice to sterilize the master and accessory gutta-percha cones.7 For safety reasons, the use of
e84
OOOOE March 2007
Dumani et al.
a strong chemical agent that acts upon microorganisms has been encouraged by many authors for disinfection of gutta-percha cones.5,7,8,14 Resilon shares similar properties with gutta-percha3,15; therefore, antimicrobial efficacy of chemical disinfection methods on Resilon should also be investigated. C albicans, ATCC 10231, is the most frequently encountered and the most virulent Candida species in experimental models examined so far.16 Furthermore, C albicans tolerates harsh ecological conditions, including high alkalinity. Oral candida species are highly resistant to the standard local root canal medicaments.17 E faecalis, ATCC 29212, a facultative gram-positive coccus, was chosen as the test species because of its implication as a possible microbial factor in therapyresistant apical periodontitis.18,19 This microorganism has also been used in several previous studies on the efficacy of endodontic irrigants.20,21 It can resist certain chemical agents that would be highly toxic to other organisms. Its persistence in endodontic infection might be aided by enhanced resistance to NaOCl.22 E faecalis and C albicans were selected for this study because they are the most resistant species in the oral cavity.18,23 However, clinically, Resilon cones directly from the package are unlikely to be contaminated with these microorganisms. Microorganisms most frequently isolated from contaminated gutta-percha cones are Staphylococcus epidermis, Staphylococcus aureus, Propionibacterium acnes, Lactobacillus spp, and Micrococcus spp, which are normal inhabitants of human skin, and Streptococcus salivarius, which belongs to the mouth microbiota and is especially found on the tongue and in saliva.7 Therefore, further studies involving other microbial species might be informative. Chlorhexidine and NaOCl are used in endodontics generally as irrigants and as intracanal medicaments. Chlorhexidine is effective against a broad range of both gram-negative and gram-positive bacteria, as well as against yeasts.11 One of the mechanisms that explains its efficacy is based on the interaction between the positive charge of the molecule and negatively charged phosphate groups on the bacterial cell wall, which allows the CHX molecule to penetrate into the microorganism.24,25 Sodium hypochlorite provides good tissue solvent action and efficiently eliminates microbes from root canals.4,20 Both CHX and NaOCl are used for chemical disinfection of gutta-percha cones. Therefore, these solutions were selected for the present study. In our study, 2% CHX solution was more effective at a 5-minute treatment than a 1-minute treatment. The results were not uniform in the groups of Resilon cones treated with this solution for 1 minute. Microbial growth was found in groups 5 and 11. Therefore, it could be concluded that 2% CHX solution was not
effective at the 1-minute treatment. Unlike our results, Cardoso et al.8 found that 2% CHX effectively disinfected gutta-percha cones contaminated with E faecalis after a 1-minute treatment. Similarly, Gomes et al.7 demonstrated that 1% and 2% CHX effectively eliminated E faecalis and C albicans in 15 seconds. But in the present study, only 2 species of microorganisms were used. Therefore, the range of microorganisms tested and the antimicrobial agents were less than the previous studies. In the present study, 1% and 5% NaOCl solutions effectively eliminated E faecalis at 1 and 5 minutes of treatments. Our results confirm findings by Senia et al.,4 who found that 45-second immersion in 5.25% NaOCl eliminated E faecalis. The antimicrobial activity of NaOCl is related to its concentration. Higher concentrations inhibit in a shorter period of time.6 Gomes et al.7 reported that 1% NaOCl eliminated E faecalis and C albicans in 20 minutes, but 5.25% eliminated the microbes in 45 seconds. In the present study, 1% and 5% NaOCl solutions were both effective against E faecalis and C albicans in 1 and 5 minutes. These results are in agreement with most previous studies.4,6,9 Chemical disinfection of gutta-percha cones is well established. It might be difficult to compare our results with other studies on gutta-percha cones because the surface texture of Resilon cones and gutta-percha cones is not similar. In addition, gutta-percha cones have antimicrobial properties,13 unlike Resilon cones, which do not.26 Even though gutta-percha and Resilon cones are usually sterile during storage, they can be easily contaminated if incorrectly manipulated. Gutta-percha and Resilon cones should be manipulated with sterilized weezers and gauzes. Unfortunately it is common to see clinicians manipulating them with their gloves. Gomes et al.7 demonstrated that 100% of the gutta-percha cones manipulated with gloves showed microbial growth. Therefore, the handling of gutta-percha cones must follow the basic principles of infection control. In addition, gutta-percha and Resilon cones that have been in contact with patients should be discarded.6,7 In conclusion, Resilon cones that were artificially contaminated with E faecalis and C albicans can be disinfected chemically, as is done for gutta-percha cones. Sodium hypochlorite, at concentrations of 1% and 5%, is an effective agent for the disinfection of Resilon cones during 1- and 5-minute treatments; however, 2% CHX was found to be effective only at the 5-minute treatment. REFERENCES 1. Kakehashi S, Stanley HP, Fitzgerald RJ. The effects of surgical exposure of dental pulps in germ-free and conventional laboratory rats. Oral Surg 1965;20:340-9.
