Antibacterial effect of low-temperature plasma on Enterococcus faecalis in dentinal tubules in vitro / 北京大学学报(医学版)

OBJECTIVE@#To construct a model of Enterococcus faecalis (E. faecalis) infection in dentinal tubules by gradient centrifugation and to evaluate the antibacterial effect of low-temperature plasma on E. faecalis in dentinal tubules.@*METHODS@#Standard dentin blocks of 4 mm×4 mm×2 mm size were prepared from single root canal isolated teeth without caries, placed in the E. faecalis bacterial solution, centrifuged in gradient and incubated for 24 h to establish the model of dentinal tubule infection with E. faecalis. The twenty dentin blocks of were divided into five groups, low-temperature plasma jet treatment for 0, 5 and 10 min, calcium hydroxide paste sealing for 7 d and 2% chlorhexidine gel sealing for 7 d. Scanning electron microscopy and confocal laser scanning microscope were used to assess the infection in the dentinal tubules and the antibacterial effect of low-temperature plasma.@*RESULTS@#The results of scanning electron microscopy and confocal laser scanning microscopy showed that after 24 h of incubation by gradient centrifugation, E. faecalis could fully enter the dentinal tubules to a depth of more than 600μm indicating that this method was time-saving and efficient and could successfully construct a model of E. faecalis infection in dentinal tubules. Low-temperature plasma could enter the dentinal tubules and play a role, the structure of E. faecalis was still intact after 5 min of low-temperature plasma treatment, with no obvious damage, and after 10 min of low-temperature plasma treatment, the surface morphology of E. faecalis was crumpled and deformed, the cell wall was seriously collapsed, and the normal physiological morphology was damaged indicating that the majority of E. faecalis was killed in the dentinal tubules. The antibacterial effect of low-temperature plasma treatment for 10 min exceeded that of the calcium hydroxide paste sealing for 7 d and the 2% chlorhexidine gel sealing for 7 d. These two chemicals had difficulty entering deep into the dentinal tubules, and therefore only had a few of antibacterial effect on the bacterial biofilm on the root canal wall, and there was also no significant damage to the E. faecalis bacterial structure.@*CONCLUSION@#Gradient centrifugation could establish the model of E. faecalis dentin infection successfully. Low-temperature plasma treatment for 10 min could kill E. faecalis in dentinal tubules effectively, which is superior to the calcium hydroxide paste sealing for 7 d and the 2% chlorhexidine gel sealing for 7 d.

Elimination of an intraradicular biofilm composed of enterococcus faecalis and candida albicans using a negative apical pressure irrigation system / Eliminación de una biopelícula intraradicular compuesta por enterococcus faecalis y candida albicans utilizando un sistema de irrigación de presión apical negativa

Endodontic treatment consists of the cleaning and disinfecting the root canal system, which is achieved using adequate mechanical instru-mentation and chemical irrigation. Endodontic microorganisms are present in root canals in the form of a biofilm, and their elimination ensures the success of endodontic treatment. Irrigation is a key factor contributing to the elimination of this intraconduct biofilm, and different irrigator agents and irrigation techniques, such as irrigation with negative apical pressure, a novel automated irrigation mechanism based on suction intraconduct, have been used. In this study, we evaluated the ability of a negative apical pressure system with different concentrations of sodium hypochlorite and durations to reduce the microbial load. Materials and Methods: An intraradicular biofilm composed of Enterococcus faecalis and Candida albicans was generated during twenty-one days of static culture on one hundred mesio-vestibular roots of upper molars with complex curvatures greater than 30°C, and the roots were classified in six groups with different concentrations and contact times of sodium hypochlorite. Subsequently, the reduction in the microbial load was measured with McFarland scale and the enumeration of colony forming units and was evaluated with scanning electronic microscopy. Results: We observed a significant difference in the reduction of the microbial load prior to instrumentation compared with postinstrumentation between the groups treated with 2.25% and 5.25% NaOCl for 30, 60 and 90 seconds of contact time (p<0.05), but we did not observe differences in the reduction of microbial load between different contact times and concentrations of sodium hypochlorite employed (p>0.05). Conclusion: Negative apical pressure is a good option for irrigation in endodontics, as it allows the passage of the irrigation fluid along the total length of the root canal and produces a better antimicrobial effect.

