Antimicrobial activity of chlorhexidine and calcium hydroxide gels: an in vitro evaluation

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1 Antimicrobial activity of chlorhexidine and calcium hydroxide gels: an in vitro evaluation Atividade antimicrobiana dos géis de clorexidina e hidróxido de cálcio: avaliação in vitro Original Research Article Artigo Original de Pesquisa Cláudia Ramos PINHEIRO 1, Celso Kenji NISHIYAMA 2, Renata Pardini HUSSNE 3, Melina Vieira BORTOLO 4, Carla Renata SIPERT 5, Sérgio Aparecido TORRES 6 ABSTRACT Objective: The aim of this study was to determine in vitro the antimicrobial activity of calcium hydroxide and chlorhexidine gels against various microorganisms. Methods: The diffusion method on double layer Müller-Hinton agar (MH) was employed and five wells were made at equidistant points. The substances were placed into the wells and the microorganisms Enterococcus faecalis, Candida albicans, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa were seeded by pour plate. Results: The gels of calcium hydroxide and chlorhexidine presented antimicrobial activity against all m icroorganisms. Association of both substances was more effective against Enterococcus faecalis and Candida albicans, when compared to calcium hydroxide gel alone, although this association produced halos with smaller diameters when compared to chlorhexidine gel used alone. Conclusions: The tested substances were effective against all microorganisms; larger inhibition halos were observed against Enterococcus faecalis and Candida albicans; the present methodology did not allow observation of a synergistic effect of this association. Key-words: bacteria; calcium; chlorhexidine; hydroxide; Endodontics. Palavras-chave: bactérias; hidróxido de cálcio; clorexidina; Endodontia. Recebido em: 08/02/2009 Aceito em: 04/05/2009 RESUMO Objetivo: O propósito deste estudo foi determinar a atividade antimicrobiana in vitro dos géis de hidróxido de cálcio e de clorexidina em diferentes microrganismos. Métodos: O método de dupla camada de difusão em agar Muller-Hinton foi empregado e cinco poços foram confeccionados a pontos equidistantes. As substâncias foram posicionadas dentro dos poços e os microrganismos Enterococcus faecalis, Candida albicans, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis e Pseudomonas aeruginosa foram inoculados sobre as substâncias. Resultados: Os géis de hidróxido de cálcio e de clorexidina apresentaram atividade antimicrobiana contra todos os microrganismos. A associação de ambas as substâncias foi mais efetiva contra Enterococcus faecalis e Candida albicans. Quando o hidróxido de cálcio foi utilizado isoladamente, produziu halos menores quando comparado com a clorexidina. Conclusões: As substâncias testadas foram efetivas contra todos os microrganismos; halos de inibição com diâmetros maiores foram encontrados contra as cepas de Enterococcus faecalis e Candida albicans. A metodologia empregada não permitiu constatar um efeito potencializador ou sinérgico dessa associação. Correspondence author: Cláudia Ramos Pinheiro Rua Capitão Gomes Duarte, apto Bauru SP Brasil Telefone: +55 (14) / (14) claudiaendo@gmail.com 1 MSc, Discipline of Endodontics, Araraquara Dental School (UNESP), SP, Brazil. 2 PhD, Head of the Department of Endodontics; Hospital for Rehabilitation of Craniofacial Anomalies University of São Paulo, Bauru, SP, Brazil. 3 PhD student, Discipline of Endodontics, Araraquara Dental School (UNESP), SP, Brazil; Endodontist of the Hospital for Rehabilitation of Craniofacial Anomalies University of São Paulo, Bauru, SP, Brazil. 4 MSc student, Discipline of Endodontics, Bauru Dental School (FOB), University of São Paulo, SP Brazil. 5 PhD student, Discipline of Oral Biology, Bauru Dental School (FOB), University of São Paulo, SP, Brazil. 6 PhD, Professor of the Discipline of Microbiology, Department of Biological Sciences, Bauru Dental School (FOB), University of São Paulo, SP, Brazil. POS Perspect. Oral Sci. v.1 n2 DEZ 2009 ISSN

2 PINHEIRO et al. CHX and gels: an in vitro evaluantion INTRODUCTION Bacterial invasion of the root canal system plays an essential role in the onset and maintenance of periapical diseases; that s why elimination of these microorganisms and possible reinfection is the goal of endodontic treatment. 1 Biomechanical preparation and root canal shaping effectively reduce the microbiota; however, they do not eliminate completely the presence of bacteria in the lateral and accessory root canals, isthmi and apical deltas, thus requiring association of proper medication between treatment sessions in addition to chemicalmechanical root canal preparation 2 in order to allow better disinfection of this area. Calcium hydroxide [ ] has antibacterial and anti-exudative activities, capacity of neutralizing the acidic ph and inducing mineralization. These characteristics are related to its alkalinity (ph around 12.5) and consequent release of calcium ions. 3 According to Lynne et al., 4 the bactericidal effect of may be at least partially assigned to its high ph, which is unfavorable for the bacteria. This substance has antimicrobial action against many anaerobic species that invade the root canal system; however, some of them are resistant to, such as Candida albicans (C. albicans) and Enterococcus faecalis (E. faecalis). 