OOOOE Volume 103, Number 3 2. Sundqvist G. Ecology of root canal flora. J Endod 1992; 18:427-30. 3. Shipper G, Orstavik 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. 4. Senia ES, Macarro RV, Mitchell JL, Lewis AG, Thomas L. Rapid sterilization of gutta-percha cones with 5.25% sodium hypochlorite. J Endod 1975;1:136-40. 5. da Motta PG, de Figueiredo CBO, Maltos SMM, Nicoli JR, Ribeiro Sobrinho AP, Maltos KLM, et al. Efficacy of chemical sterilization and storage conditions of gutta-percha cones. Int Endod J 2001;34:435-9. 6. Stabholz A, Friedman S, Helling I, Sela MN. Efficiency of different chemical agents on decontamination of gutta-percha cones. Int Endod J 1987;20:211-6. 7. Gomes BPFA, Vianna ME, Matsumoto CU, Rossi V, Zaia AA, Ferraz CC, et al. Disinfection of gutta-percha cones with chlorhexidine and sodium hypochlorite. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;100:512-7. 8. Cardoso CL, Redmerski R, Bittencourt NLR, Kotaka CR. Effectiveness of different chemical agents in rapid decontamination of gutta-percha cones. Brazil J Microbiol 2000;31;67-71. 9. Linke HAB, Chohayeb AA. Effective surface sterilization of gutta-percha points. Oral Surg 1983;55:73-7. 10. Delany GM, Patterson SS, Miller CH, Newton CW. The effect of chlorhexidine gluconate irrigation on the root canal flora of freshly extracted necrotic teeth. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1982;53:518-23. 11. Vahdaty A, Pitt Ford TR, Wilson RF. Efficacy of chlorhexidine in disinfecting dentinal tubules in vitro. Endod Dental Traumatol 1993;9:243-8. 12. Cervone F, Tronstad L, Hammond B. Antimicrobial effect of chlorhexidine in controlled release delivery system. Endod Dental Traumatol 1990;6:33-6. 13. Moorer WR, Genet JM. Evidence for antibacterial activety of endodontic gutta-percha cones. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1982;53:503-7. 14. Siqueira JF Jr, Pereira da Silva CHF, Cerqueira MOD, Lopes HP, Uzeda M. Effectiveness of four chemical solutions in eliminating Bacillus subtilis spores on gutta-percha cones. Endod Dental Traumatol 1998;14:124-6. 15. Schirrmeister JF, Meyer KM, Hermans P, Altenburger MJ,
Dumani et al. e85
16. 17.
18. 19.
20.
21.
22.
23. 24.
25. 26.
