Influence of EDTA and its Association with Benzalkonium Chloride on Enterococcus faecalis Adhesion to Dentin / Influencia del EDTA y su Asociación con Cloruro de Benzalconio en la Adhesión de Enterococcus faecalis a la Dentina

ABSTRACT: The aim of this in vitro study was to investigate the influence of ethylenediaminetetraacetic acid (EDTA) associated with the benzalkonium chloride (BAK) on the adhesion and formation of Enterococcus faecalis biofilms attached to coated dentin. Discs standard bovine dentin blocks were treated with the coating materials evaluated: Saline solution (control), 17 % EDTA, 17 % EDTA associated with 1 % BAK for 5 minutes and subsequently washed with saline solution. Afterwards, biofilms of E. faecalis (ATCC 29212) were grown on the surface of coated dentin blocks for time intervals of 1 hour and 7 days (n = 20) and were subsequently washed with phosphate-buffered saline (PBS). Bacterial viability and total biovolume were analyzed by confocal laser scanning microscopy (CLSM) using the Live/Dead technique. Nonparametric Kruskal-Wallis followed by Dunn tests were used to determine statistical differences (a = 5 %). The 17 % EDTA + 1 % BAK group showed significantly lower biovolume and bacterial viability values at the end of 1 hour (p < 0.05). After 7 days of contamination, the 17 % EDTA and 17 % EDTA + 1 % BAK groups showed similar results that differed statistically from those of the control group (p < 0.05). The saline solution group showed higher values. The use of BAK associated with EDTA on dentin blocks surfaces before exposure to contamination was able to interfere in the adhesion of E. faecalis to dentin. Also, dentin treatment by BAK associated with a chelating agent influences the secondary biofilm formation, which could have important effects on the long-term success of root canal treatment.

RESUMEN: El objetivo del estudio consistió en investigar in vitro, la influencia del ácido etilendiamino-tetraacético (EDTA) con cloruro de benzalconio (BAK) en la adhesión y formación de biopelículas de Enterococcus faecalis a la dentina. Discos de dentina bovina fueron tratadas con solución salina (control), 17 % de EDTA, 17% de EDTA asociado con 1 % de BAK durante 5 minutos y lavadas con solución salina. Las biopelículas de E. faecalis (ATCC 29212) se cultivaron sobre los discos de dentina durante intervalos de tiempo de 1 hora y 7 días (n = 20), lavados con solución salina tamponada con fosfato (PBS). La viabilidad bacteriana y el biovolumen total se analizaron mediante microscopía de barrido por láser (CLSM) utilizando la técnica Live / Dead. Se realizó prueba no paramétrica de Kruskal-Wallis, seguida por Dunn con una diferencia estadística (a = 5 %). El grupo de 17 % EDTA + 1 % BAK mostró valores significativamente menores de biovolumen y viabilidad bacteriana al final de 1 hora (p < 0,05). Después de 7 días de contaminación, los grupos de 17 % EDTA y 17 % EDTA + 1 % BAK mostraron resultados similares que diferían estadísticamente del grupo control (p < 0,05). La solución salina mostró valores más altos. La asociación de BAK con EDTA antes de la contaminación interfirió en la adhesión de E. faecalis. Además, el tratamiento de la dentina por BAK asociado con EDTA influye en la formación de biopelículas secundarias, lo que podría tener efectos importantes sobre el éxito a largo plazo del tratamiento del conducto radicular.

Efeito in vitro da laserterapia e da terapia fotodinâmica na redução de bactérias presentes em canais radiculares / In vitro effect of lasertherapy and photodynamic therapy in bacterial reduction presents in root canals

Este estudo tem por objetivo verificar in vitro o efeito bactericida da laserterapia e da terapia fotodinâmica com laser de baixa potência (660 nm e 808 nm) em bactérias presentes nos canais radiculares. Métodos: foram preparadas 60 placas de Petri com bactérias: 20 placas com Enterococcus faecalis, 20 placas com Staphylococcus aureus e 20 com Pseudomonas aeruginosa. Aleatoriamente, dividiu-se cada grupo em 10 subgrupos (duas placas cada): três subgrupos tratados com laserterapia 660 nm em doses de 150, 225 e 300J/ cm², três subgrupos tratados com terapia fotodinâmica (azul de metileno 0,2% e laser 660 nm) em doses de 150, 225 e 300J/cm²; um subgrupo tratado com laserterapia 808 nm na dose de 225J/cm², um subgrupo com terapia fotodinâmica e laser 808 nm, em dose 225J/cm²; um subgrupo tratado apenas com fotossensibilizante (FS), e um não tratado (controle). Os tratados com laserterapia e terapia fotodinâmica foram irradiados uma única vez e incubados por 24 horas. Os últimos dois não receberam irradiação. As culturas foram analisadas visualmente para verificação do halo de inibição. Nos grupos submetidos somente à laserterapia, para o grupo FS e para o grupo controle, não foram observados halos de inibição, já onde houve aplicação da TFD, tanto com L1 quanto com L2, observaram-se halos de inibição em todas as espécies bacterianas estudadas. Conclui-se que a laserterapia, não produziu efeitos bactericidas e/ou bacteriostáticos, enquanto a terapia fotodinâmica nos dois comprimentos de onda produziu halos significativos de inibição de crescimento nas três bactérias do estudo.(AU)

