Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / NTRODUCCIÓN: Streptococcus mutans (S. mutans) es una especie de bacterias cocáceas Gram positivo, presente en más del 90% de los seres humanos como miembro de la microbiota comensal de la cavidad bucal, que ha sido reportada como factor etiológico de caries dental. El potencial cariogénico de S. mutans está relacionado, entre otros, con su habilidad de producir y tolerar ambientes ácidos, formar biopelículas y adherirse tanto a la película dental adquirida (PDA), como a otros microorganismos. La expresión diferencial de estos factores de virulencia, en el complejo ambiente polimicrobiano del biofilm dental, es esencial para competir, colonizar y sobrevivir en el ecosistema oral. Mayor diversidad de cepas de S. mutans en la cavidad bucal ha sido asociada a una mayor prevalencia de caries, su permanencia se ha asociado con la habilidad de colonizar y resistir el stress ambiental, pero aún no existe consenso respecto de la relación entre la patogenicidad de las cepas aisladas de la cavidad oral de los preescolares chilenos y su experiencia de caries. Los estudios en salud bucal han determinado que uno de cada dos preescolares chilenos ha padecido caries y que el nivel socioeconómico bajo concentra la mayor carga de enfermedad. Entender la distribución y comportamiento de distintas cepas de S. mutans y su capacidad de expresar diferencialmente sus factores de virulencia, resulta útil para establecer grupos de riesgo portadores de cepas más virulentas e implementar medidas específicas de promoción, prevención y control de la caries dental. OBJETIVO: Demostrar la presencia de diferentes fenotipos de S. mutans en un grupo de preescolares chilenos, con y sin historia de caries, mediante una batería de pruebas microbiológicas convencionales. METODOLOGÍA: Mediante un examen clínico, y la aplicación de criterios de inclusión y de exclusión, se seleccionó un grupo de 12 párvulos que presentaban o no lesiones de caries. Se tomaron muestras microbiológicas mediante hisopado de superficies dentarias y mucosa oral, y a partir de ellas se aislaron colonias de S. mutans, identificadas mediante reacción en cadena de la polimerasa (PCR). Se analizó la capacidad de formar biofilms, sobrevivencia al stress ácido y resistencia ante agentes antimicrobianos de los aislados obtenidos de niños con o sin historia de caries, y se comparó la presencia de estos factores de virulencia entre los distintos aislados y la cepa de referencia de S. mutans, ATCC 38668. RESULTADOS: Aislados de S. mutans obtenidos de lesiones de caries, presentaron una mayor tolerancia al stress ácido, capacidad de formar biopelículas y de adherencia “in vitro”, que aislados obtenidos de niños sin lesiones de caries. CONCLUSIONES: Preescolares chilenos podrían ser portadores de cepas de S. mutans de alta virulencia, lo que amerita la profundización en el estudio de los factores etiológicos de la caries dental en la población infantil nacional.

Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Introducción: Streptococcus mutans está presente en más del 90% de los seres humanos como parte de la microbiota comensal de la cavidad bucal, y ha sido reportado como factor etiológico de caries dental. Mayor diversidad de genotipos de S. mutans se ha asociado con la habilidad de colonizar y resistir el stress ambiental, pero aún no existe consenso respecto de la relación entre la patogenicidad de los genotipos aislados de la cavidad oral de los preescolares chilenos y su experiencia de caries. Estudios que han usado metodologías que permiten la fenotipificación y/o genotipificación sugieren que la madre es la principal fuente de infección en niños portadores de S. mutans y que la saliva es el vehículo principal para esta transferencia. Sin embargo, la detección de genotipos que no son encontrados en las madres o en otros miembros cercanos de la familia indica que S. mutans puede ser adquirido de otras fuentes. Entender la distribución y comportamiento de distintos genotipos de S. mutans resulta útil para establecer grupos de riesgo. Materiales y métodos: Se obtuvieron muestras de saliva mediante gotario, de un grupo de 21 niños con distinta experiencia de caries. A partir de ellas se aislaron colonias de S. mutans, identificadas mediante reacción en cadena de la polimerasa (PCR). De estos aislados se identificó, mediante reacción en cadena de la polimerasa con partidores arbitrarios (AP-PCR), la presencia de distintos genotipos de S. mutans. Resultados: 1 a 5 genotipos de S. mutans colonizan la cavidad oral de los niños examinados y al menos uno de ellos es compartido por el grupo de párvulos. Conclusiones: Preescolares chilenos son portadores de distintos genotipos de S. mutans en su cavidad bucal y estos podrían ser transmitidos en forma horizontal. / Fiouch 03-14