Wrbas KT. Effectiveness of hand and rotary instrumentation for removing a new synthetic polymer-based root canal obturation material (Epiphany) during retreatment. Int Endod J 2006;39: 150-6. Odds FC. Candida and candidosis. 2nd ed. London: Baillere Tindall; 1988. Waltimo TMT, Haapasolo M, Zehnder M, Meyer J. Clinical aspects related to endodontic yeast infections. Endod Topics 2004;9:66-78. Molander A, Reit C, Dahlen G, Kvist T. Microbial status of rootfilled teeth with apical periodontitis. Int Endod J 1998;31:1-7. Sundqvist G, Figdor D, Persson S, Sjogren U. Microbiologic analysis of teeth with failed endodontic treatment and outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86-93. Ayhan H, Sultan N, Çirak M, Ruhi MZ, Bodur H. Antimicrobial effects of various endodontic irrigants on selected microorganisms. Int Endod J 1999;32:99-102. Shih M, Marshall J, Rosen S. The antibacterial efficiency of sodium hypochlorite as an endodontic irrigant. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1970;29:613-9. Gomes BPFA, Vianna ME, Berber VB, Teixeira FB, SouzaFilho FJ. In vitro antimicrobial activity of several concentrations of sodium hypochlorite and chlorhexidine gluconate in the elimination of Enterococcus faecalis. Int Endod J 2001;34:424-8. Siqueira JF Jr, Sen BH. Fungi in endodontic infections. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:632-41. Lindskog S, Pierce AM, Blomlof L. Chlorhexidine as a root canal medicament for treating inflammatory lesions in the periodontal space. Endod Dental Traumatol 1998;14:186-90. Hugo WB, Longworth AR. Some aspects of the mode of action of chlorhexidine. J Pharm Pharmacol 1964;16:665-72. Melker KB, Vertucci FJ, Rojas MF, Progulske-Fox A, Belanger M. Antimicrobial efficacy of medicated root canal filling materials. J Endod 2006;32:148-51.
Reprint requests: Oguz Yoldas, DDS, PhD Cukurova University Faculty of Dentistry Balcali Adana, Turkey [email protected]
Objective. The aim of this study was to evaluate the efficiency of chlorhexidine (CHX) and sodium hypochlorite (NaOCl) solutions on Resilon cones that were artificially contaminated with microbial samples of Enterococcus faecalis or Candida albicans at various concentrations and time exposures. Study design. Resilon cones artificially contaminated with E faecalis or C albicans were left in contact with 1% NaOCl, 5% NaOCl, and 2% CHX disinfecting solutions for 1 and 5 minutes. The cones were then individually transferred to the test tubes, which contained 10 mL of thioglycollate media, and were incubated at 37°C for 7 days. The antimicrobial activities of tested agents were determined by microbial growth. Results. All of the Resilon cones contaminated with E faecalis or C albicans could be disinfected with 1% and 5% NaOCl for 1 and 5 minutes and with 2% CHX for 5 minutes. Three of 7 Resilon cones contaminated with E faecalis and 1 of 7 Resilon cones contaminated with C albicans could not be disinfected with 2% CHX at 1 minute of treatment. Conclusions. In conclusion, these results demonstrated that 1% and 5% NaOCl solutions are effective agents for disinfecting Resilon cones in 1- or 5-minute treatments. Two percent CHX was only effective after 5 minutes of treatment. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:e82-e85)
One of the major objectives of root canal treatment is the elimination of the microorganisms from the root canal system. Microorganisms play a fundamental role in the etiology of pulp and periapical diseases.1,2 Resilon (Resilon Research LLC, Madison, CT) is a thermoplastic synthetic polymer-based root canal filling material that performs like gutta-percha in handling properties and is similar to gutta-percha in sizes.3 Gutta-percha cones and Resilon cones cannot be sterilized by the conventional processes using moist or dry heat as this may alter their structures, therefore a rapid chemical disinfection is needed.4 Chemical disinfection of guttapercha has been described in previous studies.4-9 Sodium hypochlorite (NaOCl) is the most widely used disinfecting agent in endodontic therapy for irria
