CLINICAL RESEARCH tested disinfection protocols only promoted a reversible inactivation of bacteria. They were not able to completely disrupt the E. faecalis cells. In the present study, E. faecalis was chosen as the test microorganism because it is one of the most resistant microorganisms found in infected root canals (Sedgley, et al., 2008), and it has been reported that cases with refractory endodontic treatment were associated with this bacterial strain (Stuart, et al., 2006). The analysis after performing disinfection protocols demonstrated a significant reduction in the level of CFU using an ozone gas system in relation to the high-frequency electrical pulses. This finding is consistent with the study by Virtej, et al., (2007) in which the Endox® system presented a less antimicrobial effect than sodium hypochlorite, BioPure MTAD®, and the ozone gas HealOzone® system. Although some studies, such as Cassanelli, et al. (2004), Hubbezoglu, et al. (2014), and Kaptan, et al. (2014), have demonstrated a bactericidal effect of ozone gas and high-frequency electrical pulses equipment, none of the disinfection protocols carried out in the study promoted a significant elimination of the E. faecalis cells. According to Virtej, et al., (2007), Kustarci, et al. (2009), Karale, et al. (2011), and Zan, et al. (2013), the antibacterial effect of the aqueous ozone and high-frequency electrical pulses was insufficient when compared with the sodium hypochlorite solution. In addition, in the second period (M2), 7 days after collection, there was an increase in the CFU counts when compared to the control group and immediately after the disinfection protocols. The study of Virtej, et al. (2007), observed bacterial growth after a week of incubation. It reaffirms that both protocols of disinfection were able to inactivate the bacteria but only for a short period of time. Once re-established, the environmental condition, microorganisms, and metabolic activities returned normally. Data obtained in the study of ArandaGarcia, et al. (2012), are in accordance with the considerations reported in this experiment. The authors compared the effectiveness of antibacterial Endox Plus® system and the 2.5% sodium hypochlorite solution in combination with BioPure MTAD® or EDTA on root canals infected with E. faecalis. Although the data have initially been satisfactory, all disinfection procedures allowed the bacterial recovery 7 days after treatment. It 28 Endodontic practice
The results obtained in the study reinforce the importance of finding new alternatives for greater effectiveness in the process of endodontic disinfection. demonstrated the persistence and viability of bacteria inside the root canal system. Polydorou, et al. (2006), observed bacterial growth 8 weeks after the use of the HealOzone® system. According to Cassanelli, et al. (2008), the high-frequency electrical pulses system induces pore formation and other defects in the membrane of the exposed bacteria. Nagayoshi, et al. (2004), noted that ozone gas has the power to damage the bacterial membrane by tissue oxidation, thus increasing its permeability. Therefore, both systems may exert reversible inactivation on the bacterial cell membrane. However, the restoration of bacterial environment and
REFERENCES 1. Aranda-Garcia AR, Guerreiro-Tanomaru JM, Faria-Júnior NB, et al. Antibacterial effectiveness of several irrigating solutions and the Endox Plus system - an ex vivo study. Int Endod J. 2012;45(12):1091-1096. 2. Atila-Pektaş B, Yurdakul P, Gülmez D, Görduysus O. Antimicrobial effects of root canal medicaments against Enterococcus faecalis and Streptococcus mutans. Int Endod J. 2013;46(5):413-418. 3. Bünning G, Hempel DC. Vital-fluorochromization of microorganisms using 3’, 6’-diacetylfluorescein to determine damages of cell membranes and loss of metabolic activity by ozonation. Ozone Sci Engl. 1996;18:173-181. 4. Case PD, Bird PS, Kahler WA, George R, Walsh LJ. Treatment of root canal biofilms of Enterococcus faecalis with ozone gas and passive ultrasound activation. J Endod. 2012;38(4):523-526. 5. Cassanelli C, Marchese A, Cagnacci S, Debbia EA. Alteration of membrane permeability of bacteria and yeast by high frequency alternating current (HFAC). Open Microbiol J. 2008;2:32-37.