5 Chlorhexidine (CHX) is characterized as a wide spectrum antimicrobial agent, acting against both aerobic and anaerobic bacteria and those resistant to. According to the findings of Hauman and Love, 6 CHX is a biocompatible and innocuous substance, since it does not irritate the periapical tissues, even at high concentrations. Moreover, CHX has the property of substantivity, which consists in maintaining its antibacterial action for a long lasting period when adhered to anionic substrates, being slowly released as its concentration is decreased, allowing the active principle to contact the root canal walls and dentinal tubuli Dametto et al. 11 compared the antimicrobial action of 2% CHX gel and 5.25% sodium hypochlorite (NaOCl) to reduce the presence of E. faecalis in contaminated dentin debris. In this study, the gel was more effective than NaOCl, which was unable to keep the root canal free from this microorganism in the final sample. Some studies, such as those conducted by Gomes et al. 9 and Sirén et al. 12 have tested the association between with CHX gel in an attempt to improve the properties of both. Schäfer et al. 13 investigated the efficacy of 2% CHX gel and paste against E. faecalis. Each substance was analyzed separately and in association. The efficacy of paste was not improved when CHX was added, and they concluded that 2% CHX gel was the most effective substance. Stuart et al. 14 analyzed the ability of and CHX gel to eliminate the E. faecalis. The association of and 2% CHX gel was favorable, since the ph was kept at 12.8, making the environment unfavorable to the bacteria; however, it was not more effective than 2% CHX gel, when used as an intracanal medication for a period of seven days. Gomes et al. 15 investigated the antimicrobial activity of associated with 2% CHX gel against endodontic pathogens and compared the results with the application of manipulated with sterile water and by CHX gel alone. The methodology used was the agar diffusion test. After the initial 24 hours, the inhibition halos were measured around the wells. The authors concluded that 2% CHX gel associated with showed a higher antimicrobial activity when compared to manipulated with sterile water or CHX gel alone. De Souza-Filho et al. 10 evaluated the effectiveness of 2% CHX gluconate gel, and their association with iodoform and zinc oxide powder as intracanal medications against E. faecalis, Streptococcus sanguis (S. sanguis), Streptococcus sobrinus (S. sobrinus), Streptococcus mutans (S. mutans), Phorphyromonas gingivalis (P. gingivalis) and Prevotella intermedia (P. intermedia). The authors also measured the ph changes caused by these medications. Antimicrobial activity was determined by the agar diffusion method. The zones of growth inhibition were measured and statistical analysis was performed. The ph of the pastes was measured right after preparation, 24 hours and 1 week later. The largest means of inhibition halos were produced by 2% CHX gel and the smallest values were attributed to manipulated with water. The mean ph of all medications was kept above 12.0 during the whole experiment, except for CHX gel (ph = 7.0). The results of this study showed that all medications had antimicrobial activity, but the most effective against the tested microorganisms was 2% CHX gel, followed by its association with and iodoform. Therefore, the purpose of this study was to evaluate the antimicrobial activity of chlorhexidine and calcium hydroxide gels, through an in vitro study. MATERIALS AND METHODS The antimicrobial activity was performed by the double layer technique, with a base of 10.0 ml of Müller-Hinton (MH) agar (Difco, Detroit, MI, USA) and the addition of 2% bacteriological agar (Difco) distributed on sterile Petri plates measuring 20 x 100 mm (Real Lab, Ribeirão Preto, SP, Brazil). After solidification, the sterilization of each plate was checked out. Five perforations with 6 x 4 mm were performed at equidistant points and the materials to be tested were placed in these wells (agar well method) in quadruplicate. Previously, the microorganism had been reactivated from culture stock frozen for observation of cell and colony morphology, in order to check the inoculums purity. The 14 POS Perspect. Oral Sci. v.1 n2 DEZ 2009 ISSN

