In vitro antimicrobial activity of several concentrations of sodium hypochlorite and chlorhexidine gluconate in the elimination of Enterococcus faecalis

AIM: The aim of this study was to assess, in vitro, the effectiveness of several concentrations of NaOCl (0.5%, 1%, 2.5%, 4% and 5.25%) and two forms of chlorhexidine gluconate (gel and liquid) in three concentrations (0.2%, 1% and 2%) in the elimination of E. faecalis. METHODOLOGY: A broth dilution test using 24-well cell culture plates was performed and the time taken for the irrigants to kill bacterial cells was recorded. Isolated 24 h colonies of pure cultures of E. faecalis grown on 10% sheep blood plus Brain Heart Infusion (BHI) agar plates were suspended in sterile 0.85% NaCI solution. The cell suspension was adjusted spectrophotometrically to match the turbidity of a McFarland 0.5 scale. One mL of each tested substance was placed on the bottom of wells of 24-well cell culture plates (Corning, NY), including the control group (sterile saline). Six wells were used for each time period and irrigant concentration. Two mL of the bacterial suspension were ultrasonically mixed for 10 s with the irrigants and placed in contact with them for 10, 30, and 45 s; 1, 3, 5, 10, 20, and 30 min; and 1 and 2 h. After each period of time, 1 mL from each well was transferred to tubes containing 2 mL of freshly prepared BHI + neutralizers in order to prevent a residual action of the irrigants. All tubes were incubated at 37 degrees C for 7 days. The tubes considered to have positive growth were those which presented medium turbidity during the incubation period. Data were analysed statistically by the Kruskal-Wallis test. with the level of significance set at P < 0.05. RESULTS: All irrigants were effective in killing E. faecalis. but at different times. Chlorhexidine in the liquid form at all concentrations tested (0.2%, 1% and 2%) and NaOCI (5.25%) were the most effective irrigants. However, the time required by 0.2% chlorhexidine liquid and 2% chlorhexidine gel to promote negative cultures was only 30 s and 1 min, respectively. CONCLUSIONS: Even though all tested irrigants possessed antibacterial activity, the time required to eliminate E. faecalis depended on the concentration and type of irrigant used.     

In vitro antimicrobial activity of sodium hypochlorite and chlorhexidine against selected single-species biofilms

AIM: To investigate the antimicrobial activity of 2.5% and 5.25% sodium hypochlorite and 2.0% chlorhexidine gel and liquid as endodontic-irrigating substances against selected single-species biofilms. METHODS: Single-species biofilms of Enterococcus faecalis, Staphylococcus aureus, Candida albicans, Prevotella intermedia, Porphyromonas gingivalis, Porphyromonas endodontalis and Fusobacterium nucleatum were generated on a cellulose nitrate membrane placed on agar medium. The biofilms were then immersed in the endodontic-irrigating substances for 30 s and also for 5, 10, 15, 30 and 60 min, with and without mechanical agitation. Sterile saline was used as control. After each time period, the membrane filters were then transferred to tubes containing 2 mL of fresh broth medium plus neutralizers (in order to prevent the residual action of the tested substances). The micro-organisms were suspended using a vortex, and the inoculum was serially diluted 10-fold. Aliquots of the dilutions were plated on 5% sheep blood agar medium, and incubated under adequate gaseous conditions. Colony-forming units were calculated. The samples were compared using the Friedman and Tukey test, when necessary, at a significance level of P < 0.05. RESULTS: Mechanical agitation promoted the effectiveness of the antimicrobial agents, resulting in less time to eliminate the same micro-organisms, except for S. aureus with 2.5% NaOCl. Antimicrobial agents in liquid presentation, especially 5.25% NaOCl and 2% chlorhexidine, killed the tested micro-organisms more rapidly. Saline did not inhibit the growth of any of the tested micro-organisms, with or without agitation, being statistically different (P < 0.05) from NaOCl and chlorhexidine. P. intermedia, P. gingivalis, P. endodontalis and F. nucleatum were eliminated in 30 s by all antimicrobial agents, with our without agitation, in contrast with the facultative and aerobe strains. CONCLUSIONS: Mechanical agitation improved the antimicrobial properties of the chemical substances tested using a biofilm model, favouring the agents in liquid presentation, especially 5.25% NaOCl and 2% chlorhexidine.     

