Saliva sampling methods. Cariogenic streptococci count using two different methods of saliva collection in children / Recuento de estreptococos cariogénicos a partir de dos métodos de obtención de saliva en niños

ABSTRACT The aim of this study is to compare the efficacy of two methods for collecting saliva samples from infants under 2 years of age for cariogenic streptococci (CS) count. Two collection methods were applied in 11 infants. In Method (A), saliva samples were collected by swabbing the inner cheek mucosa and floor of the mouth in figure of eight motions with a sterile cotton swab until it was soaked. In method (B), saliva samples were collected by aspiration of 1 ml of saliva with a sterile plastic syringe on the floor of the mouth, after stimulation with glove. The samples were cultured in modified Gold's broth (MSMG), and on trypticase, yeast extract, sucrose, cystine and bacitracin culture medium (TYSCB). In method (A), the swab with the sample was unloaded in situ on TYSCB and placed in PBS medium for transport. Then, 100 μl of the eluate was seeded in MSMG. In method (B) 100 μl were seeded in TYSCB and 100 μl in MSMG. Both culture media were incubated under capnophilic conditions for 48 hours at 37 °C. Colony forming units (CFU/ml) were counted by calibrated operators (kappa = 0.75). The presence of cariogenic streptococci (CS) (Streptococcus mutans-Streptococcus sobrinus) was determined by qPCR in the samples collected by both methods. The CFU/ml counts in MSMG differed significantly between methods (p = 0.021). In TYSCB, the recovery of CFU/ml was higher in method (A), without significant difference (p = 0.705). The molecular technique detected presence of CS, with no difference between collection methods. Collecting saliva samples by swabbing proved more effective in terms of recovery of microorganisms, and did not affect the detection of presence of CS by molecular techniques.

RESUMEN El objetivo de este estudio es comparar la eficacia de dos métodos de obtención de muestras salivales, en infantes menores de 2 años para el recuento de estreptococos cariogénicos (EC). Se aplicaron dos métodos de recolección en 11 infantes, el método (A), consistió en la recolección de muestras de saliva con hisopos de algodón estériles, realizando movimientos en ocho sobre la mucosa del carrillo y piso de boca, hasta embeber el hisopo. En el método (B) la recolección de las muestras se realizó por aspiración con jeringa plástica estéril en piso de boca hasta obtener 1 ml, luego de estimulación con guante. Las muestras fueron cultivadas en caldo de Gold modificado (MSMG) y medio de cultivo TYSCB (tripticasa, extracto de levadura, sacarosa, cistina y bacitracina). En (A), el hisopo con la muestra fue descargado in situ en TYSCB y colocado en medio de transporte PBS. 100 μl del eluato se sembró en MSMG. En (B) 100 μl fueron sembrados en TYSCB y 100 μl en MSMG. Ambos medios de cultivo fueron incubados en condiciones de capnofilia por 48 hs. a 37°C. El recuento de unidades formadoras de colonias (UFC/ml) se realizó por operadores calibrados (kappa= 0.75). La presencia de EC (Streptococcus mutans - Streptococcus sobrinus) fue determinada por qPCR en las muestras obtenidas por ambos métodos. Los resultados mostraron que los recuentos de UFC/ml en MSMG presentaron diferencias significativas entre ambos métodos (p=0.021) En TYSCB la recuperación de UFC/ml fue mayor en el método (A), sin observarse diferencias significativas (p=0.705). Se detectó la presencia de EC por técnica molecular, sin mostrar diferencias entre los métodos empleados. La recolección de muestra de saliva con hisopo presentó mayor eficacia en términos de recuperación de microorganismos, sin alterar la detección de presencia de EC por técnicas moleculares.

Antimicrobial and Antibiofilm Effect of Hydrogel with Origanum vulgare on Culture of Streptococcus mutans and Streptococcus sobrinus

ABSTRACT: The oral cavity is an ecosystem that provides ideal conditions for the growth of bacteria, the Streptococcus genus is important for the formation of biofilms that lead to the development of dental caries, which affects the population worldwide. The world health organization encourages the use of plants thanks to its various therapeutic actions. Origanum vulgare L. (oregano), is an aromatic plant with medicinal and culinary properties. The objective of this study was to investigate the in vitro antimicrobial and antibiofilm activity of the ethanolic extract of oregano, against the growth of Streptococcus mutans and Streptococcus sobrinus ATCC. Leaves of the plant were obtained and the ethanolic extract was made by maceration. Antimicrobial activity was evaluated using the Kirby-Bauer method and compared with 2% chlorhexidine, subsequently the extract was incorporated into a hydrogel and its effect on biofilm formation was assessed by fluorescence microscopy and the main compounds were identified. present in the extratco. The study revealed that the extract presented antimicrobial effect against both strains and at 2% it showed high antimicrobial action compared to chlorhexidine at the same concentration, with average inhibition halos of 26.3 mm and 19 mm for each microorganism analyzed, (p < 0.05). Likewise, the hydrogel prepared with 2% extract significantly eliminated the preformed Streptococcus biofilm, at 24 hours of exposure, due to the presence of a variety of chemical groups, such as sterols, triterpenes, flavonoids, flavanones, flavanonol s, lactones. sesquiterpenic, tannins and coumarins. The oregano extract presented high antimicrobial action for both species, with a greater effect towards Streptococcus mutans and an interesting antibiofilm action; These results show the importance of exploring treatment alternatives of plant origin, to be considered as interesting complementary aids in dental therapy.

