Este estudo objetivou investigar a distribuição e a relação entre os genes envolvidos com a virulência e formação de biofilme em 196 Enterococcus faecalis isolados de alimentos, clínicos e suabes cloacais de frangos de corte que receberam oocistos de Eimeria maxima e Eimeria acervulina e ração suplementada ou não com anticoccidiano. No primeiro experimento foi investigada a frequência dos genes agg, ace, tet(M), tet(L) e do operon bopABCD; e foi analisada a produção da enzima gelatinase em duas temperaturas de crescimento (36°C e 42°C). Assim como a capacidade de formar biofilme em meio de cultura suplementado com 10% de sangue, 10% de urina ou 0,75% de glicose por 70 Enterococcus faecalis isolados de frangos. Os Enterococcus faecalis isolados de frangos de corte que receberam oocistos de Eimeria maxima e Eimeria acervulina e ração suplementada ou não com anticoccidiano apresentaram capacidade de se adaptar a diferentes nichos biológicos, como por exemplo, sangue e urina. Foi observado um alto percentual dos genes dos fatores de virulência. No segundo experimento, foi avaliada a diversidade filogenética, com base no método de PCR, de 182 cepas isoladas de humanos, frangos de corte e alimentos. Estas cepas formaram quatro grupos filogenéticos (A, B, C e D) e quatro subgrupos (A1, A2, B1 e B2) com índice de similaridade entre 65 a 100%, demonstrando a adaptação de Enterococcus faecalis a diferentes ambientes. No terceiro experimento, foi determinada a capacidade de formação de biofilme de isolados clínicos e alimentares em diferentes meios de cultivo. Observou-se que o meio suplementado com 0,75% de glicose demonstrou ser o mais adequado para estabelecimento de forte aderência e, consequente formação do biofilme microbiano nos isolados de todas as origens. Em contrapartida, o meio suplementado com 10% de sangue foi o que registrou as maiores taxas de fraca formação de biofilme. Os estudos conduzidos demonstram características fenotípicas e genotípicas de Enterococcus faecalis que sugerem uma ampla adaptação ambiental entre os isolados pesquisados. / This study aimed to investigate the distribution and the relationship between genes involved in virulence and biofilm formation in 196 Enterococcus faecalis isolates from food, clinical and cloacal swabs of broilers that received oocysts of Eimeria maxima and Eimeria acervulina and diet supplemented or not with anticoccidial. The first experiment investigated the frequency of agg, ace, tet(M), tet(L) genes and bopABCD operon; production of gelatinase enzyme was analyzed at two growth temperatures (36°C and 42°C). Thus the ability to form biofilm in culture medium supplemented with 10% of blood, 10% of urine and glucose 0.75% for 70 Enterococcus faecalis isolates of chicken. The Enterococcus faecalis isolates from broilers that received oocysts of Eimeria maxima and Eimeria acervulina and supplemented or not with anticoccidial showed the ability to adapt to different biological niches, such as blood and urine. A high percentage of genes of virulence factors were observed. In the second experiment, we evaluated the phylogenetic diversity based on PCR method of 182 strains isolated from humans, broilers and food. These strains formed four phylogenetic groups (A, B, C and D) and four subgroups (A1, A2, B1 and B2) with similarity index between 65 and 100%, demonstrating the adaptation of Enterococcus faecalis to different environments. In the third experiment, the ability of biofilm formation of clinical and food isolates in different culture media was determined. It was observed that the medium supplemented with 0.75% glucose was shown to be more suitable for the establishment of strong adhesion and subsequent formation of the biofilm isolates from all sources. In other hand, medium supplemented with 10% blood was reported that the highest rates of weak biofilm formation. Studies conducted show phenotypic and genotypic characteristics of Enterococcus faecalis that suggest a broad environmental adaptation among the isolates studied.

