Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Introducción: La caries secundaria es la principal causa de fracaso de restauraciones. No sólo se relaciona con algún defecto marginal, sino que también requiere de la formación de placa bacteriana con potencial cariogénico, siendo Streptococcus mutans (S. mutans) la especie bacteriana con mayor poder patogénico. Identificarlo y cuantificarlo directamente de placa bacteriana dental es fundamental para evitar futuras lesiones. Así surgen métodos para medir presencia de S. mutans en placa dental, siendo el más utilizado el Método de “Mondadientes o Toothpick”, el cual es un método simple, económico y viable de realizar en la consulta dental, con la desventaja de requerir conocimiento y tiempo de procesamiento de laboratorio alejando al clínico de realizar este importante examen complementario. Esta investigación tiene por objetivo principal, evaluar un método para la aislación y recuento de S. mutans denominado “Técnica de Cubeta”, y correlacionarlo con el Método del Mondadientes. Material y Método: Se seleccionaron 40 pacientes de la clínica de Operatoria 4to año de la Facultad de Odontología de la Universidad de Chile. En cada uno de ellos se tomó una muestra de placa bacteriana dental de un molar restaurado con Amalgama o Resina Compuesta, con el Método del Mondadientes y en su homólogo restaurado con el mismo material con la Técnica de Cubeta. Este método consiste en una impresión directa sobre las superficies oclusales de restauraciones, mediante una cubetilla de flúor cargada con agar TYCSB. Además se tomó muestras de saliva no estimulada como control de presencia de S. mutans en la boca de cada paciente. Las muestras de saliva y mondadientes fueron sembradas en agar TYCSB. Las cubetas y las placas sembradas se incubaron a 37°C por 48 horas, para posteriormente proceder al recuento bacteriano. 2 Resultados: La Técnica de Cubeta aisló Unidades Formadoras de Colonias (UFC)/cm de S.mutans en el 87,5% de las superficies obturadas. Posee una correlación positiva y significativa (r = 0,95) con el Método del Mondadientes. Se observó una diferencia significativa entre el recuento de S.mutans en restauraciones de Resina Compuesta y Amalgama mediante la Técnica de Cubeta y el Método del Mondadientes. Conclusiones: La Técnica de Cubeta es efectiva en aislamiento y recuento de S.mutans a partir de muestras de placa bacteriana dental en restauraciones de Amalgama y Resina Compuesta.

Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Introducción En el biofilm dental se encuentran diversas bacterias, de las cuales Streptococcus mutans se considerada la de mayor potencial cariogénico. El objetivo del presente estudio es saber si la aplicación de sellante de resina en surcos profundos de dientes posteriores permanentes de pacientes adultos de alto riesgo cariogénico, disminuye el recuento de UFC/cm² de Streptococcus mutans sobre el área de surcos oclusales del diente. Materiales y métodos Se seleccionaron 38 pacientes (edad promedio 24 años) entre marzo y junio de 2013, de la clínica de Operatoria, Facultad de Odontología, Universidad de Chile,. En cada paciente, un operador tomó muestra de biofilm de la superficie oclusal de un diente posterior permanente con indicación de sellante, mediante técnica de cubeta (impresión de cara oclusal del diente usando cubetilla para flúor, cargada con medio de cultivo selectivo para Streptococcus mutans). Luego aplicó sellante en surcos del mismo diente. 30 días después tomó otra muestra con técnica de cubeta, sobre el sellante oclusal. Las muestras fueron procesadas en laboratorio de Microbiología Bucal, Departamento de Patología, Facultad de Odontología, Universidad de Chile. Un segundo operador realizó análisis estadístico. Resultados Se aislaron colonias de Streptococcus mutans desde muestras de biofilm obtenidas mediante la técnica de cubeta, según macromorfología y adherencia al agar. Se identificaron colonias haciendo pruebas bioquímicas (hidrólisis de Esculina y fermentación de Rafinosa y Melobiosa). Se cuantificaron UFC de Streptococcus mutans en área de surcos de superficies oclusales de dientes posteriores permanentes, antes (T0) y después (T1) de aplicar sellante. Usando programa ImageJ®, se sacó promedio del área oclusal de molares y premolares, y del área ocupada por sellante en dichos dientes: 10% de superficie oclusal de molares es ocupada por sellante, y 9% en premolares. Se convirtieron los datos, expresándolos 6 en cm². Comparando los resultados (test de Wilcoxon) se determinó que existe diferencia significativa (p<0.05) entre T0 (X=197,76 DS=199,42) y T1 (X=68,2 DS=102,54). Conclusión En adultos jóvenes con alto riesgo cariogénico, la aplicación de sellante de resina sin flúor en surcos de dientes posteriores permanentes sanos, contribuye a reducir la cantidad de UFC/cm² de Streptococcus mutans sobre el área de los surcos de los dientes tratados.