El tratamiento de endodoncia consiste en la limpieza y desinfección del sistema de conducto radicular, lo que se logra utilizando instrumentación mecánica adecuada y riego químico. Los microorganismos endodónticos están presentes en los conductos radiculares en forma de una biopelícula, y su eliminación asegura el éxito del tratamiento endodóntico. La irrigación es un factor clave que contribuye a la eliminación de esta biopelícula intraconductora, y se han utilizado diferentes agentes irrigadores y técnicas de irrigación, como la irrigación con presión apical negativa, un nuevo mecanismo automatizado de irrigación basado en la intraconducción de succión. En este estudio, evaluamos la capacidad de un sistema de presión apical negativa con diferentes concentraciones de hipoclorito de sodio y duraciones para reducir la carga microbiana. Material y Métodos: Se generó una biopelícula intraradicular compuesta por Enterococcus faecalisy Candida albicans durante veintiún días de cultivo estático en cien raíces mesio-vestibulares de molares superiores con curvaturas complejas superiores a 30°C, y las raíces se clasificaron en seis grupos con diferentes concentraciones y tiempos de contacto de hipoclorito de sodio. Posteriormente, la reducción en la carga microbiana se midió con la escala de McFarland y la enumeración de las unidades formadoras de colonias y se evaluó con microscopía electrónica de barrido. Resultado: Observamos una diferencia significativa en la reducción de la carga microbiana antes de la instrumentación en comparación con la postinstrumentación entre los grupos tratados con NaOCl 2.25% y 5.25% durante 30, 60 y 90 segundos de tiempo de contacto (p<0.05), pero lo hicimos No se observan diferencias en la reducción de la carga microbiana entre los diferentes tiempos de contacto y las concentraciones de hipoclorito de sodio empleado (p>0.05). Conclusión:La presión apical negativa es una buena opción para el riego en endodoncia, ya que permite el paso del líquido de riego a lo largo de todo el conducto radicular y produce un mejor efecto antimicrobiano.

Removal and killing of multispecies endodontic biofilms by N-acetylcysteine

ABSTRACT Removal of bacterial biofilm from the root canal system is essential for the management of endodontic disease. Here we evaluated the antibacterial effect of N-acetylcysteine (NAC), a potent antioxidant and mucolytic agent, against mature multispecies endodontic biofilms consisting of Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus mutans and Enterococcus faecalis on sterile human dentin blocks. The biofilms were exposed to NAC (25, 50 and 100 mg/mL), saturated calcium hydroxide or 2% chlorhexidine solution for 7 days, then examined by scanning electron microscopy. The biofilm viability was measured by viable cell counts and ATP-bioluminescence assay. NAC showed greater efficacy in biofilm cell removal and killing than the other root canal medicaments. Furthermore, 100 mg/mL NAC disrupted the mature multispecies endodontic biofilms completely. These results demonstrate the potential use of NAC in root canal treatment.

Efficacy of relacin combined with sodium hypochlorite against Enterococcus faecalis biofilms

Abstract Objective Relacin is a synthetic molecule that targets RelA, an essential protein in a conserved bacterial stress response system. It was shown to inhibit bacterial growth. The aims of this study were to evaluate the antimicrobial effect of relacin combined with sodium hypochlorite (NaOCl) on Enterococcus faecalis biofilms and to evaluate the cytotoxicity of relacin. Material and Methods 48-h E. faecalis OG1RF biofilms were treated by various concentrations of relacin in order to determine its inhibitory concentration. Then, the 48-h biofilms were treated either with 1-min NaOCl (0.01%, 0.05%) alone, or in combination of relacin. As a means of comparison, the biofilms of ΔrelA were also treated by 1-min NaOCl (0.01%, 0.05%, 0.25%). The treatment efficacy was determined by agar plate count assays. The cytotoxicity of relacin was examined on human gingival epithelial cells Ca9-22 and murine fibroblasts NIH-3T3 by a methyl thiazolyltetrazolium (MTT) assay and a lactate dehydrogenase assay. Statistical analysis was performed by one-way or two-way analysis of variance (ANOVA) with Bonferroni's post-hoc test and an independent Student's t-test. A significance level of p<0.05 was used. Results Relacin inhibited the growth of OG1RF biofilms partially at 8 mM and fully at 14 mM. The relacin (14 mM) and NaOCl combined treatment resulted in significantly higher treatment efficacy than NaOCl treatment alone. At 0.05% NaOCl, the combined treatment resulted in 5.65 (±0.19) log reduction in biofilm viability. The ΔrelA biofilms were more susceptible to NaOCl treatment than the wild type biofilms at 0.25% NaOCl. Relacin at 14 mM was not toxic to host epithelial cells and fibroblasts. Conclusions The combination of relacin with a low concentration of NaOCl was effective and not cytotoxic.

Efeito antimicrobiano do bacteriófago geneticamente modificado φEf11/φFL1C(Δ36)PnisA sobre cepas de E. faecalis em biofilme estático e em canais radiculares infectados / Antibacterial effect of a Genetically-Engineered Bacteriophage φEf11/φFL1C(Δ36)PnisA on static biofilms and on Dentin Infected with E. faecalis