RESUMEN: La cavidad oral es un ecosistema que proporciona condiciones ideales para el crecimiento de bacterias, el género Streptococcus es importante para la formación de biopelículas que conducen al desarrollo de caries dental, que afecta a la población a nivel mundial. La organización mundial de la salud, fomenta el uso de plantas gracias a sus diversas acciones terapéuticas. Origanum vulgare L. (orégano), es una planta aromática con propiedades medicinales y culinarias. El objetivo de este estudio fue investigar la actividad antimicrobiana y antibiofilm in vitro del extracto etanólico de oregano, contra el crecimiento de Streptococcus mutans y Streptococcus sobrinus ATCC. Se obtuvierón hojas de la planta y se realizó el extracto etanólico mediante maceración. La actividad antimicrobiana se evaluó mediante el método de Kirby-Bauer y se comparó con la clorhexidina al 2 %, posteriormente se incorporó el extracto en un hydrogel y se valoró su efecto sobre la formación del biofilm mediante microscopía de fluorescencia y se identificó los principales compuestos presentes en el extratco. El estudio reveló que el extracto presentó efecto antimicrobiano contra ambas cepas y al 2 % mostró alta acción antimicrobiana en comparación con la clorhexidina a la misma concentración, con halos de inhibición promedio de 26.3 mm y de 19 mm para cada microorganismo analizado, (p < 0.05). Así mismo, el hidrogel preparado con extracto al 2 %, eliminó significativamente la biopelícula preformada de Streptococcus, a las 24 horas de exposición, debido a la presencia de una variedad de grupos químicos, como esteroles, triterpenos, flavonoides, flavanonas, flavanonoles, lactonas sesquiterpénicas, taninos y cumarinas. El extracto de orégano presentó alta acción antimicrobiana para ambas especies, con mayor efecto hacia el Streptococcus mutans y una acción antibiofilm interesante; estos resultados muestran la importancia de explorar en alternativas de tratamiento de origen vegetal, para considerarse como auxiliares complementarios interesantes en la terapia dental.

Study of Bacteria Associated with Dental Caries Using a 3 Tone Disclosing Agent / 대한소아치과학회지

The aim of this study was to determine the efficacy of a 3 tone plaque disclosing gel in assessing the risk of caries related to the population of Streptococcus mutans, Streptococcus sobrinus, and Lactobacillus spp. quantified using a quantitative real-time polymerase chain reaction (qRT-PCR).15 healthy children of ages 9 – 12 years were randomly examined. The 3 tone plaque disclosing gel was applied on teeth surfaces, which changed the color to pink or red, blue or purple and light blue. Plaque was divided into 3 groups based on staining. Genomic DNA from each sample was subjected to a qRT-PCR assay for quantitative detection of target bacteria. The Kruskal-Wallis test was conducted for correlation between the color of plaque and the number of bacterial species.The levels of S. mutans, S. sobrinus, and Lactobacillus spp. were significantly different in the plaque samples of the 3 groups (p < 0.05). The proportion of S. sobrinus to S. mutans showed correlation to the color of plaque.The different color-dyed plaque was related to the number of acidogenic bacteria. The 3 tone plaque disclosing gel could be used as one of the indicators to assess the clinical risk of caries associated with the population of S. mutans, S. sobrinus, and Lactobacillus spp.

Antimicrobial effect of Prunus mume extracts against cariogenic bacteria / 대한구강보건학회지

OBJECTIVES: This study was conducted to determine whether Prunus mume extracts have an antimicrobial effect against Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus). METHODS: The study used crushed and dried Prunus mume, to which 80% methanol was added to obtain extracts. The extracts then underwent a demarcation process, sequentially using hexane, chloroform, and ethyl acetate, all of which have different polarities, followed by a reduction in pressure . The disc diffusion method was then used to measure the clear zone diameter to identify the antimicrobial effect of Prunus mume extracts using the different solvents. The methanol extracts that presented antimicrobial activity against S. mutans and S. sobrinus were then selected, and their optical densities (3, 6, 9, 12, and 24 h after cultivation) were measured to identify growth retardation effects based on extract concentration (0.01, 0.1, 1, and 5 mg/ml). RESULTS: A clear zone was observed in methanol and ethyl acetate for S. mutans when the antimicrobial effect of Prunus mume extracts of each solvent against oral microorganisms was measured via the disc diffusion method. A clear zone was observed in hexane, chloroform, methanol, and ethyl acetate, when the extracts were tested for antimicrobial activity against S. sobrinus. The extract concentration of 1 mg/ml retarded growth with a statistical significance (P<0.05) from 6 h onwards, as determined when the optical density was measured hourly and the growth curves of S. mutans and S. sobrinus were plotted. CONCLUSIONS: Prunus mume extracts retarded the growth of S. mutans and S. sobrinus with increase in time and concentration. Therefore, Prunus mume extracts hold the potential to be used for developing an oral antimicrobial agent to control dental caries.