Orientador: Marlise Inêz Klein / Resumo: O objetivo foi avaliar quais são os mecanismos de Streptococcus mutans e de Candida albicans que contribuem para aumentar a patogenicidade do biofilme misto. Biofilmes mistos e simples das cepas S. mutans UA159 (Sm) e C. albicans SC5314 (Ca) foram formados sobre discos de hidroxiapatita com película salivar, na presença de sacarose. O pH do meio de cultura foi aferido em diversas fases de desenvolvimento do biofilme. Após 43h de crescimento, foram realizadas análises bioquímicas (peso seco, proteinas, composição da matriz extracelular) e de população microbiana. A estrutura dos biofilmes foi avaliada por microscopia confocal em 19 e 43h. A expressão de genes de vias metabólicas de ambas espécies foi verificada em 28h. Os dados foram avaliados por métodos estatísticos considerando α=0,05. Verificou-se diferença do pH do meio para os três biofilmes nos tempos 19, 27 e 43h (p<0,001; ANOVA dois critérios, Tukey). Biofilmes de Sm e misto foram mais ácidos em 19 e 43h, enquanto biofilmes de Ca mantiveram o pH neutro (p>0,05). As quantidades do peso seco e de proteínas de biofilme misto foram maiores comparadas aos biofilmes simples, e menores para Ca (p=0,001; ANOVA um critério). Não houve diferença na quantidade de exopolissacarídeos solúveis entre biofilmes Sm e misto, porém o biofilme Ca apresentou menor quantidade (p<0,001; Kruskal-Wallis, Dunn). Houve maior quantidade de exopolissacarídeos insolúveis em biofilme misto (p=0,002). Verificou-se mesmo comportamento populacional pa... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective was to evaluate the mechanisms of Streptococcus mutans and Candida albicans that contribute to increasing the pathogenicity of the mixed-species biofilm. Mixed and single-species biofilms of the strains S. mutans UA159 (Sm) and C. albicans SC5314 (Ca) were formed on saliva-coated hydroxyapatite discs in the presence of sucrose. The pH values of the culture media were verified at distinct developmental phases of biofilms. After 43h of growth, the biofilms were subjected to distinct assays biochemical (dry weight, proteins, the composition of extracellular matrix) and microbiological (colony forming units), analyzes. The biofilm's structure was verified via confocal microscopy at 19 and 43h. Gene expression of metabolic ways from both species was evaluated at 28h. The data were assessed by statistical methods (α=0,05). There was a difference in the media pH for the three biofilms at times 19, 27 and 43h (p<0,001; two-way ANOVA, Tukey). Sm and mixed-species biofilms were acidic at 19 and 43h, while Ca biofilms maintained a neutral pH (p>0,05). The amounts of dry weight and proteins were higher for mixed-species biofilm compared to singlespecies biofilms, being lower for Ca (p=0,001; one-way ANOVA). The quantity of soluble exopolysaccharides was similar for Sm and mixed-species biofilms but Ca presented a lower amount than those biofilms (p<0,001; Kruskal-Wallis, Dunn). There was a higher amount of insoluble exopolysaccharides in mixed-species biofilm (p=0,002). There was no difference in Sm population in single- and mixed-species biofilms (p=0,404; Mann Whitney); however, the mixed-species biofilm presents a higher population of Ca versus the single-species biofilm (p<0,001; t-Test). The threedimensional structure analysis showed larger microcolonies in mixed-species biofilms versus Sm biofilm, and absence of these structures in Ca biofilm...(Complete abstract electronic access below) / Mestre