This study aims to verify in vitro the bactericidal effect of laser therapy and photodynamic therapy with low power laser (660 nm and 808 nm), in bacteria present in the root canals.Methods: 60 Petri dishes were prepared with bacteria: 20 plates with Enterococcus faecalis, 20 plates with Staphylococcus aureus and 20 with Pseudomonas aeruginosa. At random, each group was divided into 10 subgroups (two plates each): three subgroups treated with 660nm laser therapy at doses of 150, 225 and 300J / cm², three subgroups treated with photodynamic therapy, (0.2% methylene blue and laser 660nm) in doses of 150, 225 and 300J / cm²; a subgroup treated with 808nm laser therapy at a dose of 225J / cm², a subgroup with (photodynamic therapy and 808nm laser) at a dose of 225J / cm²; a subgroup treated only with photosensitizer(FS), and an untreated (control). Those treated with laser therapy and photodynamic therapy were irradiated only once and incubated for 24 hours. The last two received no radiation. The cultures were analyzed visually to check the inhibition zone. In the groups submitted to laser therapy only, for the FS group and for the Control group, no inhibition halos were observed, since PDT was applied, with both L1 and L2, inhibition halos were observed in all studied bacterial species. It was concluded that laser therapy did not produce bactericidal and / or bacteriostatic effects, while photodynamic therapy at both wavelengths produced significant growth inhibition halos in the three studied bacteria.(AU)

Influencia de la dentina en el efecto antibacteriano de 2 concentraciones de hipoclorito de sodio en el crecimiento de Enterococcus faecalis ATCC 29212 / Influence of the dentin on the antibacterial effect of 2 concentrations of sodium hypochlorite in the growth of Enterococcus faecalis ATCC 29212

Este estudio in vitro evaluó la influencia de la dentina sobre el efecto antibacteriano contra Enterococcus faecalis ATCC 29212 de dos concentraciones de Hipoclorito de Sodio (NaOCl) 2,5 % y 5 %. Se empleó polvo de dentina a partir de dientes humanos (84 µg/ml) y la supervivencia de la bacteria se evaluó realizando recuento de unidades formadoras de colonias (UFC) a los 10, 30 y 60 segundos. Los datos se analizaron con la prueba estadística ANOVA factorial no encontrándose diferencias estadísticamente significativas entre los grupos con dentina y sin dentina. En conclusión, la dentina en este estudio no influyó en el efecto antibacteriano del Hipoclorito de Sodio en ninguna concentración, ni en los tiempos.

This in vitro study evaluated the influence of dentin on the antibacterial effect against Enterococcus faecalis ATCC 29212 of two concentrations of Sodium Hypochlorite NaOCl 2.5 % and 5 %. Dentin powder was used from human teeth (84 mg/ml) and the survival of the bacteria was evaluated by counting colony forming units (CFU) at 10, 30 and 60 seconds. The data were analyzed with the statistical ANOVA factorial test, finding no statistically significant differences between the groups with and without dentin. In conclusion, the dentin in this study had no inhibitory effect on antibacterial activity of Sodium Hypochlorite and any concentration, nor over time.

Role of extracellular DNA in Enterococcus faecalis biofilm formation and its susceptibility to sodium hypochlorite

Abstract Objective This study investigated the role of extracellular deoxyribonucleic acid (eDNA) on Enterococcus faecalis ( E. faecalis ) biofilm and the susceptibility of E. faecalis to sodium hypochlorite (NaOCl). Methodology E. faecalis biofilm was formed in bovine tooth specimens and the biofilm was cultured with or without deoxyribonuclease (DNase), an inhibitor of eDNA. Then, the role of eDNA in E. faecalis growth and biofilm formation was investigated using colony forming unit (CFUs) counting, eDNA level assay, crystal violet staining, confocal laser scanning microscopy, and scanning electron microscopy. The susceptibility of E. faecalis biofilm to low (0.5%) or high (5%) NaOCl concentrations was also analyzed by CFU counting. Results CFUs and biofilm formation decreased significantly with DNase treatment (p<0.05). The microstructure of DNase-treated biofilms exhibited less structured features when compared to the control. The volume of exopolysaccharides in the DNase-treated biofilm was significantly lower than that of control (p<0.05). Moreover, the CFUs, eDNA level, biofilm formation, and exopolysaccharides volume were lower when the biofilm was treated with DNase de novo when compared to when DNase was applied to matured biofilm (p<0.05). E. faecalis in the biofilm was more susceptible to NaOCl when it was cultured with DNase (p<0.05). Furthermore, 0.5% NaOCl combined with DNase treatment was as efficient as 5% NaOCl alone regarding susceptibility (p>0.05). Conclusions Inhibition of eDNA leads to decrease of E. faecalis biofilm formation and increase of susceptibility of E. faecalis to NaOCl even at low concentrations. Therefore, our results suggest that inhibition of eDNA would be beneficial in facilitating the efficacy of NaOCl and reducing its concentration.