The Antibacterial Properties of Euphorbia Tirucalli Stem Extracts against Dental Caries-Related

Euphorbia tirucalli are reported to possess antibacterial activity against various microorganisms. This in vitro study aimed to evaluate the antibacterial properties of Euphorbia tirucalli stems extracts (methanol, ethanol and aqueous extracts) against dental caries-related bacteria, i.e. Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus). The antibacterial properties were determined using agar-well diffusion method at different extract concentrations (10, 20 and 30 mg/ml). Commercially available amoxicillin (10 µg) was used as positive control while the appropriate solvent served as negative control. The methanolic and ethanolic extracts of Euphorbia tirucalli stem were found to be effective against S. mutans and S. sobrinus. However, the aqueous extract of Euphorbia tirucalli stem showed no activity against both bacterial strains. The differences in the antibacterial properties in different extracts of Euphorbia tirucalli may be due to the differences in phytochemical constituents.

Effect of dietary sugars on dual-species biofilms of Streptococcus mutans and Streptococcus sobrinus – a pilot study / Efeito dos açucares da dieta em biofilme dupla espécie de Streptococcus mutans e Streptococcus sobrinus – um estudo piloto

Introduction: Frequent consumption of sugars and the presence of Streptococcus mutans and Streptococcus sobrinus are correlated with higher caries experience. Objective: The aim of this pilot study was to elucidate the effect of different fermentable carbohydrates on biomass formation and acidogenicity of S. mutans and S. sobrinus biofilms. Material and method: Single and dual-species biofilms of S. mutans ATCC 25175 and S. sobrinus ATCC 27607 were grown at the bottom of microtiter plates at equal concentrations for 24 h at 37 °C under micro-aerobic atmosphere. Carbohydrates were added at 2% concentration: maltose, sucrose, glucose and lactose. BHI Broth (0.2% glucose) was used as negative control. Acidogenicity was assessed by measuring the pH of spent culture medium after 24 h, immediately after refreshing the culture medium and for the next 1 h and 2 h. Crystal violet staining was used as an indicator of the total attached biofilm biomass after 24 h incubation. Data were analyzed by two-way ANOVA followed by Bonferroni post hoc test. Significance level was set at 5%. Result: All carbohydrates resulted in higher biomass formation in single- and dual-species biofilms when compared to the control group. Sucrose, lactose and maltose showed higher acidogenicity than the control group in both single- and dual-species biofilms after 24 h. Conclusion: These findings indicate that the type of biofilm (single- or dual-species) and the carbohydrate used may influence the amount of biomass formed and rate of pH reduction.

Introdução: O consumo frequente de açucares e a presença de Streptococcus mutans e Streptococcus sobrinus estão correlacionados com maior experiência de cárie. Objetivo: Elucidar o efeito de diferentes carboidratos fermentáveis na biomassa e acidogenicidade de biofilmes formados por S. mutans e S. sobrinus. Material e método: Biofilmes única e dupla- espécie de S. mutans ATCC 25175 e S. sobrinus ATCC 27607 em concentrações iguais cresceram no fundo de placas de microtitulação por 24 h a 37 °C em microaerofilia. Maltose, sacarose, glicose e lactose foram adicionados a 2%. BHI caldo (0.2% glicose) foi usado como controle negativo. Acidogenicidade foi avaliada por meio da medição do pH do meio de cultura após 24 h, imediatamente após troca de meio e nas próximas 1 h e 2 h. Coloração por cristal violeta foi usada como indicador do total de biomassa aderida, após 24 h de incubação. Os dados foram analisados por teste ANOVA two way e Teste de Bonferroni. O nível de significância foi de 5%. Resultado: Todos os carboidratos resultaram em maior formação de biomassa em ambos os tipos de biofilme (única ou dupla- espécie), quando comparado ao grupo controle. Sacarose, lactose e maltose mostraram maior acidogenicidade que o grupo controle após 24 h nos biofilmes única ou dupla-espécie, apenas após 24 h. Conclusão: Os achados indicam que o tipo de biofilme (única ou dupla- espécie) e o tipo de carboidrato usado podem influenciar tanto na quantidade de biomassa formada quanto na taxa de redução do pH.

