Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Introducción: E. faecalis es un microorganismo anaerobio facultativo, Gram positivo, que forma parte de la microbiota normal de la cavidad oral y del tracto gastrointestinal, forma biofilm, presenta resistencia a factores del sistema inmune y antimicrobianos. Se aísla con mayor frecuencia en fracasos endodónticos y ocasionalmente en infecciones endodónticas primarias como la Periodontitis Apical Asintomática (PAA). Su característica principal es la sobrevivencia en ecosistemas empobrecidos. Objetivo: El objetivo de este estudio fue determinar la presencia de E.faecalis en dientes con diagnóstico de PAA. Material y Método: La muestra en estudio se conformó por 30 pacientes con un diente unirradicular con diagnóstico de PAA. De cada uno de ellos se obtuvo una muestra microbiológica con el método de Schimauchi H en condiciones de aislamiento absoluto. La muestra fue depositada en un vial con 1ml de RTF a 4°C y fue llevada al laboratorio para su procesamiento antes de dos horas. La identificación de E. faecalis se realizó mediante cultivo microbiológico clásico y biología molecular mediante la Reacción en Cadena de la Polimerasa. Resultados: E.faecalis se identificó en el 63,33% (19 individuos) de las muestras microbiológicas obtenidas de dientes diagnosticados con PAA. Conclusión: El alto porcentaje de aislamiento de E. faecalis en la población estudiada, confirma que este patógeno forma parte de la microbiota habitual en la PAA.

En el presente trabajo se evaluó parcialmente las propiedades probióticas de Enterococcus faecium QPa.1, aislada por primera vez de quesos de elaboración artesanal. Para este propósito se realizaron varias pruebas, y se pudo comprobar una gran sensibilidad a la mayoria de los antibióticos ensayados, ausencia de actividad hemolítica, actividad antimicrobiana específica frente a Listeria monocytogenes, tolerancia a diferentes concentraciones de sales biliares (0,3 a 2% de bilis de buey) y resistencia a pH 2 y 3. Asimismo reveló una buena actividad proteolítica y propiedades de adherencia. Enterococcus faecium QPa.1 presenta características propias de un probiótico y tiene una buena actividad inhibitoria solo contra Listeria monocytogenes, bacteria de alto riesgo patológico para la salud humana. Por lo tanto, esta cepa seleccionada puede ser útil como probiótico o como cultivo adjunto y/o bioprotector para la elaboración de quesos regionales, preservando su valor, calidad nutritiva y seguridad sanitaria. Palabras claves: Enterococcus faecium QPa.1, probióticos, quesos / --- The present work evaluated partially probiotic properties of Enterococcus faecium QPa.1, to first time was isolated traditional cheeses. For this purpose several tests were performed, it was found very sensitivity to most antibiotics tested, no haemolytic activity, specific antimicrobial activity against Listeria monocytogenes, tolerance to different concentrations of bile salts (0,3 - 2 % oxgall) and resistance to pH 2 and 3. Also demonstrated good proteolytic activity and adherence properties. Enterococcus faecium QPa.1 presents probiotic characteristics and has a good inhibitory activity only against Listeria monocytogenes, bacteria of pathological risk to human health. Therefore, the selected strain may be useful as a probiotic culture or as adjunct and/or bioprotective cultures in cheeses traditional making, to preserving value, nutritional quality and health safety. Key words: Enterococcus faecium QPa.1, probiotics, chesses.

