María Teresa Arias-Moliz

Enterococcus faecalis Biofilms Eradication by Root Canal Irrigants

The aim of this study was to evaluate the minimal biofilm eradication concentration (MBEC) of sodium hypochlorite (NaOCl), chlorhexidine (CHX), EDTA, and citric and phosphoric acids after 1, 5, and 10 minutes of exposure to biofilms of Enterococcus faecalis. The biofilms grew in the MBEC high-throughput device for 24 hours at 37 degrees C and were exposed to 10 serial two-fold dilutions of each irrigating solution. The viable cell counts were log(10) transformed, and a concentration of an irrigant was considered to eradicate the biofilms when it produced a reduction of > or = 5 logarithmic units. NaOCl was the most effective agent, capable of eradicating the biofilms after 1 minute at a concentration of 0.00625%. CHX eradicated biofilm after 5 minutes at 2%. EDTA and citric and phosphoric acid solutions were not effective against the biofilms at any concentration or time tested.

Eradication of Enterococcus faecalis biofilms by cetrimide and chlorhexidine.

INTRODUCTION Enterococcus faecalis is the most commonly isolated bacteria from root canals of teeth with persistent periapical periodontitis. Its ability to grow as a biofilm impedes the elimination of E. faecalis by using irrigating solutions. The purpose of this study was to assess the efficacy of cetrimide and chlorhexidine (CHX), alone and in association, in combined and alternating form, in eradicating biofilms of E. faecalis. METHODS Biofilms grown in the MBEC-high-throughput device for 24 hours were exposed to irrigating solutions for 30 seconds and 1 and 2 minutes. Eradication was defined as 100% kill of biofilm bacteria. The Student t test was used to compare the efficacy of the associations of the 2 irrigants. RESULTS Cetrimide eradicated E. faecalis biofilms at concentrations of 0.5%, 0.0312%, and 0.0078% at 30 seconds and 1 and 2 minutes of contact time, respectively. CHX did not eradicate the biofilms at any of the concentrations (4% initial concentration) or times assayed. The association of 0.1% and 0.05% cetrimide with any concentration of CHX, whether in combined or alternating application, effectively eradicated E. faecalis biofilms at all the contact times tested. Eradication was also achieved with 0.02% and 0.01% cetrimide at 2 minutes. Statistical analysis revealed significantly better results with alternating rather than combined use of cetrimide and CHX (P < .05). CONCLUSIONS The associated use of cetrimide and CHX provided better results than their applications as single agents against E. faecalis biofilms, and the alternating application was significantly more effective than the combined mode of application.

Residual and Antimicrobial Activity of Final Irrigation Protocols on Enterococcus Faecalis Biofilm in Dentin

INTRODUCTION The use of root canal irrigating solutions exerting antimicrobial activity and prolonged residual activity is desirable in order to control dentin infection and delay reinfection of the root canal. The aim of this study was to evaluate the residual antimicrobial activity and the capacity to eradicate Enterococcus faecalis biofilm of different irrigating solutions, alone and in combination, in a dentin-volumetric test. METHODS Solutions of 2.5% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), 0.2% cetrimide (CTR), 17% ethylendiaminetetraacetic (EDTA), 7% maleic acid (MA), and regimens of 2.5% NaOCl followed by 17% EDTA or 7% MA and 0.2% CTR or 2% CHX were used to determine their residual activity by exposing treated dentin blocks to E. faecalis for 24 hours. Antimicrobial activity was assayed on 3-week biofilm formed on dentin blocks. Results of residual activity and antimicrobial activity were respectively expressed as the inhibition percentage of biofilm formation and the kill percentage of biofilm. RESULTS A 2% CHX and 0.2% CTR solution showed 100% biofilm inhibition; 2.5% NaOCl showed the lowest residual activity (18.10%). The kill percentage of 2.5% NaOCl and 0.2% CTR was 100% followed by 7% MA and 2% CHX, whereas 17% EDTA was the least effective (44%). Solutions of 7% MA or 17% EDTA followed by 0.2% CTR or 2% CHX showed 100% residual and antimicrobial activity. CONCLUSIONS A 0.2% CTR solution alone and the combinations in which 2% CHX or 0.2% CTR was the final irrigating solution achieved the maximum residual and antimicrobial activity.