3 PINHEIRO et al. CHX and gels: an in vitro evaluantion microorganisms were cultivated in MH broth and then they were set in the incubator (Alemmar, Rio de Janeiro, RJ, Brazil) at 37ºC for 24 hours. For the inoculum, the culture was employed, in order to standardize the final concentration of 1.5 x 108 cells/ml (0.5 of MacFarland scale, which corresponds to a concentration of 108 ml-1), through the 630-nm-wavelength spectrophotometer (Pharmacia Biotech, São Paulo, SP, Brazil). The study was performed against standard strains E. faecalis ATCC 29212, C. albicans ATCC 10231, Escherichia coli (E.coli) ATCC 25922, Staphylococcus aureus (S.aureus) ATCC 25923, Staphylococcus epidermidis (S.epidermidis) ATCC and Pseudomonas aeruginosa (P.aeruginosa) ATCC After the placement of each substance, 15.0 ml of MH agar without the inoculum were poured on one plate, taken as negative control, and the other 4 plates 15.0 ml of MH agar containing the microorganisms were poured, just to achieve a final concentration corresponding to 0.5 of McFarland scale ( cells/ml). The study evaluated the antimicrobial activity of: 2% CHX gel [2% CHX gluconate in natrosol gel (2:1)]; gel [calcium hudroxide powder in natrosol gel (2:1)]; natrosol gel and the association of 2% CHX and gels [2% chlorhexidine gluconate and calcium hydroxide powder in natrosol gel (2:2:1)]. All the substances were prepared by the same manufacturer (Specifica Pharmacy, Bauru, SP, Brazil). Zinc oxide and eugenol (SS White, Rio de Janeiro, RJ, Brazil) was used as positive control. The plates were kept at room temperature for 2 hours for pre-diffusion of the material and then incubated at 37ºC for 48 hours. After the initial 24 hours, the diffusion and inhibition halos around the wells were measured with the aid of a millimeter ruler with 0.5 mm precision. Then, 10.0 ml aliquots of triphenyltetrazolium chloride (TTC gel) (Sigma, Saint Louis MO, USA) prepared with 1% agar and 0.05% TTC were distributed on the plate with the bacterial inoculums. After solidification, the plates were again incubated at 37ºC for 30 minutes to optimize the reading of inhibition halos, following the procedures previously reported by Leonardo et al. 16 and Sipert et al. 17 RESULTS Table 1 demonstrates the mean inhibition halos obtained for the substances tested on different microorganisms for a 24-hour period. Further measurements were performed after 48 hours; at this moment, the values obtained were the same as those observed at 24 hours, only the substances continued to diffuse. Therefore inhibition halos were detected against P. aeruginosa, E. coli, S. aureus and S. epidermidis. Calcium hydroxide and 2% CHX gels individually showed antimicrobial activity against all strains investigated, which revealed larger inhibition halos against the strains of E. faecalis and C. albicans when compared to the other microorganisms. Association of 2% CHX and gels generated similar results as those achieved when the substances were tested separately; however it may be observed that this association seemed to be more effective against both E. faecalis and C. albicans, respectively, when compared to individually, but producing halos with smaller diameters against both microorganisms when compared to CHX alone. Calcium hydroxide gel or its association with 2% CHX showed diffusion halos on MH agar, detected on the control plate, which did not contain microorganisms. These values were higher when compared to the plates on which the microorganisms were plated. DISCUSSION The presence of remaining bacteria in the root canals may be reduced by using intracanal medication between the sessions of endodontic therapy. 1-3,10 This procedure is useful for achieving success in endodontic therapy. However, the efficacy of clinically employed substances may vary according to the methodology used in each study. The agar diffusion method has been widely employed to investigate the antimicrobial activity of dental materials and intracanal medications, 16,17 however this procedure does not depend only on the material Table 1 - Mean diffusion (control plate) and inhibition halos, in millimeters, for each substance tested against different microorganisms after 24 hours made in quadruplicate. Substances Microorganisms C. albicans E. coli E. faecalis S. aureus S. epidermidis P. aeruginosa Control plate 2% CHX* gel Not visible ** gel Gel*** Not visible 2% CHX + gels ZOE**** Not visible * Chlorhexidine; ** Calcium hydroxide; *** Hydroxyethylcellulose vehicle gel; **** Zinc oxide-eugenol. POS Perspect. Oral Sci. v.1 n2 DEZ 2009 ISSN