Orientador: Marlus Chorilli / Banca: Helder Ferreira Teixeira / Banca: Maria Palmira Daflon Gremião / Resumo: A cárie dentária é um importante problema de saúde coletiva ainda prevalente no Brasil e no mundo. A sua etiologia está ligada principalmente a fatores que permitam condições favoráveis ao crescimento de bactérias, principalmente do Streptococcus mutans. Recentemente, um crescente interesse tem sido observado no estudo de peptídeos, como o p1025, análogo aos fragmentos 1025-1044 da adesina celular do S. mutans, responsável pela adesão e formação do biofilme dental. Sistemas líquido-cristalinos (SLC) de liberação de fármacos, pelo fato de aumentarem a estabilidade e a eficácia de fármacos, modulando a sua ação, podem ser potenciais carreadores de peptídeos. A potencialização da ação destes sistemas pode ser conseguida com a presença de substâncias bioadesivas, para prolongar sua permanência no ambiente bucal O objetivo deste trabalho foi desenvolver e caracterizar SLC bioadesivos contendo peptídeo p1025 e avaliar sua eficácia antimicrobiana e ação sobre o biofilme dental. Foram desenvolvidos SLC constituídos por álcool cetílico etoxilado 20 OE e propoxilado 5 OP como tensoativo (PRO), ácido oleico (AO) como fase oleosa e por 5 diferentes fases aquosas formadas por água (S1), dispersões de Policarbofil® (S2), Carbopol® 974P (S3), Hidroxietilcelulose (S4) e Quitosana (S5) a 0,5%. Foram selecionados três pontos dos sistemas S1, S2, S3 e S5 fixando-se a concentração do tensoativo em 40%. Esses sistemas foram caracterizados por microscopia de luz polarizada, espalhamento de raios-X de baixo ângulo, reologia, análise térmica, análise de textura e ensaios de bioadesão in vitro, que demonstraram que as microemulsões constituídas por dispersões aquosas de Carbopol® 974P formaram mesofases líquido-cristalinas com adição de água significativamente mais estruturadas e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Dental caries is a significant public health problem still prevalent in Brazil and worldwide. The etiology is related to factors that enhance the growth of bacteria, mainly Streptococcus mutans. Recently, a great interest has been observed in studies of peptides such as peptide p1025, corresponding to residues 1025-1044 of the adhesin of S. mutans, responsible for adherence and formation of biofilm. According to the literature, solutions of this peptide inhibit by competition the bacterial biofilm adhesion to the tooth, preventing the colonization of S. mutans. Drug delivery systems, such as liquid crystalline systems (LCS) may be potential carriers of peptides because SLC can increase their stability and effectiveness. The potential action of these systems can be achieved with the presence of bioadhesive substances to prolong residence time at buccal cavity. The aim of this study was to develop and characterize bioadhesive SLC containing peptide p1025 and evaluate its antimicrobial efficacy and its action on the biofilm. It was developed five different SLC based on PPG-5-Ceteth-20 as surfactant, oleic acid as oil phase and five different aqueous phases based on water, dispersion of 0.5% Polycarbophil® (S1), Carbopol® (S2), hydroxyethylcellulose (S4) or chitosan (S5). It was selected three points of the systems S1, S2, S3 and S5 with 40% surfactant. These systems were characterized physicochemically by polarized light microscopy (MLP), SAXS, rheology, thermal analysis, TPA and in vitro bioadhesion, which showed that microemulsions composed of Carbopol® formed more structured and bioadhesive liquid crystalline mesophases with water than the other systems and it can high the retention time at buccal cavity. The incorporation of p1025 in this system demonstrated higher bioadhesive action and... (Complete abstract click electronic access below) / Mestre

