The objective of this study was to evaluate the biofilm removal efficacy of three root canal treatment systems: ProUltra® PiezoFlow™, traditional needle irrigation, and the GentleWave® system in an ex-vivo benchtop study. Twenty-four extracted maxillary and mandibular molars were selected. Teeth were all instrumented to a master apical file size #25 with 4% taper. Teeth were then randomly divided into four experimental groups and two control groups. The root canals were inoculated with a culture of Enterococcus faecalis and incubated for five weeks to form a biofilm. Each group was then treated with one of the different root canal treatment systems using 6% sodium hypochlorite (NaOCl) as per the respective manufacturer’s recommendation followed by a rinse with water. Following treatment, teeth were decoronated and roots were sectioned longitudinally. Three scanning electron microscope images were taken at the apical level per root half at 5000x magnification. Images were scored by four calibrated examiners blind to group membership using a four-point scoring system (<5% coverage, 5-33%, 34-66%, and >66%). Results were analyzed using mixed model ANOVA. All the experimental groups were significantly better than the positive control group in removing biofilm. Among the experimental groups, the GentleWave® 15/04 group was significantly better than the other groups. There was no significant difference between the GentleWave® and the ProUltra® PiezoFlow™. Traditional needle irrigation scored the worst in reducing E. faecalis biofilm. The GentleWave™ system was as effective at intracanal biofilm removal as the ProUltra® PiezoFlow™ and better than traditional needle irrigation using 6% NaOCl as an irrigant.

The study objective was to compare the antimicrobial activity of STERIPLEX™ HC with 5.25% sodium hypochlorite (NaOCl) at different dilutions (50%, 25%, 10%, 1%, 0.1%) and different time intervals (1, 3, 5 minutes) on Enterococcus faecalis. All data was analyzed using an ANOVA. The 50%, 25%, and 10% dilutions of both disinfectants reduced the colony forming unit (CFU) count to below the limit of detection (50 CFU/ml) after one minute. The 1% dilutions at each of the time intervals show NaOCl was significantly more effective than STERIPLEX™ HC (all Ps < .0001) in reducing the CFU/ml count. The 0.1% dilutions of NaOCl and STERIPLEX™ HC at 1 minute, were not different (P = 0.7808), while at 3 minutes and 5 minutes NaOCl was significantly more effective (P = 0.0098 and P < .0001, respectively).

El objetivo del presente estudio fue determinar el efecto antibacteriano de la combinación de droga 3Mix y MP y de sus combinaciones alternativas contra Enterococcus faecalis y Fusobacterium nucleatum. Se empleó el método de dilución en caldo para determinar la concentración inhibitoria mínima (CIM) y la concentración bactericida mínima (CBM) y método de difusión en agar modificado para determinar el efecto antibacteriano de los vehículos. Se emplearon dos cepas ATCC Enterococcus faecalis y Fusobacterium nucleatum, con la combinación de componentes de la droga 3Mix, 3Mix-Cefaclor(reemplazo de minociclina por cefaclor) y 3Mix-Amoxicilina(reemplazo de minociclina por cefaclor) en las siguientes concentraciones: 25µg/ml; 6,25µg/ml; 1,56µg/ml; 0,39µg/ml; 0,195µg/ml; 0,097µg/ml y macrogol(M), propilenglicol(P) y su asociación. La CIM para 3Mix, 3Mix-cefaclor fue 0,39µg/ml y 0,195µg/ml para 3Mix-Amoxicilina mientras que CBM fue >25µg/ml; 25µg/ml; 6,25µg/ml respectivamente sobre Enterococcus faecalis, para Fusobacterium nucleatum la CIM de 3Mix, 3Mix-Cefaclor fue 0,195µg/ml y ≤0,097µg/ml para 3Mix-Amoxicilina, los cuales coincidieron con CBM. Se obtuvo inhibición de crecimiento bacteriano por parte macrogol y propilenglicol+macrogol, sin embargo propilenglicol no formó halo de inhibición sobre Enterococcus faecalis ni Fusobacterium nucleatum. La Combinación de droga 3Mix, 3Mix-Cefaclor y 3Mix-Amoxicilina presentaron efectos antibacterianos contra Enterococcus faecalis y Fusobacterium nucleatum.