Indiana University-Purdue University Indianapolis (IUPUI) / Streptococcus mutans, an inhabitant of oral biofilm or dental plaque, adheres to the tooth surface via protein antigen I/II (PA I/II). Pathologic lesions of atherosclerosis (AT) and infective endocarditis (IE) harbor S. mutans. Serotypes f and k strains with collagen binding protein genes cbm and cnm are uncommon in the mouth, but these are the most prevalent S. mutans strains in AT and IE tissues and can invade endothelial cells (EC) in vitro. Tobacco use increases the risk for cardiovascular and oral diseases. Oral S. mutans encounter many substances including nicotine. Arginine is present in saliva and the EC glycocalyx that coats and protects ECs from shear forces of blood flow. Prior studies demonstrated arginine alters S. mutans biofilm. This work characterizes S. mutans serotype k strains and serotype c strains, the most prevalent in the mouth. The effects of nicotine and arginine on biofilm mass, metabolic activity and EC invasion were investigated. Biofilm production by serotypes c and k strains did not differ; there were no differences in responses to nicotine and arginine between these serotypes. Increased production of biofilm was associated with the cbm and cnm genes. Nicotine increased biofilm for all strains whereas arginine plus nicotine reduced bacteria and the extracellular polymeric substances. Previous EC invasion studies were performed with planktonic cultures of S. mutans; therefore, EC invasion by biofilm was evaluated. Significant factors for EC invasion by S. mutans are presence of the cbm gene and lack of PA I/II expression on the bacterial cell surface. Presence of the cnm gene increased EC invasion by biofilm but not planktonic cells. Planktonic cells of six strains invaded better than biofilm, whereas four strains showed increased invasion by biofilm cells. Neither nicotine nor arginine significantly altered the ability of S. mutans biofilm cells to invade ECs. Not all strains with cbm or cnm and no PA I/II expression invaded EC. A strain with PA I/II expression and without cbm and cnm genes invaded EC. While cbm, cnm and PA I/II expression are predictors of EC invasion, additional mechanisms for EC invasion by S. mutans remain to be revealed. / 2021-08-21

Background: Caries continue to be a risk to children’s health and a stagnation of improvement has been seen the last years with 5% of Swedish three year olds being caries active. Earlier studies show a connection between presence of Streptococcus mutans  in saliva and caries prevalence, and that Streptococcus mutans can be transmitted from mother to child.   Aim: To study prevalence of Streptococcus mutans in three year old children and their mothers. To gain a deeper understanding of maternal transmission of Streptococcus mutans and other factors elevating the risk of caries in children.  Methods: Saliva samples and questionnaires were collected from 150 Swedish mothers and their three year old children, and a clinical dental examination was performed. PCR-technique were used to look for Streptococcus mutans in the saliva samples. Results from the questionnaires and saliva experiments were analyzed using SPSS.  Results: The total caries prevalence including initial and manifest lesions was 8%. Streptococcus mutans was detected in 48.7% of the mothers and 6.7% of the children. Significant correlation could be seen between early introduction to sugar and caries prevalence in three year olds. No significant correlation was seen between Streptococcus mutans in children and introduction to sugar or caries prevalence.  Conclusion: In this small cohort of mother-child pairs, no correlation between children infected with S.mutans and mutans-positive mothers, as well as caries development in children could be seen.