Research Assistant, Department of Conservative Dentistry and Endodontics, University of Cukurova, Faculty of Dentistry. b Associate Professor, Department of Conservative Dentistry and Endodontics, University of Cukurova, Faculty of Dentistry. c Professor, Department of Microbiology, University of Cukurova, Faculty of Medicine. d Research Assistant, Department of Microbiology, University of Cukurova, Faculty of Medicine. Received for publication May 4, 2006; returned for revision Jun 20, 2006; accepted for publication Oct 18, 2006. 1079-2104/$ - see front matter © 2007 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2006.10.013
e82
gation and cone disinfection. It is an effective antimicrobial agent, but its effectiveness is related to concentration and exposure time.7 Another irrigant and intracanal medicament is chlorhexidine (CHX).10,11 This agent has inhibitory effects on bacteria and yeasts that are commonly found in endodontic infections.12 The aim of this study was to evaluate the effectiveness of CHX and NaOCl on Resilon cones that were artificially contaminated with the microbial samples of Enterococcus faecalis and Candida albicans at various concentrations and time exposures. MATERIAL AND METHODS One hundred nineteen Resilon cones arranged in packages containing 7 units each were sterilized using ethylene oxide. The cones were divided into 12 experimental groups and 3 control groups. The experimental groups were arranged according to the type of solution (1% NaOCl, 5% NaOCl, 2% CHX), the time of exposure to each solution (1 and 5 minutes), and the type of the microorganisms (E faecalis and C albicans). Artificial contamination of Resilon cones E faecalis ATCC 29212 and C albicans ATCC 10231, as microbial suspensions containing approximately 108 CFU/mL in trypticase soy broth, were used. For artificial contamination, 56 Resilon cones were selected for each specie. The cones were immersed in
OOOOE Volume 103, Number 3
Dumani et al. e83
Table I. Microbial growth of Enterococcus faecalis and Candida albicans after disinfection procedures E faecalis Solutions
Groups
1% 1% 5% 1% 2% 2%
Group Group Group Group Group Group
NaOCl, 1 min NaOCl, 5 min NaOCl, 1 min NaOCl, 5 min CHX, 1 min CHX, 5 min
1 2 3 4 5 6
C albicans
Number of samples
Microbial growth
7 7 7 7 7 7
0 0 0 0 3 0
20 mL of each microbial suspension (108 CFU/mL) for 30 minutes. The cones were then transferred to sterile pads in petri dishes and allowed to air-dry for 10 minutes at room temperature under a laminar flow hood. Then, for each group, 7 contaminated cones were immersed in separate glass tubes that contained 20 mL of the chemical agents at determined time intervals. The groups of Resilon cones that were artificially contaminated with E faecalis immersed in different solutions at different time periods are as follows: group 1: 1% NaOCl, 1-minute treatment; group 2: 1% NaOCl, 5-minute treatment; group 3: 5% NaOCl, 1-minute treatment; group 4: 5% NaOCl, 5-minute treatment; group 5: 2% CHX, 1-minute treatment; and group 6: 2% CHX, 5-minute treatment. The groups of Resilon cones that were artificially contaminated with C albicans immersed in different solutions at different time periods are as follows: group 7: 1% NaOCl, 1-minute treatment; group 8: 1% NaOCl, 5-minute treatment; group 9: 5% NaOCl, 1-minute treatment; group 10: 5% NaOCl, 5-minute treatment; group 11: 2% CHX, 1-minute treatment; and group 12: 2% CHX, 5-minute treatment. After immersing in chemical agents, the cones were washed for 5 minutes in 10 mL of detergent solution (3% Tween 80 and 5% sodium thiosulfate). The cones were then rinsed with 10 mL of sterile distilled water to neutralize any residual effects of the test agents. Subsequently, the cones were individually transferred to the test tubes containing 10 mL of thioglycollate media and incubated at 37°C for 7 days. All test tubes were observed at 24-hour intervals. The antimicrobial activities of the tested agents were determined by the microbial growth. After 7 days, if microbial growth occurred, 10 mL of thioglycollate media was inoculated to blood agars with 7% horse blood for E faecalis or Sabouraud dextrose agars for C albicans. The plates were incubated for 24 to 48 hours aerobically at 37°C. Microbial growth was also confirmed with Gram staining, colony morphology, and with a microbial growth identification kit (API 20 Strep, bioMeuriex SA, Marcy-l’ Etoile, France).