Orientador: Nasser Ali Daghastanli / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2012

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Enterococcus faecalis (E. faecalis), conhecidamente patógeno oportunista, tem sido frequentemente associado a infecções sistêmicas graves. É também encontrado na cavidade oral, com destaque em infecção endodôntica refratária. O objetivo deste estudo foi avaliar características moleculares de E. faecalis isolados de infecção endodôntica primária no Brasil e comparar com isolados orais e não orais de pacientes do Reino Unido e do Japão, assim como E. faecalis resistentes à vancomicina. O presente estudo também investigou o relacionamento entre E. faecalis de diferentes origens (oral e não oral) e de diferentes áreas geográficas para obter uma melhor compreensão do envolvimento dos diferentes reservatórios no surgimento e propagação de clones virulentos, aqueles que possuem genes que conferem infectividade e virulência, assim como resistência aos antibióticos. Para tal, foram estudados E. faecalis isolados em infecções endodônticas no Brasil (n = 20) e orais no Reino Unido (n = 10), e em infecções não orais no Japão (n = 9). Além disso, 20 E. faecalis isolados ambientais do Hospital Universitário de Gales (Cardiff, Reino Unido), classificados como Enterococcus resistentes à vancomicina (VRE) também foram examinados. A Concentração Inibitória Mínima (CIM) dos isolados do Brasil foi obtida pelo método de diluição em agar de acordo com as recomendações do Clinical and Laboratory Standards Institute (CLSI). Reação em cadeia da polimerase (inglês - PCR) foi a técnica empregada para detectar os genes de virulência e aqueles associados à resistência aos antibióticos, enquanto Reação de Amplificação Aleatória de DNA Polimórfico (inglês - RAPD-PCR) foi escolhida para a tipagem molecular. Dentre os genes de virulência examinados, o gene que codifica a gelatinase gelE foi o mais prevalente entre os isolados (77-100%). Entre isolados orais, foram detectados os genes agg de substâncias de agregação, esp de proteína de evasão imune, cylB de citolisina, genes de resistência à tetraciclina tetM e tetL e à eritromicina ermB com diferentes prevalências. Os isolados clínicos hospitalares do Japão apresentaram perfil genético similar aos isolados orais, mas com maior prevalência de ermB e cylB. Todas as amostras de VRE foram positivas para os genes gelE, esp, agg, vanA, ermB e tetM, 95% foram positivos para cylB e 17% positivo para tetL. Todas as amostras foram negativas para ermA, asa373, vanB, vanC1 e vanC2/3. RAPD-PCR revelou agrupamento de VRE em comparação com outros isolados. Neste estudo, os isolados de E. faecalis de infecções orais apresentaram genes de resistência à tetraciclina, um antimicrobiano frequentemente usado no tratamento local de infecções dentárias, abrindo um debate muito importante sobre o papel e a eficácia desta droga para infecções orais. Claramente, são necessários mais estudos nesta área principalmente em relação à expressão de fatores de virulência entre isolados endodônticos para melhor nortear as estratégias de tratamento. As pressões externas no microambiente dos canais radiculares podem ser responsáveis pela seleção de espécies mais resistentes e virulentas. Por fim, embora isolados orais apresentem genes de virulência fundamentais para a patogenicidade, estes foram detectados em menor incidência em comparação com os isolados não-orais e VRE. / Enterococcus faecalis is an opportunistic pathogen known to cause serious systemic infection. It is also encountered in the oral cavity and has been implicated in persistent root canal infection. The aim of this study was to evaluate a range of molecular characteristics of E. faecalis isolated from primary endondontic infections in Brazil and compare to isolates from oral and non-oral infections in patients from UK and Japan, as well as isolates of vancomycin resistant E. faecalis, VRE, from a hospital environment. The present study was undertaken to explore the relatedness of E. faecalis from different origins, oral and non oral, and from different geographic areas to gain a better understanding of the involvement of the different reservoirs in the emergence and spread of virulent clones, those that acquired a number of genes conferring infectivity and virulence and in addition antibiotic resistance. To do this, E. faecalis from oral infections in Brazilian (n=20) and UK patients (n=10), and non-oral infection in japanese patients (n=9) were studied. In addition, 20 environmental VRE isolates from the University Hospital of Wales (Cardiff, UK) were also examined. For braziliam isolates, antimicrobial susceptibility was ascertained by agar dilution, using the recommendations of the Clinical and Laboratory Standards Institute (CLSI). For all isolates, PCR with validated primers was used to detect genes associated with antibiotic resistance and virulence, whilst RAPD-PCR was used to fingerprint isolates. Of the virulence genes examined, gelatinase gene gelE was most prevalent amongst isolates (77-100%). In the case of oral isolates, the genes of aggregation substances agg, immune evasion protein esp, cytolysin cylB, tetracycline resistance tetM and tetL and erythromycin resistance ermB were detected with varying prevalence. Japanese hospital isolates had a similar genetic profile to oral isolates but with higher prevalence of ermB and cylB. All VRE strains were positive for gelE, esp, agg, vanA, ermB and tetM, 95% were positive for cylB and 17% positive for tetL. All isolates were negative for ermA, asa373 vanB, vanC1 and vanC2/3. RAPD-PCR revealed clustering of VRE compared with other isolates. In this study, isolates of E. faecalis from oral infections showed antibiotic resistance genes for tetracycline, an agent used in the local treatment of dental infection. This opens up a much-needed debate on the role and efficacy of this antibiotic for oral infections. Clearly, more research in this area is required particularly in relation to the possession and expression of virulence factors in the root canal environment, to better inform our management strategies. Environmental pressures in root canals may be responsible for the selection of more resistant species and the possession of virulence determinants. This knowledge is important in guiding procedures for controlling the increasing problem of antibiotic resistance amongst clinically relevant bacteria. Furthermore, whilst oral isolates had genes that could contribute to pathogenicity, these were detected at lower incidence compared with non-oral and VRE isolates.