A presença de micro-organismos no sistema de canais radiculares (SCR) tem sido apontada como uma das principais causas de insucesso da terapia endodôntica. A capacidade de formar biofilme e penetrar nos túbulos dentinários são fatores de sobrevivência que favorecem a perpetuação de micro-organismos no interior do SCR. Terapias que promovam a desorganizazão de biofilmes e eliminação de bactérias dentro dos túbulos dentinários são fundamentais para a desinfecção endodôntica. Uma terapia descoberta há um século, denominada de Bacteriofagoterapia, baseia-se na utilização de vírus capazes de infectar e matar bactérias. Esta abordagem antimicrobiana tem recebido bastante atenção atualmente por representar uma alternativa para o tratamento de doenças causadas por bactérias multirresistentes aos antibióticos. O objetivo deste estudo foi avaliar a eficácia de um bacteriófago modificado geneticamente, φEf11/φFL1C(Δ36)PnisA, para eliminar biofilmes de duas cepas de E. faecalis: JH2-2 (sensível à vancomicina e resistente ao ácido fusídico e à rifampicina) e V583 (resistente à vancomicina). Este estudo foi dividido em dois experimentos distintos. No primeiro experimento, biofilmes estáticos de 48 horas de cepas de E. faecalis JH2-2 (pMSP3535 nisR / K) ou V583 (pMSP3535 nisR / K) formados em lâminulas de vidro (coverslips) foram inoculados por suspensão do bacteriófago φEf11/φFL1C(Δ36)PnisA. Após 48 horas de incubação, a biomassa bacteriana foi fotografada por microscopia confocal e as células viáveis foram quantificadas por medição de unidades formadoras de colônias (UFC). No segundo experimento, segmentos radiculares de dentes humanos extraídos foram cimentados em dispositivos vedáveis de duas câmaras para formar modelos ex vivo com dentina infectada, contendo solução tampão na câmara inferior. Os modelos foram inoculados com uma suspensão de E. faecalis V583 ou E. faecalis JH2-2. Após sete dias de incubação a 37°C, adicionou-se ao canal de cada segmento de dentina infectada dos grupos 2 e 5 uma suspensão do fago geneticamente modificado, φEf11/φFL1C(Δ36)PnisA e manteve-se a incubação por mais 72 horas. Os segmentos de dentina foram instrumentados com Gates Glidden e a solução tampão foi aliquotada para semeadura e contagem de UFC e aferição do título residual de células de E. faecalis. Os resultados do primeiro experimento mostraram uma diminuição de 10-100 vezes (p≤ 0,05) das células viáveis (UFC / biofilme) após tratamento com bacteriófago, o que foi consistente com a comparação das imagens de biofilme tratado e não tratado visualizadas com projeções máximas da série Z. No segundo experimento a titulação de E. faecalis verificada após tratamento com o bacteriófago foi reduzida em 18% para os modelos infectados com JH2-2 e em 99% (p≤ 0,05) nos modelos infectados com V583. Com base nesses resultados, pode-se concluir que a biomassa dos biofilmes de E. faecalis, tanto sensíveis quanto resistentes à vancomicina, foi significantemente reduzida após a infecção pelo bacteriófago φEf11/φFL1C(Δ36)PnisA. Além disso, o tratamento da dentina infectada por E. faecalis com bacteriófago φEf11/φFL1C(Δ36)PnisA resultou em diminuição da população bacteriana residual de cepas sensíveis e resistentes à vancomicina, alcançando significância estatística no grupo que utilizou a cepa V583.

Residual microorganisms in the root canal system (RCS) have been identified as the main cause of endodontic therapy failure. The ability to form a biofilm and penetrate the dentin tubules are survival factors that favor the perpetuation of microorganisms within the RCS. Therapies that promote the disorganization of biofilms and elimination of bacteria within the dentinal tubules are essential for endodontic disinfection. A therapy discovered a century ago called Bacteriophage Therapy is based on the use of viruses capable of infecting and killing bacteria. Recently, this antimicrobial approach has been receiving considerable attention since it represents an alternative for the treatment of diseases caused by multiresistant antibiotic bacteria. The aim of this study was to evaluate the efficacy of a genetically engineered bacteriophage, φEf11/φFL1C(Δ36)PnisA, to disrupt biofilms of two Enterococcus faecalis strains: JH2-2 (vancomycin sensitive) and V583 (vancomycin resistant). This study was divided into two separate experiments. In the first experiment, 24 hour static biofilms of E. faecalis strains JH2-2(pMSP3535 nisR/K) and V583(pMSP3535 nisR/K) formed on cover slips were inoculated with bacteriophage φEf11/φFL1C(Δ36)PnisA. After 24 and 48 hours incubation, the bacterial biomass was imaged by confocal microscopy and viable cells were quantified by colony forming unit (CFU) measurement. In the second experiment, extracted human dentin root segments were cemented into sealable two-chamber devices, fabricated from syringe needle caps to form in vitro infected-dentin models. The models were inoculated with an overnight suspension of either E. faecalis V583 (vancomycin resistant strain) or E. faecalis JH2-2 (fusidic acid and rifampin resistant, vancomycin sensitive strain). After 7 days of incubation at 37°C, a suspension of a genetically engineered phage, φEf11/φFL1C(Δ36)PnisA, was added to the root canal of each infected dentin segment, and the incubation was continued for an additional 72-hours. Dentin was harvested from the walls of each root canal and assayed for the residual titer of E. faecalis cells. The results from the first experiment showed a 10-100-fold fewer decrease in viable cells (CFU/biofilm) after bacteriophage treatment, which was consistent with comparisons of treated and untreated biofilm images visualized as max projections of the Z-series. On the second experiment, the recovered E. faecalis titer was reduced by 18% for the JH2-2 infected models, and by 99% for the V583 infected models. These results suggest that the biomass of E. faecalis biofilms, both sensitive and resistant to vancomycin, was significantly reduced after infection by bacteriophage φEf11/φFL1C(Δ36)PnisA. In addition, treatment of E. faecalis-infected dentin with the phage resulted in the decrease of the residual bacterial population for both susceptible and vancomycin resistant strains, reaching statistical significance in strain V583 group.