Resistance to the antibiotic bacitracin in Enterococcus faecalis strain AR01/DGVS is conferred by the genes bcrABD that are under the control of the regulatory protein BcrR. The N-terminal domain of BcrR has similarity to the helix-turn-helix motif of DNA-binding proteins and topological modelling predicts that the C-terminal domain contains four transmembrane α-helices. These data have led to the hypothesis that BcrR is a novel transmembrane transcriptional regulator which acts to both sense bacitracin and initiate transcription of bcrABD. The transcription of bcrABD in the presence of bacitracin results in the expression of a putative ABC transporter, BcrAB, that may function by the efflux of bacitracin from the cell membrane. The aim of this study was to further characterise the mechanism of bacitracin resistance in E. faecalis and to conduct structural and functional studies with BcrR. A series of bcrA-lacZ transcriptional fusions were created to investigate the regulation of bcrABD transcription by BcrR and map the bcrABD promoter. We determined that BcrR activates bcrA-lacZ expression in response to bacitracin directly and not indirectly via the build-up of cell wall stress or cell wall precursors. A 69-bp region of the bcrABD promoter was required for activation of bcrA-lacZ expression. The introduction of mutations into this sequence demonstrated that two inverted repeat regions (viz. 5�-CTGACA(N)₆GTGTC-3�) were required for bcrA-lacZ expression. Furthermore, the creation of a bcrR::kan insertion mutant and a bcrR-lacZ transcriptional fusion revealed that bcrR is required for high-level bacitracin resistance in AR01/DGVS and that BcrR does not act to auto-regulate bcrR-lacZ expression. To study BcrR function, we overproduced BcrR with a C-terminal hexa-histidine tag in Escherichia coli membranes, extracted the protein with n-dodecyl-β-D-maltoside, and subsequently purified it via Ni�⁺-NTA affinity and gel filtration chromatography to apparent homogeneity. Purified BcrR was reconstituted into liposomes and BcrR binding to bcrABD promoter DNA was analysed using gel-shift assays. We demonstrated that BcrR binds the bcrABD promoter in both the absence and presence of Zn�⁺-bacitracin and that the presence of the inverted repeat regions is necessary for binding. Purification of BcrR has also enabled us to commence structural studies toward obtaining the three dimensional structure of BcrR. We obtained crystals of BcrR that diffracted poorly to a resolution of 19 Å. To characterise the function of the putative ABC type transporter (exporter) of bacitracin, BcrAB, we conducted transport assays with purified bacitracin A, fluorescently labelled FITC-bacitracin A, and radioactive ⁶�Ni�⁺-bacitracin. Transport assays conducted with radioactive ⁶�Ni�⁺-bacitracin and inverted membrane vesicles of E. coli, in which BcrABD had been recombinantly overproduced, displayed uptake of ⁶�Ni�⁺-bacitracin, suggesting that BcrAB functions as a transporter of bacitracin. We conclude that BcrR is a transmembrane DNA-binding protein that functions as both a bacitracin sensor and regulator of bcrABD expression. We propose that BcrR binds to inverted repeat regions in bcrABD promoter to regulate bcrABD expression. Expression of bcrABD results in the production of BcrAB, which appears to be capable of bacitracin efflux (i.e. uptake in inverted membrane vesicles), conferring high-level bacitracin resistance to E. faecalis.

Resistance to the antibiotic bacitracin in Enterococcus faecalis strain AR01/DGVS is conferred by the genes bcrABD that are under the control of the regulatory protein BcrR. The N-terminal domain of BcrR has similarity to the helix-turn-helix motif of DNA-binding proteins and topological modelling predicts that the C-terminal domain contains four transmembrane α-helices. These data have led to the hypothesis that BcrR is a novel transmembrane transcriptional regulator which acts to both sense bacitracin and initiate transcription of bcrABD. The transcription of bcrABD in the presence of bacitracin results in the expression of a putative ABC transporter, BcrAB, that may function by the efflux of bacitracin from the cell membrane. The aim of this study was to further characterise the mechanism of bacitracin resistance in E. faecalis and to conduct structural and functional studies with BcrR. A series of bcrA-lacZ transcriptional fusions were created to investigate the regulation of bcrABD transcription by BcrR and map the bcrABD promoter. We determined that BcrR activates bcrA-lacZ expression in response to bacitracin directly and not indirectly via the build-up of cell wall stress or cell wall precursors. A 69-bp region of the bcrABD promoter was required for activation of bcrA-lacZ expression. The introduction of mutations into this sequence demonstrated that two inverted repeat regions (viz. 5�-CTGACA(N)₆GTGTC-3�) were required for bcrA-lacZ expression. Furthermore, the creation of a bcrR::kan insertion mutant and a bcrR-lacZ transcriptional fusion revealed that bcrR is required for high-level bacitracin resistance in AR01/DGVS and that BcrR does not act to auto-regulate bcrR-lacZ expression. To study BcrR function, we overproduced BcrR with a C-terminal hexa-histidine tag in Escherichia coli membranes, extracted the protein with n-dodecyl-β-D-maltoside, and subsequently purified it via Ni�⁺-NTA affinity and gel filtration chromatography to apparent homogeneity. Purified BcrR was reconstituted into liposomes and BcrR binding to bcrABD promoter DNA was analysed using gel-shift assays. We demonstrated that BcrR binds the bcrABD promoter in both the absence and presence of Zn�⁺-bacitracin and that the presence of the inverted repeat regions is necessary for binding. Purification of BcrR has also enabled us to commence structural studies toward obtaining the three dimensional structure of BcrR. We obtained crystals of BcrR that diffracted poorly to a resolution of 19 Å. To characterise the function of the putative ABC type transporter (exporter) of bacitracin, BcrAB, we conducted transport assays with purified bacitracin A, fluorescently labelled FITC-bacitracin A, and radioactive ⁶�Ni�⁺-bacitracin. Transport assays conducted with radioactive ⁶�Ni�⁺-bacitracin and inverted membrane vesicles of E. coli, in which BcrABD had been recombinantly overproduced, displayed uptake of ⁶�Ni�⁺-bacitracin, suggesting that BcrAB functions as a transporter of bacitracin. We conclude that BcrR is a transmembrane DNA-binding protein that functions as both a bacitracin sensor and regulator of bcrABD expression. We propose that BcrR binds to inverted repeat regions in bcrABD promoter to regulate bcrABD expression. Expression of bcrABD results in the production of BcrAB, which appears to be capable of bacitracin efflux (i.e. uptake in inverted membrane vesicles), conferring high-level bacitracin resistance to E. faecalis.

