Effect of chlorhexidine/thymol and fluoride varnishes on dental biofilm formation in vitro

This study evaluated the effect of chlorhexidine/thymol (CHX/T) and fluoride (F) varnishes on biofilm formation in vitro. Hydroxyapatite discs coated with varnish were first immersed in saline for 0, 3, 7 or 14 d, then immersed in pasteurized saliva. The discs were incubated for 20 h with a bacterial suspension containing Actinomyces naeslundii, Fusobacterium nucleatum, Streptococcus oralis, and Veillonella dispar. Uncoated discs were used as controls. Growth of bacteria on the discs was evaluated by culture and by scanning electron microscopy (SEM). Bacterial vitality was examined by fluorescence staining. In the CHX/T-treated group, bacterial accumulation was delayed, and the total number of bacteria was significantly lower than in the controls. In the F-treated group, the total number of bacteria did not differ from the control, although the number of S. oralis was lower. Bacterial vitality in the CHX/T and F groups did not differ from that in the controls. The total number of bacteria on the CHX/T-treated discs immersed in saline was significantly higher than that on the non-immersed discs. Biofilm development was inhibited by the CHX/T varnish but not by the F varnish. The effect of the CHX/T varnish decreased following the immersion of discs in saline.     

胞外多糖对粪肠球菌单菌种生物膜形成的影响

目的:观察粪肠球菌单菌种生物膜的动态形成过程,探讨胞外多糖在粪肠球菌生物膜形成过程中的分布和作用。方法:粪肠球菌在盖玻片上形成单菌种生物膜,采用荧光技术标记胞外多糖和细菌,荧光显微镜观察生物膜结构和胞外多糖的分布;蒽酮法测定粪肠球菌在游离和粘附状态下产生水溶性胞外多糖(Water soluble exopolysaccharides,WSE)和水不溶性胞外多糖(Water insoluble exopolysaccharides,WIE)的能力变化。结果:粪肠球菌能在玻片表面粘附形成生物膜,生物膜中胞外多糖分布与菌落中的细菌分布一致,相互包裹成熟形成网络状结构。不同培养时间、不同生存状态粪肠球菌合成WSE和WIE的能力不同,差异有显著性(P<0.05)。粘附菌产生WSE的能力往往低于浮游菌,但是粘附菌产生WIE均高于浮游菌,差异具有统计学意义(P<0.05)。结论:胞外多糖在粪肠球菌生物膜形成过程中发挥重要作用。     

Antimicrobial efficacy of chlorhexidine and two calcium hydroxide formulations against Enterococcus faecalis

The purpose of this study was to investigate the efficacy of chlorhexidine (CHX) and calcium hydroxide (Ca(OH2) against Enterococcus faecalis in vitro. Extracted single-rooted human teeth were instrumented up to size 40. After removal of the smear layer, an inoculum of E. faecalis was inserted into the root canals. After incubation, the inoculum was removed and the root canals were filled with one of three different disinfectants: Ca(OH2 paste, CHX 2%, and a mixture of CHX and Ca(OH2 paste (n = 10 in each group). Control teeth were filled with water of standardized hardness (n = 10). The teeth were then incubated for 3 days. After incubation, each root canal was instrumented, and the removed dentin was examined microbiologically. CHX was significantly more effective against E. faecalis than was Ca(OH2 paste or a mixture of CHX with Ca(OH2 paste (p < 0.05). There was no increase in the efficiency of Ca(OH2 paste when CHX was added (p > 0.05). The results suggest that CHX is effective in the elimination of E. faecalis from dentinal tubules under the conditions of this study.     

In vitro antimicrobial activity of auxiliary chemical substances and natural extracts on Candida albicans and Enterococcus faecalis in root canals

Objective:

The aim of this study was to evaluate the antimicrobial activity of auxiliary chemical substances and natural extracts on Candida albicans and Enterococcus faecalis inoculated in root canals.

Material and Methods:

Seventy-two human tooth roots were contaminated with C. albicans and E. faecalis for 21 days. The groups were divided according to the auxiliary chemical substance into: G1) 2.5% sodium hypochlorite (NaOCl), G2) 2% chlorhexidine gel (CHX), G3) castor oil, G4) glycolic Aloe vera extract, G5) glycolic ginger extract, and G6) sterile saline (control). The samples of the root canal were collected at different intervals: confirmation collection, at 21 days after contamination; 1^st collection, after instrumentation; and 2^nd collection, seven days after instrumentation. Microbiological samples were grown in culture medium and incubated at 37º C for 48 hours.