Groups Group Group Group Group Group Group
7 8 9 10 11 12
Number of samples
Microbial growth
7 7 7 7 7 7
0 0 0 0 1 0
Control groups Seven Resilon cones were individually cultivated in thioglycollate media to confirm sterility with ethylene oxide (control group 1, negative control). Seven Resilon cones contaminated with E faecalis and 7 Resilon cones contaminated with C albicans were individually cultivated in thioglycollate media to control the artificial contamination procedures (control group 2, positive control). For washing controls, 7 Resilon cones contaminated with E faecalis and 7 Resilon cones contaminated with C albicans were washed for 5 minutes with 10 mL of detergent solution (3% Tween 80 and 5% sodium thiosulfate), then rinsed with 10 mL of sterile distilled water and cultivated in thioglycollate media (control group 3, washing control). RESULTS Data obtained from experimental groups are summarized in Table I. In group 5, 3 of 7 Resilon cones contaminated with E faecalis could not be disinfected with 2% CHX at 1 minute of treatment. In addition, in group 11, one of 7 Resilon cones contaminated with C albicans could not be disinfected with 2% CHX at 1 minute of treatment. All the other Resilon cone groups contaminated with C albicans and E faecalis were totally disinfected. All positive controls and washing controls had microbial growth within the first 24 hours. Negative controls did not have any microbial growth for 1 week. DISCUSSION The disinfection of gutta-percha cones has been considered unnecessary because they are produced under aseptic conditions and have antimicrobial properties.13 However, there is a risk of gutta-percha contamination by aerosols and physical sources during the storage process.5,7 Gutta-percha cones cannot be sterilized by heat. Therefore, a chemical agent should be used in routine endodontic practice to sterilize the master and accessory gutta-percha cones.7 For safety reasons, the use of
e84
OOOOE March 2007
Dumani et al.
a strong chemical agent that acts upon microorganisms has been encouraged by many authors for disinfection of gutta-percha cones.5,7,8,14 Resilon shares similar properties with gutta-percha3,15; therefore, antimicrobial efficacy of chemical disinfection methods on Resilon should also be investigated. C albicans, ATCC 10231, is the most frequently encountered and the most virulent Candida species in experimental models examined so far.16 Furthermore, C albicans tolerates harsh ecological conditions, including high alkalinity. Oral candida species are highly resistant to the standard local root canal medicaments.17 E faecalis, ATCC 29212, a facultative gram-positive coccus, was chosen as the test species because of its implication as a possible microbial factor in therapyresistant apical periodontitis.18,19 This microorganism has also been used in several previous studies on the efficacy of endodontic irrigants.20,21 It can resist certain chemical agents that would be highly toxic to other organisms. Its persistence in endodontic infection might be aided by enhanced resistance to NaOCl.22 E faecalis and C albicans were selected for this study because they are the most resistant species in the oral cavity.18,23 However, clinically, Resilon cones directly from the package are unlikely to be contaminated with these microorganisms. Microorganisms most frequently isolated from contaminated gutta-percha cones are Staphylococcus epidermis, Staphylococcus aureus, Propionibacterium acnes, Lactobacillus spp, and Micrococcus spp, which are normal inhabitants of human skin, and Streptococcus salivarius, which belongs to the mouth microbiota and is especially found on the tongue and in saliva.7 Therefore, further studies involving other microbial species might be