Antibiofilm Effect of Citric Acid-modified Chlorhexidine Gluconate on a Dual-species Biofilm
Article Sidebar
Published:
Oct 12, 2021
Keywords:
Chlorhexidine, Citric acid, Enterococcus faecalis, Candida albicans,Biofilms
Main Article Content
Abstract
Objective: The purpose of this study was to compare the antibiofilm effect of chlorhexidine gluconate (CHX) added with citric acid (CA) on bacterial-fungal biofilm.
Methods: Dentin slices were sectioned from the crown of extracted human third molars. After sterilization, samples were inoculated with Enterococcus faecalis(E. faecalis) and Candida albicans(C. albicans) for 14 days to establish a bacterial-fungal biofilm. All samples were randomly divided into four treatment groups: phosphate-buffered saline (PBS) (negative control), 2% CHX, 10% CA, and 10% citric acid-modified 2% chlorhexidine gluconate (CAmCHX). Each dentin slice was treated with one of the solutions for 1 min. Following treatment, samples were labeled with a fluorescent viability stain to identify live and dead cells. The proportion of dead cells to total cells was analyzed with confocal laser scanning microscopy (CLSM), and biofilm removal efficacy was evaluated with scanning electron microscopy (SEM).
Results:10% CA and CAmCHX groups demonstrated a higher proportion of dead cells to total cells than the PBS group (P<0.05). No significant difference was observed between the 10% CA and CAmCHX groups (P>0.05). SEM images revealed less remaining biofilm in 10% CA and CAmCHX groups. Whereas, in 2% CHX and PBS groups, the biofilmstructure was intact.
Conclusions: Within the limitations of this study, CAmCHX demonstrated an antibiofilm effectagainst E. faecalisand C. albicansbiofilm on the surface of dentin slices.
Keywords: Chlorhexidine, Citric acid, Enterococcus faecalis, Candida albicans,Biofilms
Article Details
How to Cite
Leelapornpisid, W., Dewi, A. ., Chaiariyakul, D. ., Upara, C., & Louwakul, P. (2021). Antibiofilm Effect of Citric Acid-modified Chlorhexidine Gluconate on a Dual-species Biofilm. Chiang Mai Dental Journal, 42(3), 42–48. Retrieved from https://he01.tci-thaijo.org/index.php/cmdj/article/view/250283
Section
Original article
References
1. Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol. 1965;20:340-9.
2. Flemming HC, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S. Biofilms: an emergent form of bacterial life. Nat Rev Microbiol. 2016;14:563-75.
3. Narayanan LL, Vaishnavi C. Endodontic microbiology. J Conserv Dent. 2010;13:233-9.
4. Sedgley CM, Lennan SL, Appelbe OK. Survival of Enterococcus faecalis in root canals ex vivo. Int Endod J. 2005;38:735-42.
5. Lleo MM, Tafi MC, Canepari P. Nonculturable Enterococcus faecalis cells are metabolically active and capable of resuming active growth. Syst Appl Microbiol. 1998;21:333-9.
6. Pinheiro ET, Gomes BP, Ferraz CC, Sousa EL, Teixeira FB, Souza-Filho FJ. Microorganisms from canals of root-filled teeth with periapical lesions. Int Endod J. 2003;36:1-11.
7. O'Donnell LE, Millhouse E, Sherry L, Kean R, Malcolm J, Nile CJ, et al. Polymicrobial Candida biofilms: friends and foe in the oral cavity. FEMS Yeast Res. 2015;15:1-14.
8. Torabinejad M. Winter 2011 endodontics: collegues for excellence newsletter: root canal irrigants and disinfectants [monograph on the internet]. AAE; 2011 [cited 20 Nov 2020]. Available from: https://www.aae.org/specialty/newsletter/root-canal-irrigants-disinfectants/
9. Khademi AA, Mohammadi Z, Havaee A. Evaluation of the antibacterial substantivity of several intra-canal agents. Aust Endod J. 2006;32:112-5.
10. Fernández M, Pérez G, Villagómez M, Villagómez G, Báez T, Lara G. In vitro study of erosion caused by EDTA on root canal dentin. Revista Odontológica Mexicana. 2012;16:8-13.
11. Karkehabadi H, Yousefifakhr H, Zadsirjan S. Cytotoxicity of endodontic irrigants on human periodontal ligament cells. Iran Endod J. 2018;13:390-4.