Thesis (M.Sc.)--University of Alberta, 2010. / Title from PDF file main screen (viewed on Apr. 30, 2010). A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Food Science and Technology, Department of Agricultural, Food and Nutritional Science, University of Alberta. Includes bibliographical references.

SANTIAGO, Adriana Kelly de Sousa. Avaliação in vitro da efetividade de diferentes pas antibióticas utilizadas para curativos endodônticos sobre o E. faecalis. 2013. 48 f. Dissertação (Mestrado em Odontologia) - Universidade Federal do Ceará. Faculdade de Farmácia, Odontologia e Enfermagem, Fortaleza, 2013. / Submitted by denise santos (denise.santos@ufc.br) on 2013-10-21T11:31:49Z No. of bitstreams: 1 2013_dis_akssantiago.pdf: 897004 bytes, checksum: 12ecc6e8524770b8355227a887a95a45 (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2013-10-21T13:51:55Z (GMT) No. of bitstreams: 1 2013_dis_akssantiago.pdf: 897004 bytes, checksum: 12ecc6e8524770b8355227a887a95a45 (MD5) / Made available in DSpace on 2013-10-21T13:51:55Z (GMT). No. of bitstreams: 1 2013_dis_akssantiago.pdf: 897004 bytes, checksum: 12ecc6e8524770b8355227a887a95a45 (MD5) Previous issue date: 2013 / There is an agreement that in dental treatments with incomplete rizogenese it is necessary an intracanal medication with high antibacterial effect because the use of files and standard protocol based on instrumentation must be avoided to protect the fragile dental element from extra hazard. Several endodontic dressings have been used with this purpose, alone or associated to a copious irrigation procedure. However, it is not yet standardized in the literature a unique medication with protracted and excellent antimicrobial effect, associated to a feasible handling and insertion into the duct. The purpose of this study was to evaluate the in vitro effectiveness of different antimicrobial pastes currently used as intracanal medication against the pathogen Enterococcus faecalis by means of agar disk diffusion method. Accordingly, it was created a polypropylene device to simulate the release characteristics of the main root canal. The efficacy tests were performed over a period of 30 days. The formulations studied were distributed in triplicate just as follows: Triantibiotic Paste (metronidazole, ciprofloxacin and minocycline); Biantibiotic Paste (metronidazole and ciprofloxacin); Ciprofloxacin Paste; Amoxilin Paste; Calcium hydroxide paste; 0.9% saline (control group). All pastes were still allotted into 2 categories: open and closed apex. The devices remained in the medium at 37ºC over 30 days (static). Samples used for the measurement of biologic response were collected in pre-fixed times: H1(1st hour), H6(6th hour), H24(24th hour), D3(3rd day), D7(7th day), D14(14th day) and D30(30th day) in the course of thirty days. At every settled time, 20μL of solution was removed from each device and embedded on sterile paper discs with 6mm of diameter, being the equivalent volume replaced with fresh release medium. After, the discs were transferred with sterile tweezersg to the surface of agar-BHI medium previously inoculated with bacteria over petri plaques. From the devices with closed apex, it was removed 20μL of solution from above the paste surface, avoiding any directly touch on it. This collect aimed to evaluate the antimicrobial effect of different preparations inside the root canal. From the devices with open apex it was removed 20μL of the solution in which the plastic device was submerged, in order to measure the action of the diffused antibiotic to the periapical region through the apex. From the inhibition halos obtained in each case it was possible to rank the formulations in order of effectiveness. The Amoxicillin paste presented an initial antimicrobial effect more robust, keeping it throughout the experiment. However from the second week there was no more significant difference between this and the other antibiotic pastes (triantibiotic and biantibiotic). These pastes had an initial effect less representative than amoxicillin, however the effect became similar from the second week. Nevertheless, calcium hydroxide paste had a discreet effect initially and it was completely ceased already after the 3rd day. Even if Amoxicillin paste presented a superior result among the studied pastes, it was noticed color changes associated to degradation signs after two weeks. The findings show that these antibiotics pastes posed an excellent pharmacological effect, apart from calcium hydroxide. It can be concluded that all the antibiotics pastes, among them the pastes of amoxicillin, triantibiotic and biantibiotic were effective against E. faecalis. As a result its use as dressing for teeth with incomplete risogenesis in may be a good alternative. Among the advantages it is the microbial effectiveness, as well as