Estudo da capacidade fagocítica de macrófagos humanos frente a diferentes cepas de Enterococcus faecalis / Study of phagocytic ability of human macrophages against different strains of Enterococcus faecalis

Enterococcus faecalis (E. faecalis) é um microrganismo presente em lesões endodônticas persistentes, mostrando maior resistência do que outras bactérias ao Hidróxido de Cálcio, um medicamento alcalino que consegue eliminar diversos microrganismos durante o tratamento endodôntico. Assim, os objetivos desse estudo foram: (a) avaliar a resposta de E. faecalis isolados de canal radicular, após estresse alcalino, quanto sobrevivência, crescimento, alteração do pH, resistência/susceptibilidade antimicrobiana e formação de biofilme sobre discos de dentina; (b) avaliar a capacidade fagocítica e produção de óxido nítrico (NO) por macrófagos humanos, frente a bactérias E. faecalis de canais radiculares, submetidas a estresse alcalino; (c) avaliar a expressão de TLR2 e CD14 na superfície dos macrófagos desafiados com as diferentes cepas bacterianas. As cepas utilizadas foram: ATCC4083 (CANAL 1) e uma cepa clínica, obtida por nós, a partir de uma lesão endodôntica primária (CANAL 2), ambas isoladas de canais radiculares; e ATCC29212 isolada de urina (URINA), utilizada como controle. O estresse alcalino foi obtido através da inoculação das bactérias em meio BHIalcalino por 4, 24, 48 e 72 horas. As bactérias alcalino-resistentes foram semeadas em ágar, com ou sem troca do meio, e quantificadas por CFU/mL. A susceptibilidade antimicrobiana das diferentes cepas, estressadas ou não (controle), foi determinada pelo Etest; e o biovolume do biofilme foi quantificado microscopicamente. Para avaliar a capacidade fagocítica, macrófagos obtidos a partir de monócitos do sangue periférico foram desafiados com as diferentes cepas, estressadas ou não em meio BHI-alcalino, por 30 minutos, na proporção 5:1 (bactéria/macrófago), e corados com Laranja de Acridina. Foi contado o total de macrófagos com bactérias internalizadas, considerando o número de bactérias internalizadas por célula (<5 e =5). A concentração de NO foi medida em sobrenadantes, através da reação de Griess, e a expressão de TLR2 e CD14 pelos macrófagos foi analisada por citometria de fluxo. Os resultados revelaram que Enterococcus oriundos de canal radicular foram menos resistentes ao estresse alcalino e mais susceptíveis aos antibióticos testados, do que as bactérias oriundas de urina. A falta de nutrientes foi um fator determinante para o crescimento bacteriano de todas as cepas. O biovolume dos biofilmes foi semelhante para todas as cepas estudadas, e não foi alterado após exposição ao BHI-alcalino. Na presença de bactérias submetidas ao estresse alcalino, houve um menor número de macrófagos com bactérias internalizadas, em comparação ao controle. No entanto, a produção de NO e a expressão de TLR2 e CD14 não foram alteradas. Independentemente da cepa utilizada e da presença de estresse alcalino, a maioria dos macrófagos apresentavam-se com =5 bactérias internalizadas por célula. Na ausência de estresse, as cepas de urina resultaram em maior produção de NO que aquelas oriundas do canal radicular; entretanto, a produção deste gás foi semelhante entre as cepas após estresse alcalino. A partir desses resultados, podemos concluir que bactérias E. faecalis de urina diferem daquelas oriundas do canal radicular, principalmente quanto a susceptibilidade/resistência microbiana; assim sugerimos que estudos envolvendo o campo da Endodontia devam ser realizados com cepas oriundas de canal radicular, preferencialmente que de urina. Concluiu-se ainda que um ambiente alcalino associado a falta de nutrientes pode reduzir o crescimento de E. faecalis. Adicionalmente, o estresse alcalino pode levar a alterações na estrutura da parede de E. faecalis, o que dificulta o seu reconhecimento, reduzindo sua fagocitose, mas não a sua capacidade de ativar a produção de NO, pelos macrófagos. Assim, uma medicação intracanal a base de hidróxido de cálcio associada a restaurações coronais muito bem adaptadas, para se evitar infiltração, é fundamental em tratamentos endodônticos. No entanto, os efeitos do estresse alcalino, nos Enterococcus alcalino-resistentes, podem prejudicar sua fagocitose, contribuindo para sua persistência na doença endodôntica.(AU)