Orientador: Rogério de Castilho Jacinto / Coorientadora: Carolina Simonetti Lodi / Banca: Luciano Tavares Angelo Cintra / Banca: João Eduardo Gomes Filho / Banca: Francisco Montagner / Resumo: O presente estudo teve como objetivo avaliar a atividade antimicrobiana dos cimentos endodônticos, com ou sem associação do uso prévio de hidróxido de cálcio em um modelo "in vitro" de biofilme. Espécimes de dentina bovina (240) foram colocadas e deixas em contato direto com inoculo de E. faecalis (ATCC 51299) por 14 dias, para induzir a formação do biofilme. Em seguida, metade das espécimes foram incubadas (37⁰C e 5% CO2) em contato com um dos seguintes cimentos: AH Plus, Acroseal e Sealapex por 2, 7 e 14 dias, e a outra metade foi tratada com solução de hidróxido de cálcio por 14 dias e incubada em contato com os cimentos AH Plus, Acroseal e Sealapex por 2, 7 e 14 dias. Cada grupo continha um n = 8. Após cada período experimental, as amostras foram agitadas e as suspensões formadas foram diluídas em série e triplamente plaqueadas em ágar m-Enterococcus. As unidades formadoras de colônias foram contadas, e os dados foram analisados estatisticamente usando os testes one-way ANOVA, Shapiro-Wilk e Kruskal-Wallis (p <0,05) para determinar o potencial antimicrobiano. Foi observada diferença estatisticamente significante entre os grupos com e sem o tratamento com Hidróxido de Cálcio, para todos os cimentos avaliados. Entretanto, nenhum dos cimentos testados foi capaz de eliminar completamente o biofilme. Ao comparar os cimentos, Sealapex reduziu E. faecalis após 7 dias, enquanto AH Plus e Acroseal mostraram atividade antimicrobiana apenas no 14º dia experimental. Em conclusão, o u... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present study aimed at evaluating the antimicrobial activity of endodontic sealers, with or without prior use associationof calcium hydroxide in an in vitro biofilm model. Bovine dentin specimens (240) were placed and left in contact with inoculum of E. faecalis(ATCC 51299) for 14 days, to induce biofilm formation. Then, half of the specimens were incubated (37⁰C and 5% CO2) in contact with one of the following sealers AH Plus, Acroseal and Sealapex for 2, 7 and 14 days, and the other half were treated with calcium hydroxide solution for 14 days, and then incubated in contact with the sealers AH Plus, Acroseal and Sealapex for 2, 7 and 14 days. Each group comprised a n=8. After each experimental time the samples were agitated, and the suspensions formed were serially diluted, and triple plated onto m-Enterococcus agar. Colony forming units were counted, and the data were statistically analyzed using ANOVA, Shapiro-Wilk and Kruskal-Wallis one-way tests (p<0.05) to determine antimicrobial potential. A statistically significant difference was observed between the groups with and without the treatment with Calcium Hydroxide, for all sealers evaluated. However, neither of the sealers tested were able to completely eliminate the biofilm.When comparing the sealers, Sealapex reduced E. faecalisafter 7 days, while AH Plus and Acroseal showed antimicrobial activity only on the 14th experimental day. In conclusion, previous use of calcium hydroxide helped to decrease Enterococcus faecalisbiofilm of the sealers studied in all experimental times. (Complete abstract electronic access below) / Doutor

Objective: To assess biofilm proliferation within the sealer-dentin interfaces of methacrylate resin-based sealers, self-etch (SE) and total-etch (TE), and an epoxy resin-based sealer (EP). Methods: Standardized human root specimens were filled with the test materials and were aged for 1 week, 1, 3 or 6 months in saline (n=3/group). Monoclonal biofilms of Enterococcus faecalis were grown on the specimens for 7 days in continuous media reactor. The extent of biofilm proliferation of E. faecalis within the sealer-dentin interface for each material at each incubation period was assessed using fluorescence microscopy of dihydroethidium-stained specimens. Results: TE had less biofilm proliferation than EP and SE (p<0.01). Deeper biofilm proliferation was detected in SE and EP specimens aged for 1 and 3 months than those aged for 1 week or 6 months (p<0.05). Conclusion: Self-etch and epoxy resin-based sealers were more susceptible to interfacial biofilm proliferation than total-etch system at shorter incubation periods.