the feasible manipulation during the clinical procedure. When comparing biantibiotic and triantibiotic pastes, the first showed to be almost equally effective, being a great choice for the clinical treatments of anterior teeth, where the pigmentation caused by minocycline antibiotic could present a drawback in the final result of the treatment. / Existe um consenso de que no tratamento de dentes com rizogênese incompleta é necessária uma medicação intracanal com máximo efeito antibacteriano, pois o uso de limas e o protocolo convencional de instrumentação devem ser evitados para que não torne o elemento dental ainda mais frágil. Diversos curativos endodônticos têm sido utilizados com este objetivo, bem como o uso de uma irrigação mais copiosa e com irrigante mais efetivo. No entanto, ainda não está padronizada na literatura uma medicação com efeito antibiótico prolongado e efetivo, além de fácil manipulação e inserção no conduto. O objetivo deste trabalho foi avaliar in vitro a eficácia de diferentes pastas antimicrobianas utilizadas como medicação intracanal sobre o patógeno Enterococcus faecalis utilizando o método de disco - difusão em Ágar. Para isso, foi confeccionado um dispositivo de polipropileno que possui características semelhantes ao canal radicular principal. Os testes de eficácia foram realizados por um período de 30 dias. Foram analisadas as seguintes formulações: Pasta triantibiótica (metronidazol, ciprofloxacino e minociclina); Pasta biantibiótica (metronidazol e ciprofloxacino); Pasta de Ciprofloxacino; Pasta de Amoxicilina; Pasta de hidróxido de cálcio; Solução fisiológica 0,9% (grupo controle). Todas as pastas foram ainda alocadas em duas categorias: ápice aberto e ápice fechado e ensaiadas em triplicata. Os dispositivos permaneceram em estufa a 37ºC durante 30 dias (sem agitação), sendo as coletas para aferição da resposta biológica realizadas em períodos preestabelecidos: H1(1ª hora), H6(6ª hora), H24(24ª hora), D3(3º dia), D7(7º dia), D14(14º dia) e D30(30º dia) no decorrer de 30 dias. A cada coleta foram retirados de cada dispositivo 20µL de solução e depositados sobre discos de papel de filtro estéreis com 6 mm de diâmetro. Em seguida, os discos de papel embebidos foram transferidos com pinça estéril para a superfície das placas de petri previamente semeadas com a bactéria. Dos dispositivos com ápice fechado foram removidos 20µL da solução que estava sobre a superfície da pasta, tendo o cuidado para não tocá-la. Este procedimento teve o intuito de medir a ação antimicrobiana direta dos diferentes preparados no interior do conduto radicular. A partir dos dispositivos com ápice aberto foram retirados 20µL da solução na qual o dispositivo plástico encontrava-se imerso, com o intuito de medir a ação do preparado difundido à região periapical. A partir dos halos de inibição obtidos, foi possível observar quais preparados foram mais eficazes com respeito ao seu efeito antibacteriano. A pasta de amoxicilina apresentou o maior efeito antimicrobiano inicial, mantendo-o durante todo o experimento, no entanto, a partir da segunda semana não houve mais diferença estatística entre as pastas antibióticas e o efeito tornou-se semelhante. As pastas triantibiótica e biantibiótica tiveram efeito inicial menor do que o da Amoxicilina, porém o efeito foi igualado a partir da segunda semana. Por outro lado, a pasta de hidróxido de cálcio teve um efeito discreto inicialmente, o qual foi totalmente cessado já após o 3° dia. Ainda que a pasta de Amoxicilina tenha apresentado os melhores resultados dentre as pastas estudadas, foi evidenciado o aparecimento de cor escurecida associada a sinais de degradação após duas semanas. Este estudo mostrou que as pastas antibióticas tiveram um excelente efeito farmacológico, ao contrário da pasta de hidróxido de cálcio. Pode-se concluir que as pastas antibióticas, dentre elas as pastas de Amoxicilina, triantibiótica e biantibiótica apresentaram um excelente efeito sobre o micro-organismo E. faecalis. Portanto, seu uso como medicação intracanal nos casos de dentes com rizogênese incompleta pode ser uma ótima alternativa, estando ainda a facilidade de manipulação durante o procedimento clínico atrelada à eficácia microbicida. Quando comparadas, a pasta biantibiótica mostrou-se quase que igualmente efetiva a triantibiótica, podendo ser uma ótima escolha em casos clínicos de tratamento de dentes anteriores, sempre e quando a pigmentação causada pelo fármaco Minociclina possa comprometer o resultado final do tratamento. Pôde-se concluir que as pastas antibióticas, dentre elas as pastas de amoxicilina, triantibiótica e biantibiótica apresentaram excelente efeito inibitório sobre o micro-organismo E. faecalis. Portanto, o seu uso como medicação intracanal nos casos de dentes com rizogênese incompleta pode ser uma excelente alternativa.