informative. Chlorhexidine and NaOCl are used in endodontics generally as irrigants and as intracanal medicaments. Chlorhexidine is effective against a broad range of both gram-negative and gram-positive bacteria, as well as against yeasts.11 One of the mechanisms that explains its efficacy is based on the interaction between the positive charge of the molecule and negatively charged phosphate groups on the bacterial cell wall, which allows the CHX molecule to penetrate into the microorganism.24,25 Sodium hypochlorite provides good tissue solvent action and efficiently eliminates microbes from root canals.4,20 Both CHX and NaOCl are used for chemical disinfection of gutta-percha cones. Therefore, these solutions were selected for the present study. In our study, 2% CHX solution was more effective at a 5-minute treatment than a 1-minute treatment. The results were not uniform in the groups of Resilon cones treated with this solution for 1 minute. Microbial growth was found in groups 5 and 11. Therefore, it could be concluded that 2% CHX solution was not
effective at the 1-minute treatment. Unlike our results, Cardoso et al.8 found that 2% CHX effectively disinfected gutta-percha cones contaminated with E faecalis after a 1-minute treatment. Similarly, Gomes et al.7 demonstrated that 1% and 2% CHX effectively eliminated E faecalis and C albicans in 15 seconds. But in the present study, only 2 species of microorganisms were used. Therefore, the range of microorganisms tested and the antimicrobial agents were less than the previous studies. In the present study, 1% and 5% NaOCl solutions effectively eliminated E faecalis at 1 and 5 minutes of treatments. Our results confirm findings by Senia et al.,4 who found that 45-second immersion in 5.25% NaOCl eliminated E faecalis. The antimicrobial activity of NaOCl is related to its concentration. Higher concentrations inhibit in a shorter period of time.6 Gomes et al.7 reported that 1% NaOCl eliminated E faecalis and C albicans in 20 minutes, but 5.25% eliminated the microbes in 45 seconds. In the present study, 1% and 5% NaOCl solutions were both effective against E faecalis and C albicans in 1 and 5 minutes. These results are in agreement with most previous studies.4,6,9 Chemical disinfection of gutta-percha cones is well established. It might be difficult to compare our results with other studies on gutta-percha cones because the surface texture of Resilon cones and gutta-percha cones is not similar. In addition, gutta-percha cones have antimicrobial properties,13 unlike Resilon cones, which do not.26 Even though gutta-percha and Resilon cones are usually sterile during storage, they can be easily contaminated if incorrectly manipulated. Gutta-percha and Resilon cones should be manipulated with sterilized weezers and gauzes. Unfortunately it is common to see clinicians manipulating them with their gloves. Gomes et al.7 demonstrated that 100% of the gutta-percha cones manipulated with gloves showed microbial growth. Therefore, the handling of gutta-percha cones must follow the basic principles of infection control. In addition, gutta-percha and Resilon cones that have been in contact with patients should be discarded.6,7 In conclusion, Resilon cones that were artificially contaminated with E faecalis and C albicans can be disinfected chemically, as is done for gutta-percha cones. Sodium hypochlorite, at concentrations of 1% and 5%, is an effective agent for the disinfection of Resilon cones during 1- and 5-minute treatments; however, 2% CHX was found to be effective only at the 5-minute treatment. REFERENCES 1. Kakehashi S, Stanley HP, Fitzgerald RJ. The effects of surgical exposure of dental pulps in germ-free and conventional laboratory rats. Oral Surg 1965;20:340-9.