Three Streptococcal strains: S. milleri P213, S. milleri P35 and S. milleri B200 and three enterococcal strains: E. faecalis 123, E. faecalis 126 and E. faecium were used to test for vancomycin resistance. Two strains were used as reference strains that were already characterized as vancomycin resistant. E. faecium BM4147 was used as a VanA control and E. faecalis ATCC was used as a VanB control. Susceptibility of each strain to this antibiotic was tested by disk-diffusion assay and the MIC values for the strains were found to be between 5 - 10 ug/ml and for the VanA control, the MIC was > 64 ug/ml and for the VanB control was 32 ug/ml. These MIC values indicate that S. milleri P213, S. milleri P35, S. milleri B200, E. faecalis 123, E. faecalis 126, and E. faecium are all of the VanC phenotype. All strains were tested for lysis by means of addition of vancomycin (10 ug/ml) to the bacterial cultures. Lytic curves were constructed and the VanB control was found to be most autolytic upon addition of vancomycin and E. faecalis 123 was the least autolytic. However, under normal conditions in phosphate buffer, lytic curves showed that S. milleri P213 was the most autolytic and the VanA control, the least autolytic. PCR assays were performed to detect specific antibiotic resistant genes. Primers were selected from Dukta-Malen et al., 1995. The VanA primer yielded amplification of 732 bp for only the VanA control DNA and the VanB primer set yielded products for the VanB control DNA. S. milleri P213, P35, B200 and E. faecalis 123 and 126, and E. faecium DNA were amplified with the VanC primers. This supports the results obtained in MIC that these strains are possibly VanC resistant strains. Amplified VanA control and that of E. faecalis 126 were thereafter sequenced. VanA control amplicon was correctly amplified since it showed homology to E. faecium BM4147 as well as the VanB amplicons which was found to be homologous to the transposon Tn1549 found on the well-characterized E. faecalis strain which is known to harbour the VanB vancomycin-resistant genes. Whilst E. faecalis 126 which represented the VanC phenotype showed 96% homology to E. gallinarum BM4147 which is a well-characterized glycopeptide-resistant enterococci belonging to the VanC phenotype. Southern blots were performed using specific primers as a probe to verify whether the gene sequences for the specific genotype were present in these strains and results confirmed those found in the PCR assays and in DNA sequencing. The peptidoglycan precursors of each strain were arrested in vancomycin (20 ug/ml) to block transpeptidation and transglycosylation steps of peptidoglycan synthesis and bacitracin (100 ug/ml) was used to amplify precursors at the transglycosylation step. Precursors were extracted and analysed by reverse-phase HPLC. UDP-MurNAc-tetrapeptides cell wall precursors, which are found abundantly in vancomycin-resistant strains, were found in large proportions in all strains, except in E. faecalis 123 when arrested with vancomycin. This precursor has a noticeably decreased affinity for vancomycin, hence contributing to its resistance. The precursor accumulated when arrested with bacitracin, was, UDPMurNAc-tetrapeptide in all strains except in E. faecalis 126. UDP-MurNAc-pentapeptides were also found in moderate amounts in most strains. The molecular masses of the peptidoglycan precursors obtained from mass spectrometry correctly identified them. This confirmed that the bacterial strains investigated were in fact resistant to the antibiotic vancomycin and this study shows that results obtained from conventional phenotypical screening methods reliably correlated with the genotypes classified using more advanced techniques such as PCR, southern blot/hybridisation and DNA sequencing. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Objective: To assess biofilm proliferation within the sealer-dentin interfaces of methacrylate resin-based sealers, self-etch (SE) and total-etch (TE), and an epoxy resin-based sealer (EP). Methods: Standardized human root specimens were filled with the test materials and were aged for 1 week, 1, 3 or 6 months in saline (n=3/group). Monoclonal biofilms of Enterococcus faecalis were grown on the specimens for 7 days in continuous media reactor. The extent of biofilm proliferation of E. faecalis within the sealer-dentin interface for each material at each incubation period was assessed using fluorescence microscopy of dihydroethidium-stained specimens. Results: TE had less biofilm proliferation than EP and SE (p<0.01). Deeper biofilm proliferation was detected in SE and EP specimens aged for 1 and 3 months than those aged for 1 week or 6 months (p<0.05). Conclusion: Self-etch and epoxy resin-based sealers were more susceptible to interfacial biofilm proliferation than total-etch system at shorter incubation periods.

Thesis (M.S.D.)--Indiana University School of Dentistry, 2010. / Title from PDF t. p. (viewed July 28, 2010) Advisor(s): Mychel Vail, Chair of the Research Committee, Richard Gregory, Joseph Legan, Kenneth Spolnik, Susan Zunt. Curriculum vitae. Includes abstract. Includes bibliographical references (leaves 108-120).