Residual Effectiveness of Final Irrigation Regimens on Enteroccus faecalis-infected Root Canals

INTRODUCTION The application of a final irrigating solution that remains active over a fairly long period of time stands as one strategy for preventing bacterial recolonization or eliminating the bacteria that persist after root canal treatment. The aim of this study was to evaluate the residual antimicrobial activity of four final irrigation regimens in root canals contaminated with Enterococcus faecalis. METHODS Biofilms of E. faecalis were grown in uniradicular roots for 4 weeks. After preparing the roots chemomechanically, four final irrigation regimens were applied: (1) group EDTA-NaOCl, 17% EDTA followed by 5.25% sodium hypochlorite (NaOCl); (2) group MA-NaOCl, 7% maleic acid (MA) followed by 5.25% NaOCl; (3) group EDTA-CHX + CTR, 17% EDTA followed by 2% chlorhexidine (CHX) + 0.2% cetrimide (CTR); and (4) group MA-CHX + CTR, 7% MA followed by 2% CHX + 0.2% CTR. Samples were collected for 60 days to denote the presence of bacterial growth. The Fisher exact test was used to compare the percentages of specimens without E. faecalis regrowth. RESULTS All root canals in which the final irrigant was 5.25% NaOCl yielded positive cultures on the fifth day. Groups EDTA-CHX + CTR and MA-CHX + CTR with a final irrigation of 2% CHX + 0.2% CTR showed respective percentages of samples without regrowth of 72.1% and 66.8% at 60 days. There were no statistically significant differences between these groups. CONCLUSIONS The combination of 2% CHX + 0.2% CTR would be an effective alternative final irrigation regimen given its antimicrobial action over time.

Antimicrobial substantivity over time of chlorhexidine and cetrimide.

INTRODUCTION To reach an adequate control of dentin infection and to promote success in endodontic therapy, antimicrobial irrigating solutions with confirmed substantivity are recommended. The aim of the present study was to evaluate the antimicrobial substantivity against Enterococcus faecalis of a dentin-volumetric unit exposed for 1 minute to chlorhexidine (CHX) and cetrimide (CTR). METHODS Standardized coronal dentin blocks of human molars, with and without collagen, were treated for 1 minute with 0.2% and 2% CHX and 0.2% CTR. Afterwards, they were exposed to E. faecalis suspension to determine the antimicrobial substantivity over a period of 60 days. Results were analyzed by means of Kaplan-Meier survival analysis (P < .05). RESULTS A direct relationship was seen between CHX concentration and survival time, and the most statistically significant results were obtained in specimens with collagen. CTR showed intermediate survival values close to those of 2% CHX. CONCLUSIONS The present study shows that 2% CHX used for 1 minute provides the longest substantivity followed by 0.2% CTR when applied to a dentin-volumetric model.

Bactericidal activity of phosphoric acid, citric acid, and EDTA solutions against Enterococcus faecalis

OBJECTIVES The objectives of this study were to evaluate the minimal bactericidal concentration (MBC) for Enterococcus faecalis of phosphoric acid, citric acid, and ethylenediaminetetraacetic acid (EDTA) solutions, and to determine the contact time required for 2.5% and 5% phosphoric acid, 10% and 25% citric acid, and 17% EDTA to exert bactericidal activity. STUDY DESIGN Bactericidal activity was tested by means of the dilution neutralization method in accordance with BS-EN-1040:2005 norm, using contact times of 0.5 to 60 minutes. RESULTS The MBCs of citric and phosphoric acid were 20% and 2.5%, respectively. EDTA solution lacks bactericidal activity, even after 60 minutes of contact. The 2.5% and 5% phosphoric acid solutions required 5- and 3-minute contact times, and the 10% and 25% citric acid solutions required 10- and 3-minute contact times, respectively. CONCLUSIONS Phosphoric acid revealed bactericidal activity against E. faecalis and required less time than citric acid to exert its activity.

Antimicrobial Activity of a Sodium Hypochlorite/Etidronic Acid Irrigant Solution

INTRODUCTION The aim of this study was to evaluate the antimicrobial activity of a 2.5% sodium hypochlorite (NaOCl)/9% etidronic acid (HEBP) irrigant solution on Enterococcus faecalis growing in biofilms and a dentinal tubule infection model. METHODS The antimicrobial activity of the solutions 2.5% NaOCl and 9% HEBP alone and associated was evaluated on E. faecalis biofilms grown in the Calgary biofilm model (minimum biofilm eradication concentration high-throughput device). For the dentinal tubule infection test, the percentage of dead cells in E. faecalis-infected dentinal tubules treated with the solutions for 10 minutes was measured using confocal laser scanning microscopy and the live/dead technique. Available chlorine and pH of the solutions were also measured. Distilled water was used as the control. Nonparametric tests were used to determine statistical differences. RESULTS The highest viability was found in the distilled water group and the lowest in the NaOCl-treated dentin (P < .05). Both NaOCl solutions killed 100% of the E. faecalis biofilms and showed the highest antimicrobial activity inside dentinal tubules, without statistical differences between the 2 (P < .05). The HEBP isolated solution killed bacteria inside dentinal tubules but did not present any significant effect against E. faecalis biofilms. The incorporation of HEBP to NaOCl did not cause any loss of available chlorine within 60 minutes. CONCLUSIONS HEBP did not interfere with the ability of NaOCl to kill E. faecalis grown in biofilms and inside dentinal tubules.