Enterococcus faecalis (E. faecalis) is an microorganism present in persistent endodontic lesions, with greater resistance than other bacteria to the calcium hydroxide, an alkaline intracanal dressing which eliminate several bacterial species during endodontic treatment. The objectives of this study were: (a) to evaluate the response of E. faecalis, isolated from root canal, under alkaline-stress, starvation, antimicrobial resistance/susceptibility and biofilm formation on dentin disks; (b) to evaluate the phagocytic ability and the nitric oxide (NO) concentration of human macrophages against root canal E. faecalis isolates submitted to alkaline stress; (c) to evaluate the intensity of TLR2 and CD14 expression on the surface of macrophages challenged with the different bacterial strains. The bacterial strains used were: ATCC 4083 (CANAL 1) and a clinical strain, obtained by us, from a primary endodontic lesion (CANAL 2), both isolated from pulpless teeth; and ATCC29212, isolated from urine (URINE), was a reference for comparison. All strains were inoculated in alkaline-BHI broth for 4, 24, 48 and 72 hours. The alkalineresistant bacteria were seeded in agar and quantified by CFU/mL. Antimicrobial susceptibility of bacterial strains, stressed or not (control) was determined by the Etest and the biovolume after biofilm formation was quantified by microscopy. To evaluate the phagocytic ability, macrophages obtained by culture of peripheral blood monocyte, were challenged with bacterial strains, stressed or not in BHI-alkaline for 30 minutes at 5:1 ratio (bacteria/macrophages) and stained with Acridine Orange. The total of macrophages with internalized bacteria and also the number of internalized bacteria per cell (<5 and =5) were counted. The NO concentration in the supernatants was measured by Griess reaction and the intensity of TLR2 and CD14 expression on the surface of macrophages was also analyzed by flow cytometry. Results shows less resistance to alkaline stress in root canal strains and less resistance to tested antibiotics when compared with urine enterococci. The lack of nutrient was a determining factor for the bacterial growth in all enterococci strains. The biovolume of biofilm formed by all strains were similar, and were not altered after exposure to an alkaline-BHI. In the presence of alkaline-stressed bacteria, there was a smaller number of macrophages with internalized bacteria, when compared to the control. The NO production or the TLR2 and CD14 expression were not altered. Regardless of the strain or alkaline environment, the number of macrophages that showed =5 internalized bacteria per cell was higher. Without an alkaline-stress the NO production results higher in the urine strain, when compared with the root canal strains, however, was not modificated after the exposure of bacteria to alkalinestress. We conclude that root-canal strains have different features when compared with urine enterococci, with the main differences being evident in their resistance/susceptibility to antibiotics; thus, we suggest that researches with aims directed to interpreting responses to endodontic treatment should be conducted with strains from root-canals. Besides, an alkaline environment associated to a starvation condition can reduce bacterial growth. Additionally, alterations in the structure of bacterial cell wall after alkali-stressing possibly made their recognition difficult, reducing their ability to be phagocytized, but not their ability to activate NO production. Therefore, intracanal medication with calcium hidroxyde dressing and coronal restorations, to prevent infiltration, should be critical in treatments of endodontics infections. However, the impact of alkaline stress, in alkaline-resistant enterococci, can impair the phagocytosis, contributing to their persistence in endodontic disease.(AU)

Ex vivo evaluation of three instrumentation techniques on E. faecalis biofilm within oval shaped root canals

The objective of the present study was to assess the effectiveness of reciprocating instrumentation in disinfecting oval-shaped root canals infected with Enterococcus faecalis. Forty-five human lower premolars were infected with a culture of E. faecalis (ATCC 29212) for 28 days. Five other teeth that were neither contaminated nor instrumented were used as controls. The 45 specimens were divided into three experimental groups (n = 15) based on the root canal preparation technique used: manual (K-type, Dentsply Maillefer, Ballaigues, Switzerland); rotary (MTwo, VDW GmbH, Munich, Germany); and reciprocating (Reciproc R50, VDW GmbH, Munich, Germany) instruments. During chemomechanical preparation, 21 mL of 2.5% NaOCl was used as the irrigating solution. Microbiological sampling was performed before (S1) and immediately after (S2) the chemomechanical preparation using sterilized paper points. Specimens were then cleaved, and 0.02 g of dentine chips was collected from the root thirds to verify the presence of microorganisms in dentinal tubules. All three preparation techniques reduced the number of microorganisms in the root canal lumen and dentine chips from the root thirds, but no significant differences were observed between the three groups (p > 0.05). Reciprocating instrumentation with Reciproc R50 was effective in reducing the number of microorganisms within the root canal system. Although this technique involves the use of only one file to perform the root canal therapy, it is as effective as conventional rotary instrumentation in reducing theE. faecalis biofilm from the root canal system. However, further clinical investigations are warranted in order to ratify these results.

