The astounding complexity of microbial communities limits the ability to study the role of genomic diversity in shaping the community composition at the species level. With the advancement and increased affordability of high-throughput sequencing methods, it is increasingly recognized that genomic diversity at the sub-species level plays an important role in selection during microbial community succession. Recent studies using the cpn60 universal target (UT) have shown that it is a high-resolution tool that provides superior resolution in comparison to 16S rRNA based tools and can predict genome relatedness. However, studies to characterize the nature and degree of genome content differences predicted by cpn60 UT sequence relationships have not been conducted. In this work, we focused on the Enterococcus community obtained from feces of healthy pigs. Enterococci are both accessible with selective culture, and interesting since the genus includes members that are significant human pathogens and others that are used as probiotics. Previous work has shown that cpn60 UT sequences can resolve pig fecal Enterococcus faecalis and E. hirae into phylogenetically and phenotypically distinct ecotypes. The utility of cpn60 UT sequences for resolution of Enterococcus species was first established in the year 2000, and this demonstration included 17 Enterococcus species. We have expanded the analysis to include all currently recognized Enterococcus species and confirmed that cpn60 UT sequences provide higher resolution than 16S rRNA sequences for identification of Enterococcus species. In addition, we showed that cpn60 UT sequences could resolve sub-groups in E. faecium consistent with results obtained from comparison of whole genome sequences. GTG rep PCR based clusters for E. faecalis and E. hirae isolates were generally consistent with the cpn60 defined Enterococcus ecotypes observed in our previous study, suggesting that cpn60 UT sequences predict overall genomic relationships. Results from analysis of CRISPR sequences provided insights into the extensive networking and transfer of genetic material that takes place within the intestinal Enterococcus community. We conducted whole genome sequencing of representative isolates to characterize further the genomic diversity in cpn60 defined E. hirae ecotypes to determine the nature and degree of genome content differences predicted by cpn60 UT sequences. Differences in phosphotransferase systems, amino acid metabolism pathways for glutamine, proline and selenocystiene, potassium-transporting ATPases, copper homeostasis systems and putative prophage associated sequences, CRISPRs and antibiotic resistance genes were observed. Results from in vitro growth competition assays showed that isolates from E. hirae-1 and E. hirae-2 were able to out-compete isolates from the E. hirae-3 ecotype, consistent with the relatively low abundance of E. hirae-3 relative to E. hirae-1 and E. hirae-2 previously observed in the pig fecal microbiome, and with observed gene content differences between the ecotypes. Results presented in this thesis provide a genomic basis for the definition of ecotypes within E. hirae and confirm the utility of the cpn60 UT sequence for high resolution profiling of complex microbial communities.

El objetivo del presente estudio fue comparar la efectividad antibacteriana de la pasta de hidróxido de calcio con clorhexidina al 2% y la pasta de hidróxido de calcio con yodopovidona al 1%, frente al Enterococcus faecalis. El diseño de contrastación fue experimental. Se distribuyeron 10 placas Petri que contenían agar Müller Hinton a 40° C, sobre las cuales fue inoculada la bacteria Enterococcus faecalis. Además, estas fueron divididas de manera aleatoria en 3 segmentos cada una de acuerdo al tipo de pasta medicamentosa que se aplicó: grupo P1 (hidróxido de calcio + clorhexidina al 2%), grupo P2 (hidróxido de calcio + yodopovidona al 1%) y el grupo P3 o control (hidróxido de calcio + agua destilada). Finalmente, se procedió a la lectura de halos de inhibición a las 24 horas, 48 horas, 7 días, 14 días. Los datos fueron procesados a través del análisis de Tukey para determinar la diferencia de medias entre los grupos experimentales y el análisis de ANOVA con un nivel de significancia del 95%, utilizando el programa SPSS 20. Se concluyó que la pasta de hidróxido de calcio con clorhexidina al 2% fue más efectiva que la pasta de hidróxido de calcio con yodopovidona al 1% frente al crecimiento in vitro del Enterococcus faecalis.