Quorum sensing LuxS/autoinducer-2 inhibits Enterococcus faecalis biofilm formation ability

Abstract Objective: To investigate the relation between biofilm formation ability and quorum sensing gene LuxS/AI-2. Materials and Methods: Enterococcus faecalis (E. faecalis) standard strain ATCC 29212 was used in the study. Long flanking homology polymerase chain reaction method was used to build the LuxS gene knockout strain. Sequential culture turbidity measurement and CFU counting were used to assess the proliferation ability of E. faecalis after the depletion of LuxS. 96-well plate assay was used to quantify the biofilm formation ability; CLSM was used to observe the attached bacteria areas, while scanning electron microscopy (SEM) was performed to observe biofilm microstructure conditions. Results: LuxS gene knockout strains were successfully constructed and identified. The results showed that proliferation ability of E. faecalis was not affected by the depletion of the luxS gene, and the biofilm formation ability of ΔLuxS 29212 significantly decreased (P<0.05). Conclusions: Collectively, our studies provide the LuxS gene's key role in controlling biofilm formation of E. faecalis, which presented a negative regulation, and furthermore, providing us a possible way to conquer the persistent apical periodontitis.

Effect of ultrasound streaming on the disinfection of flattened root canals prepared by rotary and reciprocating systems

Abstract New technical and scientific developments have been advocated to promote the success of the endodontic treatment. In addition to rotary and reciprocating systems, irrigating solution agitation has been suggested and passive ultrasonic irrigation (PUI) is the most used. Objective: To evaluate, in vitro, the effect of ultrasound streaming (US) in the disinfection of flattened root canal systems prepared by the ProTaper, BioRaCe and Reciproc systems, utilizing the microbiological culture. Methodology: Extracted human mandibular incisors (n=84) were used. Suspensions of Enterococcus faecalis (ATCC 29212) were standardized and inserted along with the teeth immersed in brain-heart infusion (BHI) broth. The contamination was made following a protocol during 5 days. The teeth were randomly divided into six groups: G1, ProTaper Universal; G2, ProTaper Universal with US; G3, BioRaCe; G4, BioRaCe with US; G5, Reciproc; and G6, Reciproc with US. Irrigation was performed with saline solution. After biomechanical preparation, microbiological samples were performed with sterilized paper points, which were diluted and spread on BHI agar; after 48 h, the colony forming units (CFU/mL) were counted for each sample. Results: Groups using ultrasonic agitation presented a greater antibacterial effect than the other ones, even using saline solution as irrigant. The ProTaper Universal system showed the best antibacterial activity of the tested systems (median of 0 CFU/mL with and without surfactant or ultrasonic activation [PUI]). Even with PUI, Reciproc (median of 2.5 CFU/mL with PUI and 5 without it) could not reduce as many colonies as ProTaper Universal without US. The BioRaCe system had greater bacterial reduction when using US (median of 0 CFU/mL with PUI and 30 without it). Conclusions: US promoted greater reduction in the number of bacteria in the flattened root canals prepared with nickel-titanium mechanized systems. Regarding the instruments used, the ProTaper Universal system was the most effective in reducing the bacterial number.

Antimicrobial activity of ozone and NaF-chlorhexidine on early childhood caries

Abstract An early childhood carie (ECC) is an extremely destructive form of tooth decay. The aim of this study was to investigate the action of ozone (O3), and the association of sodium fluoride (NaF) with chlorhexidine (CHX) on bacteria related to ECC. Overnight culture of the bacteria was performed. On exponential phase the suspension was adjusted (101-108 CFU/mL). A drop (10μL) of each concentration of bacteria was applied on sheep blood agar plates and treated with O3 (2, 20, 200, and 2,000 ppm); after 18 hours, recovery analysis of CFU verified the reduction of bacterial activity. For NaF-CHX, sterile 96-well plates were prepared and divided into groups: G1 (150 µL TSB); G2 (20 µL of bacteria + 25 µL CHX + 25 µL NaF); and G3 (150 µL TSB + 20 µL of bacteria + 50 µL water). The plates were verified by analysis of the optical density (0, 12, 14, 16, and 18 hours). The data from O3 test were submitted to ANOVA and Tukey’s test (p < 0.05). For the data from NaF-CHX, the ANOVA 2-way and Bonferroni’s test (p < 0.05) were used. The number of CFU/mL showed death > 3log10 (99.9%) for all bacteria (ozone ≥ 20ppm), while the combination of NaF-CHX was more effective (p < 0.001) compared to each substance tested alone and the control group. The antimicrobial agents tested were able to inhibit all bacteria tested; O3 seemed to be a good alternative for controlling progression of carious lesions, while the association of NaF-CHX showed to be a good antimicrobial with easy and inexpensive application.