Molecular detection of virulence factors among food and clinical Enterococcus faecalis strains in South Brazil

The present report aimed to perform a molecular epidemiological survey by investigating the presence of virulence factors in E. faecalis isolated from different human clinical (n = 57) and food samples (n = 55) in Porto Alegre, Brazil, collected from 2006 to 2009. In addition, the ability to form biofilm in vitro on polystyrene and the β-haemolytic and gelatinase activities were determined. Clinical strains presented a higher prevalence of aggregation substance (agg), enterococcal surface protein (esp) and cytolysin (cylA) genes when compared with food isolates. The esp gene was found only in clinical strains. On the other hand, the gelatinase (gelE) and adherence factor (ace) genes had similar prevalence among the strains, showing the widespread occurrence of these virulence factors among food and clinical E. faecalis strains in South Brazil. More than three virulence factor genes were detected in 77.2% and 18.2% of clinical and food strains, respectively. Gelatinase and β-haemolysin activities were not associated with the presence of gelE and cylA genes. The ability to produce biofilm was detected in 100% of clinical and 94.6% of food isolates, and clinical strains were more able to form biofilm than the food isolates (Student's t-test, p < 0.01). Results from the statistical analysis showed significant associations between strong biofilm formation and ace (p = 0.015) and gelE (p = 0.007) genes in clinical strains. In conclusion, our data indicate that E. faecalis strains isolated from clinical and food samples possess distinctive patterns of virulence factors, with a larger number of genes that encode virulence factors detected in clinical strains.

Biofilm formation on polystyrene under different temperatures by antibiotic resistant Enterococcus faecalis and Enterococcus faecium isolated from food

The ability of antibiotic resistant E. faecalis and E. faecium isolated from food to form biofilm at different temperatures in the absence or presence of 0.75% glucose was evaluated. A synergistic effect on biofilm at 10 °C, 28 °C, 37 °C and 45 °C and glucose was observed for E. faecalis and E. faecium.

Avaliação da comunidade microbiana, com ênfase para a espécie Enterococus faecalis, em dentes decíduos com necrose pulpar através de técnicas fenotípicas e moleculares / Evaluation of microbiota with emphasis on species of Enterococcus faecalis, in primary teeth root canals with pulp necrosis, using conventional and molecular microbiological techniques

Infecção endodôntica em dentes decíduos tem sido pouca avaliada, apesar da influência destes sobre a dentição permanente. No presente estudo foi avaliada a microbiota, com ênfase na espécie de Enterococcus faecalis, de canais radiculares de dentes decíduos com diagnóstico de necrose pulpar, utilizando-se técnicas microbiológicas convencionais e moleculares. Para tanto, um estudo do tipo seccional, clínico e laboratorial foi desenvolvido, sendo a coleta do material endodôntico realizada na clínica de Odontopediatria da Faculdade de Odontologia da UFRJ. Para o estudo, 244 crianças saudáveis foram examinadas no período de um ano, e destas, 43 se enquadravam nos critérios de inclusão. O material foi coletado do canal radicular utilizando-se cones estéreis de papel, dos quais dois foram inoculados em caldo seletivo-indicador Enterococcosel e os outros dois em TSB-DMSO sob congelamento a -20º C. A identificação de E. faecalis foi realizada a partir do cultivo inicial no caldo e subcultivo em agar sangue para observação de colônias bacterianas características. Testes bioquímicos e enzimáticos e a Reação em Cadeia da Polimerase (PCR) para o gene do rRNA 16S também foram utilizados para identificação da espécie. A técnica de Eletroforese em Gel com Gradiente Desnaturante (DGGE) foi utilizada para avaliação do perfil da comunidade microbiana presentes nos espécimes clínicos. Os resultados mostraram que dos 43 espécimes clínicos obtidos, 18 foram excluídos devido à contaminação no controle. Entre os 25 casos estudados, 10 foram positivos no caldo de enterococcosel, sendo cinco (20%) positivos para a espécie E. faecalis nos testes fenotípicos e na PCR. Outros cinco espécimes foram positivos no caldo, mas as amostras bacterianas não apresentaram bioquímica compatível com a espécie E. faecalis, e foram então submetidas ao seqüenciamento de um fragmento do gene do rRNA 16S. Foram identificados Lactobacillus plantarum (4 amostras) e Lactobacillus rhamnosus (1 amostra). Não houve relação dos dados clínicos com a presença de E. faecalis (p > 0,05). A técnica de DGGE mostrou uma comunidade polimicrobiana nos 25 espécimes analisados e, considerando-se o número de bandas no gel, um número ≥ 20 foi relacionado com pacientes com idade ≤ 4 anos e  20 bandas com pacientes com mais de 4 anos de idade (p  0,05). Relação significativa também foi observada entre idade  4 anos e cárie em dente posterior, assim como entre idade  4 anos e cárie em dente anterior. Trauma como causa de infecção endodôntica foi significativa entre crianças com 4 anos de idade. Os resultados demostram a presença de E. faecalis em infecções endodônticas com necrose pulpar em dentição decídua, confirmando sua presença na cavidade oral destes pacientes. Adicionalmente, a técnica de DGGE mostrou uma comunidade polimicrobiana, indicando associação entre idade do paciente e doença cárie.