It is well established that bacteria cause pulpal and periradicular disease (Kakehashi et al. 1965). Of the bacteria recovered from failing root canals, Enterococcus faecalis is one of the most prevalent species (Molander et al. 1998; Sundqvist et al. 1998). Many laboratory studies have investigated the effectiveness of root canal irrigants and medicaments against E. faecalis. Most used planktonic cultures, which are not representative of the in vivo growth conditions of an infected root canal system, where bacteria grow as a biofilm adhering to the dentinal wall (Nair 1987). Organisation of bacteria within biofilms confers a range of phenotypic properties that are not evident in their planktonic counterparts, including a markedly reduced susceptibility to antimicrobial killing (Wilson 1996). Objectives: The aims of this study were: 1) To compare the efficacy of commonly used endodontic medicaments against E. faecalis cultured as a biofilm. The medicaments tested were Ledermix paste, calcium hydroxide, Odontopaste, 0.2% chlorhexidine gel and 50:50 combinations of Ledermix/calcium hydroxide and Odontopaste/calcium hydroxide. 2) To compare the antimicrobial effect achieved through exposure to endodontic medicaments with that achieved by exposure to a constant concentration of sodium hypochlorite for varying times. Methods: A biofilm was established using a continuous flow cell. E. faecalis inoculum was introduced into the flow cell and allowed to establish on human dentine slices over 4 weeks. Each test medicament was introduced into the flow cell for a period of 24 or 48 hours, while sodium hypochlorite was evaluated after 1, 10, 30 and 60 minutes. Biofilms were harvested by sonication in sterile PBS. Cellular protein levels were measured to quantitate the amount of biofilm harvested. Cellular viability was determined using serial plating. The number of colony forming units was then adjusted for cellular protein levels to allow treatment protocols to be compared. Qualitative SEM analyses of the biofilm was performed following exposure to each test agent. Results: Sodium hypochlorite was the only agent that achieved total bacterial elimination. Ledermix and Odontopaste had no significant effect on the E. faecalis biofilm, while calcium hydroxide and 50:50 combinations of calcium hydroxide with either Ledermix or Odontopaste were able to reduce viability by > 99%. Conclusion: When used in isolation, antibiotic containing medicaments had no appreciable effect on the viability of Enterococcus faecalis. Sodium hypochlorite remains the gold standard for bacterial elimination in root canal therapy. / Thesis (D.Clin.Dent.) -- University of Adelaide, School of Dentistry, 2009

It is well established that bacteria cause pulpal and periradicular disease (Kakehashi et al. 1965). Of the bacteria recovered from failing root canals, Enterococcus faecalis is one of the most prevalent species (Molander et al. 1998; Sundqvist et al. 1998). Many laboratory studies have investigated the effectiveness of root canal irrigants and medicaments against E. faecalis. Most used planktonic cultures, which are not representative of the in vivo growth conditions of an infected root canal system, where bacteria grow as a biofilm adhering to the dentinal wall (Nair 1987). Organisation of bacteria within biofilms confers a range of phenotypic properties that are not evident in their planktonic counterparts, including a markedly reduced susceptibility to antimicrobial killing (Wilson 1996). Objectives: The aims of this study were: 1) To compare the efficacy of commonly used endodontic medicaments against E. faecalis cultured as a biofilm. The medicaments tested were Ledermix paste, calcium hydroxide, Odontopaste, 0.2% chlorhexidine gel and 50:50 combinations of Ledermix/calcium hydroxide and Odontopaste/calcium hydroxide. 2) To compare the antimicrobial effect achieved through exposure to endodontic medicaments with that achieved by exposure to a constant concentration of sodium hypochlorite for varying times. Methods: A biofilm was established using a continuous flow cell. E. faecalis inoculum was introduced into the flow cell and allowed to establish on human dentine slices over 4 weeks. Each test medicament was introduced into the flow cell for a period of 24 or 48 hours, while sodium hypochlorite was evaluated after 1, 10, 30 and 60 minutes. Biofilms were harvested by sonication in sterile PBS. Cellular protein levels were measured to quantitate the amount of biofilm harvested. Cellular viability was determined using serial plating. The number of colony forming units was then adjusted for cellular protein levels to allow treatment protocols to be compared. Qualitative SEM analyses of the biofilm was performed following exposure to each test agent. Results: Sodium hypochlorite was the only agent that achieved total bacterial elimination. Ledermix and Odontopaste had no significant effect on the E. faecalis biofilm, while calcium hydroxide and 50:50 combinations of calcium hydroxide with either Ledermix or Odontopaste were able to reduce viability by > 99%. Conclusion: When used in isolation, antibiotic containing medicaments had no appreciable effect on the viability of Enterococcus faecalis. Sodium hypochlorite remains the gold standard for bacterial elimination in root canal therapy. / Thesis (D.Clin.Dent.) -- University of Adelaide, School of Dentistry, 2009

Thesis (Ph.D.) -- McMaster University, 1999. / Includes bibliographical references (leaves 216-244). Also available via World Wide Web.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references (leaves 71-76). Also available in electronic version. Access restricted to campus users.