Effect of addition of nano-hydroxyapatite on physico-chemical and antibiofilm properties of calcium silicate cements

ABSTRACT Objective Mineral Trioxide Aggregate (MTA) is a calcium silicate cement composed of Portland cement (PC) and bismuth oxide. Hydroxyapatite has been incorporated to enhance mechanical and biological properties of dental materials. This study evaluated physicochemical and mechanical properties and antibiofilm activity of MTA and PC associated with zirconium oxide (ZrO2) and hydroxyapatite nanoparticles (HAn). Material and Methods White MTA (Angelus, Brazil); PC (70%)+ZrO2 (30%); PC (60%)+ZrO2 (30%)+HAn (10%); PC (50%)+ZrO2 (30%)+HAn (20%) were evaluated. The pH was assessed by a digital pH-meter and solubility by mass loss. Setting time was evaluated by using Gilmore needles. Compressive strength was analyzed by mechanical test. Samples were radiographed alongside an aluminum step wedge to evaluate radiopacity. For the antibiofilm evaluation, materials were placed in direct contact with E. faecalis biofilm induced on dentine blocks. The number of colony-forming units (CFU mL-1) in the remaining biolfilm was evaluated. The results were submitted to ANOVA and the Tukey test, with 5% significance. Results There was no difference in pH levels of PC+ZrO2, PC+ZrO2+HAn (10%) and PC+ZrO2+HAn (20%) (p>0.05) and these cements presented higher pH levels than MTA (p<0.05). The highest solubility was observed in PC+ZrO2+HAn (10%) and PC+ZrO2+HAn (20%) (p<0.05). MTA had the shortest initial setting time (p<0.05). All the materials showed radiopacity higher than 3 mmAl. PC+ZrO2 and MTA had the highest compressive strength (p<0.05). Materials did not completely neutralize the bacterial biofilm, but the association with HAn provided greater bacterial reduction than MTA and PC+ZrO2 (p<0.05) after the post-manipulation period of 2 days. Conclusions The addition of HAn to PC associated with ZrO2 harmed the compressive strength and solubility. On the other hand, HAn did not change the pH and the initial setting time, but improved the radiopacity (HAn 10%), the final setting time and the E. faecalis antibiofilm activity of the cement.

In vitro effectiveness of Brazilian brown propolis against Enterococcus faecalis

The aim of this study was to evaluate the in vitro antimicrobial activity of Brazilian brown propolis as an intracanal medication againstEnterococcus faecalis. Thirty dentin discs prepared from intact freshly extracted bovine maxillary central incisors were infected withE. faecalis for 21 days. The specimens were distributed into six groups according to the medicament used as follows: G1- calcium hydroxide paste; G2- Carbowax 400 (control group); G3- 20% brown propolis paste; G4- 40% brown propolis paste; G5- 20% brown propolis paste + calcium hydroxide paste; and G6- 40% brown propolis paste + calcium hydroxide paste. The experimental pastes were placed into the canal lumen and left for 14 days. After each period, irrigation was performed with sterile saline to remove the medicament, and the canals were dried with sterile paper points. The dentin chips were removed from the canals with sequential sterile round burs at low speed and were immediately collected in separate test tubes containing BHI broth. The tubes were incubated at 37°C, and microbial growth was analyzed by spectrophotometry after 15 days. All the experimental medications significantly reduced the number of viable bacteria. The G4 and G5 pastes were more effective than the G1 paste, with 35.8%, 41%, and 21.3% antibacterial activity, respectively. Brazilian brown propolis shows antibacterial capacity againstE. faecalis.

A laboratory assessment of bacterial leakage in MTA apical plugs exposed to phosphate-buffered saline / Avaliação laboratorial da infiltração bacteriana em plugs apicais de mta expostos ao tampão fosfato-salino

O presente estudo avaliou a influência da exposição do agregado de trióxido mineral (MTA) – com e sem cloreto decálcio (CaCl2) – ao tampão fosfato-salino (PBS) sobre a microinfiltração apical. Sessenta segmentos radiculares foram divididos em 4 grupos experimentais (n=15). As cavidades apicais foram preenchidas com MTA, com ou sem CaCl2, e os canais radiculares receberam uma bolinha de algodão umedecida ou PBS, como medicação intracanal: 1) MTA/bolinha de algodão umedecida; 2) MTA/PBS; 3) MTA+10%CaCl2/ bolinha de algodão umedecida; 4) MTA+10% CaCl2/PBS. Após 2 meses, a penetração de E. faecalis ao longo dos plugs apicais foi avaliada. As amostras foram observadas semanal -mente durante 70 dias e a infiltração detectada através da turbidez do meio em contato com os segmentos radiculares. Dentes pertencentes aos grupos controle (n=2) foram mantidos completamente impermeáveis ou sem plug apical. A análise de sobrevivência e a comparação das curvas foram realizadas por meio dos testes Kaplan-Meier e Log-rank (p<0.05), respectiva -mente. Todas as amostras do grupo controle positivo apresentaram evidência de infiltração dentro de 24h, enquanto nenhuma amostra do grupo controle negativo apresentou infiltração aolongo dos 70 dias. Não houve diferença significativa entre os grupos experimentais (p=0.102). O uso do PBS como medicação intracanal pode melhorar a capacidade de selamento do MTA,mas não é capaz de impedir a infiltração bacteriana. A adição de CaCl2 ao MTA não melhora sua capacidade de selamento.