Endodontic infections in primary teeth have been poorly evaluated despite their influence on permanent dentition. In the present study we assessed the microbiota, with emphasis on species of Enterococcus faecalis, in primary teeth root canals with pulp necrosis, using conventional and molecular microbiological techniques. Thus, a cross-sectional study of clinical and laboratory data was developed. The material was collected at the endodontic clinic of Pediatric Dentistry, Faculty of Dentistry, UFRJ. For the study, 244 healthy children were examined during one year, and of these, 43 met the inclusion criteria. Material was collected from root canals using sterile paper cones. Four paper cones were used for each canal: two were inoculated in the selective broth indicator Enterococcosel and anothers two placed in TSB-DMSO and frozen at -20 ° C until required. Identification of E. faecalis was initially made using culture in broth and subculture on blood agar for observational characteristics of bacterial colonies. Biochemical and enzymatic analysis as well as Polymerase Chain Reaction (PCR) for the 16S rRNA gene were also used for species identification. The Gradient Gel Electrophoresis Agents denaturants (DGGE) technique was used to assess the profile of the microbial communities present in the clinical specimens. Eighteen (18) of the 43 clinical specimens obtained were excluded due to contamination. Among the 25 cases studied, 10 were positive for the species E. faecalis in Enterococcosel broth, five (20%) were positive in the phenotypic tests and PCR. Another five specimens were positive in the broth medium, but the bacterial samples showed no biochemical species compatible with E. faecalis, and so were subjected to sequencing of a fragment of the 16S rRNA gene. Lactobacillus plantarum were identified (4 samples) and Lactobacillus rhamnosus (1 sample). There was no statistically significant correlation of clinical data with the presence of E. faecalis (p> 0.05). The DGGE analysis showed a community polymicrobial. About 20 bands or more were associated with patients ≤ 4 years old and that lesser 20 bands were associated with patients over 4 years old (p  0,05). A significant relationship was also found between patients > 4 years old and caries in posterior teeth, as well as between patients  4 years and anterior tooth caries. Trauma as the cause of endodontic infection was significant among children  4 years old. The results show the presence of E. faecalis in endodontic infections with pulp necrosis in the primary dentition, confirming its presence in the oral cavity of patients. Additionally, DGGE showed a polymicrobial community in 25 samples obtained, suggesting an association between patient age and tooth decay.

Efetividade de diferentes sistemas de preparo do canal radicular na eliminação de Enterococcus faecalis / Effectiveness of diferente root canal preparation systems against Enterococcus faecalis

O objetivo deste estudo consiste em avaliar a efetividade de diferentes sistemas empregados no preparo biomecânico, dentre eles: Self-adjusting file (SAF), TiLOS Anatomic Endodontic Technology System (TiLOS) e Mtwo sobre a eliminação de Enterococcus faecalis cultivados em canais radiculares. Para isso, foram selecionados 47 prés-molares inferiores humanos, os quais foram contaminados por Enterococcus faecalis (ATCC 29212) durante 21 dias e divididos em três grupos: GI - SAF com irrigação contínua; GII - EndoEze Tilos e irrigação com auxílio de agulhas NaviTip; GIII - MTwo e irrigação com NaviTip a cada troca de instrumento. A irrigação foi realizada com solução de NaOCl a 2,5%. Coletas bacterianas foram realizadas em três tempos experimentais: coleta da contaminação (S1), posterior ao preparo dos canais radiculares (S2) e após sete dias do preparo biomecânico (S3). Os dados em UFC foram submetidos aos testes ANOVA e Tukey ou Kruskal-Wallis e Dunn com P<0.05. Na análise dos resultados, foi observada redução de E.faecalis após preparo (P < .05) em todos os grupos, sem diferença significante entre os grupos (P > .05). A coleta final demonstrou aumento bacteriano similar em todos os grupos, esclarecendo a viabilidade das bactérias incubadas. Dessa forma, conclui-se que o preparo dos canais radiculares realizado com SAF, EndoEze Tilos e Mtwo contribuem na desinfecção do canal radicular, sem, todavia, eliminar o E. faecalis do sistema de canais radiculares.

The aim of this study was to evaluate the effectiveness of different chemo-mechanical systems for root canal preparation Self-adjusting File (SAF), TiLOS Anatomic Endodontic Technology System (TiLOS), and Mtwo against Enterococcus faecalis in the canal. Forty seven human mandibular bicuspids were contaminated with E. faecalis (ATCC 29212) for 21 days and divided into groups according to preparation methodÇ SAF group with continuous irrigationç TiLOS;Endo-Eze group and irrigation with a NaviTip needle Mtwo group and irrigation eith NaviTip. The irrigant used was 2.5% NaOCl. Bacterial harvests were performed after the contamination period (initial harvest), immediately post-preparation, and seven days after chemo-mechanical preparation. Data were expressed as CFU and subjected to ANOVA and Tukey or Kruskal-Wallis and Dunn tests at P < 0.05. Reduction in E.faecalis was observed after preparation (P< .05), with no significant differences between the groups (P> .05). The final harvest demonstrated statistically similar increase in the bacterial counts in all groups. Root canal preparation using SAF, TiLOS;Endo-Eze, and Mtwo systems contribute for the disinfection of the root