Thesis (M.S.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Sept. 3, 2009). Includes bibliographical references (p. 44-49).

Made available in DSpace on 2014-08-13T14:50:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-03-11Bitstream added on 2014-08-13T18:01:16Z : No. of bitstreams: 1 000731950_20150311.pdf: 325288 bytes, checksum: 324db53ef4e3d775e299c6c4c92a035e (MD5) Bitstreams deleted on 2015-03-12T12:02:42Z: 000731950_20150311.pdf,Bitstream added on 2015-03-12T12:03:13Z : No. of bitstreams: 1 000731950.pdf: 1695739 bytes, checksum: 36607b7a254d4ab1aee0e89e07392e36 (MD5) / O objetivo do estudo foi avaliar a ação das soluções de hipoclorito de sódio e clorexidina associadas à cetramida e do QMiX, produto comercial que combina clorexidina, EDTA e cetramida, sobre Enterococcus faecalis (ATCC 29212) em fase planctônica e biofilme. As soluções avaliadas foram: G1: Hipoclorito de sódio (NaOCl) a 2,5%; G2: NaOCl a 2,5% + cetramida (CTR) a 0,2%; G3: Clorexidina (CLX) a 2%; G4: CLX a 2% + CTR a 0,2%; G5: CTR a 0,2%; G6: QMiX. Como controle negativo foi utilizada solução salina. O estudo foi dividido em dois capítulos. No capítulo 1, foi realizada a avaliação da atividade antibacteriana sobre os micro-organismos em fase planctônica e biofilme. Para o contato direto com E. faecalis em fase planctônica, 50 μl da suspensão bacteriana foram colocados em tubos contendo 1,45 mL de cada solução irrigadora ou associações e, após os períodos de 1 e 3 minutos foram utilizadas soluções neutralizantes da ação antimicrobiana dos irrigantes. Em seguida, foi realizada diluição decimal seriada e plaqueamento em meio Tryptic Soy agar (TSa). Para a indução dos biofilmes foram utilizados blocos de dentina bovina como substrato no período de 14 dias. Os biofilmes foram colocados em contato com as soluções avaliadas, e transferidos para tubos de ensaio contendo soluções neutralizantes. Após agitação por 60 segundos para a suspensão das células remanescentes, foi realizada diluição decimal seriada e plaqueamento em TSa em triplicata. A incubação foi realizada em microaerofilia a 37°C por 24 horas e a análise efetuada por meio da contagem de UFC mL-1. Os resultados foram submetidos aos testes ANOVA e Tukey, com nível de significância de 5%. Todas as soluções irrigadoras e associações avaliadas eliminaram completamente os micro-organismos em fase planctônica. Sobre biofilme, somente NaOCl e NaOCl+CTR apresentaram 100% de eliminação bacteriana... / The aim of this study was to evaluate the action of conventional endodontic solutions such as sodium hypochlorite and chlorhexidine associated with cetrimide and QMiX, a commercial product composed by chlorhexidine, EDTA and cetrimide, on biofilm and planktonic phase of Enterococcus faecalis (ATCC 29212). The evaluated solutions were: G1: 2.5% Sodium Hypochlorite (NaOCl); G2: 2.5% NaOCl + 0.2% cetrimide (CTR); G3: 2% Chlorhexidine (CLX) 2%; G4: 2% CLX + 0.2% CTR; G5: 0.2% CTR; G6: QMiX. Saline solution was used as negative control. The study was divided into two chapters. In Chapter 1, the antibacterial activity of the solutions on microorganisms in planktonic phase and biofilm was evaluated. For direct contact with E. faecalis in planktonic phase, 50 μL of bacterial suspension were placed in tubes containing 1.45 mL of each irrigation solution or associations and after 1 and 3 minutes, neutralizing solutions were used to stop of the antimicrobial action of endodontic solutions. Decimal serial dilutions were performed and the suspensions were plated in Tryptic Soy agar medium (TSa). Dentin bovine blocks were used as substrate for induction of biofilms for 14 days. The biofilms were placed in contact with the solutions evaluated and transferred to test tubes containing