substrate may have promoted the return of their physiological conditions. The results obtained in the study reinforce the importance of finding new alternatives for greater effectiveness in the process of endodontic disinfection. Perhaps, one of the possibilities would be the association of irrigating solutions with the tested equipment because the damage to the bacterial membrane promoted by them can facilitate and potentiate the antimicrobial action. According to the results of the present study, both ozone gas and the highfrequency electrical pulses were not effective to eliminate E. faecalis, immediately after the disinfection protocol and after 7 days. EP
of high-frequency electrical pulses on organic tissue in root canals. Int Endod J. 2005;38(8):531-538. 14. Lima SM, Sousa MG, Freire MS, Almeida, et al. Immune Response Profile against Persistent Endodontic Pathogens Candida albicans and Enterococcus faecalis In Vitro. J Endod. 2015;41(7):1061-1065. 15. Mehrvarzfar P, Saghiri MA, Asatourian A, et al. Additive effect of a diode laser on the antibacterial activity of 2.5% NaOCl, 2% CHX and MTAD against Enterococcus faecalis contaminating root canals: an in vitro study. J Oral Sci. 2011;53(3):355-360. 16. Nagayoshi M, Fukuizumi T, Kitamura C, Yano J, Terashita M, Nishihara T. Efficacy of ozone on survival and permeability of oral microorganisms. Oral Microbiol Immunol. 2004;19(4):240-246. 17. Polydorou O, Pelz K, Hahn P. Antibacterial effect of an ozone device and its comparison with two dentin-bonding systems. Eur J Oral Sci. 2006;114(4):349-353. 18. Rôças IN, Siqueira JF, Santos KRN. Association of Enterococcus faecalis with different forms of periradicular diseases. J Endod. 2004;30(5):315-320.
6. Cassanelli C, Roveta S, Cavallini F, Marchese A, Debbia EA, Armanino R. Bactericidal effect of endox against various pathogens. 14th ECCMID; 2004; Prague, Czech Rep. Abstr. P480.
19. Sedgley CM, Lee EH, Martin MJ, Flannagan SE. Antibiotic resistance gene transfer between Streptococcus gordonii and Enterococcus faecalis in root canals of teeth ex vivo. J Endod. 2008;34(5):570-574.
7. Evans M, Davies JK, Sundqvist G, Figdor D. Mechanisms involved in the resistance of Enterococcus faecalis to calcium hydroxide. Int Endod J. 2002;35(3):221-228.
20. Siqueira JF Jr, Rôças IN, Favieri A, Lima KC. Chemomechanical reduction of the bacterial population in the root canal after instrumentation and irrigation with 1%, 2.5%, and 5.25% sodium hypochlorite. J Endod. 2000;26(6):331-334.
8. Figdor D, Davies JK, Sundqvist G. Starvation survival, growth and recovery of Enterococcus faecalis in human serum. Oral Microbiol Immunol. 2003;18(4):234-239. 9. Hubbezoglu I, Zan R, Tunc T, Sumer Z. Antibacterial Efficacy of Aqueous Ozone in Root Canals Infected by Enterococcus faecalis. Jundishapur J Microbiol. 2014;7(7):e11411. 10. Kaptan F, Güven EP, Topcuoglu N, Yazici M, Külekçi G. In vitro assessment of the recurrent doses of topical gaseous ozone in the removal of Enterococcus faecalis biofilms in root canals. Niger J Clin Pract. 2014;17(5):573-578. 11. Karale R, Thakore A, Shetty V. An evaluation of antibacterial efficacy of 3% sodium hypochlorite, high-frequency alternating current and 2% chlorhexidine on Enterococcus faecalis: An in vitro study. J Conserv Dent. 2011;14(1):2-5. 12. Kustarci A, Sümer Z, Altunbaş D, Koşum S. Bactericidal effect of KTP laser irradiation against Enterococcus faecalis compared with gaseous ozone: an ex vivo study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009;107(5):e73-e79. 13. Lendini M, Alemanno E, Migliaretti G, Berutti E. The effect
21. Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis: its role in root canal treatment failure and current concepts in retreatment. J Endod. 2006;32(2):93-98. 22. Vianna ME, Zilio DM, Ferraz CC, Zaia AA, de Souza-Filho FJ, Gomes BP. Concentration of hydrogen ions in several calcium hydroxide pastes over different periods of time. Braz Dent J. 2009;20(5):382-388. 23. Virtej A, MacKenzie CR, Raab WH, Pfeffer K, Barthel CR. Determination of the performance of various root canal disinfection methods after in situ carriage. J Endod. 2007;33(8):926-929. 24. Zan R, Hubbezoglu I, Sümer Z, Tunç T, Tanalp J. Antibacterial effects of two different types of laser and aqueous ozone against Enterococcus faecalis in root canals. Photomed Laser Surg. 2013;31(4):150-154. 25. Zhang C, Du J, Peng Z. Correlation between Enterococcus faecalis and persistent intraradicular infection compared with primary intraradicular infection: a systematic review. J Endod. 2015;41(8):1207-1213.
Volume 10 Number 1