Evaluation of the antimicrobial activities of chlorhexidine gluconate,sodium hypochlorite and octenidine hydrochloride in vitro

The objective of this study was to compare the antimicrobial activity of sodium hypochlorite (NaOCl), chlorhexidine gluconate (CHX) and octenidine hydrochloride (OCT) in different concentrations against endodontic pathogens in vitro. Agar diffusion procedure was used to determine the antimicrobial activity of the tested materials. Enterococcus faecalis, Candida albicans and the mixture of these were used for this study. In the agar diffusion test, 5.25% NaOCl exhibited better antimicrobial effect than the other concentrations of NaOCl for all strains. All concentrations of OCT were effective against C. albicans and E. faecalis. Some 0.2% CHX was ineffective on all microorganisms. Antibacterial effectiveness of all experimental solutions decreased on the mixture of all strains. Decreasing concentrations of NaOCl resulted in significantly reduced antimicrobial effect.     

Dissolution of pulp tissue by aqueous solution of chlorhexidine digluconate and chlorhexidine digluconate gel

AIM: To evaluate the activity of various root canal irrigants on bovine pulp tissue. METHODOLOGY: The irrigants tested were: 0.5, 1.0 and 2.5% sodium hypochlorite; 2% aqueous solution of chlorhexidine digluconate; 2% chlorhexidine digluconate gel (Natrosol); and distilled water as control. Bovine pulp fragments were weighed and placed in contact with 20 mL of each tested substance in a centrifuge at 150 r.p.m. until total dissolution. Dissolution speed was calculated by dividing pulp weight by dissolution time. Statistical analysis was performed using the Kruskal-Wallis test. RESULTS: Distilled water and both solutions of chlorhexidine did not dissolve the pulp tissue within 6 h. Mean dissolution speeds for 0.5, 1.0 and 2.5% sodium hypochlorite solutions were 0.31, 0.43 and 0.55 mg min(-1), respectively. The solvent ability of chlorhexidine solutions was similar to that of distilled water. The results for sodium hypochlorite solutions, chlorhexidine solutions and distilled water were statistically different (P>0.01). CONCLUSIONS: Both chlorhexidine preparations and distilled water were not able to dissolve pulp tissue. All sodium hypochlorite solutions were efficient in dissolving pulp tissue; the dissolution speed varied with the concentration of the solution.     

Efficacy of various concentrations of NaOCl and instrumentation techniques in reducing Enterococcus faecalis within root canals and dentinal tubules

AIM: To evaluate the efficacy of 0.5%, 2.5% and 5.25% sodium hypochlorite (NaOCl) as intracanal irrigants associated with hand and rotary instrumentation techniques against Enterococcus faecalis within root canals and dentinal tubules. METHODOLOGY: A total of 180 extracted human premolar teeth were infected for 21 days with E. faecalis. The specimens were divided into 12 groups, as follows: group 1: 5.25% NaOCl + Hybrid technique (Valdrighi et al. 1998); group 2: 5.25% NaOCl + nickel-titanium (NiTi) rotary technique 4 mm shorter than the apex (by FOP-UNICAMP); group 3: 5, 25% NaOCl + NiTi rotary technique (Hero 642); group 4: 2.5% NaOCl +Hybrid technique; group 5: 2.5% NaOCl + NiTi rotary technique 4 mm shorter than the apex; group 6: 2.5% NaOCl + NiTi rotary technique (Hero 642); group 7: 0.5% NaOCl + Hybrid technique; group 8: 0.5% NaOCl + NiTi rotary technique 4 mm shorter than the apex; group 9: 0.5% NaOCl + NiTi rotary technique (Hero 642); group 10: sterile saline solution + Hybrid technique; group 11: sterile saline solution + NiTi rotary technique 4 mm shorter than the apex; group 12: sterile saline solution + NiTi rotary technique (Hero 642). Canals were sampled before and after preparation. After serial dilution, samples were plated onto brain heart infusion (BHI) agar, and the colony forming units (CFU) that were grown were counted. The teeth were sectioned into three thirds and dentine chips were removed from the canals with conical burs. The samples obtained with each bur were immediately collected into test tubes containing BHI broth, and were incubated at 37 degrees C and plated onto BHI agar. The CFU were counted and analysed. RESULTS: At all depths and thirds of the root canals and for all techniques used, 5.25% NaOCl was shown to be the most effective irrigant solution tested when dentinal tubules were analysed, followed by 2.5% NaOCl. No differences among concentrations in cleaning the canals were found. CONCLUSIONS: Especially at higher concentrations, NaOCl, was able to disinfect the dentinal tubules, independent of the canal preparation technique used.     