Streptococcus mutans is a member of the dental plaque and is the principal causative agent of dental caries. It can metabolize a wide array of sugars which results in the production of acid that causes demineralization of the tooth surface. S. mutans can also persist for extended periods of starvation, which may occur in different niches within the oral cavity. Previous studies have shown that mucin prolonged the survival of S. mutans in batch cultures and biofilms. Our laboratory has shown that the pyruvate dehydrogenase (pdh) operon is upregulated in stationary phase and is important for prolonged survival during nutrient starvation in chemically defined medium (CDM). In this study, I found that mucin enabled S. mutans to grow in a minimal chemically defined medium (MCDM) containing glucose and lacking all amino acids. A pdh mutant was found to be impaired in survival in CDM or MCDM supplemented with mucin. Additionally, I have shown that a galactose utilization mutant was impaired in survival in CDM with mucin and no exogenous glucose; galactose is the most abundant utilizable sugar residue in mucin. Mucin, supplemented in minimal medium with sucrose, also enabled S. mutans to form biofilms. Survival of the biofilms was pdh independent. Although biofilm formation was not seen in the absence of sucrose, cells did adhere to the glass coverslip; their survival was found to be pdh dependent. Using a previously constructed reporter strain, pdh expression was observed in a majority of cells in this condition. The expression of the pdh operon was also monitored in batch cultures. The starting inoculum size affected the percentage of fluorescent cells and the outcome of survival in media (CDM or MCDM) supplemented with mucin and no sugar. Greater than 50% of the stationary phase population expressed pdh in CDM with galactose. Less than 1% expressed pdh in CDM with glucose. However, galactose was unable to prolong survival of S. mutans in batch cultures in contrast to the effect of mucin. These results show that mucin may have a metabolic role in promoting long term survival of S. mutans. However it is possible that different components of mucin can be utilized in different conditions. / Microbiology and Immunology

No / Streptococcus mutans was shown to produce two extracellular proteases capable of degrading both gelatin and collagen-like substrates. These enzymes have molecular masses of 52 and 50 kDa when analysed by SDS-PAGE. Both enzymes were inhibited by EDTA, but not by a range of other inhibitors with different specificities, indicating that they are metalloproteases. The activity of EDTA-inactivated enzymes could be restored by the addition of manganese and zinc. The identical inhibition and restoration profiles of the two enzymes suggest that one of the proteases may be a degradation product of the other.

No / Isogenic mutants of Streptococcus mutans LT11, deficient in the production of the wall-associated protein antigens A and B, were generated by recombinant DNA technology. The hydrophobicity, adherence, and aggregation of the mutants were compared with those of the parent strain. These studies indicated that hydrophobicity, adherence, and saliva- or sucrose-induced aggregation were unaltered in the A- mutant but that hydrophobicity and adherence to saliva-coated hydroxylapatite were greatly reduced in the B- mutant whilst sucrose-dependent adherence and aggregation were increased. To determine whether these changes correlated with changes in the mutated gene product alone, the levels of a number of cell wall antigens were determined in each of the mutants. The loss of antigen A resulted in significantly reduced levels of wall-associated lipoteichoic acid, and loss of antigen B resulted in reductions in both antigen A and lipoteichoic acid. Data presented here thus suggest that changes in the expression of one wall antigen can have a dramatic effect on the levels of others.