Este estudo objetivou investigar a distribuição e a relação entre os genes envolvidos com a virulência e formação de biofilme em 196 Enterococcus faecalis isolados de alimentos, clínicos e suabes cloacais de frangos de corte que receberam oocistos de Eimeria maxima e Eimeria acervulina e ração suplementada ou não com anticoccidiano. No primeiro experimento foi investigada a frequência dos genes agg, ace, tet(M), tet(L) e do operon bopABCD; e foi analisada a produção da enzima gelatinase em duas temperaturas de crescimento (36°C e 42°C). Assim como a capacidade de formar biofilme em meio de cultura suplementado com 10% de sangue, 10% de urina ou 0,75% de glicose por 70 Enterococcus faecalis isolados de frangos. Os Enterococcus faecalis isolados de frangos de corte que receberam oocistos de Eimeria maxima e Eimeria acervulina e ração suplementada ou não com anticoccidiano apresentaram capacidade de se adaptar a diferentes nichos biológicos, como por exemplo, sangue e urina. Foi observado um alto percentual dos genes dos fatores de virulência. No segundo experimento, foi avaliada a diversidade filogenética, com base no método de PCR, de 182 cepas isoladas de humanos, frangos de corte e alimentos. Estas cepas formaram quatro grupos filogenéticos (A, B, C e D) e quatro subgrupos (A1, A2, B1 e B2) com índice de similaridade entre 65 a 100%, demonstrando a adaptação de Enterococcus faecalis a diferentes ambientes. No terceiro experimento, foi determinada a capacidade de formação de biofilme de isolados clínicos e alimentares em diferentes meios de cultivo. Observou-se que o meio suplementado com 0,75% de glicose demonstrou ser o mais adequado para estabelecimento de forte aderência e, consequente formação do biofilme microbiano nos isolados de todas as origens. Em contrapartida, o meio suplementado com 10% de sangue foi o que registrou as maiores taxas de fraca formação de biofilme. Os estudos conduzidos demonstram características fenotípicas e genotípicas de Enterococcus faecalis que sugerem uma ampla adaptação ambiental entre os isolados pesquisados. / This study aimed to investigate the distribution and the relationship between genes involved in virulence and biofilm formation in 196 Enterococcus faecalis isolates from food, clinical and cloacal swabs of broilers that received oocysts of Eimeria maxima and Eimeria acervulina and diet supplemented or not with anticoccidial. The first experiment investigated the frequency of agg, ace, tet(M), tet(L) genes and bopABCD operon; production of gelatinase enzyme was analyzed at two growth temperatures (36°C and 42°C). Thus the ability to form biofilm in culture medium supplemented with 10% of blood, 10% of urine and glucose 0.75% for 70 Enterococcus faecalis isolates of chicken. The Enterococcus faecalis isolates from broilers that received oocysts of Eimeria maxima and Eimeria acervulina and supplemented or not with anticoccidial showed the ability to adapt to different biological niches, such as blood and urine. A high percentage of genes of virulence factors were observed. In the second experiment, we evaluated the phylogenetic diversity based on PCR method of 182 strains isolated from humans, broilers and food. These strains formed four phylogenetic groups (A, B, C and D) and four subgroups (A1, A2, B1 and B2) with similarity index between 65 and 100%, demonstrating the adaptation of Enterococcus faecalis to different environments. In the third experiment, the ability of biofilm formation of clinical and food isolates in different culture media was determined. It was observed that the medium supplemented with 0.75% glucose was shown to be more suitable for the establishment of strong adhesion and subsequent formation of the biofilm isolates from all sources. In other hand, medium supplemented with 10% blood was reported that the highest rates of weak biofilm formation. Studies conducted show phenotypic and genotypic characteristics of Enterococcus faecalis that suggest a broad environmental adaptation among the isolates studied.