Results:

The results were submitted to the Kruskal-Wallis and Dunn (5%) statistical tests. NaOCl and CHX completely eliminated the microorganisms of the root canals. Castor oil and ginger significantly reduced the number of CFU of the tested bacteria. Reduction of CFU/mL at the 1^st and 2^nd collections for groups G1, G2, G3 and G4 was greater in comparison to groups G5 and G6.

Conclusion:

It was concluded that 2.5% sodium hypochlorite and 2% chlorhexidine gel were more effective in eliminating C. albicans and E. faecalis, followed by the castor oil and glycolic ginger extract. The Aloe vera extract showed no antimicrobial activity.     

Antimicrobial activity of endodontic sealers on Enterococcus faecalis

Enterococcus faecalis has been shown to be highly resistant once established in the root canal system and may play an important role in endodontic failures. The purpose of this study was to evaluate the antimicrobial activity of four root canal sealers on E. faecalis. Seventeen blood-agar plates were inoculated with E. faecalis using the Lawn technique. Five discs were placed on each plate, four with sealers--Sealapex, Roth 811, Kerr EWT, and AH-Plus--and an ampicillin disc as the control. The plates were incubated at 37 degrees C. The zones of inhibition were measured at 24 and 48 h. Analysis using a one-way ANOVA and Tukey test showed a statistically significance difference (p < 0.05) between all four groups of sealers. Roth 811 showed the largest zone of inhibition (1.1 mm), followed by Sealapex (0.8 mm) and Kerr EWT (0.5 mm), whereas AH-Plus had no antimicrobial activity. There was no difference in the zones of inhibition between the 24- and 48-h time periods.     

In vitro Enterococcus faecalis biofilm formation on five adhesive systems

Objective: To determine the E. faecalis biofilm formation on the surface of five adhesive systems (AS) and its relationship with roughness. Study Design: The formation of E. faecalis biofilms was tested on the surface of four dual-cure AS: AdheSE DC, Clearfil DC Bond, Futurabond DC and Excite DSC and one light-cure antimicrobial AS, Clearfil Protect Bond, after 24 hours of incubation, using the MBEC high-throughput device. Results: E. faecalis biofilms grew on all the adhesives. The least growth of biofilm was on Excite DSC, Clearfil Protect Bond, and the control. Futurabond DC resulted in the greatest roughness and biofilm amount. There was a close relationship between the quantity of biofilm and roughness, except for Clearfil Protect Bond, which showed little biofilm but high roughness. Conclusion: None of the tested AS prevented E. faecalis biofilm formation, although the least quantity was found on the surface of Clearfil Protect Bond. Key words:Adhesive systems, biofilm, Enterococcus faecalis, roughness.     

五种冲洗液对根管内粪肠球菌抗菌效果的体外实验

目的评价五种冲洗液对根管内粪肠球菌的杀菌效果。方法建立粪肠球菌根管内感染模型,将40个感染根管标本随机分为5组,每组8颗牙。器械预备时分别采用5.25%NaClO（A组）、2.5%NaClO（B组）、1%NaClO（C组）、17%EDTA（D组）、17%EDTA＋1%NaClO（E组）冲洗根管。冲洗前、冲洗后即刻及冲洗后72h分别取样,37℃下CO2孵育箱中培养,48h后计数菌落数（CFU/ml）。结果冲洗后5组根管内的粪肠球菌量均显著下降（P〈0.05）,其中A组与其余各组间差异均有显著性（P〈0.01）;B组与C组、D组间差异有显著性（P〈0.05）,与E组间差异无显著性（P〉0.05）;C组与D组、E组间差异有显著性（P〈0.05）;D组与E组间差异有显著性（P〈0.05）。根管冲洗后培养72h均有细菌生长。结论5.25%NaClO抗菌效果最强;17%EDTA＋1%NaClO的抗菌效果优于1%NaClO,与2.5%NaClO抗菌效果相近似。     

Nd:YAG激光和Er:YAG激光对根管内粪肠球菌杀灭效果的研究

［摘要］ 目的 观察Nd:YAG激光、Er:YAG激光及两者联用对根管内粪肠球菌的杀灭效果。方法 选取离体牙建立粪肠球菌感染模型,随机分为五组：A组：生理盐水组;B组：1%次氯酸钠溶液组;C组：Nd:YAG激光组;D组：Er:YAG激光组;E组：Nd:YAG激光+Er:YAG激光组。通过细菌培养和扫描电镜观察对比各组的杀菌效果。结果 细菌培养和扫描电镜结果显示：A组杀菌率最低,B组杀菌率最高,E组次高。除C组和D组间杀菌率相近外（P>0.05）,各组间均有统计学差异（P<0.05）。结论 Nd:YAG激光和Er:YAG激光均有一定的根管杀菌效果,两者联用效果更佳,可作为传统消毒方法的补充。     