4 PINHEIRO et al. CHX and gels: an in vitro evaluantion toxicity to a given microorganism, but may also be influenced by the diffusion and affinity of the material in the culture medium. A material presenting easier diffusion will produce larger zones of inhibition of bacterial growth. 10,14,18 That s why no statistical analysis was performed in this study. Since the diameter of the inhibition zone does not necessarily reflect the efficacy of the antimicrobial agent, the activity of each material was evaluated considering the presence or absence of inhibition zone, as previously reported. 16,17 Calcium hydroxide has been used as intracanal medication for a long time, due to its antimicrobial property, ability to induce hard tissue formation when placed in the root canal system, alkaline ph (12.5) and dissociation into calcium and hydroxyl ions, making the environment unfavorable for bacterial proliferation. In fact, the substance acts by contact and its diffusion in agar is well delimited, being influenced by the vehicle employed. 8,9,19 Chlorhexidine is a cationic salt (bisguanidine) with wide antibacterial action, which presents the ability to adhere to anionic substrates for an extended period, which is called substantivity. 8 Two percent CHX gel was employed in the present study also because it was demonstrated to be innocuous to periradicular tissues. 20 In this in vitro study, it was evaluated the antimicrobial activity of and CHX, separately or in association, using as vehicle natrosol gel, allowing a better standardization. Its formula contains hydroxyethylcellulose, 0.1% butylhydroxytoluene (BHT) and 0.1% ethylenediaminetetraacetic acid disodium (EDTA), which makes this substance biocompatible, inert, non-ionic and water-soluble, as mentioned by Miyamoto et al. 21 The values presented on Table 1 indicate that there was antimicrobial action of all substances against the microorganisms investigated. Inhibition halos were detected against E. coli, S. aureus, S. epidermidis, P. aeruginosa, C. albicans and E. faecalis, however diffusion halos were only observed around groups containing, considering this substance has radiopacity, whereas for 2% CHX gel, these halos were only observed on plates on which the microorganisms were inoculated, since CHX is a transparent material and the gel used as vehicle is cristaline. 21 Calcium hydroxide gel or its association with 2% CHX gel showed visible diffusion halos in MH agar. This was clearly observed in the control plate, which did not contain microorganisms and in which values were higher than those observed in inoculated plates. The presence of this diffusion halo would influence or even impair the reading of antimicrobial activity, problem partly solved by addition of TTC gel. 16,17,21 Table 1 also reveals that gel showed antimicrobial activity against all strains tested, with observation of larger inhibition halos against E. faecalis and C. albicans, respectively (20.62 and mm), when compared to the other microorganisms. These data are in agreement with the findings of Lynne et al., 4 Schäfer et al. 22 and Estrela et al., 23 who conducted in vitro studies to evaluate the antimicrobial activity of the aforementioned substance in culture medium, reading the diameters of diffusion and inhibition halos after 24 hours. However, they disagree with the findings of Gomes et al., 8 Orstavikt et al., 24 Heling et al. 25 and Sukawat et al., 26 who employed different methodologies and observed that was unable to eliminate E. faecalis from the infected dentinal tubules and thus questioning the efficacy of this medication to reduce the pathogenic microbiota in the root canals. The results indicate that 2% CHX gel had antimicrobial efficacy against all microorganisms tested, showing higher values for inhibition halos against strains of E. faecalis and C. albicans, respectively (32.12 and mm), when compared to the other microorganisms. These results were similar to those observed in a clinical study conducted by Lima et al, 27 which demonstrated that 2% CHX completely eliminated the biofilm produced by the colonization of E. faecalis. Portenier et al. 28 observed that CHX was as effective as a substance made of isometric tetracycline, acid and detergent MTAD to completely eradicate E. faecalis from the infected dentinal tubuli. Our findings also agree with an in vitro study conducted by Lin et al., 29 which employed a similar methodology as in the present study, reporting that CHX inhibited the growth of E.faecalis; however our results are different from those observed by Gomes et al. 8 in an in vivo study, in which the E.faecalis was persistent in instrumented root canals medicated with or 2% CHX gel after several treatment sessions. The association of and 2% CHX gel was effective both against E. faecalis and C. albicans, with observation of inhibition halos of and mm, respectively, when compared to alone (20.62 and 19.00); however, this association produced smaller halos for both microorganisms when compared to CHX alone (32.12 and mm). These results agree with the studies conducted by Stuart et al., 14 who analyzed the ability of and CHX separately or in association, to eliminate E. faecalis from infected canals, and concluded that 2% CHX gel was more effective to eradicate the pathogen than the substances employed in combination. The methodology employed does not allow concluding whether there was potentiating or synergism of the substances, even though the association of both reduced the inhibition halo produced by CHX individually, partly corroborating with the findings of Gomes et al. 8 Zinc oxide and eugenol showed excellent antimicrobial activity against all microorganisms tested 17,30 because it has the capacity of diffusing in MH agar, producing a distinct inhibition halo, always taken as positive control in studies with this kind of methodology. Thus, according to the methodology presented in this study, it was possible to conclude that the tested 16 POS Perspect. Oral Sci. v.1 n2 DEZ 2009 ISSN