In vitro antibacterial activity and major bioactive components of Cinnamomum verum essential oils against cariogenic bacteria, Streptococcus mutans and Streptococcus sobrinus

Objective: To evaluate the antibacterial activity of Cinnamomum verum (C. verum) from 32 different essential oils against cariogenic bacteria, Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus). Methods: The antibacterial activities of each essential oil were individually investigated against S. mutans and S. sobrinus. The essential oil of C. verum was selected for further evaluation against S. mutans and S. sobrinus. Gas chromatography mass spectrometry was used to determine the major constituents of C. verum essential oil. In addition, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration of the most effective constituent was investigated. Results: The essential oil from C. verum exhibited the greatest antibacterial activity. Gas chromatography mass spectrometry analysis revealed that the major components of C. verum essential oil were cinnamaldehyde (56.3%), cinnamyl acetate (7.1%) and β-phellandrene (6.3%). The MIC of cinnamaldehyde was measured using broth dilution assays. The MIC of cinnamaldehyde was 0.02% (v/v) against both bacterial strains tested. The minimum bactericidal concentration of cinnamaldehyde against S. mutans and S. sobrinus were 0.2% and 0.1% (v/v), respectively. Conclusions: The essential oil of C. verum and its major component cinnamaldehyde possessed considerable in vitro antibacterial activities against cariogenic bacteria, S. mutans and S. sobrinus strains. These results showed that the essential oil of C. verum and its bioactive component, cinnamaldehyde, have potential for application as natural agents for the prevention and treatment of dental caries.

Development of Quantitative Real-Time PCR Primers for Detection of Streptococcus sobrinus

The purpose of this study was to develop Streptococcus sobrinus-specific qPCR primers based on the nucleotide sequence of the RNA polymerase β-subunit gene (rpoB). The specificity of the primers was determined by conventional polymerase chain reaction (PCR) with 12 strains of S. sobrinus and 50 strains (50 species) of non-S. sobrinus bacteria. The sensitivity of the primers was determined by quantitative real-time PCR (qPCR) with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of S. sobrinus ATCC 33478(T). The specificity data showed that the S. sobrinus-specific qPCR primers (RTSsob-F4/RTSsob-R4) detected only the genomic DNAs of S. sobrinus strains with a detection limit of up to 4 fg of S. sobrinus genomic DNA. Our results suggest that the RTSsob-F4/RTSsob-R4 primers are useful in detecting S. sobrinus with high sensitivity and specificity for epidemiological studies of dental caries.

The effects of hesperidin in caries prevention in rats / 实用口腔医学杂志

Objective:To investigate the anticaries effects of hesperidin in rats.Methods:The MIC of hesperidin against Streptococ-cus sobrinus(S.sobrinus)was explored with disc diffusion method.The rats with artificial caries were administered with 1/2 MIC hes-peridin,0.12%Chlorhexidine and distilled water respectively.The results of S.sobrinus level,Keyes scoring and DIAGNOdent exam-ination were used to evaluate the effects of hesperidin on S.sobrinus and caries development.Results:The MIC of hesperidin agaist S. sobrinus was 8 mg/ml.The S.sobrinus level was not statistically different between hesperidin group and negative control group(P>0. 05).Keyes scores of grade E of smooth and pit and fissure caries in hesperidin group were lower than in negative control(P<0.05), those of grade Ds and Dm of pit and fissure caries in hesperidin group were lower than in negative control(P<0.05 ),while higher than in chlorhexidine group(P<0.05).Hesperidin showed significant anti-caries effect(P<0.05)examined by DIAGNOdent.Con-clusion:Hesperidin at 1/2 MIC has anti-caries effect in rats without influence of the oral microecological balance.

Synergistic effect of xylitol and ursolic acid combination on oral biofilms

OBJECTIVES: This study was designed to evaluate the synergistic antibacterial effect of xylitol and ursolic acid (UA) against oral biofilms in vitro. MATERIALS AND METHODS: S. mutans UA 159 (wild type), S. mutans KCOM 1207, KCOM 1128 and S. sobrinus ATCC 33478 were used. The susceptibility of S. mutans to UA and xylitol was evaluated using a broth microdilution method. Based on the results, combined susceptibility was evaluated using optimal inhibitory combinations (OIC), optimal bactericidal combinations (OBC), and fractional inhibitory concentrations (FIC). The anti-biofilm activity of xylitol and UA on Streptococcus spp. was evaluated by growing cells in 24-well polystyrene microtiter plates for the biofilm assay. Significant mean differences among experimental groups were determined by Fisher's Least Significant Difference (p < 0.05). RESULTS: The synergistic interactions between xylitol and UA were observed against all tested strains, showing the FICs < 1. The combined treatment of xylitol and UA inhibited the biofilm formation significantly and also prevented pH decline to critical value of 5.5 effectively. The biofilm disassembly was substantially influenced by different age of biofilm when exposed to the combined treatment of xylitol and UA. Comparing to the single strain, relatively higher concentration of xylitol and UA was needed for inhibiting and disassembling biofilm formed by a mixed culture of S. mutans 159 and S. sobrinus 33478. CONCLUSIONS: This study demonstrated that xylitol and UA, synergistic inhibitors, can be a potential agent for enhancing the antimicrobial and anti-biofilm efficacy against S. mutans and S. sobrinus in the oral environment.