Effect of the quorum‐sensing luxS gene on biofilm formation by Enterococcus faecalis

Enterococcus faecalis is the species of bacterium most frequently isolated from the root canals of teeth that exhibit chronic apical periodontitis refractory to endodontic treatment. In this study, we evaluated the effect of the S‐ribosylhomocysteine lyase (luxS) quorum‐sensing gene on E. faecalis biofilm formation by constructing a knockout mutant. The biofilms formed by both E. faecalis and its luxS mutant strain were evaluated using the MTT method. Important parameters that influence biofilm formation, including cell‐surface hydrophobicity and the nutrient content of the growth medium, were also studied. Biofilm structures were observed using confocal laser scanning microscopy (CLSM), and expression of biofilm‐related genes was investigated using RT‐PCR. The results showed that the luxS gene can affect biofilm formation, whereas it does not affect the bacterial growth rate. Deletion of the luxS gene also increased cell‐surface hydrophobicity. Biofilm formation was accelerated by the addition of increasing concentrations of glucose. The CLSM images revealed that the luxS mutant strain tends to aggregate into distinct clusters and relatively dense structures, whereas the wild‐type strain appears confluent and more evenly distributed. All genes examined were up‐regulated in the biofilms formed by the luxS mutant strain. The quorum‐sensing luxS gene can affect E. faecalis biofilm formation.     

Antimicrobial activity of nanoemulsion on cariogenic planktonic and biofilm organisms

IntroductionNanoemulsions (NE) are a unique class of disinfectants produced by mixing a water immiscible liquid phase into an aqueous phase under high shear forces. NE have antimicrobial properties and are also effective anti-biofilm agents.Materials and methodsThe effectiveness of nanoemulsion and its components was determined against Streptococcus mutans and Lactobacillus casei by live/dead staining. In vitro antimicrobial effectiveness of nanoemulsion against planktonic S. mutans, L. casei, Actinomyces viscosus, Candida albicans and mixed culture was determined by a serial dilution technique to obtain minimum inhibitory concentration and minimum bactericidal concentration (MIC/MBC). In addition, efficacy was investigated by kinetics of killing, adherence and biofilm assays.ResultsCompared to its components, nanoemulsion showed notable antimicrobial activity against biofilm organisms, up to 83.0% kill within 1 min. NE dilutions ranging from 243 to 19683 were effective against planktonic S. mutans, L. casei, A. viscosus, C. albicans and mixed culture of these four strains as shown through MIC/MBC assays. NE showed antimicrobial activity against planktonic cells at high dilutions, confirmed by time kill studies. The level of adhesion on glass surface was reduced by 94.2–99.5% in nanoemulsion treated groups (p < 0.001). 4-Day-old S. mutans, L. casei, A. viscosus, C. albicans and mixed cultures biofilms treated with NE showed reductions of bacterial counts with decreasing dilutions (p < 0.001).ConclusionThese results suggest that nanoemulsion has effective anti-cariogenic activity against cariogenic microorganisms and may be a useful medication in the prevention of caries.     

Antibacterial effect of calcium hydroxide combined with chlorhexidine on Enterococcus faecalis: a systematic review and meta-analysis

Objective

Enterococcus faecalis (E. faecalis) is the most frequently isolated strain in failed endodontic therapy cases since it is resistant to calcium hydroxide (CH). Whether a combination of CH and chlorhexidine (CHX) is more effective than CH alone against E. faecalis is a matter of controversy. Thus, the aim of this study was to conduct a systematic review and meta-analysis of the literature.

Material and Methods

A comprehensive search in PubMed, EMbase, EBSCOhost, The Cochrane Library, SciELO, and BBO databases, Clinical trials registers, Open Grey, and conference proceedings from the earliest available date to February 1, 2013 was carried out and the relevant articles were identified by two independent reviewers. Backward and forward search was performed and then inclusion and exclusion criteria were applied. The included studies were divided into "comparisons" according to the depth of sampling and dressing period of each medicament. Meta-analysis was performed using Stata software 10.0. The level of significance was set at 0.05.

Results

Eighty-five studies were retrieved from databases and backward/forward searches. Fortyfive studies were considered as relevant (5 in vivo, 18 in vitro, 18 ex vivo, and 4 review articles). Nine studies were included for meta-analysis. Inter-observer agreement (Cohen kappa) was 0.93. The included studies were divided into 21 comparisons for meta-analysis. Chi-square test showed the comparisons were heterogeneous (p<0.001). Random effect model demonstrated no significant difference between CH/CHX mixture and CH alone in their effect on E. faecalis (p=0.115).