Master of Science / Department of Biology / Helmut Hirt / Enterococcus faecalis, a gram-positive member of the mammalian gastrointestinal flora, emerged as an important contributor to nosocomial infections and antibiotic resistance gene transfer. Lipoteichoic acid (LTA), a vital component of gram-positive cell walls, has been reported to function in numerous cellular processes, ranging from maintenance of cation homeostasis and virulence to modulating function and presentation of wall proteins such as adhesins and autolysins. Interestingly, LTA can be covalently modified by the addition of D-alanyl ester residues, which appear to help regulate its function by altering surface charge. In E. faecalis the process of esterification is catalyzed by four proteins encoded by the dlt operon. Mutants lacking a functional dlt operon display the inability to incorporate D-alanyl residues on LTA and are thus deficient in their ability to regulate the anionic charge of the outer envelope in response to extracellular cues. Recent evidence suggests that two-component systems are responsible for sensing environmental conditions and regulating dlt operon expression. Utilizing a reporter construct with the upstream promoter region of dlt fused to lacZ, we were able to determine how extracellular stimuli affect transcription of this operon by measuring [Beta]-galactosidase activity. Furthermore, we were able to identify specific response regulators important for bile salt, magnesium and polymyxin B signaling.

Orientador: Fabio Luiz Camargo Villela Berbert / Resumo: O sucesso do tratamento endodôntico depende da eliminação de microrganismos do sistema de canais radiculares. As soluções irrigadoras e métodos mecânicos são incapazes de eliminar completamente as bactérias, que penetram profundamente nos túbulos dentinários e sistema de canais radiculares. Novos métodos de descontaminação intracanal, como o laser, têm sido estudados. Este estudo ex vivo foi realizado para avaliar o efeito antibacteriano de um laser diodo de alta potência, a irrigação convencional e combinação destas técnicas em dentes contaminados com biofilme de Enterococcus faecalis. Sessenta e cinco dentes unirradiculados com canal único foram selecionados, suas coroas foram removidas e as raizes padronizadas em 15mm e foram preparados com limas manuais e Reciproc R50. Posteriormente foram preparados para o estudo microbiológico. Seus forames apicais foram selados com resina fotopolimerizável e a superfícies externa radicular foi impermeabilizada com adesivo epoxi e os espécimes foram fixados em placas de 24-poços e esterilizados com óxido de etileno. Cinquenta e cinco foram contaminados com E. faecalis. Após 21 dias de incubação, os espécimes foram divididos em três grupos experimentais (n = 15): NaOCl, Solução salina + laser (SS+laser), e NaOCl + laser; e dois grupos controle (n = 10): Controle positivo (C+) e controle negativo (C-). No grupo NaOCl os espécimes foram irrigados com 5 mL de hipoclorito de sódio-NaOCl a 2,5%; no grupo SS+laser, foram irrigados com 5 mL de ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The success of the endodontic treatment depends on the elimination of microorganisms from the root canal system. The irrigant solutions and mechanical methods are incapable of eliminating bacteria, which penetrate deeply into the dentinal tubules and root canal systems. New methods of intracanal decontamination, like lasers, have been studied. This ex vivo study was done to evaluate the antibacterial effect of a high-power diode laser, conventional irrigation and the combination of these techniques in teeth contaminated with Enterococcus faecalis biofilm. Sixty-five uniradicular teeth with single canal were selected, their crowns were removed, and roots were standardized with 15 mm and were prepared with manual files and Reciproc R50. Afterwards, they were prepared for microbiological study. Their apical foramen was sealed with light cured resin and their root external surfaces were sealed with Epoxi adhesive and the specimens were attached to 24-well microplates and sterilized with ethylene oxide. Fifty-five specimens were contaminates with E. faecalis. After 21 days of incubation, the specimens were divided into three experimental groups (n = 15): NaOCl, Saline + laser (SS+laser), and NaOCl + laser; and two control groups (n = 10): Positive control (C+) and negative control (C-). In the NaOCl group, the specimens were irrigated with 5 mL of 2,5% sodium hypochlorite - NaOCl; in the SS+laser group, they were irrigated with 5 mL of saline and then irradiated with highpower dio... (Complete abstract click electronic access below) / Mestre