This study evaluated the influence of the exposure of mineral trioxide aggregate (MTA) - with and without calcium chloride(CaCl2) -to phosphate-buffered saline (PBS) on apical microleakage. Sixty root segments were divided into 4 experimental groups (n=15). Apical cavities were filled with MTA with or without CaCl2, and the root canals dressed with a moistened cotton pellet or PBS: 1) MTA/cotton pellet; 2) MTA/PBS; 3) MTA+10%CaCl2/cotton pellet; 4) MTA+10%CaCl2/PBS. After 2months, E. faecalis penetration was analyzed a long the apical plugs. Samples were observed weekly for 70 days, and leakage was detected by turbidity of the medium in contact with the root segment. Teeth in the control groups (n=2) were either made completely impermeable or kept without an apical plug. The Kaplan–Meier method was used to analyze survival and the Log-rank test was used to compare the survival curves (p<0.05). All specimens in the positive control group showed evidence of leakage within 24h, while none in the negative control group showed leakage up to 70 days. There was no statisticall y significant difference among the experimental groups (p=0.102).The use of PBS as intracanal dressing may improve MTA sealing ability, but cannot prevent bacterial leakage. The addition of CaCl2 to the MTA did not improve MTA sealing ability.

The antimicrobial effects of Citrus limonum and Citrus aurantium essential oils on multi-species biofilms

The aim of this study was to evaluate the effects of Citrus limonum and Citrus aurantium essential oils (EOs) compared to 0.2% chlorhexidine (CHX) and 1% sodium hypochlorite (NaOCl) on multi-species biofilms formed by Candida albicans, Enterococcus faecalis and Escherichia coli. The biofilms were grown in acrylic disks immersed in broth, inoculated with microbial suspension (106 cells/mL) and incubated at 37°C / 48 h. After the biofilms were formed, they were exposed for 5 minutes to the solutions (n = 10): C. aurantium EO, C. limonum EO, 0.2% CHX, 1% NaOCl or sterile saline solution [0.9% sodium chloride (NaCl)]. Next, the discs were placed in sterile 0.9% NaCl and sonicated to disperse the biofilms. Tenfold serial dilutions were performed and the aliquots were seeded onto selective agar and incubated at 37°C / 48 h. Next, the number of colony-forming units per milliliter was counted and analyzed statistically (Tukey test, p ≤ 0.05). C. aurantium EO and NaOCl inhibited the growth of all microorganisms in multi-species biofilms. C. limonum EO promoted a 100% reduction of C. albicans and E. coli, and 49.3% of E. faecalis. CHX was less effective against C. albicans and E. coli, yielding a reduction of 68.8% and 86.7%, respectively. However, the reduction of E. faecalis using CHX (81.7%) was greater than that obtained using C. limonum EO. Both Citrus limonum and Citrus aurantium EOs are effective in controlling multi-species biofilms; the microbial reductions achieved by EOs were not only similar to those of NaOCl, but even higher than those achieved by CHX, in some cases.

New trends in dentistry: plant extracts against Enterococcus faecalis. The efficacy compared to chlorhexidine

Enterococcus faecalis is an important pathogen associated with endodontic diseases, and its elimination and control are of paramount importance, as it represents one of the major causes of failure in the treatment of endodontic disease. Twenty-five plant extracts obtained from Brazilian forests were found to be effective against planktonic E. faecalis and were subjected to two traditional antibacterial assays, the microdilution broth assay (MDBA) and the disk diffusion assay (DDA), using chlorhexidine (CHX) as a control. Seven out of 25 extracts showed significant antibacterial activity and were tested in a biofilm assay, and three of these extracts were subjected to chemical fractionation. Residues were tested for their antibacterial activity, and the first chemical findings were described based on thin layer chromatography (TLC). Extracts obtained from Ipomoea alba, Symphonia globulifera and Moronobea coccinea showed significant bactericidal activity in the MDBA. The same I. alba and S. globulifera extracts, as well as the extract obtained from Connarus ruber var. ruber, showed significant activity in the DDA. RH2O obtained from Psidium densicomum and Stryphnodendron pulcherrimum showed better antibacterial activity compared to the respective crude extracts and CHX. TLC analysis showed that phenolic compounds and triterpenes represent the first findings of chemical groups that may occur in all species. The results of the present study include the discovery of six active extracts against planktonic E. faecalis and support further testing via assays involving biofilm formation, as well as the determination of the compounds' chemical profiles, as their activity was significantly better than that observed for CHX.

Ex-vivo Effect of Intracanal Medications Based on Ozone and Calcium Hydroxide in Root Canals Contaminated with Enterococcus faecalis

This ex vivo study evaluated the antibacterial effect of intracanal medications in root canals contaminated with Enterococcus faecalis. Fifty single-rooted human teeth were contaminated with E. faecalis (ATCC 29212) and incubated at 37°C for 21 days. The specimens were randomly divided into 5 groups according to the intracanal medication used: OZ-PG: ozonized propylene glycol; CH/CPMC: calcium hydroxide/camphorated paramonochlorophenol; OZ-PG/CH ozonized PG/CH; PC: positive control group (no medication); and NC: negative control group (no contamination). The samples were collected after 7 days (post-medication) and 14 days (final). Bacterial growth was checked by counting the colony-forming units (CFU). OZ-PG and CH/CPMC reduced significantly the CFU counts compared with PC in the post-medication and final samples, with no statistically significant differences among them. On the other hand, OZ-PG/CH did not reduce significantly the number of bacteria compared with PC. In conclusion, among the evaluated medications OZ-PG and CH/CPMC were the most effective against E. faecalis.