Biofilms are highly organized bacterial structures that are attached to a surface. They are ubiquitous in nature and may be detrimental, causing numerous types of illnesses in living organisms. Biofilms in the human oral cavity are the main cause of dental caries and periodontal diseases and can act as a source for pathogenic organisms to spread within the body and cause various types of systemic diseases. Streptococcus mutans is the primary etiological agent of dental caries, the single most chronic childhood disease. In many cases, quorum sensing (QS) is required for initial formation and subsequent development of biofilms and the signaling molecule autoinducer 2 (AI- 2) has been well studied as an inter-species QS signaling molecule. However, recent reports also suggest that AI-2-mediated signaling is important for intra-species biofilm formation in both Gram-negative and positive bacteria. Therefore, there is significant interest in understanding the role of different QS signals such as AI-2 in oral biofilm formation. Microfluidic devices provide biomimetic environments and offer a simple method for executing multiple stimuli experiments simultaneously, thus, can be an extremely powerful tool in the study of QS in biofilms. In this study, we report conditions that support the development of S. mutans biofilms in microchannel microfluidic devices, and the effects of extracellular addition of chemically synthesized (S)-4,5-dihydroxy-2,3-pentanedione (DPD; precursor of AI-2) on mono-species S. mutans luxS (AI-2 deficient strain) biofilm formation using a gradient generating microfluidic device. S. mutans wild type (WT) and luxS biofilms were developed in nutrient rich medium (25% brain heart infusion medium, BHI + 1% sucrose) for up to 48 h. Maximum biofilm formation with both strains was observed after 24 h, with distinct structure and organization. No changes in S. mutans luxS biofilm growth or structure were observed upon exposure to different concentrations of AI-2 in a gradient generating device (0 to 5 M). These results were also validated by using a standard 96-well plate assay and by verifying the uptake of AI-2 by S. mutans luxS. Our data suggest that extracellular addition of AI-2 does not complement the luxS deletion in S. mutans with respect to biofilm formation.

Type II toxin-antitoxin (TA) systems are composed of a stable toxin and its cognate unstable antitoxin that impedes the toxin through direct interaction. The human oral pathogen Streptococcus mutans uses a quorum-sensing peptide (CSP) as a stress-inducible pheromone to synchronize gene expression in response to specific stressors. The objectives of this study were to investigate the role of S. mutans MazEF TA in cell survival and characterize the functionality of CSP-inducible chromosomal type II TAs. Our results suggest that MazEF represents a stress-response element. Interestingly, S. mutans negatively regulates its MazEF system under high-cell-density environment that is characteristic of oral biofilms. S. mutans also encodes a novel chromosomal type II TA involved in biofilm formation and development of dormant persister cells. The results from this study suggest a complex interplay between quorum-sensing (signal), type II TA activation (response), and persister formation (phenotype) as a reaction to environmental perturbations.

Type II toxin-antitoxin (TA) systems are composed of a stable toxin and its cognate unstable antitoxin that impedes the toxin through direct interaction. The human oral pathogen Streptococcus mutans uses a quorum-sensing peptide (CSP) as a stress-inducible pheromone to synchronize gene expression in response to specific stressors. The objectives of this study were to investigate the role of S. mutans MazEF TA in cell survival and characterize the functionality of CSP-inducible chromosomal type II TAs. Our results suggest that MazEF represents a stress-response element. Interestingly, S. mutans negatively regulates its MazEF system under high-cell-density environment that is characteristic of oral biofilms. S. mutans also encodes a novel chromosomal type II TA involved in biofilm formation and development of dormant persister cells. The results from this study suggest a complex interplay between quorum-sensing (signal), type II TA activation (response), and persister formation (phenotype) as a reaction to environmental perturbations.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1985. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Rijksuniversiteit te Utrecht.

Thesis (doctoral)--Lunds Universitet, Malmö, 1988. / Extra t.p. with thesis statement inserted. Includes bibliographical references.