L’argument selon lequel les antibiotiques bactéricides stimulent la formation d'espèces réactives de l’oxygène (ROS pour reactive oxygen species) chez certaines bactéries en augmentant leur activité respiratoire est sujet à discussions. Enterococcus faecalis a des propriétés intéressantes pour tester cette hypothèse. La respiration ne s’observe qu’en présence d'hème ou de fumarate. En l'absence d'hème, E. faecalis produit du superoxyde extracellulaire par l’autoxydation d’une demethylmenaquinone (DMK), un composant de sa chaîne respiratoire associé à sa membrane. En raison de cette propriété, E. faecalis est soupçonné de jouer un rôle dans la cancérogenèse colorectale. Nous montrons dans cette étude que les β–lactamines amplifient significativement la production de ROS. Cette augmentation, dépendante de DMK, n'est observée qu'en l'absence de respiration. Nos résultats pourraient également fournir une explication quant au risque accru de cancer colorectal observé chez des patients traités par des β–lactamines, comme le montrent de récentes études cliniques. / The proposal that bactericidal antibiotics stimulate the formation of reactive oxygen species (ROS) by increasing respiration is still a matter of debate. Enterococcus faecalis has interesting properties to test this hypothesis. Respiration occurs only in the presence of heme or fumarate. In the absence of heme, E. faecalis produces extracellular superoxide through autoxidation of demethylmenaquinone (DMK), a component of its respiratory chain. Due to this ability, E. faecalis is suspected to play a role in colorectal carcinogenesis. We show in this study that β–lactam antibiotics increase significantly the production of ROS. This boost of ROS formation is DMK–dependent and only observed in the absence of respiration. Our results could provide a mechanistic explanation for the observed increased risk of colorectal cancer by β–lactam antibiotics in several recent nested case–control studies.

Indiana University-Purdue University Indianapolis (IUPUI) / For regenerative endodontic procedures (REPs) to be successful an elimination of bacteria from the root canal system must be accomplished. Many different medicaments with antibacterial properties have been used to obtain complete disinfection. Double antibiotic paste (DAP) containing a mixture of ciprofloxacin and metronidazole has been shown to be a promising intracanal medicament. The addition of a radiopaque filler such as zirconium oxide to DAP may affect the antibacterial properties of DAP as well as allow precise placement and radiographic visualization of its position in the canal system. The aim of the proposed study was to evaluate the direct antibacterial properties of zirconium oxide radiopacifier combined with DAP (RoDAP) against a multispecies biofilm from a bacterial isolate from an infected immature tooth with a necrotic pulp and a known single species biofilm. 4x4 mm radicular dentin specimens (n = 112) obtained from human extracted teeth were used prepared and sterilized prior to use. A multispecies clinical bacterial isolate from an immature tooth with a necrotic pulp and a single species Enterococcus faecalis isolate were obtained. These bacterial isolates were used to inoculate dentin slabs and grown for 3 weeks. The dentin slabs were treated for 1 week with 1.0-mg/mL and 10- mg/mL RoDAP, 1.0-mg/mL DAP, and two placebo pastes consisting of methyl cellulose (MC) and methyl cellulose combined with zirconium oxide (RoMC), respectively, as well as two no-treatment controls. Following treatment, the grown biofilm was detached and spiral plated. The plated biofilm cells were cultured for 24 hours and each group examined using a colony counter to determine bacterial numbers (CFUs/mL). Data analysis, using a 5.0-percent significance level was conducted using one-way ANOVA followed by pair-wise group comparisons. Both 1.0-mg/mL and 10 mg/mL RoDAP demonstrated significant antibacterial effects against bacterial isolates from an immature tooth with a necrotic pulp as well as an E. faecalis isolate. The precise application of RoDAP confirmed radiographically with its direct antibacterial properties may be beneficial for intracanal disinfection during REPs.