Periradicular repair after two-visit endodontic treatment using two different intracanal medications compared to single-visit endodontic treatment

The number of appointments necessary to treat infected root canals is one of the most controversial issues in endodontics. This study evaluated, in dogs, the response of the periradicular tissues to the endodontic treatment of infected root canals performed in a single visit or in two visits, using different interappointment dressings. Periradicular lesions were induced by inoculating Enterococcus faecalis in the root canals. After confirming that a periradicular lesion developed, the root canals were treated within one or two visits, using either ozonized oil or calcium hydroxide in camphorated paramonochlorophenol (CMCP) as an intracanal medication. After 6 months, the animals were sacrificed and the specimens were processed for histological and histobacteriological analysis. The root canals treated in a single visit showed a success rate of 46 percent. When a calcium hydroxide/CMCP-based interappointment intracanal medication was used, 74 percent of the cases were categorized as success. In cases where ozonized oil was used as the intracanal medication, a success rate of 77 percent was observed. These results of the present study demonstrated that the two-visit treatment offered a higher success rate compared to one-visit therapy. In addition, ozonized oil may potentially be used as an intracanal medication.

O número de sessões necessárias para tratar um canal radicular infectado é um dos assuntos mais controversos da endodontia. O objetivo deste estudo foi analisar, em cães, a resposta dos tecidos perirradiculares ao tratamento endodôntico de canais infectados em uma ou duas consultas, usando diferentes medicamentos entre as sessões. Lesões perirradiculares foram induzidas pela inoculação de Enterococcus faecalis nos canais. Após a confirmação do desenvolvimento de uma lesão perirradicular, os canais foram tratados em uma ou duas sessões, usando óleo ozonizado ou hidróxido de cálcio associado ao paramonoclorofenol canforado (PMCC) como medicação intracanal. Após 6 meses, os animais foram sacrificados e os espécimes processados para análise histológica e histobacteriológica. Os canais tratados em sessão única apresentaram uma taxa de sucesso de 46 por cento dos casos. Quando a medicação usada entre as sessões foi o hidróxido de cálcio associado com o PMCC, 74 por cento dos casos resultaram em sucesso. Nos casos em que o óleo ozonizado foi usado, uma taxa de sucesso de 77 por cento foi observada. Esses achados demonstraram que o tratamento em duas sessões oferece uma taxa de sucesso mais alta quando comparado à terapia em uma sessão. Além disso, o óleo ozonizado mostrou potencial para ser usado como medicação intracanal.

Reporte del primer caso de infección por Enterococcus faecalis resistente a vancomicina en un servicio de medicina interna / Report of first ERV infection in an internal medicine service

El presente trabajo describe la primera infección por ERV en un paciente inmunodeprimido pesquisado en el Servicio de Medicina del Hospital San José, de Santiago. Su tratamiento y el manejo epidemiológico con las medidas que se adoptaron para la no ocurrencia de nuevos casos.

This paper describes the first ERV infection in an immunocompromised patient detected in the Internal Medicine Service of San José Hospital in Santiago, Chile. We report on the therapy given, the endemiologic measures taken to avoiding the emergence of new cases.

Estudo da translocaçäo bacteriana após sutura primária de colo com e sem limpeza mecânica: trabalho experimental em cäes / Bacteria translocation study after primary colocolonic suture with and without mechanical purging in dogs

O objetivo do presente trabalho foi investigar a ocorrencia de translocaçäo bacteriana em cäes submetidos a suturas colocólicas primárias, no colo descendente, com e sem limpeza intestinal. Para o estudo, foram utilizados três grupos de oito animais. O Grupo I (obs. de 1 a 8) foi utilizado como grupo-controle. Nesse grupo, os animais foram submetidos à laparotomia sob anestesia geral, exposiçäo das alças intestinais ao ar ambiente da sala cirúrgica pelo período de cinco minutos. No Grupo II (obs. de 9 a 16), realizou-se colotomia e sutura colocólica primária, sem limpeza do conteúdo cólico e fechamento da parede abdominal. No Grupo III (obs. de 17 a 24), realizou-se colotomia, limpeza do reto, limpeza do colo por meio de lavagem mecânica transoperatória retrógrada, seguida de sutura colocólica terminoterminal e fechamento da parede abdominal. Todos os animais foram mantidos em observaçäo pelo período de 24 horas. Após esse período foi realizada a coleta de amostras de sangue e amostras de linfonodo mesentérico, do fígado, baço, rim e pulmäo para realizaçäo de culturas. As culturas foram realizadas em meios específicos para Escherichia coli e Enterococcus faecalis. Os resultados foram: näo ocorreu translocaçäo bacteriana para o sangue portal nos Grupos II e III. Ocorreu translocaçäo bacteriana para os linfonodos, fígado, baço, rim e pulmäo nos Grupos II e III. Observaram-se índices de translocaçäo bacteriana mais elevados no Grupo II do que no Grupo III, porém sem significância estatística. A Escherichia coli foi a bactéria isolada nas culturas positivas dos Grupos II e III