Antimicrobial activity of sodium hypochlorite and chlorhexidine by two different tests

The aim of this study was to evaluate the antimicrobial capacity of sodium hypochlorite (1% and 5%) and chlorhexidine (0.12%, 0.5% and 1%) with or without the addition of organic material (bovine serum albumin, BSA) against some bacterial samples (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Porphyromonas gingivalis and Fusobacterium nucleatum) using two activity tests (contact and diffusion agar tests). In the contact test (first model), bacterial samples were kept in contact with each irrigating solution for different time intervals: immediately (t(0)), 5 min (t(5)), 15 min (t(15)) and 30 min (t(30)). The agar diffusion test was the second model used. In half the specimens, 0.5% BSA was added to simulate organic tissue present in the root canal. Bacterial growth was evaluated for each microorganism and activity test. Each test was repeated 10 times. In the contact test, 0.12% chlorhexidine solution (CHX) did not eliminate E. faecalis at any tested time. CHX at 0.5% eliminated all strains except E. faecalis after immediate contact. All strains were eliminated by 1% CHX, 1% NaOCl and 5% NaOCl. BSA did not interfere with the antimicrobial activity of the irrigating solutions. In the agar diffusion test, all solutions exhibited zones of antimicrobial activity; however, BSA interfered with the antimicrobial activity of NaOCl and CHX. Under the condition of the contact test, the 0.12% CHX was ineffective in eliminating E. faecalis, while 0.5% CHX, 1% CHX, 1% NaOCl and 5% NaOCl showed antibacterial effectiveness against all the tested bacterial strains. The addition of an organic load interfered with the accuracy of the agar diffusion test.     

In vitro evaluation of the effectiveness of irrigants and intracanal medicaments on microorganisms within root canals

AIM: To evaluate in vitro the effectiveness of sodium hypochlorite (NaOCl), chlorhexidine (CHX) and five intracanal medicaments on microorganisms within root canals. METHODOLOGY: Ninety-six human single-rooted extracted teeth were used. After removing the crowns, canal preparation was completed and the external root surfaces were coated with epoxy resin. Following sterilization, the teeth were contaminated with Candida albicans and Enterococcus faecalis, and were incubated at 37 +/- 1 degrees C for 7 days. The teeth were divided according to the irrigant solution or intracanal medicament: group 1, sterile physiologic solution (SPS) and calcium hydroxide (Ca(OH)2) paste; group 2, SPS and camphorated paramonochlorophenol (CPMC); group 3, SPS and tricresol formalin; group 4, SPS and CaOH2 + CPMC paste; group 5, SPS and PMC furacin; group 6, 2.5% NaOCl without intracanal medication; group 7, 2.0% CHX without intracanal medication and group 8, SPS without intracanal medication (control group). Microbiological samples were collected with sterile paper points, and bacterial growth was determined. The data were submitted to the analysis of variance (anova, P = 0.05). RESULTS: For C. albicans, groups 3 and 8 were statistically less effective than groups 1, 2, 4 and 5 (Kruskal-Wallis (K-W) = 65.241; gl = 7; P = 0.001). For E. faecalis, groups 6 and 8 were statistically less effective than groups 1-4 and 7 (K-W = 61.048; gl = 7; P = 0.001). CONCLUSIONS: Ca(OH)2 + CPMC paste was the most effective intracanal medicament for the elimination of the two microorganisms; 2.0% CHX solution was more effective than 2.5% NaOCl against E. faecalis.     