Enterococcus faecalis is a well known nosocomial drug resistant pathogen that is responsible for urinary tract infections, bacteremia, wound infections and endocarditis through the formation of biofilms. It has been shown that 68 genes present within the core genome of E. faecalis are upregulated in biofilm formation. One of those 68 genes is a putative seleniumdependent molybdenum hydroxylase (SDMH). Adjacent to this gene are a series of open reading frames that have been postulated to play a role in the maturation of a labile selenium cofactor. The biosynthesis of this labile cofactor has yet to be studied at either the genetic or biochemical level. The addition of selenium to growth medium caused a significant increase in biofilm density and extracellular hydrogen peroxide by wild type E. faecalis. By site-directed mutagenesis gene products encoded in the SDMH operon were shown to be necessary for the selenium-dependent biofilm formation as well as extracellular hydrogen peroxide production. This biofilm and peroxide phenotype is inhibited both by tungsten or auranofin in wild type E. faecalis suggesting the SDMH is a necessary enzyme for selenium-dependent biofilm and peroxide formation. These results show that the gene products encoded within the SDMH operon are necessary for a selenium-dependent biofilm formation as well as extracellular hydrogen peroxide production. These mutants will provide the basis for defining the synthesis of the labile selenium cofactor and allow for an expanded understanding of the biological use of selenium.

A ampliação do conhecimento sobre as formas de comunicação, controle e regulação existentes em bactérias traz luz aos avanços no combate das infecções hospitalares que são responsáveis por inúmeros prejuízos relacionados à saúde pública em todo planeta. DUMOULIN et al (2013), descreveram o regulador transcricional (RT) ElrR, que regula positivamente a transcrição do gene elrA, um fator de virulência de Enterococcus faecalis. ElrA apresenta grande similaridade com as internalinas de Listeria monocytogenes, que facilitam a invasão da bactéria ao hospedeiro. ElrR é considerada como pertencente à família Rgg-like de RT exclusivo de bactérias Gram positivas. Por vários motivos a família Rgg foi inserida à superfamília RNPP, gerando a superfamília RRNPP de RT. Os RRNPP fazem parte de um sistema de regulação por quorum sensing (QS), um sistema de comunicação célula-célula dependente de densidade celular, com função associada na ativação ou inibição da expressão de proteínas relacionadas, dentre outros, à virulência, formação de biofilme e esporulação. Para a melhor compreensão do mecanismo de como ocorre a ativação da transcrição do fator de virulência ElrA, este trabalho apresenta resultados de expressão heteróloga em E. coli e purificação das proteínas ElrR e ElrA, bem como resultados de experimentos biofísicos que caracterizam algumas propriedades estruturais e biológicas destas proteínas. Utilizando técnicas de cromatografia, espectroscopia de dicroísmo circular (CD), anisotropia de fluorescência, espalhamento dinâmico de luz (DLS), cristalografia de raios X e ressonância plasmônica de superfície (SPR), foi possível a obtenção da estrutura tridimensional de ElrR e de indícios da interação com uma região de 25bp do DNA. Realizou-se ainda, em colaboração com Dra. Pascale Serror, a tentativa de obtenção da molécula autoindutora (AI) de ElrR. São apresentados primeiros resultados da obtenção heteróloga de ElrA, sua purificação e cristalização, com importantes características que permitirão a continuação da investigação deste fator de virulência. ElrR é composta somente por alfa-hélices e apresenta-se dimérico em solução. Apesar da similaridade estrutural dos RRNPP, a identidade da sequência entre ElrR e os outros membros é extremamente baixa, o que motivou a resolução das fases cristalográficas experimentalmente. A estrutura de ElrR apresenta-se similar às homólogas, porém, com maior interface de interação entre os protômeros, que