Conclusions

According to the evidence available now, mixing CH with CHX does not significantly increase the antimicrobial activity of CH against E. faecalis. It appears that mixing CH with CHX does not improve its ex vivo antibacterial property as an intracanal medicament against E. faecalis. Further in vivo studies are necessary to confirm and correlate the findings of this study with the clinical outcomes.     

Polyspecies biofilm formation on implant surfaces with different surface characteristics

Objective:

To investigate the microbial adherence and colonization of a polyspecies biofilm on 7 differently processed titanium surfaces.

Material and Methods:

Six-species biofilms were formed anaerobically on 5-mm-diameter sterilized, saliva-preconditioned titanium discs. Material surfaces used were either machined, stained, acid-etched or sandblasted/acid-etched (SLA). Samples of the latter two materials were also provided in a chemically modified form, with increased wettability characteristics. Surface roughness and contact angles of all materials were determined. The discs were then incubated anaerobically for up to 16.5 h. Initial microbial adherence was evaluated after 20 min incubation and further colonization after 2, 4, 8, and 16.5 h using non-selective and selective culture techniques. Results at different time points were compared using ANOVA and Scheffé post hoc analysis.

Results:

The mean differences in microorganisms colonizing after the first 20 min were in a very narrow range (4.5 to 4.8 log CFU). At up to 16.5 h, the modified SLA surface exhibited the highest values for colonization (6.9±0.2 log CFU, p<0.05) but increasing growth was observed on all test surfaces over time. Discrepancies among bacterial strains on the differently crafted titanium surfaces were very similar to those described for total log CFU. F. nucleatum was below the detection limit on all surfaces after 4 h.

Conclusion:

Within the limitations of this in vitro study, surface roughness had a moderate influence on biofilm formation, while wettability did not seem to influence biofilm formation under the experimental conditions described. The modified SLA surface showed the highest trend for bacterial colonization.     

Effects of varnishes containing chlorhexidine on the human dental plaque flora

This study describes the effects of varnishes containing 0%, 10%, 20%, or 40% chlorhexidine diacetate on the microflora of human fissure dental plaque. Sandarac, a natural resin, was used as the varnish base. Ten subjects, each with at least four sound fissures harboring high levels of Streptococcus mutans, participated in the study. The fissures in each of the individuals were randomly assigned to four experimental groups, in each of which one of the varnishes was tested. The varnish treatment consisted of a single application of a small amount of varnish onto the fissures. Apart from the selected fissures, the rest of the dentition was left untreated. All chlorhexidine-containing varnishes selectively suppressed S. mutans in fissure plaque, and had no effect on total viable counts or on the numbers of Actinomyces viscosus/naeslundii and Streptococcus sanguis beyond one week. The extent of the suppression depended upon the concentration of chlorhexidine in the varnish, 40% chlorhexidine varnish giving the greatest suppression of S. mutans. At 22 weeks, after a single treatment with varnish containing 40% chlorhexidine, mean S. mutans counts were more than ten times lower than in the control or 10%-chlorhexidine varnish group. At that time, S. mutans was still undetectable in five out of ten experimental fissures in this group. The results suggested that sandarac varnishes containing high concentrations of chlorhexidine can be used successfully for long-term suppression of S. mutans in dental fissures.     

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.     

Effect of chlorhexidine on denture biofilm accumulation

Purpose: Adequate denture hygiene can prevent and treat infection in edentulous patients, who are frequently elderly and have difficulty brushing their teeth. This study evaluated the efficacy of complete denture biofilm removal using a chlorhexidine solution in two concentrations: 0.12% and 2.0%. Materials and Methods: Sixty complete denture wearers participated in a trial for 21 days after receiving brushing instructions. They were distributed into three groups, according to the tested solution and regimen (n = 20): (G1) Control (daily overnight soaking in water); (G2) daily immersion at home in 0.12% chlorhexidine for 20 minutes after dinner; and (G3) a single immersion in 2.0% chlorhexidine for 5 minutes at the end of the experimental period, performed by a professional. Biofilm coverage area (%) was quantified on the internal surface of maxillary dentures at baseline and after 21 days. Afterward, the differences between initial and posttreatment results were compared by means of the Kruskal‐Wallis test (α= 0.05). Results: Median values for biofilm coverage area after treatment were: (G1) 36.0%; (G2) 5.3%; and (G3) 1.4%. Differences were significant (KW = 35.25; p < 0.001), although G2 and G3 presented similar efficacy in terms of biofilm removal. Conclusions: Both chlorhexidine‐based treatments had a similar ability to remove denture biofilm. Immersion in 0.12% or 2.0% chlorhexidine solutions can be used as an auxiliary method for cleaning complete dentures.     

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.