The properties and applications of chlorhexidine in endodontics

Microorganisms and their by‐products are considered to be the major cause of pulp and periradicular pathosis. Hence, a major objective in root canal treatment is to disinfect the entire root canal system, which requires that all contents of the root canal system be eliminated as possible sources of infection. This goal may be accomplished using mechanical instrumentation and chemical irrigation, in conjunction with medication of the root canal system between treatment sessions. To reduce or eliminate bacteria, various irrigation solutions have been advocated. Chlorhexidine is a cationic molecule, which can be used during treatment. It has a wide range of antimicrobial activity. Its cationic structure provides a unique property named substantivity. The purpose of this paper is to review the structure and mechanism of action of CHX, its antibacterial and antifungal activity, its effect on biofilm, its substantivity (residual antibacterial activity), its tissue solvent ability, its interaction with calcium hydroxide and sodium hypochlorite, its anticollagenolytic activity, its effect on coronal and apical leakage of bacteria, its toxicity and allergenicity and the modulating effect of dentine and root canal components on its antimicrobial activity. A Medline search was performed from 1981 to the end of March 2008 and was limited to English‐language papers. The keywords searched on Medline were ‘chlorhexidine AND endodontics’, ‘chlorhexidine AND root canal therapy’, ‘chlorhexidine AND substantivity’ and ‘chlorhexidine AND toxicity’. The reference lists of each article were manually checked for additional articles of relevance.     

Evaluation of the colour change in enamel and dentine promoted by the interaction between 2% chlorhexidine and auxiliary chemical solutions

To evaluate the colour change in enamel and dentine, promoted by interaction of 2% chlorhexidine gluconate (CHX) with 5.25% sodium hypochlorite (NaOCl) and 17% ethylenediaminetetraacetic acid (EDTA). Fragments containing enamel and dentine were obtained from the crowns of extracted bovine incisors. Before and after immersion of the samples in the substances, they were evaluated with reference to the colour of the enamel and dentine. The values obtained in numerical scores were subjected to statistical analysis using Wilcoxon test. A colour change in the enamel and dentine in groups treated with CHX gel + NaOCl and CHX gel + NaOCl + EDTA, and a change in colour only in the dentine in groups treated with CHX solution + NaOCl and CHX solution + NaOCl + EDTA. When used prior to NaOCl, CHX has the ability to induce a colour change in dental structures.     

Scanning electron microscopic study of the cleaning ability of chlorhexidine as a root-canal irrigant

AIM: To evaluate in vitro the cleaning of root-canal walls after irrigation with different irrigants. METHODOLOGY: A total of 36 recently extracted human teeth were divided into four experimental groups according to the irrigating solution used: saline; 2% chlorhexidine; 2.5% sodium hypochlorite; and 2.5% sodium hypochlorite + EDTA. The cleaning of the apical, middle and coronal thirds of the root canals was evaluated by scanning electron microscope examination using a 4-point scoring system. RESULTS: The best cleaning was obtained using 2.5% sodium hypochlorite and EDTA, followed by 2.5% sodium hypochlorite only (P < 0.05), whose cleaning was similar to chlorhexidine only in the cervical third. Cleaning by saline and 2% chlorhexidine was worse than the other two groups and was similar in all thirds. Better cleaning was found in the cervical and middle thirds for all groups with the worst results in the apical third. CONCLUSIONS: The apical third of the root canals was not cleaned as well as the middle and coronal thirds. Cleaning by chlorhexidine and saline was inferior compared to the cleaning by sodium hypochlorite with and without EDTA.     

Antimicrobial activity of different concentrations of NaOCl and chlorhexidine using a contact test

The purpose of this study was to analyze the in vitro antimicrobial activity of sodium hypochlorite (1% and 5%) and chlorhexidine (0.12%, 0.5% and 1%). Bacterial samples (ATCC) of Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Porphyromonas gingivalis and Fusobacterium nucleatum were submitted to a contact test. Solutions were evaluated at different time intervals: immediately, 5 min, 15 min, and 30 min after contact and repeated 10 times. The results of the contact test showed that 0.12% chlorhexidine did not eliminate E. faecalis at any time interval, while 0.5% and 1% chlorhexidine and 1% and 5% sodium hypochlorite did. These results permit us to conclude that to obtain better antimicrobial activity, chlorhexidine in a concentration greater than 0.12% should be used.     