Antimicrobial activity of fractions and subfractions of Elaeagia utilis against microorganisms of importance in dental caries

La caries dental es una enfermedad infecciosa multifactorial que conduce a la destrucción del tejido duro dental. El principal objetivo de la investigación en plantas medicinales es la búsquedade compuestos con actividad antimicrobiana para su posterior uso en estrategias de prevención o control de enfermedades infecciosas. El objetivo de este estudio fue evaluar la actividad antimicrobiana de fracciones y subfracciones obtenidas de la planta Elaeagia utilis contra Streptococcus mutans, Streptococcus sobrinus y Lactobacillus acidophilus. El materialvegetal fue colectado en la ciudad de Albán (Cundinamarca-Colombia) situada a una altitud de 2245 metros sobre el nivel del mar. Mediante el método de maceración en frio de hojas de E. utilis se obtuvieron dos extractos, uno en éter de petróleo y otro en etanol. Del extracto etéreo se obtuvieron fraccionesmediante cromatografía en columna al vacío y al extracto etanólico se le realizó fraccionamiento líquido/líquido continuo. La evaluación de la actividad antimicrobiana de las fracciones ysubfracciones se realizó por el método de difusión en pozo. A una concentración de 10 mg/pozo, múltiples fracciones obtenidas de los dos extractos presentaron actividad antimicrobianasobre S. mutans, S. sobrinus y L. acidophilus. De las fracciones del extracto etanólico se destaca la fracción diclorometano, por presentar mayor actividad antimicrobiana, razón por lo cual se subfracciona y se obtienen tres subfracciones con actividad inhibitoria.La subfracción más activa fue eOH:H2O (Bp) con una concentración mínima inhibitoria de 0.1 mg/pozo sobre las 3bacterias en estudio. En esta subfracción se determinaron terpenos, sesquiterpenlactonas y compuestos fenólicos simples. En conclusión, en este estudio se presenta el potencial antimicrobiano de fracciones y subfracciones obtenidas de extractos dehojas de E. utilis contra microorganismos de importancia encaries dental.

Dental caries is a multifactorial infectious disease that leads tothe destruction of dental hard tissue. The main goal of researchinto medicinal plants is to seek compounds with antimicrobialactivity for subsequent use in prevention strategies and controlof infectious diseases. The aim of this study was to evaluate theantimicrobial activity of fractions and subfractions obtainedfrom Elaeagia utilis against Streptococcus mutans, Streptococ-cus sobrinus and Lactobacillus acidophilus. The plant materialwas collected in the town of Alban (Cundinamarca, Colombia),which is located at an altitude of 2245 meters above sea level.Two extracts were obtained by cold maceration of E. utilis leavesin (a) petroleum ether extract and (b) ethanol extract. Fractionswere obtained from the petroleum ether extract by column vacu-um chromatography, and from the ethanol extract by continuousliquid / liquid partitioning. The antimicrobial activity of frac-tions and subfractions was evaluated by the well diffusionmethod. At a concentration of 10 mg /well, several fractions fromboth extracts showed antimicrobial activity against S. mutans, S.sobrinus and L. acidophilus. Among the ethanol extract frac-tions, the dichloromethane fraction had notably greaterantimicrobial activity. It was sub-partitioned, yielding three sub-fractions with inhibitory activity, of which the most active wasMeOH: H2O (Bp) with minimum inhibitory concentration 0.1mg /well on the 3 study bacteria. Terpenes, sesquiterpenlactonesand simple phenolic compounds were identified in it. In conclu-sion, this study shows the antimicrobial potential of fractionsand subfractions obtained from extracts of E. utilis leaves againstbacteria that are important in dental caries.

Chemical and botanical characterization of Chilean propolis and biological activity on cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus

Propolis is a non-toxic natural substance with multiple pharmacological properties including anticancer, antioxidant, fungicidal, antibacterial, antiviral, and anti-inflammatory among others. The aim of this study was to determine the chemical and botanical characterization of Chilean propolis samples and to evaluate their biological activity against the cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. Twenty propolis samples were obtained from beekeeping producers from the central and southern regions of Chile. The botanical profile was determined by palynological analysis. Total phenolic contents were determined using colorimetric assays. Reverse phase HPLC and HPLC-MS were used to determine the chemical composition. The minimum inhibitory concentration (MIC) was determined on S. mutans and S. sobrinus. All propolis samples were dominated by structures from native plant species. The characterization by HPLC/MS, evidenced the presence of quercetin, myricetin, kaempferol, rutine, pinocembrin, coumaric acid, caffeic acid and caffeic acid phenethyl ester, that have already been described in these propolis with conventional HPLC. Although all propolis samples inhibited the mutans streptococci growth, it was observed a wide spectrum of action (MIC 0.90 to 8.22 µgmL-1). Given that results it becomes increasingly evident the need of standardization procedures, where we combine both the determination of botanical and the chemical characterization of the extracts. Research conducted to date, describes a promising effectiveness of propolis in the prevention of caries and other diseases of the oral cavity, making it necessary to develop studies to identify and understand the therapeutic targets or mechanisms of molecular action of the various compounds present on them.