12. Machado-Silveiro LF, Gonzalez-Lopez S, Gonzalez-Rodriguez MP. Decalcification of root canal dentine by citric acid, EDTA and sodium citrate. Int Endod J. 2004;37:365-9.
13. Georgopoulou M, Kontakiotis E, Nakou M. Evaluation of the antimicrobial effectiveness of citric acid and sodium hypochlorite on the anaerobic flora of the infected root canal. Int Endod J. 1994;27:139-43.
14. Wayman BE, Kopp WM, Pinero GJ, Lazzari EP. Citric and lactic acids as root canal irrigants in vitro. J Endod. 1979;5:258-65.
15. Clegg MS, Vertucci FJ, Walker C, Belanger M, Britto LR. The effect of exposure to irrigant solutions on apical dentin biofilms in vitro. J Endod. 2006;32:434-7.
16. Fidalgo TK, Barcelos R, Portela MB, Soares RM, Gleiser R, Silva-Filho FC. Inhibitory activity of root canal irrigants against Candida albicans, Enterococcus faecalis and Staphylococcus aureus. Braz Oral Res. 2010;24:406-12.
17. White RR, Hays GL, Janer LR. Residual antimicrobial activity after canal irrigation with chlorhexidine. J Endod. 1997;23:229-31.
18. Waltimo TM, Orstavik D, Siren EK, Haapasalo MP. In vitro susceptibility of Candida albicans to four disinfectants and their combinations. Int Endod J. 1999;32:421-9.
19. Gomes BP, Vianna ME, Zaia AA, Almeida JF, Souza-Filho FJ, Ferraz CC. Chlorhexidine in endodontics. Braz Dent J. 2013;24:89-102.
20. Gonzalez-Lopez S, Camejo-Aguilar D, Sanchez-Sanchez P, Bolanos-Carmona V. Effect of CHX on the decalcifying effect of 10% citric acid, 20% citric acid, or 17% EDTA. J Endod. 2006;32:781-4.
21. Dewi A, Upara C, Chaiariyakul D, Louwakul P. Smear layer removal from root canal dentin and antimicrobial effect of citric acid-modified chlorhexidine. Eur Endod J. 2020;5(3):257-63.
22. Rocas IN, Hulsmann M, Siqueira JF, Jr. Microorganisms in root canal-treated teeth from a German population. J Endod. 2008;34:926-31.
23. Distel JW, Hatton JF, Gillespie MJ. Biofilm formation in medicated root canals. J Endod. 2002;28:689-93..
24. Shen Y, Stojicic S, Haapasalo M. Bacterial viability in starved and revitalized biofilms: comparison of viability staining and direct culture. J Endod. 2010;36:1820-3.
25. Kishen A, Shrestha A, Del Carpio-Perochena A. Validation of biofilm assays to assess antibiofilm efficacy in instrumented root canals after syringe irrigation and sonic agitation. J Endod. 2018;44:292-8.
26. Pavlovic V, Zivkovic S. Chlorhexidine as a root canal irrigant-antimicrobial and scanning electron microscopic evaluation. Srp Arh Celok Lek. 2010;138:557-63.
27. Elakanti S, Cherukuri G, Rao VG, Chandrasekhar V, Rao AS, Tummala M. Comparative evaluation of antimicrobial efficacy of QMix 2 in 1, sodium hypochlorite, and chlorhexidine against Enterococcus faecalis and Candida albicans. J Conserv Dent. 2015;18:128-31.
28. Chandra SS, Miglani R, Srinivasan MR, Indira R. Antifungal efficacy of 5.25% sodium hypochlorite, 2% chlorhexidine gluconate, and 17% EDTA with and without an antifungal agent. J Endod. 2010;36:675-8.
29. Akbas MY, Kokumer T. The prevention and removal of biofilm formation of Staphylococcus aureus strains isolated from raw milk samples by citric acid treatments. Int J Food Sci Technol. 2015;50(7):1666-72.
30. Faot F, Cavalcanti YW, Mendonca e Bertolini M, Pinto Lde R, da Silva WJ, Cury AA. Efficacy of citric acid denture cleanser on the Candida albicans biofilm formed on poly(methyl methacrylate): effects on residual biofilm and recolonization process. BMC Oral Health. 2014;14:77.