ARRUDA, Theodora Thays Prado. Perfil de sensibilidade de cepas planctônicas e biofilmes de enterococcus faecalis frente a desafios antimicrobianos. 2007. 141 f. Dissertação (Mestrado em Microbiologia Médica)- Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2007. / Submitted by denise santos (denise.santos@ufc.br) on 2012-01-05T16:48:36Z No. of bitstreams: 1 2007_dis_ttparruda.pdf: 3211660 bytes, checksum: 8202e4aa912c156153c5126023e376da (MD5) / Approved for entry into archive by Eliene Nascimento(elienegvn@hotmail.com) on 2012-02-01T14:26:22Z (GMT) No. of bitstreams: 1 2007_dis_ttparruda.pdf: 3211660 bytes, checksum: 8202e4aa912c156153c5126023e376da (MD5) / Made available in DSpace on 2012-02-01T14:26:22Z (GMT). No. of bitstreams: 1 2007_dis_ttparruda.pdf: 3211660 bytes, checksum: 8202e4aa912c156153c5126023e376da (MD5) Previous issue date: 2007 / Enterococcus faecalis has been suggested to be an important etiological agent in endodontic failure. It has been found in the root canal system in a perceptual ranging from 22% to 77% and it has been associated to organisms structured in biofilms. The aim of the present study was to evaluate, in vitro, the effectiveness of endodontic sealers, 2.5% sodium hypochlorite, and 0.5% Lippia sidoides essential oil in eliminating E. faecalis biofilms. Clinical material was collected from 37 patients with root canal chronic infections and 14 Enterococcus faecalis strains were isolated (37.8%). Biofilms from a reference (ATCC) and a clinical multirresistant strain (isolate 12 ) were grown for 8 days. This period was selected based on a chronological study of the Enterococcus faecalis biofilm development through Atomic Force Microscopy. It was verified that there was a significant reduction of the bacteria number when the biofilms were exposed to the endodontic sealers related to the control for the two tested strains (p<0.001). Analyzing the ATCC strain, it was seen that the Epiphany® endodontic sealer presented similar action compared to Endofill® (p>0.05), similar result was found for Isolate 12. When the strains susceptibilities against the sealers was compared it was verified that the isolate 12 was less susceptible than the ATCC strain (p<0.001). It was verified that the 0.5% essential oil solution of Lippia siodides presented a similar action to 2.5% sodium hypochlorite when the biofilm of ATCC strain and isolate 12 were exposed for 10 minutes to this substances (p<0.001). Comparing the susceptibility of the two strains to the solutions tested, there was no difference between them (p>0.05) / Enterococcus faecalis foi sugerido como sendo um importante agente etiológico do insucesso endodôntico. Foi encontrado no sistema de canais radiculares em um percentual de 22% a 77% e foi associado com a formação de estruturas chamadas biofilmes. O objetivo do presente estudo foi avaliar, in vitro, a efetividade de cimentos endodônticos, hipoclorito de sódio a 2,5% e solução do óleo essencial da Lippia sidoides a 0,5% na eliminação de biofilmes de E. faecalis. Material clínico foi coletado de 37 pacientes com infecções crônicas do canal radicular e 14 cepas de Enterococcus faecalis foram isoladas (37,8%). Biofilmes de uma cepa de coleção de cultura (ATCC) e de uma cepa clínica multiresistente (Isolado 12) foram incubados por 8 dias. Esse período foi selecionado baseado em estudo cronológico do desenvolvimento de biofilmes de Enterococcus faecalis através de Microscopia de Força Atômica. Foi verificado que houve uma redução significativa no número de bactérias quando os biofilmes foram expostos aos cimentos endodônticos em relação ao controle para as duas cepas testadas (p<0.001). Analisando a cepa ATCC, foi verificado que o cimento endodôntico Epiphany® apresentou ação similar ao cimento Endofill® (p>0.05); resultado semelhante foi encontrado para o isolado 12. Quando a susceptibilidade das cepas frente aos cimentos endodônticos foi comparada verificou-se que o Isolado 12 foi menos susceptível comparado à cepa ATCC (p<0.001). Observou-se que a solução do óleo essencial de Lippia siodides a 0,5% apresentou ação similar ao hipoclorito de sódio a 2,5% quando os biofilmes das duas cepas foram expostos por 10 minutos a essas substâncias (p<0.001). Comparando a susceptibilidade das duas cepas às soluções testadas, não houve diferença entre elas (p>0.05)