neutralizing solution. After agitation for 60 seconds to suspend the remaining cells, decimal serial dilutions were performed and the suspensions were cultivated in triplicate on TSA plates. The incubation was held in microaerophilic at 37 ° C for 24 hours and the analysis was performed by counting CFU mL-1. The results were submitted to ANOVA and Tukey test, with significance level of 5%. All irrigating solutions and associations evaluated completely eradicated the microorganisms in planktonic phase. On biofilm, only NaOCl and NaOCl+CTR showed 100%- of bacterial elimination. CLX showed similar activity to CLX+CTR, CTR and QMiX after...

Orientador: Elizeu Antonio Rossi / Banca: Célia Maria de Sylos / Banca: Alice Yoshiko Tanaka / Resumo: Os alimentos probióticos representam uma classe muito importante para a manutenção do estado de saúde. No mercado, esses produtos são predominantemente iogurtes e leites fermentados, havendo uma demanda por alimentos probióticos não-lácteos para ampliar as opções dos consumidores vegetarianos, bem como atender às necessidades dos indivíduos intolerantes à lactose. Nesse contexto, a soja apresenta-se como um adequado substituto do leite, em virtude das suas qualidades nutricionais e funcionais. O presente estudo teve como objetivo o desenvolvimento de uma sobremesa potencialmente probiótica mista de tofu e extrato hidrossolúvel de soja, fermentado com Enterococcus faecium CRL 183. No total, foram produzidas três formulações de sobremesa probiótica, com diferentes proporções de tofu e fermentado de soja: "A" (50%/ 50%), "B" (70%/ 30%) e "C" (80%/ 20%). Todas apresentavam as mesmas porcentagens de NaHCO3, creme de soja, polpa de morango, aroma de leite em pó, corante natural carmim de colchonilha, goma carragena e açúcar refinado. Testes sensoriais de aceitação e de intenção de compra foram realizados com 65 consumidores visando a escolha da melhor formulação, à qual foi submetida a análises físico-químicas e microbiológicas nos tempos de 2, 14 e 28 dias de estocagem a ±5ºC e, também, a análise sensorial nos tempos de 4, 14 e 28 dias. A composição centesimal e o valor calórico também foram determinados. Os dados foram avaliados através de análise de variância e teste de médias de Tukey (p<0,05). A sobremesa "A" foi considerada a melhor em termos sensoriais, principalmente quanto à intenção de compra, uma vez que 47% dos consumidores afirmaram que "certamente ou provavelmente comprariam" o produto. Sendo assim, ela foi selecionada para prosseguir o estudo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The probiotic foods represent a very important class for the maintenance of health state. At the market, these products are predominantly yogurts and fermented milks. There is a demand for non-dairy probiotic foods to increase the vegetarian options and attend the individuals with lactose intolerance. In this context, soy based food appears as an ideal substitute because its nutritional and functional qualities. The present study aimed the development of a potentially probiotic dessert added with tofu and aqueous extract of soy fermented with Enterococcus faecium CRL 183. Three formulations were produced with different proportions of tofu and fermented soy product: "A" (50% / 50%), "B" (70% / 30%) and "C" (80% / 20 %). All the desserts contained the same percentages of NaHCO3, soy cream, strawberry pulp, flavor milk powder, natural colouring agent, carrageenan gum, and sugar. Sensory tests for acceptance and purchase intent were performed to choose the best formulation, which was studied by physico-chemical, microbiological and rheological analysis after 2, 14 and 28 days of storage (± 5 º C) and sensory analysis after 4, 14 and 28 days. The chemical composition