31. Souza JGS, Cordeiro JM, Lima CV, Barao VAR. Citric acid reduces oral biofilm and influences the electrochemical behavior of titanium: An in situ and in vitro study. J Periodontol. 2019;90:149-58.
32. Malheiros CF, Marques MM, Gavini G. In vitro evaluation of the cytotoxic effects of acid solutions used as canal irrigants. J Endod. 2005;31:746-8..
33. Scelza MZ, de Noronha F, da Silva LE, Mauricio M, Gallito MA, Scelza P. Effect of citric acid and ethylenediaminetetraacetic acid on the surface morphology of young and old root dentin. Iran Endod J. 2016;11:188-91.
34. Calt S, Serper A. Time-dependent effects of EDTA on dentin structures. J Endod. 2002;28:17-9.
35. Serper A, Calt S. The demineralizing effects of EDTA at different concentrations and pH. J Endod. 2002;28:501-2.
36. Zehnder M. Root canal irrigants. J Endod. 2006;32:389-98.
37. Wang Z, Shen Y, Haapasalo M. Effectiveness of endodontic disinfecting solutions against young and old Enterococcus faecalis biofilms in dentin canals. J Endod. 2012;38:1376-9.
2. Flemming HC, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S. Biofilms: an emergent form of bacterial life. Nat Rev Microbiol. 2016;14:563-75.
3. Narayanan LL, Vaishnavi C. Endodontic microbiology. J Conserv Dent. 2010;13:233-9.
4. Sedgley CM, Lennan SL, Appelbe OK. Survival of Enterococcus faecalis in root canals ex vivo. Int Endod J. 2005;38:735-42.
5. Lleo MM, Tafi MC, Canepari P. Nonculturable Enterococcus faecalis cells are metabolically active and capable of resuming active growth. Syst Appl Microbiol. 1998;21:333-9.
6. Pinheiro ET, Gomes BP, Ferraz CC, Sousa EL, Teixeira FB, Souza-Filho FJ. Microorganisms from canals of root-filled teeth with periapical lesions. Int Endod J. 2003;36:1-11.
7. O'Donnell LE, Millhouse E, Sherry L, Kean R, Malcolm J, Nile CJ, et al. Polymicrobial Candida biofilms: friends and foe in the oral cavity. FEMS Yeast Res. 2015;15:1-14.
8. Torabinejad M. Winter 2011 endodontics: collegues for excellence newsletter: root canal irrigants and disinfectants [monograph on the internet]. AAE; 2011 [cited 20 Nov 2020]. Available from: https://www.aae.org/specialty/newsletter/root-canal-irrigants-disinfectants/
9. Khademi AA, Mohammadi Z, Havaee A. Evaluation of the antibacterial substantivity of several intra-canal agents. Aust Endod J. 2006;32:112-5.
10. Fernández M, Pérez G, Villagómez M, Villagómez G, Báez T, Lara G. In vitro study of erosion caused by EDTA on root canal dentin. Revista Odontológica Mexicana. 2012;16:8-13.
11. Karkehabadi H, Yousefifakhr H, Zadsirjan S. Cytotoxicity of endodontic irrigants on human periodontal ligament cells. Iran Endod J. 2018;13:390-4.
12. Machado-Silveiro LF, Gonzalez-Lopez S, Gonzalez-Rodriguez MP. Decalcification of root canal dentine by citric acid, EDTA and sodium citrate. Int Endod J. 2004;37:365-9.
13. Georgopoulou M, Kontakiotis E, Nakou M. Evaluation of the antimicrobial effectiveness of citric acid and sodium hypochlorite on the anaerobic flora of the infected root canal. Int Endod J. 1994;27:139-43.
14. Wayman BE, Kopp WM, Pinero GJ, Lazzari EP. Citric and lactic acids as root canal irrigants in vitro. J Endod. 1979;5:258-65.
15. Clegg MS, Vertucci FJ, Walker C, Belanger M, Britto LR. The effect of exposure to irrigant solutions on apical dentin biofilms in vitro. J Endod. 2006;32:434-7.
16. Fidalgo TK, Barcelos R, Portela MB, Soares RM, Gleiser R, Silva-Filho FC. Inhibitory activity of root canal irrigants against Candida albicans, Enterococcus faecalis and Staphylococcus aureus. Braz Oral Res. 2010;24:406-12.