Purpose: The purpose was to examine the maternal influences on the development of infant oral biofim and dominant bacterial strains of at risk populations. Methods: The study used a cross-sectional design to examine factors influencing biofilm colonization and the identification of bacterial strains transmitted from mother to child. Participants were enrolled in Children’s Health Involving Parents of Greater Richmond (CHIP). Plaque and saliva samples were collected from mothers and their children ages 6-36 months. The colonized oral bacteria strains of the mother infant dyads were then compared. Oral bacterial strain identification was completed using the HOMIM Forsythe microbe identification array. Examination for concordant strains was done using the statistical boot strap shuffle in Excel. Results: Forty-one CHIP families were involved in the pilot study. Participants were predominantly non-white , less than high school education 46.3%, and their average age was 29.1 years. Mothers had a caries prevalence of 87.8% and the infant’s caries rate was 26.7%. To date n=14 pairs of the n=41 samples have been processed and analyzed using the HOMIM microarray. Twelve paired samples were not processed due to non-detectable levels of bacterial DNA. Fifteen samples are currently being processed by HOMIM Forsyth. Predominate species transferred from mother to child include S. Oralis, S. parasanguinous, S. mitis, Slakia, and S. anginosis. 425 unique strains of bacteria were analyzed on the array with a maternal concurrence rate of 33%. Conclusion: When comparing total bacterial populations in the oral environment a concurrence of transmission from mother to child was 33%. Higher rates of vertical transmission were observed in S. Oralis, S. sanguinous, and Slakia.

Orientador: Alessandra Nara de Souza Rastelli / Resumo: A inativação fotodinâmica é descrita como terapêutica promissora na inativação de micro-organismos bucais. A curcumina é um fotossensibilizador com característica hidrofóbica, e que pode comprometer a sua eficiência. Dessa forma, torna-se relevante o desenvolvimento de estudos que verifiquem o seu efeito quando conjugada a micelas poliméricas. O objetivo deste estudo foi avaliar a efetividade da inativação fotodinâmica em biofilme de Streptococcus mutans utilizando-se curcumina, conjugada ou não, a micelas como fotossensibilizador, irradiado por luz LED. Realizou-se a caracterização das micelas poliméricas nas concentrações de 0.1 e 0.5% de curcumina, por meio do Potencial Zeta, determinação do espalhamento de luz dinâmico, microscopia eletrônica de transmissão e a investigação dos mecanismos envolvidos na reação fotodinâmica. Após, foi selecionada a melhor concentração a ser utilizada na inativação fotodinâmica sobre biofilme de Streptococcus mutans. As concentrações inibitória mínima (CIM) e bactericida mínima (CBM) foram determinadas. Foram induzidos em placa de 96 poços biofilmes single espécie. Os biofilmes foram irradiados uniformemente com sistema de iluminação a LED (Biotable, MMO) em comprimento de onda de 460 nm com dose/densidade de energia de 15 J/cm2 . Unidades formadoras de colônia (UFC/mL), a partir da CIM e CBM foram obtidas. Por meio de microscopia confocal utilizando-se corante BacLight® LIVE/DEAD foi avaliado a viabilidade celular nos biofilmes. Diferente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Photodynamic inactivation is described as promising therapy in the inactivation of oral microorganisms. Curcumin is a hydrophobic photosensitizer, which can compromise its efficiency. Thus, the development of studies that verify its effect when conjugated to polymeric micelles becomes relevant. The objective of this study was to evaluate the effectiveness of photodynamic inactivation in Streptococcus mutans biofilm using curcumin, conjugated or not, to micelles as photosensitizer, irradiated by LED light. The characterization of the polymeric micelles in the 0.1% and 0.5% concentrations of curcumin was carried out by Zeta Potential, determination of dynamic light scattering, transmission electron microscopy and investigation of the mechanisms involved in the photodynamic reaction. After that, the best concentration to be used in photodynamic inactivation on Streptococcus mutans biofilm was selected. Minimum inhibitory concentrations (MIC), minimal bactericidal (MBC) were determined. Single-species biofilms were induced in 96-well plate. The biofilms were uniformly irradiated with a LED illumination system (Biotable, MMO) at wavelength of 460 nm with dose or energy density of 15 J/cm2. Colony forming units (CFU / mL) from CIM and CBM were obtained. By means of confocal microscopy using BacLight® LIVE/DEAD dye, cell viability in biofilms was evaluated. Different groups were analyzed: FSM+L+ (Photosensitizer conjugate micelles Pluronic + Light), FSD+L+ (Photosensitizer in 10% DM... (Complete abstract click electronic access below) / Mestre