Resumo Este estudo ex vivo avaliou o efeito antibacteriano de medicações intracanal em canais radiculares contaminados com Enterococcus faecalis. Cinquenta dentes humanos unirradiculares foram contaminados com E. faecalis (ATCC 29212) e incubados a 37°C durante 21 dias. Os espécimes foram aleatoriamente divididos em diferentes grupos de acordo com a medicação intracanal utilizada: PG-OZ: propilenoglicol ozonizado; HC/PMCC: hidróxido de cálcio/paramonoclorofenol canforado; PG-OZ/CH; CP: controle positivo (sem medicação); e CN: controle negativo (sem contaminação). As amostras foram coletadas após 7 dias (pós-medição) e 14 dias (final). O crescimento bacteriano foi verificado através da contagem das unidades formadoras de colônias (UFC). PG-OZ e HC/PMCC reduziram estatisticamente o número de bactérias quando comparados com o CP nas amostras pós-medição e final, sem diferenças estatísticas entre si. Por outro lado, PG-OZ/HC não reduziu significativamente o número de bactérias em comparação com o CP. Em conclusão, entre as medicações avaliadas, PG-OZ e HC/PMCC foram as mais eficazes contra E. faecalis. .

Biofilm biomass disruption by natural substances with potential for endodontic use

This study evaluated the in vitro effects of four natural substances on the biomass of bacterial biofilms to assess their potential use as root canal irrigants. The following substances and their combinations were tested: 0.2% farnesol; 5% xylitol; 20% xylitol; 0.2% farnesol and 5% xylitol; 0.2% farnesol, 5% xylitol, and 0.1% lactoferrin; 5% xylitol and 0.1% lactoferrin; and 20 mM salicylic acid. The crystal violet assay was used to evaluate the effects of these substances on the biomass of biofilms formed by Enterococcus faecalis and Staphylococcus epidermidis. All substances except for 20 mM salicylic acid and 20% xylitol reduced biofilm mass when compared to controls. The combination of farnesol and xylitol was the most effective agent against E. faecalis ATCC 29212 (p < 0.05). Farnesol combined with xylitol and lactoferrin was the most effective against biofilms of the endodontic strain of E. faecalis MB35 (p < 0.05). Similarly, combinations involving farnesol, xylitol, and lactoferrin reduced the biomass of S. epidermidis biofilms. In general, farnesol, xylitol, and lactoferrin or farnesol and xylitol reduced biofilm biomass most effectively. Therefore, it was concluded that combinations of antibiofilm substances have potential use in endodontic treatment to combat biofilms.

A preliminary study of the antibacterial potential of cetylpyridinium chloride in root canals infected by E. faecalis

The aim of this preliminary study was to verify the antibacterial potential of cetylpyridinium chloride (CPC) in root canals infected by Enterococcus faecalis. Forty human maxillary anterior teeth were prepared and inoculated with E. faecalis for 60 days. The teeth were randomly assigned to the following groups: 1: Root canal preparation (RCP) + 0.1% CPC with positive-pressure irrigation (PPI, Conventional, NaviTip®); 2: RCP + 0.2% CPC PPI; 3: RCP + 2.5% NaOCl PPI; 4: RCP + 2.5% NaOCl with negative-pressure irrigation system (NPI, EndoVac®); 5: Positive control; and 6: Negative control. Four teeth of each experimental group were evaluated by culture and 4 by scanning electron microscopy (SEM). In all teeth, the root canals were dried and filled with 17% EDTA (pH 7.2) for 3 min for smear layer removal. Samples from the infected root canals were collected and immersed in 7 mL of Letheen Broth (LB), followed by incubation at 37°C for 48 h. Bacterial growth was analyzed by turbidity of culture medium and then observed with a UV spectrophotometer. The irrigating solutions were further evaluated for antimicrobial effect by an agar diffusion test.The statistical data were treated by means, standard deviation, Kruskal-Wallis test and analysis of variance. Significance level was set at 5%. The results showed the presence of E. faecalis after root canal sanitization. The number of bacteria decreased after the use of CPC. In the agar diffusion test, CPC induced large microbial inhibition zones, similar to 2% chlorhexidine and large than 2.5% NaOCl. In conclusion, cetylpyridinium chloride showed antibacterial potential in endodontic infection with E. faecalis.