Evaluation of the antifungal activity of four solutions used as a final rinse in vitro

The aim of the study was to compare the antifungal activity of 1.3% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), MTAD and Tetraclean as a final rinse against Candida albicans in a human tooth model in vitro. Ninety extracted human maxillary central and lateral incisor teeth were randomly divided into four groups each with 20 teeth, a positive and a negative control each with five teeth. After preparing the root canals, teeth were inoculated with Candida albicans (ATCC 10261) and incubated for 72 h. Teeth were divided into four experimental groups according to the irrigation solution as follows: NaOCl, CHX, MTAD and Tetraclean. After culturing aliquots from the experimental teeth on Sabouraud 4% dextrose agar, colony‐forming units were counted. The results showed that 1.3% NaOCl and 2% CHX were equally effective and significantly superior to MTAD and Tetraclean (P < 0.05). Furthermore, antifungal efficacy of Tetraclean was significantly superior to MTAD (P < 0.05).     

Comparison of the in vivo antimicrobial effectiveness of sodium hypochlorite and chlorhexidine used as root canal irrigants: a molecular microbiology study

Introduction

The purpose of this clinical study was to compare the antimicrobial effects of 2.5% sodium hypochlorite (NaOCl) and 0.12% chlorhexidine digluconate (CHX) when used as irrigants during treatment of teeth with apical periodontitis.

Methods

Forty-seven single-rooted single-canal teeth with necrotic pulps and asymptomatic apical periodontitis were selected for this study according to stringent inclusion/exclusion criteria. Bacterial samples were taken at the baseline (S1) and after (S2) chemomechanical preparation using 2.5% NaOCl (n = 30) or 0.12% CHX (n = 17) as the irrigant. Bacterial, archaeal, and fungal presence was evaluated by broad-range polymerase chain reaction (PCR), whereas bacterial identifications were performed by a closed-ended reverse-capture checkerboard approach targeting 28 candidate endodontic pathogens.

Results

All S1 samples were PCR positive for bacterial presence but negative for both archaea and fungi. Both NaOCl- and CHX-based protocols were significantly effective in reducing the bacterial levels and number of taxa. No significant differences were observed between them in all tested parameters including the incidence of negative PCR results in S2 (40% for NaOCl vs 47% for CHX, p = 0.8), reduction in the number of taxa per canal (p = 0.3), and reduction in the bacterial levels (p = 0.07). The most prevalent taxa in S2 samples from the NaOCl group were Propionibacterium acnes, Streptococcus species, Porphyromonas endodontalis, and Selenomonas sputigena. In the CHX group, the most prevalent taxa in S2 were Dialister invisus, Actinomyces israelii, Prevotella baroniae, Propionibacterium acidifaciens, and Streptococcus species.

Conclusions

Treatment protocols using irrigation with either NaOCl or CHX succeded in significantly reducing the the number of bacterial taxa and their levels in infected root canals, with no significant difference between these substances.     

Effects of 2% chlorhexidine and 5.25% sodium hypochlorite on gutta-percha cones studied by atomic force microscopy

AIM: To compare the effects of 2% chlorhexidine (CHX) and 5.25% sodium hypochlorite (NaOCl) on gutta-percha (GP) cone structure using atomic force microscopy (AFM). METHODOLOGY: Two standardized GP cones were sectioned 3 mm from the tip, attached to a glass base and immersed in 2% CHX or 5.25% NaOCl for 1, 5, 10, 20 and 30 min. Untreated GP cones were used as control. Topography and elasticity analyses were performed on 12 different regions located between 1 and 2 mm from the tip. Root mean square (RMS) parameters for contact mode imaging and force modulation microscopy variations were measured. The differences between RMS values were tested by anova with Fisher's protected LSD test for multiple comparisons. RESULTS: There was no deterioration in the topography and physical properties studied when 2% CHX was used in comparison with the control (P < 0.05). The RMS parameter for topography increased after 10 min of 5.25% NaOCl exposure in comparison with the control (P < 0.05). In addition, 5.25% NaOCl increased the elasticity of the GP cone after an immersion time of 1 min in comparison with the control (P < 0.05). CONCLUSIONS: Two per cent CHX did not change GP cone structure following up to 30 min exposure. Conversely, 5.25% NaOCl caused elastic changes after 1 min exposure.