O objetivo do presente estudo foi avaliar, in vitro, o efeito de diferentes tempos de pré-irradiação do fotossensibilizador na terapia fotodinâmica em biofilmes formados por Streptococcus mutans e Candida albicans, por meio da avaliação da carga microbiana. Os fatores em estudo foram: tempos de pré-irradiação do fotossensibilizador em 3 níveis (1, 2 ou 5 minutos). Para o controle do biofilme dentário cariogênico com aPDT foi utilizado o azul de metileno (0,01%) associado ao laser de diodo (&lambda;=660 nm). O digluconato de clorexidina (CHX a 0,12%) e a solução salina foram utilizados como controle positivo e negativo, respectivamente. O delineamento do estudo foi realizado em blocos completos e casualizados, sendo a amostra composta por 15 culturas de biofilmes de S. mutans, divididas aleatoriamente em 5 grupos e 15 culturas de C. albicans, também divididas em 5 grupos. O experimento foi realizado em triplicata (n=3) e as variáveis de resposta foram obtidas por meio de análise quantitativa da viabilidade bacteriana, expressa em unidades formadoras de colônia (UFC) por mm2 da área do espécime. Os dados obtidos foram analisados com o auxílio do teste one-way ANOVA e pós-teste de Tukey. Todas as análises foram efetuadas por meio do programa Graph Pad Prism 4.0, com nível de significância de 5%. Para o grupo de S. mutans, apenas a solução salina apresentou diferença estatisticamente significante quando comparada aos demais tratamentos (p<0.05), ou seja, o tratamento com aPDT, independentemente do tempo de irradiação aplicado, foi semelhante ao tratamento com CHX e ambos foram mais eficazes na redução do biofilme cariogênico, em comparação à solução salina. Para o grupo de C. albicans não houve diferença estatística entre os grupos (p>0.05). Portanto, pode-se concluir que o tratamento com aPDT diminuiu o número de UFCs de S. Mutans de forma semelhante à CHX, independentemente do tempo de pré-irradiação aplicado. Não foi possível constatar nenhum efeito desta terapia e dos diferentes tempos de pré-irradiação sobre o biofilme de C. albicans. Desta forma, o tempo de pré-irradiação de 1 minuto pode ser utilizado com o objetivo de reduzir a carga microbiana de S. Mutans. / The aim of the present study was to evaluate, in vitro, the effect of different pre-irradiation times of the photosensitizer in photodynamic therapy in biofilms formed by on by Streptococcus mutans and Candida albicans, through the evaluation of the microbial load. The factors under study were: times of pre-irradiation of the photosensitizer in 3 levels (1, 2 or 5 minutes). For the control of the cariogenic dental biofilm with aPDT, methylene blue (0.01%) was used in association with the diode laser (&lambda;=660 nm). Chlorhexidine digluconate (0.12% CHX) and saline were used as positive and negative controls, respectively. The study design was carried out in complete and randomized blocks. The sample consisted of 15 S. mutans biofilms cultures, randomly divided into 5 groups and 15 C. albicans cultures, also divided into 5 groups. The experiment was performed in triplicate (n = 3) and the response variables were obtained through quantitative analysis of bacterial viability, expressed in colony forming units (CFU) per mm2 of the specimen area. The data were analyzed with the aid of the ANOVA one-way test and Tukey\'s post-test. All analyzes were performed using the Graph Pad Prism 4.0 program, with a significance level of 5%. For the S. mutans group, only the saline solution presented a statistically significant difference when compared to the other treatments (p <0.05), that is, the treatment with aPDT, irrespective of the irradiation time applied, was similar to the treatment with CHX and both were more effective in reducing cariogenic biofilm compared to saline. For the group of C. albicans there was no statistical difference between the groups (p> 0.05). Therefore, it can be concluded that the treatment with aPDT reduced the number of CFUs of S. Mutans in a similar way to CHX, independently of the pre-irradiation time applied. No effect of this therapy or of the different pre-irradiation times on the C. albicans biofilm could be observed. In this way, the pre-irradiation time of 1 minute can be used to reduce the microbial load of S. mutans.