Quantitative analysis of S. mutans and S. sobrinus cultivated independently and adhered to polished orthodontic composite resins

In Orthodontics, fixed appliances placed in the oral cavity are colonized by microorganisms. OBJECTIVE: The purpose of this study was to quantitatively determine the independent bacterial colonization of S. mutans and S. sobrinus in orthodontic composite resins. MATERIAL AND METHODS: Seven orthodontic composite adhesives for bonding brackets were selected and classified into 14 groups; (GIm, GIs) Enlight, (GIIm, GIIs) Grengloo, (GIIIm, GIIIs) Kurasper F, (GIVm, GIVs) BeautyOrtho Bond, (GVm, GVs) Transbond CC, (GVIm, GVIs) Turbo Bond II, (GVIIm, GVIIs) Blugloo. 60 blocks of 4x4x1 mm of each orthodontic composite resin were made (total 420 blocks), and gently polished with sand-paper and ultrasonically cleaned. S. mutans and S. sobrinus were independently cultivated. For the quantitative analysis, a radioactive marker was used to codify the bacteria (³H) adhered to the surface of the materials. The blocks were submerged in a solution with microorganisms previously radiolabeled and separated (210 blocks for S. mutans and 210 blocks for S. sobrinus) for 2 hours at 37ºC. Next, the blocks were placed in a combustion system, to capture the residues and measure the radiation. The statistical analysis was calculated with the ANOVA test (Sheffè post-hoc). RESULTS: Significant differences of bacterial adhesion were found amongst the groups. In the GIm and GIs the significant lowest scores for both microorganisms were shown; in contrast, the values of GVII for both bacteria were significantly the highest. CONCLUSIONS: This study showed that the orthodontic composite resin evaluated in the GIm and GIs, obtained the lowest adherence of S. mutans and S. sobrinus, which may reduce the enamel demineralization and the risk of white spot lesion formation.

Polymerase Chain Reaction for detection of Streptococcus mutans and Streptococcus sobrinus in dental plaque of children from Cartagena, Colombia / Reacción en cadena de la polimerasa para la detección de Streptococcus mutans y Streptococcus sobrinus en placa dental de preescolares de Cartagena, Colombia

Objectives: To detect the presence of Streptococcus mutans and Streptococcus sobrinus in dental plaque of children from Cartagena and correlate it to dental caries precavity stages, applying a standardized PCR-based technique for epidemiological purposes. Methods: Descriptive study using a non-probabilistic sample of 50 children between 3 and 5 years of age, preschoolers from a Caribbean population in Colombia. Criteria for selection were that children should exhibit plaque accumulations on the surface of the cervical margins of the rearmost molars, and placed in one of two study groups: carious lesions and sound surfaces. Dental plaque samples from both groups were subjected to molecular analysis and statistical analysis was applied to determine the difference between the two groups using the frequencies of presence of S. mutans, S. sobrinus or both in the two groups applying Fisher's exact test for association between the presence of microorganisms and the state of the tooth surface from where the dental plaque was taken. Results: The frequency of S. mutans in carious lesions was 76% and 24% in healthy surfaces. The frequency of S. sobrinus in carious lesions was 81.9% and 18.1% in caries-free surfaces. There was statistical significance between the presence of S. mutans and the presence of caries (p=0.001) and between the presence of S. sobrinus (p=0.02) and the presence of caries. There was no statistical significance between the presence of caries and the simultaneous presence of both microorganisms (p=0.08). Conclusions: The presence of S. mutans and S. sobrinus in dental plaque samples is highly prevalent and associated to non cavitated carious lesions, being the molecular identification of these microorganisms by PCR a sensitive, fast, and easy to use detection method for the mutans group of oral bacteria.