formam o dímero. O sítio de ligação do AI, em ElrR, apresenta-se mais amplo, com cavidade maior que as demais estruturas estudadas, conservando vários dos resíduos apresentados nos homólogos que realizam a estabilização do AI. Os altos fatores de temperatura dos cristais de ElrR, adicionado a anisotropia dos átomos, de uma das estruturas obtidas, apresenta a grande flexibilidade desse RT. Os indícios de interação entre ElrR e DNA aqui apresentados, obtidos por SPR e anisotropia de fluorescência, apresentam que ElrR liga especificamente ao fragmento proposto do DNA, ainda na ausência do AI. A não cristalização do complexo (ElrR-DNA), adicionada a alta flexibilidade apresentada na estrutura e a instabilidade observada na ligação ao DNA (por SPR) apontam para a obrigatoriedade da molécula de regulação (AI) para que o complexo ElrR-DNA seja estável. / The enhancing of the knowledge about communication, control and regulation in bacteria bring possibilities on the advance of hospital-acquired infections control responsible for various prejudices related to public health worldwide. DUMOULIN, et al (2013) described ElrR, a transcriptional regulator (TR), that positively regulates transcription of the elrA gene, which codifies a virulence factor of Enterococcus faecalis. ElrA shows high similarity with Listeria monocytogeneses internalins, which facilitates host invasion by these bacteria. ElrR are considered belonging to Rgg-like TR family exclusive of Gram positive bacteria. Several reasons include the Rgg family into the RNPP superfamily, generating the RRNPP superfamily of TR. The RRNPP are controlled by a quorum sensing (QS) regulation system, a cell-cell communication system based on cellular density that activates or inhibits the expression of proteins related with virulence, biofilm formation, sporulation, and others. For a better understanding of the transcription activation mechanism of ElrA, this work shows ElrR and ElrA heterologous expression in E. coli and purification of these proteins, as well as biophysics assays to characterize some structural and biological features of both proteins. Using chromatography, circular dichroism (CD), fluorescence anisotropy, dynamic light scattering (DLS), X-ray crystallography and surface plasmon resonance (SPR) technics, it was possible to obtain the tridimensional structure of ElrR, and evidences of ElrR-DNA complex formation, confirming DNA interaction site of ElrR with a 25 bp fragment. In collaboration with Dr. Pascale Serror, we attempted to achieve the ElrR auto-induction (AI) molecule. Also, results of the heterologous obtainment of ElrA are presented, as well as ElrA purification and crystallization, presenting important characteristics which will allow the further investigation of this virulence factor in near future. ElrR is composed by alpha-helices presenting dimeric fold in solution. Despite the similarity between the RRNPP members, the low identity of ElrR to the other members motivates the experimental crystallographic phases solution. ElrR structure is very similar to the homologous structures, presenting a higher interface between the protomers that compose the dimer. Its AI binding site is wider than the other structures studied, conserving several amino acid residues presented at the homologous proteins, that stabilizes the AI molecule. High temperature factors of the amino acid residues showed in all the obtained ElrR crystallographic structures plus the anisotropy of the atoms in one of those structures show the high flexibility of this TR. The evidence of the ElrR-DNA complex presented in this study, obtained by SPR and fluorescence anisotropy, indicates that ElrR binds at the proposed DNA site even in the absence of the AI molecule. The failure to obtain the ElrR-DNA complex crystals added to the high flexibility presented at some places of the structure and the observed instability at the formed complex (observed at SPR) suggest the mandatory need of the AI molecule to create a stable ElrR-DNA complex.