DANTAS, Thereza Cristina Farias Botelho. Terapia fotodinâmica e plasma de baixa temperatura e pressão como tratamentos alternativos contra biofilmes endodônticos patogênicos. 2015. 116 f. Tese (Doutorado em Enfermagem) - Faculdade de Farmácia, Odontologia e Enfermagem, Universidade Federal do Ceará, Fortaleza, 2015. / Submitted by denise santos (denise.santos@ufc.br) on 2016-03-01T15:37:55Z No. of bitstreams: 1 2015_tese_tcfbdantas.pdf: 3995317 bytes, checksum: fba97baf6756229d3df0bdc25b16145f (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2016-03-01T15:39:58Z (GMT) No. of bitstreams: 1 2015_tese_tcfbdantas.pdf: 3995317 bytes, checksum: fba97baf6756229d3df0bdc25b16145f (MD5) / Made available in DSpace on 2016-03-01T15:39:58Z (GMT). No. of bitstreams: 1 2015_tese_tcfbdantas.pdf: 3995317 bytes, checksum: fba97baf6756229d3df0bdc25b16145f (MD5) Previous issue date: 2015 / Photodynamic Antimicrobial Therapy (PACT) and Low Pressure Cold Plasma emerged as an effective adjunctive procedure to conventional endodontic treatment, especially in case of persistent infection. This study was divided into four chapters, which objectives were: Chapter 1) investigate the antibacterial effects of photodynamic antimicrobial chemotherapy (PACT), with different concentrations of toluidine blue-O (TBO), at three different exposure times, over suspensions of Enterococcus faecalis, using a fluorescence probe – Dihydrorhodamine 1, 2,3 for detecting the release of ROS. Chapter 2) study the antimicrobial effect of PACT mediated by Toluidine blue-O activated by red light (LumaCare® LC122) on Enterococcus faecalis and Candida albicans biofilms. Chapter 3) a tissue-tolerable-plasma (TTP) was tested for its antimicrobial activity against mature biofilm of a key endodontic bacterium Enterococcus faecalis. Chapter 4) evaluate the anti-biofilm efficacy of PACT and TTP applied on saliva-coated-teeth with 2-week E. faecalis (ATCC 29212) biofilm, treated with PACT and TTP for 3 different exposure times (1, 2 and 5 minutes) and compared with 2.5% NaOCl irrigation for 5 minutes. In chapters 2 and 3 biofilms were formed on saliva-coated hydroxyapatite discs using batch culture method at 37°C, 5% CO2. BHI broth was changed daily. In chapter 2, mature E. faecalis and C. albicans biofilms were subjected to PACT using TBO (100 μg/mL), using a non-coherent red light source (LumaCare®, 630 nm, 2 mm distance) and energy density of 118.9 J/cm2, 237.8 J/cm2 and 594.5 J/cm2 (chapter 2). Using the same growth conditions, mature E. faecalis biofilms were subject to TTP on chapter 3. The results were expressed by counting colony forming units (cfu) and group means were compared using 1-way ANOVA. The anti-biofilm effect of PACT and TTP improved as exposure time was increased, reaching the maximum effect after 5 minutes of treatment for both therapies. After 5 min of exposure, there is a significant reduction in cfu numbers in PACT and TTP treatments (p<0.05), but neither treatment was as effective as 2.5% NaOCl irrigation. Using root canals in vitro model (chapter 4) TTP was better than PACT, at 5 minutes of exposure. Bacterial killing was confirmed by CLSM/COMSTAT and SEM analysis / A terapia fotodinâmica antimicrobiana (TFDA) e o plasma de baixa temperatura e pressão (PBTP) surgem como tratamentos coadjuvantes à terapia endodôntica convencional, diante do surgimento de cepas resistentes e complexidades anatômicas do canal radicular. Esse estudo foi dividido em 4 capítulos, cujos objetivos foram: Capítulo 1) avaliar o efeito da TFDA mediada pelo TBO em diferentes concentrações e tempos de exposição à luz na produção de EROS e a viabilidade celular em culturas planctônicas de E. faecalis. Capítulo 2) avaliar os efeitos da TFDA realizada com AOT e fonte de luz vermelha, em três diferentes tempos de exposição na viabilidade de biofilmes maduros de E. faecalis e C. albicans. Capítulo 3) avaliar os efeitos do PBTP, em três diferentes tempos de exposição, na viabilidade de biofilmes maduros de E. faecalis. Capítulo 4) comparar o efeito da TFDA e do PBTP em canais radiculares infectados com biofilmes maduros de E. Faecalis, nos mesmos tempos de exposição para as duas terapias. Nos capítulos 2 e 3, os biofilmes foram crescidos em discos de hidroxiapatita, imersos em Brain-heart infusion broth (E. faecalis por 15 dias) ou RPMI 1640 medium (C. albicans por 72 horas). Os biofilmes maduros foram submetidos à TFDA mediada pelo fotossentitizador AOT (100 μg/mL) e fonte de luz vermelha (LumaCare®, 630 nm, 2 mm de distância), com densidades de energia de 118,9 J/cm2, (1 min) 237,8 J/cm2 (2 min) e 594,5 J/cm2 (5 min) (capítulo 2). Nas mesmas condições, biofilmes maduros de E. faecalis foram também submetidos à ação do PBTP (capítulo 3). Tanto a TFDA como PBTP apresentaram-se eficazes como agentes antimicrobianos em um efeito dose- dependente. No capítulo 4, 96 dentes unirradiculares foram preparados com instrumentação mecanizada. Os espécimes foram divididos em 8 grupos: 1-3 – dentes submetidos à TFDA; 4-6 dentes submetidos ao PBTP e dois grupos-controle – hipoclorito de sódio a 2,5% (positivo) e dentes sem tratamento (negativo). Os dados foram tabulados e tratados estatisticamente pelo método ANOVA (p<0,05). Houve uma redução da microbiota em todos os grupos de tratamento, com diferença estatisticamente significante entre os grupos tratados pelo PBTP e TFDA, no tempo de exposição de 5 minutos. Conclui-se então que a TFDA e o PBTP, nos parâmetros testados, apresentaram atividade antimicrobiana eficaz contra os biofilmes estudados, nos dois modelos in vitro apresentados.

Made available in DSpace on 2014-06-11T19:24:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-03-21Bitstream added on 2014-06-13T19:11:10Z : No. of bitstreams: 1 chavezandrade_gm_me_arafo.pdf: 525015 bytes, checksum: e3ea9f8a7b0458b73d6f8fccb2f04314 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo deste estudo foi avaliar a eficácia da Irrigação Ultrassônica Passiva (IUP) na limpeza e eliminação Enterococcus faecalis de canais radiculares, em comparação à Irrigação Manual Convencional (IMC). O estudo foi dividido em dois capítulos. No capítulo I, foram utilizadas raízes de 75 dentes unirradiculados humanos extraídos. Após o preparo biomecânico, os espécimes foram divididos em cinco microplacas de cultura e esterilizados em óxido de etileno. Os canais radiculares foram contaminados com Enterococcus faecalis (ATCC 29212) e incubados a 37ºC durante 21 dias. As microplacas com os espécimes foram divididas em cinco grupos de acordo com o protocolo de irrigação utilizado (n=15): G1- IUP com solução salina; G2- IUP com NaOCl 1%; G3- IMC com solução salina; G4- IMC com NaOCl 1%; G5- controle (sem irrigação). Foram realizadas três coletas microbiológicas: inicial (após 21 dias de contaminação do canal), imediatamente após irrigação e final (após 7 dias dos tratamentos realizados). Após diluições decimais seriadas e semeadura, foi determinado o número de unidades formadoras de colônia por mililitro de solução (UFC/mL). No capítulo II, foram utilizadas raízes de dentes artificiais unirradiculados. Após o preparo dos canais radiculares, foram realizados quatro canais laterais nos terços apical e médio da raiz, nas superfícies vestibular e lingual. Posteriormente, os canais foram preenchidos com uma solução de contraste radiológico. Os espécimes foram divididos aleatoriamente em três grupos de acordo com o protocolo de irrigação (n=08): GI- IMC com agulha 30G, GII- IUP1 com fluxo intermitente, GIII- IUP2 com fluxo contínuo. Antes e após irrigação, os dentes foram radiografados no sentido proximal utilizando... / The aim of this study was to evaluate the cleaning efficacy and elimination of Enterococcus faecalis from the root canal provided by passive ultrasonic irrigation (PUI) compared with conventional needle irrigation (CNI). The study was divided into two chapters. In chapter I, seventy-five extracted human single-rooted teeth were used. After root canal instrumentation, specimens were randomly divided into cell culture microplates. The microplates containing the specimens were wrapped and sterilized by ethylene oxide. Root canals were inoculated with Enterococcus faecalis (ATCC 29212) and incubated at 37ºC for 21 days. The microplates containing the roots were randomly divided in five groups, according to the irrigation method: G1- PUI with saline solution, G2- PUI with 1% NaOCl, G3- CNI with saline solution, G4- CNI with 1% NaOCl, G5- control (no irrigation). Microbiological samples were collected at three time points: initial (21 days after inoculation), post-irrigation (immediately