O objetivo deste estudo preliminar foi verificar o potencial antibacteriano de cloreto de cetilpiridínio (CCP) em canais radiculares infectados por E. faecalis. Quarenta dentes anteriores de humanos foram preparados e inoculados com E. faecalis por 60 dias. Os dentes foram aleatoriamente distribuídos como se segue: 1. Preparo do canal radicular (PCR) + CCP 0,1% com sistema de pressão positiva de irrigação (PPI, convencional, Navitip®); 2. PCR + CPC 0,2% PPI; 3. PCR + NaOCl 2,5% PPI, 4. PCR + NaOCl 2,5% com sistema de pressão negativa de irrigação (PNI, EndoVac®); 5 e 6. Controles positivos e negativos. Quatro dentes de cada grupo experimental foram avaliados por cultura e quatro por microscopia eletrônica de varredura (MEV). Em todos os dentes, os canais foram secos e preenchidos com EDTA 17% (pH 7,2) durante 3 min. As amostras dos canais radiculares infectados foram coletadas e imersas em 7 mL Letheen Broth (LB), seguido de incubação a 37° C durante 48 h. O crescimento bacteriano foi analisado pela turvação do meio de cultura, e mensurados por meio de um espectrofotometro (UV). As soluções irrigantes foram ainda avaliadas em teste de difusão em ágar. A análise estatística utilizou de média, desvio padrão,teste de Kruskal-Wallis e análise de variância. O nível de significância foi de 5%. Os resultados mostraram a presença de E. faecalis posterior ao processo de desinfecção do canal radicular. O cloreto de cetilpiridínio mostrou reduzir o número de bactérias. No teste de difusão em ágar, o CPC determinou inibição microbiana, com resultados semelhantes à CHX a 2% e maiores do que o hipoclorito de sódio a 2,5%. O cloreto de cetilpiridínio demonstrou potencial antibacteriano em infecção endodôntica por E. faecalis.

The antimicrobial effect of iodine-potassium iodide after cleaning and shaping procedures in mesial root canals of mandibular molars

The aim of this study was to evaluate the antibacterial activity of 2% iodine potassium iodide (IKI) used as a final rinse after the cleaning and shaping procedures in mesial root canals of mandibular molars infected with Enterococcus faecalis. Seventy two mandibular first molars were used. The root canals were infected with Enterococcus faecalis for 30 days. After the infection procedures, the root canals were cleaned and shaped by using the ProTaper rotary system and manual files. The teeth were randomly assigned to four experimental groups (N=18). In group 1, the root canals were irrigated with sterile distilled water (control). In group 2, the root canals were irrigated with 1% Sodium hypochlorite (NaOCl) during instrumentation. In group 3, root canals were irrigated with 1% NaOCl during instrumentation and a five minute final irrigation using 2 % IKI. In group 4, the root canals were irrigated with 1% NaOCl during instrumentation and a 15 minutes final irrigation with 2 % IKI. Bacteria colony-forming units (CFU) from root canals were semi-quantified and the presence of negative cultures among the groups was compared using Fisher’s test (p < 0,05). The order of effectiveness was: 1% NaOCl plus 2% IKI for 15 minutes (95%), 1% NaOCl plus 2% IKI for 5 minutes (44%), 1% NaOCl (17%) and sterile distilled water (0%). Fisher’s exact test showed a significant difference among the groups (p<0.05). It was concluded that under in vitro conditions, IKI was able to eliminate the Enterococcus faecalis from infected dentin significantly in a 15-minute time frame after the cleaning and shaping procedures.

El objetivo del presente trabajo fue determinar el efecto in vitro del yoduro de potasio yodado al 2% posterior a la preparacion quimiomecanica en conductos radiculares infectados con Enterococcus faecalis. Para este estudio, se emplearon 72 primeras molares inferiores permanentes de humanos, los cuales fueron infectados con Enterococcus faecalis ATCC 29212. Los conductos fueron preparados mediante instrumentacion rotatoria y distribuidos de manera aleatoria en cuatro grupos de acuerdo al irrigante empleado: Grupo 1, agua destilada esteril; Grupo 2, NaOCl al 1%; Grupo 3: NaOCl al 1% IKI al 2% durante cinco minutos; y, Grupo 4: NaOCl al 1% mas IKI al 2% durante 15 minutos. Se tomaron muestras pre y postoperatorias de los conductos y se realizo la semicuantificacion microbiologica de las unidades formadoras de colonias de las bacterias. Fue comparada la presencia de cultivos negativos en los grupos mediante el test de Fisher utilizando un nivel de significancia de p < 0.05. El orden de efectividad para la desinfeccion de los conductos radiculares de mayor a menor fue: NaOCl al 1 % mas IKI al 2% durante 15 minutos (95%), NaOCl al 1% mas IKI al 2% durante 5 minutos (44%), NaOCl al 1% (17%) y agua destilada (0%). Se concluye, que bajo las condiciones in vitro de este estudio, el yoduro de potasio yodado empleado despues de la instrumentacion fue capaz de eliminar significativamente a la bacteria Enterococcus faecalis en un tiempo de 15 minutos.

Evaluation of the antimicrobial effect of super-oxidized water (Sterilox®) and sodium hypochlorite against Enterococcus faecalis in a bovine root canal model