O preparo do canal radicular visa a remoção de bactérias que podem causar a patologia periapical, e os instrumentos endodônticos estão sendo constantemente aprimorados para tornar o tratamento do canal radicular mais fácil, rápido e seguro. Assim, novos instrumentos precisam ser avaliados quanto ao seu desempenho. O objetivo deste estudo foi avaliar a remoção bacteriana dos sistemas Wave One Gold e One Shape New Generation comparando-os com seus sistemas predecessores. Cinquenta e seis canais disto vestibulares de molares superiores esterilizados por oxido de etileno foram contaminados com Enterococcus faecalis por 21 dias, e então uma amostra bacteriana inicial foi coletada e paqueada em M-enterococcus agar para contagem bacteriana em unidades formadoras de colônias. Os espécimes foram aleatoriamente divididos em quatro grupos de acordo com a instrumentação (n=12): Wave One Gold Primary, One Shape New Generation 25/.06, Wave One Primary e One Shape 25/.06, e os outros 8 canais não contaminados foram o controle de assepsia. Todos os grupos utilizaram água destilada como irrigante. Nova coleta foi feita imediatamente após a instrumentação e aos 7 dias. A redução bacteriana foi calculada em porcentagem, e então feita análise intragrupo pelo teste de Wilcoxon e entre grupos por Kruskal Wallis e teste de Dunn, todos com significância de 5%. Todos os sistemas reduziram significativamente a carga bacteriana do canal radicular tanto na coleta imediata quanto aos 7 dias (p<0,05). Houve aumento do número de bactérias 7 dias após o preparo quando comparado com a coleta imediata (p<0,05). A análise entre grupos mostrou que Wave One Gold e One Shape New Generation promoveram maior redução bacteriana que os sistemas Wave One e One Shape (p<0,05), sem diferença significativa entre Wave One Gold e One Shape New Generation ou entre Wave One e One Shape (p>0,05). Conclui-se que Wave One Gold e One Shape New Generation promoveram maior remoção bacteriana do que seus sistemas predecessores. / The root canal preparation aims to remove the bacteria that can cause periapical pathologies, and endodontic instruments are constantly being improved to make root canal treatment easier, faster and safer. Thus, new instruments need to be evaluated for their performance. The aim of this study was to evaluate the bacterial removal promoted by Wave One Gold and One Shape New Generation systems in comparison to that of their predecessor systems. Forty-six distobuccal root canals of upper molars sterilized with ethylene oxide were infected with Enterococcus faecalis for 21 days, and then root canal initial bacterial sampling was collected and plated on M-enterococcus agar to bacterial count in colony forming unities. The specimens were randomly divided into four groups according to the instrumentation (n=12): Wave One Gold Primary, One Shape New Generation 25/.06, Wave One Primary and One Shape 25/.06, and the other 8 uncontaminated canals were used as asepsis control. All groups used distilled water as irrigant. New sampling was obtained immediately after instrumentation and at 7 days. The bacterial reduction was calculated in percentage, after that intra-group analysis was carried out by Wilcoxon test, and inter-group analysis by Kruskal-Wallis complemented by Dunn\'s test, all at 5% significance. All the systems significantly reduced the bacterial amount in the root canal in both immediate and at 7 days sampling (p<0.05). The bacterial amount increased at 7 days after preparation comparing to immediate sampling (p<0.05). The analysis between groups showed that Wave One Gold and One Shape New Generation promoted greater bacterial reduction than Wave One and One Shape systems (p<0.05). No significant difference was found between Wave One Gold and One Shape New Generation or between Wave One and One Shape (p>0.05). It can be concluded that Wave One Gold and One Shape New Generation promote greater bacterial removal than their predecessor systems.