after irrigation), and final (7 days after irrigation). After serial decimal dilutions, the inocula were seeded and was determined the number of CFU/mL. In chapter II, single-rooted artificial teeth were used. After root canal instrumentation, four lateral canals were performed in the middle and apical thirds. After, the root canals were filled with a contrast solution. The roots were randomly divided into three groups, according to the irrigation technique (n=8): GI- IMC with 30-gauge needle, GII- IUP1 with intermittent flow and GIII- IUP2 with continuous flow. Before and after irrigation, the roots were radiographed using a digital radiographic system. The areas of the... (Complete abstract click electronic access below)

Orientador: Juliane Maria Guerreiro Tanomaru / Banca: Idomeo Bonetti Filho / Banca: Celso Luiz Caldeira / Resumo: O objetivo deste estudo foi avaliar a eficácia da Irrigação Ultrassônica Passiva (IUP) na limpeza e eliminação Enterococcus faecalis de canais radiculares, em comparação à Irrigação Manual Convencional (IMC). O estudo foi dividido em dois capítulos. No capítulo I, foram utilizadas raízes de 75 dentes unirradiculados humanos extraídos. Após o preparo biomecânico, os espécimes foram divididos em cinco microplacas de cultura e esterilizados em óxido de etileno. Os canais radiculares foram contaminados com Enterococcus faecalis (ATCC 29212) e incubados a 37ºC durante 21 dias. As microplacas com os espécimes foram divididas em cinco grupos de acordo com o protocolo de irrigação utilizado (n=15): G1- IUP com solução salina; G2- IUP com NaOCl 1%; G3- IMC com solução salina; G4- IMC com NaOCl 1%; G5- controle (sem irrigação). Foram realizadas três coletas microbiológicas: inicial (após 21 dias de contaminação do canal), imediatamente após irrigação e final (após 7 dias dos tratamentos realizados). Após diluições decimais seriadas e semeadura, foi determinado o número de unidades formadoras de colônia por mililitro de solução (UFC/mL). No capítulo II, foram utilizadas raízes de dentes artificiais unirradiculados. Após o preparo dos canais radiculares, foram realizados quatro canais laterais nos terços apical e médio da raiz, nas superfícies vestibular e lingual. Posteriormente, os canais foram preenchidos com uma solução de contraste radiológico. Os espécimes foram divididos aleatoriamente em três grupos de acordo com o protocolo de irrigação (n=08): GI- IMC com agulha 30G, GII- IUP1 com fluxo intermitente, GIII- IUP2 com fluxo contínuo. Antes e após irrigação, os dentes foram radiografados no sentido proximal utilizando ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the cleaning efficacy and elimination of Enterococcus faecalis from the root canal provided by passive ultrasonic irrigation (PUI) compared with conventional needle irrigation (CNI). The study was divided into two chapters. In chapter I, seventy-five extracted human single-rooted teeth were used. After root canal instrumentation, specimens were randomly divided into cell culture microplates. The microplates containing the specimens were wrapped and sterilized by ethylene oxide. Root canals were inoculated with Enterococcus faecalis (ATCC 29212) and incubated at 37ºC for 21 days. The microplates containing the roots were randomly divided in five groups, according to the irrigation method: G1- PUI with saline solution, G2- PUI with 1% NaOCl, G3- CNI with saline solution, G4- CNI with 1% NaOCl, G5- control (no irrigation). Microbiological samples were collected at three time points: initial (21 days after inoculation), post-irrigation (immediately after irrigation), and final (7 days after irrigation). After serial decimal dilutions, the inocula were seeded and was determined the number of CFU/mL. In chapter II, single-rooted artificial teeth were used. After root canal instrumentation, four lateral canals were performed in the middle and apical thirds. After, the root canals were filled with a contrast solution. The roots were randomly divided into three groups, according to the irrigation technique (n=8): GI- IMC with 30-gauge needle, GII- IUP1 with intermittent flow and GIII- IUP2 with continuous flow. Before and after irrigation, the roots were radiographed using a digital radiographic system. The areas of the... (Complete abstract click electronic access below) / Mestre