5 PINHEIRO et al. CHX and gels: an in vitro evaluantion substances were effective against all microorganisms; larger inhibition halos were observed against E. faecalis and C. albicans. The present methodology did not allow observation of a synergistic effect of this association. ACKNOWLEDGEMENTS The authors wish to thank the technical help provided by André Luis da Silva e Dalva Riberio from the Microbiology Laboratory of University of São Paulo, Bauru, SP, Brazil. REFERENCES 1. Vianna ME, Horz HP, Conrads G, Zaia AA, Souza- Filho FJ, Gomes BP. Effect of root canal procedures on endotoxins and endodontic pathogens. Oral Microbiol Immunol 2007; 22(6): Vianna ME, Gomes BP. Efficacy of sodium hypochlorite combined with chlorhexidine against Enterococcus faecalis in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 107(4): Allotta RC, Ribeiro MS, de Lima Machado ME. Determination of the minimum inhibitory concentration of four medicaments used as intracanal medication. Aust Endod J 2007; 33(3): Lynne RE, Liewehr FR, West LA, Patton WR, Buxton TB, Mcpherson JC. In vitro antimicrobial activity of various medication preparations on Enterococcus faecalis in root canal dentin. J Endod 2003; 29(3): Basrani B, Tjaderhane L, Santos JM, Pascon E, Grad H, Lawrence HP et al. Efficacy of chlorhexidine and calcium hydroxide-containing medicaments against Enterococcus faecalis in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003; 96(5): Hauman CH, Love RM. Biocompatibility of dental materials used in contemporary endodontic therapy: a review. Part 1. Intracanal drugs and substances. Int Endod J 2003; 36(2): Fava LRG, Conde MC, Siqueira JF Jr. Use of chlorhexidine in Endodontics: Current and future perspectives. J Bras Clin Odontol Int 2002; 5(8): Gomes BP, Souza SF, Ferraz CC, Teixeira FB, Zaia AA, Valdrighi L et al. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro. Int Endod J 2003; 36(4): Gomes BP, Sato E, Ferraz CC, Teixeira FB, Zaia AA, De Souza- Filho FJ. Evaluation of time required for recontamination of coronally sealed canals medicated with calcium hydroxide and chlorhexidine. Int Endod J 2003; 36(9): de Souza-Filho FJ, Soares Ade J, Vianna ME, Zaia AA, Ferraz CC, Gomes BP. Antimicrobial effect and ph of chlorhexidine gel and calcium hydroxide alone and associated with other materials. Braz Dent J 2008; 19(1): Dametto FR, Ferraz CC, de Almeida Gomes BP, Zaia AA, Teixeira FB, de Souza-Filho FJ. In vitro assessment of the immediate and prolonged antimicrobial action of chlorhexidine gel as an endodontic irrigant against Enterococcus faecalis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005; 99(6): Sirén EK, Haapasalo MP, Waltimo TM, Orstavik D. In vitro antibacterial effect of calcium hydroxide combined with chlorhexidine or iodine potassium iodide on Enterococcus faecalis. Eur J Oral Sci 2004; 112(4): Schäfer E, Bössmann K. Antimicrobial efficacy of chlorhexidine and two calcium hydroxide formulations against Enterococcus faecalis. J Endod 2005; 31(1): 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): Gomes BP, Ferraz CC, Zaia AA, Sena NT, Vianna ME, de Souza-Filho FJ. In vitro evaluation of the antimicrobial activity of calcium hydroxide combined with chlorhexidine gel used as intracanal medicament. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006; 102(4): Leonardo MR, da Silva LA, Tanomaru Filho M, Bonifácio KC, Ito IY. In vitro evaluation of antimicrobial activity of sealers and pastes used in endodontics. J Endod 2000; 26(7): Sipert CR, Hussne RP, Nishiyama CK, Torres SA. In vitro antimicrobial activity of Fill Canal, Sealapex, Mineral Trioxide Aggregate, Portland cement and EndoRez. Int Endod J 2005; 38(8): Thorn RM, Greenman J, Austin AJ. In vitro method to assess the antimicrobial activity and potential efficacy of novel types of wound dressings. J Appl Microbiol 2005; 99(4): Stevens RH, Grossman LI. Evaluation of the antimicrobial potential of calcium hydroxide as an intracanal medicament. J Endod 1983; 9(9): Komorowski R, Grad H, Wu XY, Friedman S. Antimicrobial substantivity of chlorhexidine-treated bovine root dentin. J Endod 2000; 26(6): POS Perspect. Oral Sci. v.1 n2 DEZ 2009 ISSN

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