O objetivo foi avaliar através da Microscopia Confocal de Varredura a Laser (MCVL) e cultura microbiológica (CM) a influência do veículo propilenoglicol e água destilada e agitação ultrassônica (U) na atividade antimicrobiana e penetrabilidade de pastas de hidróxido de cálcio (HC), em diferentes tempos de manutenção da medicação intra-canal. Para isso cento e noventa e três tubos de dentina bovina padronizados foram infectados com Enterococcus faecalis em caldo BHI (Brain Heart Infusion) utilizando um novo protocolo de contaminação com dois ciclos de centrifugação por 5 dias. Durante o período experimental os espécimes foram divididos em 8 grupos de acordo com o veículo da pasta de HC, água destilada (Ad) e propilenoglicol (prop), com o uso do ultrassom, e tempo experimental e de 7 e 15 dias: Grupo1- HC+pro+15d, Grupo 2 HC+prop+U+15d, Grupo3 HC+prop+7d, Grupo 4 HC+prop+U+7d Grupo5- HC+Ad+15d, Grupo 6 HC+Ad+U+15d, Grupo7 HC+Ad+7d, Grupo8 HC+Ad+U+7d . A agitação ultrassônica foi realizada durante 1 minuto nas direções vestíbulo-lingual e mésio-distal, com o auxílio de um inserto liso. As medicações permaneceram no interior dos espécimes durante cada período experimental. A MCVL analisou as bactérias viáveis (verde) e mortas (vermelho), com o auxílio do corante Live and Dead® nos tubos de dentina após o período de medicação. A contagem de Unidades Formadoras de Colônia (UFCs) foi realizada a partir da cultura microbiológica (CM). As raspas de dentina foram coletadas e diluídas para semeadura em placas de Petri com ágar BHI. Para a penetração foi utilizado o corante Rodamina B durante a manipulação das pastas de HC e as análises feitas por MCVL. O grupo 3 e 7 mostraram a maior viabilidade bacteriana nos tubos de dentina contaminados e os demais grupos as menores. A agitação ultrassônica (U) reduziu significativamente a viabilidade bacteriana, nos diferentes períodos. A pasta de HC + Prop+U 7 e 15 dias, mostraram-se mais eficazes, seguidas pela pasta HC+Ad+U+15. Concluiu-se que todas as pastas demonstraram ação antimicrobiana contra E. faecalis e com melhor desempenho quando potencializadas com ultrassom, mesmo em períodos de 7 dias, podendo ser de escolha clínica um curativo que permaneça menor tempo no interior do canal com a mesma efetividade, otimizando o tratamento endodôntico. / The purpose was to evaluate using Confocal Laser Scanning Microscopy (CLSM) and microbiological cultures (MC) of infected dentin the influence of veicol propylene glycol (prop) or distilled water (Ad) and ultrasonic agitation (U) on the antimicrobial potential and penetrability of calcium hydroxide (CH) pastes). one hundred ninetythree cylindrical dentin specimens were infected with Enterococcus faecalis in BHI broth using a new contamination protocol for 5 days with centrifugations. During the experimental period, the specimens were divided into 8 groups and dressed with the following during 7 or 15 days: Group1- HC+pro+15d, Group 2 HC+prop+U+15d, Group 3 HC+prop+7d, Group 4 HC+prop+U+7d Group 5- HC+Ad+15d, Group 6 HC+Ad+U+15d, Group 7 HC+Ad+7d, Group 8 HC+Ad+U+7d. The ultrasonic activation was made during 1 minute in both directions (buccal-lingual / mesio-distal), with the aid of a plain point insertion. The medications remained in the root canals for each experimental period. The CLSM analyzed the viable (green) and dead (red)bacteria in the infected dentinal tubules, with Live and Dead dye. The colony forming units (CFUs) count was made possible with the MC method. The dentinal wall debris were collected and diluted to be seeded on Petri BHI-agar plates. For the penetration test, the dye Rodamine B was added to CH pastes during the manipulation and analysed by CLSM. The groups 3 and 7 showed the greatest bacteria viability in the infected dentinal tubes and the other groups the lowest,. The pastes HC + Prop+U 7 and 15 days performed more efficacy, followed by HC+Ad+U+15d paste . We conclude that all the paste tested demostrated antimicrobial activity against E. faecalis from calcium hydroxide paste with propylene glycol and distilled water when leveraged with ultrasound, even in periods of seven days. Therefore, a dressing that remains less time inside the root canal with the same effectiveness can be the choice in clinical practice, optimizing the endodontic treatment.