Objetivos: Detectar la presencia de Streptococcus mutans y Streptococcus sobrinus en placa dental de niños de Cartagena y relacionarlo con la caries dental en estadíos precavitacionales, estandarizándose la técnica basada en PCR para detectar a nivel epidemiológico la presencia de estos microorganismos. Métodos: Estudio de tipo descriptivo, a partir de una muestra no probabilística de 50 niños entre 3 y 5 años de los hogares infantiles de una población del Caribe colombiano. Como criterios de selección se tuvo en cuenta que los niños presentaran acumulaciones de placa dental sobre la superficie de los márgenes cervicales de los últimos molares. El estudio se dividió en dos grupos: lesiones cariosas y superficies sanas. Las mediciones se realizaron a partir de muestras de placa dental, sometidas a análisis molecular. El análisis estadístico se realizó a partir de las frecuencias de presencia de S. mutans, S. sobrinus o ambos en las lesiones cariosas y superficies sanas, utilizando la prueba exacta de Fisher para la asociación entre la presencia de los microorganismos y el estado de la superficie dental donde se encontró la placa. Resultados: La frecuencia de S. mutans para las lesiones cariosas fue 76% y 24% en superficies sanas. Con respecto a S. sobrinus, la frecuencia en lesiones cariosas fue 81.9% y 18.1% en superficies sin caries. La presencia de caries estuvo asociada significativamente en forma individual con S. mutans (p=0.001) o con S. sobrinus (p=0.02), pero no fue significante con la presencia simultánea de los dos microorganismos (p=0.08). Conclusiones: La presencia de S. mutans y S. sobrinus en muestras de placa bacteriana se considera un factor de asociación con la presencia de lesiones de caries precavitacionales, siendo la identificación molecular por PCR un método sensible, rápido y de fácil uso para la detección de bacterias del grupo mutans.

Antimicrobial Effects of Ursolic Acid against Mutans Streptococci Isolated from Koreans

Ursolic acid is a triterpenoid compound present in many plants. This study examined the antimicrobial activity of ursolic acid against mutans streptococci (MS) isolated from the Korean population. The antimicrobial activity was evaluated by the minimum inhibitory concentration (MIC) and time kill curves of MS. The cytotoxicity of ursolic acid against KB cells was tested using an MTT assay. The MIC90 values of ursolic acid for Streptococcus mutans and Streptococcus sobrinus isolated from the Korean population were 2 microg/ml and 4 microg/ml, respectively. Ursolic acid had a bactericidal effect on S. mutans ATCC 25175T and S. sobrinus ATCC 33478T at > 2 x MIC (4 microg/ml) and 4 x MIC (8 microg/ml), respectively. Ursolic acid had no cytotoxic effect on KB cells at concentrations at which it exerted antimicrobial effects. The results suggest that ursolic acid can be used in the development of oral hygiene products for the prevention of dental caries.

Anticariogenic activity of the active fraction from isertia laevis against S. mutans and S. sobrinus: comparison of two extraction methods

Dental caries is considered a multi-factorial, infectious, chronic, localized, post-eruptive, transmissible disease that leads to the destruction of dental hard tissue. The recognition of Streptococcus mutans as the major bacterial species involved in dental caries has led to the implementation of prevention and control measures for eliminating or reducing it in oral cavity. The main goal of research on medicinal plants is the search for substances or compounds with antimicrobial activity. The aim of this study was to evaluate the antimicrobial activity of fractions obtained by two methods from Isertia laevis against S. mutans and S. sobrinus. The plant material was collected in Medina (Colombia), at an elevation of 550 meters above sea level. From the ethanol extract of leaves of I. laevis, fractions were obtained by two methods: extraction by column vacuum chromatography (CVC) and extraction by continuous liquid / liquid partitioning (CLLP). The evaluation of the antimicrobial activity of fractions against S. mutans and S. sobrinus was performed by well diffusion and bioautography assays. From the CVC technique, only the methanol and methanol-dichloromethane fractions showed activity against S. mutans and S. sobrinus, with a minimum inhibitory concentration of 2 mg/well. From the CLLP technique, only the dichloromethane fraction showed activity against both microorganisms, with a minimum inhibitory concentration of 1 mg/well. Compounds C1 and C2 were isolated from the three active fractions, and showed a minimum inhibitory concentration of 0.4 mg/well for S. mutans and S. sobrinus, with zones of inhibition measuring 6.5 and 6.2 mm, respectively. In conclusion: 1) the three active fractions of I. laevis showed activity against S. mutans and S. sobrinus, 2) compounds C1 and C2 were present equally in the three active fractions showing activity against the two bacteria, 3) compounds C1 and C2 may be triterpenoid and/or steroidal saponin structures, and 4) the two extraction methods lead equally to obtaining the active fractions.