ABSTRACT Ideally root canal irrigants should have, amongst other properties, antimicrobial action associated with a lack of toxicity against periapical tissues. Sodium hypochlorite (NaOCl) is a widely used root canal irrigant, however it has been shown to have a cytotoxic effect on vital tissue and therefore it is prudent to investigate alternative irrigants. Sterilox's Aquatine Alpha Electrolyte® belongs to the group of the super-oxidized waters; it consists of a mixture of oxidizing substances, and has been suggested to be used as root canal irrigant. Super-oxidized waters have been shown to provide efficient cleaning of root canal walls, and have been proposed to be used for the disinfection of medical equipment. OBJECTIVE: To compare the antimicrobial action against Enterococcus faecalis of NaOCl, Optident Sterilox Electrolyte Solution® and Sterilox's Aquatine Alpha Electrolyte® when used as irrigating solutions in a bovine root canal model. METHODOLOGY: Root sections were prepared and inoculated with E. faecalis JH2-2. After 10 days of incubation the root canals were irrigated using one of three solutions (NaOCl, Optident Sterilox Electrolyte Solution®and Sterilox's Aquatine Alpha Electrolyte®) and subsequently sampled by grinding dentin using drills. The debris was placed in BHI broth and dilutions were plated onto fresh agar plates to quantify growth. RESULTS: Sodium hypochlorite was the only irrigant to eliminate all bacteria. When the dilutions were made, although NaOCl was still statistically superior, Sterilox's Aquatine Alpha Electrolyte® solution was superior to Optident Sterilox Electrolyte Solution®. CONCLUSIONS: Under the conditions of this study Sterilox's Aquatine Alpha Electrolyte® appeared to have significantly more antimicrobial action compared to the Optident Sterilox Electrolyte Solution® alone, however NaOCl was the only solution able to consistently eradicate E. faecalis in the model.

A model system to study antimicrobial strategies in endodontic biofilms

The purpose of this work was to develop a model system to study antimicrobial strategies in endodontic biofilms. Enterococcus faecalis suspension was colonized in 10 human root canals. Five milliliters of Brain Heart Infusion (BHI) were mixed with 5 mL of the bacterial inoculums (E. faecalis) and inoculated with sufficient volume to fill the root canal during 60 days. This procedure was repeated every 72 h, always using 24-h pure culture prepared and adjusted to No. 1 MacFarland turbidity standard. Biofilm formation was analyzed by scanning electron microscopy (SEM). E. faecalis consistently adhered to collagen structure, colonized dentin surface, progressed towards the dentinal tubules and formed a biofilm. The proposed biofilm model seems to be viable for studies on antimicrobial strategies, and allows for a satisfactory colonization time of selected bacterial species with virulence and adherence properties.

Survival & detection of the faecal indicator bacterium Enterococcus faecalis in water stored in traditional vessels.

BACKGROUND & OBJECTIVE: Enterococci are used as indicators of faecal pollution and are typically detected using agar-based growth media incubated under standard aerobic conditions. However, such conditions may not be fully effective in enumerating injured bacteria, due to their sensitivity to reactive oxygen species (ROS) under aerobic conditions. Investigations were carried out to assess the extent of sub-lethal damage and ROS-sensitivity on different strains of Enterococcus faecalis in water stored in traditional brass and earthern vessels by enumerating the bacteria under standard aerobic conditions and under conditions designed to neutralize the effect of ROS. METHODS: Pure cultures of E. faecalis were maintained for up to 48 h in brass and earthern vessels and enumerated on various selective and non-selective media either under (i) standard aerobic conditions, (ii) aerobic conditions in a growth medium supplemented with the peroxide scavenger sodium pyruvate, (iii) anaerobic conditions using unsupplemented medium; and (iv) anaerobic conditions in a growth medium supplemented with sodium pyruvate, the latter being regarded as ROS-neutralized conditions. RESULTS: Counts of E. faecalis decreased substantially after storage for 12 h in water kept in the brass vessel but not in the earthern vessel. However, the decrease in counts depended upon the growth medium and the conditions used for enumeration, with a non-selective medium giving the highest count under ROS-neutralized conditions. While the counts obtained on various selective media were also enhanced under ROS-neutralized conditions, they remained lower than those of the non-selective medium. INTERPRETATION & CONCLUSION: Our study showed that growth conditions where reactive oxygen species are neutralized, were effective in enhancing the colony count of stressed E. faecalis, irrespective of the type of medium used for enumeration.

Colonizaçåo vaginal pelo Enterococcus faecalis: prevalência e manifestaçöes citológicas e microbiológicas / Vaginal colonizatin by Enterococcus faecalis: prelance and cytologic and microbiologic findings

Os autores estudaram corrimentos vaginais de 113 pacientes (idade entre 2-52 anos), para investigar a prevalência de Enterococcus faecalis e sua associaçåo com achados citológicos. Cinco faixas etárias foram estudadas: 0-12 anos, 13-18 anos, 19-30 anos, 31-40 anos e maior de 40 anos. A prevalência de Enterococcus faecalis foi maior em pacientes com 0-12 anos (22,8 por cento), 19-30 (40,9 por cento) e 31-40 (22,8 por cento) e menor nas faixas de 13-18 anos (9,0 por cento) e maior de 40 anos (4,5 por cento). A presença de Enterococcus faecalis nas secreçöes vaginais analisadas nåo foi associada com aumento de leucócitos polimorfonucleares, de histiócitos, ou de células epiteliais descamativas. Por outro lado, a colonizaçåo de Sthaphylococcus aureus e de Staphylococcus epidermidis foi menor na presença de Enterococcus faecalis, em pacientes com 0-12 anos (p<0,05) e 19-30 (p<0,05 para a colonizaçåo pelo Staphylococcus aureus e p<0,01 para a colonizaçåo pelo Staphylococcus epidermidis). O mesmo ocorreu com a colonizaçåo de Escherichia coli, em mulheres com 19-30 anos (p<0,05). Os significados destes achados såo discutidos