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.     

The influence of organic load on the antimicrobial activity of different concentrations of NaOCl and chlorhexidine in vitro

AIM: To evaluate bacterial growth after contact with sodium hypochlorite (NaOCl; 1 and 5%) and chlorhexidine (CHX; 0.12, 0.5 and 1%) in vitro with or without the addition of organic material (bovine serum albumin (BSA) 0.5%). METHODOLOGY: Bacterial samples (American Type Culture Collection (ATCC)) of Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Porphyromonas gingivalis and Fusobacterium nucleatum were kept in contact with each irrigating solution for varying intervals of time: immediately (t0), 5 min (t5), 15 min (t15) and 30 min (t30). Each test was repeated 10 times. In half of the specimens, 0.5% BSA was added as organic material in an attempt to simulate the organic tissue present in the root canal system. Bacterial growth under appropriate condition of incubation was evaluated and compared for each microorganism at all time intervals. RESULTS: A 0.12% CHX solution did not eliminate E. faecalis at any time interval. One percent CHX eliminated all strains, as did both NaOCl concentrations. BSA did not interfere substantially with the antimicrobial activity of any of the irrigating solutions. CONCLUSIONS: Under the condition of this study, a 0.12% CHX solution was ineffective at killing E. faecalis.     

Antimicrobial activity of varying concentrations of sodium hypochlorite on the endodontic microorganisms Actinomyces israelii, A. naeslundii, Candida albicans and Enterococcus faecalis

AIM: To determine the resistance of microorganisms associated with refractory endodontic infections to sodium hypochlorite used as a root canal irrigant. METHODOLOGY: Two strains each of Actinomyces naeslundii, Candida albicans and Enterococcus faecalis were tested as late logarithmic phase inocula, against sodium hypochlorite adjusted to 0.5, 1.0, 2.5 and 5.25% w/v. Contact times used were 0, 10, 20, 30, 60 and 120 s. In the case of E. faecalis, additional experiments used contact times of 1.0, 2.0, 5.0, 10.0 and 30.0 min. Anti-microbial action was halted by sodium thiosulphate addition. Survivors were measured primarily using viable counts on drop plates. Additionally, pour plates were used to count low colony-forming units (cfu) and dilutions to 10(-6) were used to count high cfu. RESULTS: All concentrations of NaOCl lowered cfu below the limit of detection after 10 s in the case of A. naeslundii and C. albicans. However, E. faecalis proved to be more resistant to NaOCl. Using 0.5% NaOCl for 30 min reduced cfu to zero for both strains tested. This compares with 10 min for 1.0%, 5 min for 2.5% and 2 min for 5.25% (P < 0.001). Regression analysis for the dependent variable log(e)(count + 1) with log(e)(time + 1) and concentration as explanatory variables gave rise to a significant interaction between time and concentration (P < 0.001). CONCLUSION: The published association of E. faecalis with refractory endodontic infection may result, at least partially, from high resistance of this species to NaOCl. This does not appear to be the case with A. naeslundii or C. albicans.     

基于重水拉曼技术评价次氯酸钠对粪肠球菌抑菌效能的研究

目的 系统评价重水拉曼技术在耐药性研究领域的可行性及次氯酸钠对粪肠球菌的抑菌效能.方法 1)采用吸光度测定、重水拉曼技术评价粪肠球菌在不同浓度重水中生长状态及对重水的吸收规律,评价重水拉曼技术在细菌耐药性研究方面的普适性.2)采用肉汤稀释法,结合吸光度测定次氯酸钠对粪肠球菌的最小抑菌浓度(MIC);采用重水拉曼技术测定...     

次氯酸钠对粪肠球菌杀菌效果的体外研究

目的体外评价次氯酸钠(NaOCl)对粪肠球菌的杀菌效果及浓度依赖关系。方法采用纸片扩散法药敏实验观察NaOCl对粪肠球菌的抑菌环大小(mm),直接接触法观察NaOCl对粪肠球菌的灭活时间。结果5.25%NaOCl的抑菌环直径最大,4.00%与2.50%、1.00%与0.50%差异无显著性。5.25%NaOCl在30s内可杀死全部粪肠球菌,2.50%NaOCl杀死粪肠球菌需要15min,随浓度的下降,杀死细菌所需要的时间延长。结论高浓度NaOCl的效果优于低浓度NaOCl,2.50%NaOCl基本可满足临床要求。     

Evaluation of chemical interactions of maleic acid with sodium hypochlorite and chlorhexidine gluconate

Introduction

The elimination of microorganisms from the root canal system necessitates the use of combination of irrigating solutions to enhance their antimicrobial property. The combination of irrigants and their interaction sometimes could be detrimental to the outcome of the root canal therapy. The purposes of this study were (1) to evaluate the interaction between 7% maleic acid (MA) and 2% chlorhexidine gluconate solution (CHX) and to find out the availability of individual irrigant and (2) to determine the free available chlorine content when 7% MA was mixed with 2.5% sodium hypochlorite (NaOCl) solution.

Methods

Interaction between MA and CHX was assessed by high-performance liquid chromatography. Available chlorine content in NaOCl was evaluated by the standard iodine/thiosulfate titration method.

Results

It was observed that more than 90% free MA and CHX were available when MA was combined with CHX. It was also observed that there was no precipitate formation when 7% MA was mixed with 2% CHX. Available chlorine content decreased significantly in the MA/NaOCl mixture.

Conclusions

There were no adverse interactions or precipitate formation observed when MA was combined with CHX, but the available chlorine content was reduced when NaOCl was mixed with MA.     