Residual Activity of Chelating Agents and their Combinations with Cetrimide on Root Canals Infected with Enterococcus faecalis

INTRODUCTION The use of chelating agents in endodontic treatment is required to remove the smear layer and to achieve better disinfection within the root canal system. The aim of this study was to evaluate the residual antimicrobial activity of 17% ethylenediaminetetraacetic acid (EDTA), 7% maleic acid (MA), and 10% lactic acid (LA) alone and combined with 0.2% cetrimide (CTR) on infected root canals with Enterococcus faecalis. METHODS Biofilms of E. faecalis were grown in uniradicular roots for 4 weeks. After preparation the roots were randomly divided into 6 groups (n = 15) according to the final irrigation regimen as follows: 5 mL of 17% EDTA; 5 mL of 17% EDTA + 0.2% CTR; 5 mL of 7% MA; 5 mL of 7% MA + 0.2% CTR; 5 mL of 10% LA; and 5 mL of 10% LA + 0.2% CTR. Samples were collected for 60 days to denote the presence of bacterial growth. Fisher and Pearson χ(2) tests were applied to compare the percentages of specimens without E. faecalis regrowth at all the time periods. RESULTS There were significant differences in bacterial regrowth between the groups with and without CTR. There were not significant differences over time among the 3 groups with CTR or among the 3 without CTR. CONCLUSIONS The residual activity against E. faecalis of 7% MA + 0.2% CTR, 17% EDTA + 0.2% CTR, and 10% LA + 0.2% CTR was greater than the use of chelating agents alone.

Antimicrobial activity of alexidine alone and associated with N-acetylcysteine against Enterococcus faecalis biofilm

The purpose of this study was to assess the efficacy of alexidine (ALX), alone and combined with N-acetylcysteine (NAC), in eradicating two Enterococcus faecalis strain biofilms. The biofilms of E. faecalis ATCC 29212 and the clinical isolate E. faecalis D1 were grown in the MBEC-high-throughput device for 24 h and were exposed to five twofold dilutions of ALX (2%–0.007 8%) alone and combined with 100 mg⋅mL−1 NAC, for 1 and 5 min. Eradication was defined as 100% kill of biofilm bacteria. The Student’s t-test was used to compare the efficacy of the associations of the two irrigants. After 1-min contact time, ALX eradicated the biofilms at all concentrations except for 0.007 8% and 0.015 6%–0.007 8% with E. faecalis ATCC 29212 and E. faecalis D1, respectively. Similar results for eradication and concentration were obtained when it was combined with 100 mg⋅mL−1 NAC. After 5 min of contact time, ALX alone and combined with NAC eradicated all enterococci biofilms. ALX showed antimicrobial properties against the two E. faecalis strain biofilms tested at very low concentrations, and its combined use with NAC was not seen to enhance its activity.

Eradication of enterococci biofilms by lactic acid aloneand combined with chlorhexidine and cetrimide

Objective: The antimicrobial activity of lactic acid (LA) alone or in combination with chlorhexidine (CHX) and cetrimide (CTR) against three Enterococcus faecalis strains, E. faecalis ATCC 29212, E. faecalis EF-D1 and E. faecalis U-1765, one Enterococcus durans strain and one dual-species biofilm was investigated. Study Design: The irrigating solutions tested were 20%, 15%, 10%, 5% and 2.5% LA, alone and in combination with 2% CHX and with 0.2% CTR. The biofilms were grown in the MBECTM high-throughput device for 24 hours and exposed to the solutions for 30 seconds and 1 minute. “Eradication” was defined as 100% bacterial kill. Results: Twenty percent LA eradicated all enterococci biofilms after 30 seconds contact time. The association of LA + 0.2% CTR achieved better results than LA alone, in contrast with the results obtained using LA + 2% CHX. E. durans was eradicated by all the tested solutions at 1 minute. The dual-species biofilm, E. faecalis ATCC 29212 + E. durans, gave intermediate values of the pure cultures. Conclusions: LA is capable of eradicating enterococci biofilm at a concentration of 20%. The combination of lower concentrations with 0.2% CTR achieved eradication after 1 minute. Key words:Biofilm, cetrimide, chlorhexidine, enterococcus durans, enterococcus faecalis.