and caloric value were also determined. Data were evaluated by analysis of variance and Tukey test (p <0,05). The dessert "A" was considered the best sensorially, especially regarding the purchase intent, since that 47% of the panelists said they "would certainly or probably buy this product". Thus, it was chosen for further study. The composition of the formulation "A" had 72.2% of moisture, 4.4% of protein, 3.1% of fat, 0.6% of ash and 19.6% of carbohydrate, and the caloric value was 124 3 kcal / 100g. In this formulation, the strains of E. faecium CRL 183 and L. helveticus 416 remained viable throughout the experimental period, they exhibited... (Complete abstract click electronic access below) / Mestre

Summary Objectives and hypothesis: This thesis concerns the study of plasmids of vancomycin- resistant enterococci isolated from feces of American crows in the years 2012 - 2013 period. The hypothesis is that, in various environments, there is one or more types of epidemiologically significant vanA gene-carrying plasmids that are capable of horizontally spread. Methods: Based on PFGE method the number and size of plasmids were detected in selected isolates of vancomycin-resistant E. faecium. Using PCR method the isolates were subjected to detection of genes of replicases, relaxases and toxin-antitoxin system of plasmid-bound resistance genes. Using 19 primers were characterized types of Tn1546. Results: Of the 12 tested vancomycin-resistant isolates of E. faecium the following number and size of plasmids was proven using PFGE method: 2 isolates contained two plasmids (17%), 3 isolates contained three plasmids (25 %), 5 isolates contained four plasmids (42 %) and 2 isolates contained five plasmids (17 %). All isolates (n = 12) were then subjected to the detection of genes of replicases, relaxases and toxin-antitoxin system for typing of plasmids from each plasmid families. RepA_N family of plasmids: genes characterizing plasmids related to pRUM: rep17 in 11 isolates (92 %), gene Axe-Txe was detected in 5 isolates (42 %) genes characterizing plasmids related to pLG1: rep20 in 7 isolates (58 %) genes characterizing plasmids related to pAD1: relpAD1 gene was detected in one isolate (8 %) Inc18 family of plasmids: genes characterizing plasmids related to pIL501: rep1 gene detected in one case (8 %) genes characterizing plasmids related to pRES25: rep2 gene in 2 isolates (17 %) genes characterizing plasmids related to pEF1: relpEF1 detected in 11 isolates (92 %) pHTB family of plasmids: genes characterizing plasmids related to pHTB: rep22 gene was detected in 4 isolates (33%) and in 2 isolates gene relpHTB was detected (17%) RCR family of plasmids: genes characterizing plasmids related to pRI: positive detection of Rep14 gene in 8 isolates (67%) and in 4 isolates relpRI gene was detected Small theta-replicating plasmids: genes characterizing plasmids related to pEF418 plasmids: rep18a gene in 2 isolates (17%) genes characterizing plasmids related to pB82: rep18b gene was detected in one isolate (8%) genes characterizing plasmids related to pCIZ2: relpCIZ2 gene was detected in 9 isolates tested (75%) Types of transposon Tn1546 Using the PCR method types of Tn1546 were characterized. In 4 isolates (n = 12; 33 %) Tn1546 was characterized as a F3 type. In one isolate (8 %) Tn1546 was characterized as a type F5, in one isolate (8 %) as a type PP-16. In 6 isolates Tn1546 was untypeable. Most likely these are new, yet unknown types. Conclusion: This is the first study of plasmids of vancomycin-resistant isolates E. faecium isolated from feces of American crows. These results emphasize not only a high proportion of plasmids in individual isolates, but also a high proportion of genes with horizontal transfer.