OOOOE Volume 103, Number 3 2. Sundqvist G. Ecology of root canal flora. J Endod 1992; 18:427-30. 3. Shipper G, Orstavik 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. 4. Senia ES, Macarro RV, Mitchell JL, Lewis AG, Thomas L. Rapid sterilization of gutta-percha cones with 5.25% sodium hypochlorite. J Endod 1975;1:136-40. 5. da Motta PG, de Figueiredo CBO, Maltos SMM, Nicoli JR, Ribeiro Sobrinho AP, Maltos KLM, et al. Efficacy of chemical sterilization and storage conditions of gutta-percha cones. Int Endod J 2001;34:435-9. 6. Stabholz A, Friedman S, Helling I, Sela MN. Efficiency of different chemical agents on decontamination of gutta-percha cones. Int Endod J 1987;20:211-6. 7. Gomes BPFA, Vianna ME, Matsumoto CU, Rossi V, Zaia AA, Ferraz CC, et al. Disinfection of gutta-percha cones with chlorhexidine and sodium hypochlorite. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;100:512-7. 8. Cardoso CL, Redmerski R, Bittencourt NLR, Kotaka CR. Effectiveness of different chemical agents in rapid decontamination of gutta-percha cones. Brazil J Microbiol 2000;31;67-71. 9. Linke HAB, Chohayeb AA. Effective surface sterilization of gutta-percha points. Oral Surg 1983;55:73-7. 10. Delany GM, Patterson SS, Miller CH, Newton CW. The effect of chlorhexidine gluconate irrigation on the root canal flora of freshly extracted necrotic teeth. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1982;53:518-23. 11. Vahdaty A, Pitt Ford TR, Wilson RF. Efficacy of chlorhexidine in disinfecting dentinal tubules in vitro. Endod Dental Traumatol 1993;9:243-8. 12. Cervone F, Tronstad L, Hammond B. Antimicrobial effect of chlorhexidine in controlled release delivery system. Endod Dental Traumatol 1990;6:33-6. 13. Moorer WR, Genet JM. Evidence for antibacterial activety of endodontic gutta-percha cones. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1982;53:503-7. 14. Siqueira JF Jr, Pereira da Silva CHF, Cerqueira MOD, Lopes HP, Uzeda M. Effectiveness of four chemical solutions in eliminating Bacillus subtilis spores on gutta-percha cones. Endod Dental Traumatol 1998;14:124-6. 15. Schirrmeister JF, Meyer KM, Hermans P, Altenburger MJ,
Dumani et al. e85
16. 17.
18. 19.
20.
21.
22.
23. 24.
25. 26.
Wrbas KT. Effectiveness of hand and rotary instrumentation for removing a new synthetic polymer-based root canal obturation material (Epiphany) during retreatment. Int Endod J 2006;39: 150-6. Odds FC. Candida and candidosis. 2nd ed. London: Baillere Tindall; 1988. Waltimo TMT, Haapasolo M, Zehnder M, Meyer J. Clinical aspects related to endodontic yeast infections. Endod Topics 2004;9:66-78. Molander A, Reit C, Dahlen G, Kvist T. Microbial status of rootfilled teeth with apical periodontitis. Int Endod J 1998;31:1-7. Sundqvist G, Figdor D, Persson S, Sjogren U. Microbiologic analysis of teeth with failed endodontic treatment and outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86-93. Ayhan H, Sultan N, Çirak M, Ruhi MZ, Bodur H. Antimicrobial effects of various endodontic irrigants on selected microorganisms. Int Endod J 1999;32:99-102. Shih M, Marshall J, Rosen S. The antibacterial efficiency of sodium hypochlorite as an endodontic irrigant. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1970;29:613-9. Gomes BPFA, Vianna ME, Berber VB, Teixeira FB, SouzaFilho FJ. In vitro antimicrobial activity of several concentrations of sodium hypochlorite and chlorhexidine gluconate in the elimination of Enterococcus faecalis. Int Endod J 2001;34:424-8. Siqueira JF Jr, Sen BH. Fungi in endodontic infections. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:632-41. Lindskog S, Pierce AM, Blomlof L. Chlorhexidine as a root canal medicament for treating inflammatory lesions in the periodontal space. Endod Dental Traumatol 1998;14:186-90. Hugo WB, Longworth AR. Some aspects of the mode of action of chlorhexidine. J Pharm Pharmacol 1964;16:665-72. Melker KB, Vertucci FJ, Rojas MF, Progulske-Fox A, Belanger M. Antimicrobial efficacy of medicated root canal filling materials. J Endod 2006;32:148-51.
Reprint requests: Oguz Yoldas, DDS, PhD Cukurova University Faculty of Dentistry Balcali Adana, Turkey [email protected]