La caries dental es considerada una enfermedad infecciosa multifactorial, cronica, localizada, poseruptiva y transmisible que conlleva a la destruccion del tejido dental duro. El claro reconocimiento de Streptococcus mutans como la principal especie bacteriana implicada en caries dental, ha conducido a la implementacion de medidas de prevencion y control para la eliminacion o disminucion de este microorganismo en cavidad oral. El objetivo fundamental de la investigacion en plantas medicinales, es la busqueda de sustancias o compuestos con actividad antimicrobiana para ser utilizadas en el control o prevencion de enfermedades infecciosas. En este sentido, en salud bucal muchas sustancias obtenidas de plantas han mostrado actividad antimicrobiana. El objetivo de este estudio fue evaluar la actividad antimicrobiana de fracciones obtenidas de la planta Isertia laevis mediante dos metodologias contra S. mutans y S. sobrinus. El material vegetal se colecto en el municipio de Medina (Cundinamarca- Colombia) situado a una altura de 550 metros sobre el nivel del mar. A partir del extracto etanolico de hojas de I. laevis se obtuvieron fracciones mediante dos metodologias, extraccion por cromatografia en columna al vacio (CCV) y extraccion por fraccionamiento liquido/liquido continuo (FLLC). La evaluacion de la actividad antimicrobiana de las fracciones frente a S. mutans y S. sobrinus se realizo por el metodo de difusion en pozo y bioautografico. De las fracciones obtenidas por CCV, solamente las fracciones metanol y metanol-diclorometano presentaron actividad antimicrobiana sobre S. mutans y S. sobrinus, con una concentracion minima inhibitoria de 2 mg/pozo. De las fracciones obtenidas por FLLC solamente la fraccion diclorometano presento actividad antimicrobiana sobre S. mutans y S. sobrinus, con una concentracion minima inhibitoria de 1 mg/pozo. De las 3 fracciones activas se aislaron los compuestos C1 y C2, que presentaron una concentracion minima inhibitoria de 0.4 mg/pozo tanto para S. mutans como para S. sobrinus con halos de inhibicion, respectivamente, de 6.5 y 6.2 mm. En conclusion, 1. Las fracciones metanol y diclorometano obtenidas por CCV y la fraccion diclorometano obtenida por FLLC de hojas de I. laevis presentaron actividad antimicrobiana sobre S. mutans y S. sobrinus; 2. Los compuestos C1 y C2 presentes por igual en las tres fracciones activas tuvieron accion inhibitoria sobre las dos bacterias en evaluacion; 3. Las pruebas quimicas cualitativas para los compuestos C1 y C2 indican que posiblemente corresponden a estructuras de saponinas triterpenicas y/o esteroidales; y 4. Las dos metodologias de extraccion conducen por igual a la obtencion de las fracciones activas.

Differential gene expression between Streptococcus sobrinus 6715 and its fluoride-resistant strains / 上海第二医科大学学报

Objective To detect the differential gene expression between Streptococcus sobrinus(S.sobrinus) 6715 and its fluoride-resistant strains. Methods The fluoride-resistant strains of S.sobrinus 6715 was induced by increasing the concentration of fluoride step by step.Total RNA of both S.sobrinus 6715 and its fluoride-resistant strains was extracted,mRNA was separated and purificated,and then cDNA was obtained by reversed transcription.Suppression subtractive hybridization(SSH) technology was used to detect the differential gene expression between them.The differential gene expression fragments were cloned and compared with the GenBank by BLAST.Results After comparing with the GenBank by BLAST,it was identified that there were two differential gene expression fragments,fruA and SMU.438c. Conclusion The cDNA subtractive lib of differential gene expression between S.sobrinus 6715 and its fluoride-resistant strains was successfully constructed through SSH,which paves a way for the further study of fluoride-resistant mechanism.

Detection of Streptococcus sobrinus by polymerase chain reaction / 实用口腔医学杂志

Objective: To develop a PCR method for detecting S treptococcus sobrinus (S.s) in conventional culture. Method: A pair of specific primers were designed from the dexA gene of S.s , the genome DNA of 12 strains of Mutans Streptococci (8 serotypes from a ～h) and 23 species of the bacteria which were commonly found in oral cavity wer e tested by PCR amplification, the PCR products were identified by electrophore sis. Result: Only S.s of serotype d and g could produ ce 277 bp DNA fragments, and the PCR method could detect less than 1 000 copies of S.s. Conclusion: PCR method is specific and sen sitive in the detection of S.s.

The effects of Streptococcus sobrinus on corrosion behavior of metal-ceramic alloys / 实用口腔医学杂志

Objective:To study the corrosion resistance of four metal-ceramic alloys in the presence of Streptococcus sobrinus, and to compare the effects of bacteria action in liquid and solid environment.Methods:Four metal-ceramic alloys were exposured to BHIA or BHI media with or without Streptococcus sobrinus and served as different groups. After inoculating 10 weeks, their I_ corr ,E_ corr ,E_ tp through cyclic polarization curves were obtained through Corrview 2 corrosion analyses software;and their surface changes after electrochemical corrosion were observed by field emission scanning electron microscopy (FESEM).Results:The E_ corr of four alloys were decreased in presence of oral microorganism, and the declines were more remarkable in BHI with bacteria. SEM observation showed the pittings in their surface.The corrosion resistance in BHI without bacteria were significant increased than that in the blank control. Conclusion:Streptococcus sobrinus can promote the corrosion behavior of metal-ceramic alloys and the corrosion resistance is more worse in BHI than BHIA with bacteria.

The initial adherence of Streptococci isolates from the children with rampant caries / 实用口腔医学杂志

0.05). S.sobrinus strains had significantly lower initial adhesive ability to saliva coated hydroxyapatite in the absence of sucrose than S.mutans (P