17. White RR, Hays GL, Janer LR. Residual antimicrobial activity after canal irrigation with chlorhexidine. J Endod. 1997;23:229-31.
18. Waltimo TM, Orstavik D, Siren EK, Haapasalo MP. In vitro susceptibility of Candida albicans to four disinfectants and their combinations. Int Endod J. 1999;32:421-9.
19. Gomes BP, Vianna ME, Zaia AA, Almeida JF, Souza-Filho FJ, Ferraz CC. Chlorhexidine in endodontics. Braz Dent J. 2013;24:89-102.
20. Gonzalez-Lopez S, Camejo-Aguilar D, Sanchez-Sanchez P, Bolanos-Carmona V. Effect of CHX on the decalcifying effect of 10% citric acid, 20% citric acid, or 17% EDTA. J Endod. 2006;32:781-4.
21. Dewi A, Upara C, Chaiariyakul D, Louwakul P. Smear layer removal from root canal dentin and antimicrobial effect of citric acid-modified chlorhexidine. Eur Endod J. 2020;5(3):257-63.
22. Rocas IN, Hulsmann M, Siqueira JF, Jr. Microorganisms in root canal-treated teeth from a German population. J Endod. 2008;34:926-31.
23. Distel JW, Hatton JF, Gillespie MJ. Biofilm formation in medicated root canals. J Endod. 2002;28:689-93..
24. Shen Y, Stojicic S, Haapasalo M. Bacterial viability in starved and revitalized biofilms: comparison of viability staining and direct culture. J Endod. 2010;36:1820-3.
25. Kishen A, Shrestha A, Del Carpio-Perochena A. Validation of biofilm assays to assess antibiofilm efficacy in instrumented root canals after syringe irrigation and sonic agitation. J Endod. 2018;44:292-8.
26. Pavlovic V, Zivkovic S. Chlorhexidine as a root canal irrigant-antimicrobial and scanning electron microscopic evaluation. Srp Arh Celok Lek. 2010;138:557-63.
27. Elakanti S, Cherukuri G, Rao VG, Chandrasekhar V, Rao AS, Tummala M. Comparative evaluation of antimicrobial efficacy of QMix 2 in 1, sodium hypochlorite, and chlorhexidine against Enterococcus faecalis and Candida albicans. J Conserv Dent. 2015;18:128-31.
28. Chandra SS, Miglani R, Srinivasan MR, Indira R. Antifungal efficacy of 5.25% sodium hypochlorite, 2% chlorhexidine gluconate, and 17% EDTA with and without an antifungal agent. J Endod. 2010;36:675-8.
29. Akbas MY, Kokumer T. The prevention and removal of biofilm formation of Staphylococcus aureus strains isolated from raw milk samples by citric acid treatments. Int J Food Sci Technol. 2015;50(7):1666-72.
30. Faot F, Cavalcanti YW, Mendonca e Bertolini M, Pinto Lde R, da Silva WJ, Cury AA. Efficacy of citric acid denture cleanser on the Candida albicans biofilm formed on poly(methyl methacrylate): effects on residual biofilm and recolonization process. BMC Oral Health. 2014;14:77.
31. Souza JGS, Cordeiro JM, Lima CV, Barao VAR. Citric acid reduces oral biofilm and influences the electrochemical behavior of titanium: An in situ and in vitro study. J Periodontol. 2019;90:149-58.
32. Malheiros CF, Marques MM, Gavini G. In vitro evaluation of the cytotoxic effects of acid solutions used as canal irrigants. J Endod. 2005;31:746-8..
33. Scelza MZ, de Noronha F, da Silva LE, Mauricio M, Gallito MA, Scelza P. Effect of citric acid and ethylenediaminetetraacetic acid on the surface morphology of young and old root dentin. Iran Endod J. 2016;11:188-91.
34. Calt S, Serper A. Time-dependent effects of EDTA on dentin structures. J Endod. 2002;28:17-9.
35. Serper A, Calt S. The demineralizing effects of EDTA at different concentrations and pH. J Endod. 2002;28:501-2.
36. Zehnder M. Root canal irrigants. J Endod. 2006;32:389-98.
37. Wang Z, Shen Y, Haapasalo M. Effectiveness of endodontic disinfecting solutions against young and old Enterococcus faecalis biofilms in dentin canals. J Endod. 2012;38:1376-9.