Evaluation of the effectiveness of sodium hypochlorite used with three irrigation methods in the elimination of Enterococcus faecalis from the root canal, in vitro

The effectiveness of 4.0% sodium hypochlorite (NaOCl) used with three irrigation methods in the elimination of Enterococcus faecalis from the root canal was tested in vitro . Root canals contaminated with E. faecalis were treated as follows: (i) irrigation with 2 mL of NaOCl solution and agitation with hand files; (ii) irrigation with 2 mL of NaOCl solution and ultrasonic agitation; (iii) irrigation with NaOCl alternated with hydrogen peroxide. Contaminated canals irrigated with sterile saline solution served as the control. Paper points used to sample bacteria from the root canals were transferred to tubes containing 5 mL of brain heart infusion (BHI) broth. Tubes were incubated and the appearance of broth turbidity was indicative of bacteria remaining in the root canal. There were no statistically significant differences between the experimental groups. However, NaOCl applied by the three methods tested, was significantly more effective than the saline solution (control group) in disinfecting the root canal.     

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.     

The effect of sodium hypochlorite on Enterococcus faecalis when grown on dentine as a single‐ and multi‐species biofilm

Enterococcus faecalis is often involved in the aetiology of apical periodontitis after endodontic treatment. This project aimed to establish, on dentine in vitro, a multi‐species biofilm containing E. faecalis, and to determine if the organism had an increased resistance to sodium hypochlorite compared with an axenic biofilm. Biofilms were established on dentine discs in flow cells with either E. faecalis alone (axenic) or together with Fusobacterium nucleatum and Streptococcus sanguinis. Following treatment with either 0.9% sodium hypochlorite or saline, the viability of E. faecalis was determined by serial plating and qualitative analysis was performed by scanning electron microscopy and confocal laser scanning microscopy. Viable counts indicated that 0.9% NaOCl is highly effective against E. faecalis grown alone and as part of a multi‐species biofilm (P = 0.0005 and P = 0.001, respectively). No significant difference in its survival in the two biofilm types was found (P = 0.8276).     

次氯酸钠对根管内粪肠球菌杀菌效果的体外实验

目的评价次氯酸钠对根管内粪肠球菌的杀菌效果。方法将45个离体前磨牙的感染根管标本分为6组，1、2组用5．25％及2．5％次氯酸钠冲洗，3组用0．9％NaCl冲洗，4、5组在根管预备时辅以5．25％及2．5％次氯酸钠冲洗，6组在根管预备时辅以0．9％Nacl冲洗。冲洗前、冲洗后即刻及冲洗后72h分别取样培养。结果6组根管内的细菌量均显著下降。1、2组差异无统计学意义（P〉0．05），但均好于3组（P〈0．05）。4、5和6组差异均有统计学意义（P〈0．05）。根管冲洗后培养72h均有细菌生长。结论2．5％次氯酸钠基本可达到更高浓度的灭菌效果，但是经过机械预备和次氯酸钠化学消毒后的根管内仍有细菌残留。     

In vitro evaluation of the antimicrobial activity of chlorhexidine and sodium hypochlorite

The aim of this study was to investigate in vitro the antimicrobial activity of 0.2%, 1%, and 2% chlorhexidine gluconate (CHX gel and CHX liquid), against endodontic pathogens and compare the results with the ones achieved by 0.5%, 1%, 2.5%, 4%, and 5.25% sodium hypochlorite (NaOCl). A broth dilution test was performed, and the timing for irrigants to kill microbial cells was recorded and statistically analyzed. Both 2.0% gel and liquid formulations eliminated Staphylococcus aureus and Candida albicans in 15 seconds, whereas the gel formulation killed Enterococcus faecalis in 1 minute. All tested irrigants eliminated Porphyromonas endodontalis, Porphyromonas gingivalis, and Prevotella intermedia in 15 seconds. The timing required for 1.0% and 2.0% CHX liquid to eliminate all microorganisms was the same required for 5.25% NaOCl. The antimicrobial action is related to type, concentration, and presentation form of the irrigants as well as the microbial susceptibility.     

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.     

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.     

Antimicrobial efficacy of ozonated water, gaseous ozone, sodium hypochlorite and chlorhexidine in infected human root canals

AIM: To determine the antimicrobial efficacy of ozonated water, gaseous ozone, sodium hypochlorite and chlorhexidine in human root canals infected by Enterococcus faecalis. METHODOLOGY: Thirty human maxillary anterior teeth were prepared and inoculated with E. faecalis for 60 days. Eppendorf tubes were connected to the coronal portion of the teeth. Urethane hoses were attached to the tubes and to the entrance of a peristaltic pump. The exit of the apparatus corresponded to the apical portion of the root canals. The test irrigating solutions were ozonated water, gaseous ozone, 2.5% sodium hypochlorite (NaOCl), 2% chlorhexidine that circulated at a constant flow of 50 mL min(-1) for 20 min. Samples from the root canals were collected and immersed in 7 mL Letheen Broth (LB), followed by incubation at 37 degrees C for 48 h. Bacterial growth was analysed by turbidity of the culture medium and subculture on a specific nutrient broth. A 0.1 mL inoculum obtained from LB was transferred to 7 mL of brain heart infusion and incubated at 37 degrees C for 48 h. Bacterial growth was checked by turbidity of the culture medium carried out in triplicate. RESULTS: No solution used as an irrigant over a 20-min contact time demonstrated an antimicrobial effect against E. faecalis. CONCLUSION: The irrigation of infected human root canals with ozonated water, 2.5% NaOCl, 2% chlorhexidine and the application of gaseous ozone for 20 min was not sufficient to inactivate E. faecalis.