Molecular mechanisms of inhibiting glucosyltransferases for biofilm formation in Streptococcus mutans / 国际口腔科学杂志·英文版

Glucosyltransferases (Gtfs) play critical roles in the etiology and pathogenesis of Streptococcus mutans (S. mutans)- mediated dental caries including early childhood caries. Gtfs enhance the biofilm formation and promotes colonization of cariogenic bacteria by generating biofilm extracellular polysaccharides (EPSs), the key virulence property in the cariogenic process. Therefore, Gtfs have become an appealing target for effective therapeutic interventions that inhibit cariogenic biofilms. Importantly, targeting Gtfs selectively impairs the S. mutans virulence without affecting S. mutans existence or the existence of other species in the oral cavity. Over the past decade, numerous Gtfs inhibitory molecules have been identified, mainly including natural and synthetic compounds and their derivatives, antibodies, and metal ions. These therapeutic agents exert their inhibitory role in inhibiting the expression gtf genes and the activities and secretion of Gtfs enzymes with a wide range of sensitivity and effectiveness. Understanding molecular mechanisms of inhibiting Gtfs will contribute to instructing drug combination strategies, which is more effective for inhibiting Gtfs than one drug or class of drugs. This review highlights our current understanding of Gtfs activities and their potential utility, and discusses challenges and opportunities for future exploration of Gtfs as a therapeutic target.

Typing of Streptococcus mutans strains isolated from caries free and susceptible subjects by multilocus enzyme electrophoresis

This study was evaluated the clonal diversity of Streptococcus mutans in caries-free and caries-active subjects using MLEE. Strains from caries-free subjects were grouped in a single taxon. Unrooted dendrogram showed that different strains clustered in four different clades, also showed that more than one clonal type can be found in a same individual.

Inhibition of dextransucrase activity in Streptococcus mutans by plant phenolics.

Streptococcus mutans is responsible for causing dental caries in humans and utilizes sucrose for its growth. The dextransucrase (EC 2.4.1.5) is responsible for sucrose metabolism, which exhibits both hydrolytic and glucosyltransferase activities. In this study, we examined the effects of the plant phenols, namely gallic, tannic and syringic acids and aqueous extracts of certain traditionally used chewing sticks (Acacia arabica, Azadirachta indica, Pongamia pinnata and Salvadora persica) for prevention of dental caries on hydrolytic activity of dextransucrsae in S. mutans. Gallic acid (4-5 mM) produced 80-90% inhibition of the enzyme, while tannic acid (0.2 mM) and syringic acid (5 mM) inhibited the enzyme activity 80% and 48%, respectively in vitro. The aqueous extracts of chewing sticks produced 35-40% inhibition of dextransucrase activity at 5 mg phenol concentration. Kinetic analysis revealed mixed-type of enzyme inhibition by polyphenols, where both Km and Vmax were altered. The value of Ki for tannic, gallic and syringic acids were 0.35, 1.6 and 1.94 mM, respectively. The enzyme inhibition by polyphenols was optimum at pH 7-7.5, while by plant extract was maximum at pH 5-6. These results suggest that plant polyphenols may find potential applications in the prevention and control of dental caries by inhibiting dextransucrase activity in S. mutans.

Acción antimicrobiana in vitro de la miel de abejas sobre los microorganismos cariogénicos estreptococos del grupo mutans / In vitro antimicrobial activity of honey on mutans streptococci

En el presente estudio fue evaluada la actividad antimicrobiana in vitro de cuatro muestras de mieles producidas en nuestro país, sobre los recuentos de estreptococos del grupo mutans, en escolares con alto riesgo de caries dental. Se obtuvieron muestras de saliva de 20 escolares, con edades entre 12 y 14 años, pertenecientes a la ciudad de Temuco, Región de La Araucanía (Chile). El recuento de estreptococos del grupo mutans en saliva fue estimado por el método microbiológico semi-cuantitativo Linoscreen®. La evaluación de la acción antibacteriana de la miel se realizó en 9 escolares que presentaron los recuentos más elevados de estreptococos del grupo mutans, utilizando concentraciones de miel entre 5 por ciento y 35 por ciento. Los datos mostraron que el 100 por ciento de los niños analizados poseían colonias de estreptococos del grupo mutans en su saliva. Además, se verificó que el 45 por ciento (9/20) de los escolares se encontraba en la categoría de alta actividad cariogénica. Con este estudio, también se comprobó que la miel de abejas posee actividad antimicrobiana sobre las bacterias estreptococos del grupo mutans y que no existían diferencias significativas entre las 4 mieles utilizadas, con relación a su capacidad antibacteriana (p>0.05). Por otra parte, este estudio también permitió demostrar que a mayor concentración de miel utilizada mayor era la reducción de las bacterias cariogénicas. En conclusión, en el presente estudio se demostró la acción antimicrobiana in vitro de la miel sobre los recuentos de bacterias cariogénicas estreptococos del grupo mutans. Sin embargo, serán necesarios futuros estudios para identificar y evaluar los componentes de la miel de abejas, responsables de esta propiedad.

The aim of the present issue was to evaluate the antibacterial activity of four commercial honeys against mutans streptococci in schoolchildren with high risk of caries development. We have investigated 20 schoolchildren from Temuco city (Chile), aged 12 -14 years. Saliva samples were obtained by stimulation with solid paraffin. The count of mutans streptococci in saliva was estimated by microbiological method. The antimicrobial activity of honey was evaluated by dilution in TYCBS medium in 9 schoolchildren (> 500.000 cfu/mL). Four concentrations of honey were used ranging from 5 to 35 percent. The presence of S. mutans was detected in 100 percent of schoolchildren. Our data shown that honey present high antimicrobial activity against S. mutans, being the dilutions at 30 and 35 percent that shown the better inhibitory effect (p<0.001). No differences between 4 honeys in relation to antibacterial activity were observed (p>0.05). In conclusion, our data showed that honeybees present in vitro antibacterial activity against S. mutans. However, future studies are necessary to characterize the honey components, responsible for this property.

[In vitro reduction in colonization of Streptococcus mutans by honey beeswax ethyl acetate extract]

Dental plaque is composed of bacterial derived extracellular polysaccharide known as glucan which is synthesized by Streptococcus mutans. Natural substances that could inhibit the plaque formation of the bacteria have a significant importance. This investigation has evaluated the honey beeswax extract effect on the Gft production, the key enzyme of S. mutans colonization factor for the first time. In this experimental study extraction of the sample conducted with ethyl acetate and methanol solutions in the Clevenger extractor. The ethyl acetate soluble fraction was separated in the first step and after the evaporation of the first solute, the 70% methanol as inactive solvent was added and the water mixture was used as a second solution, then materials were separated with dH[2]O. Minimum inhibitory concentration [MIC] of the honey beeswax extract was assessed by Broth diffusion method. Examination of cell adherence [Biofilm Inhibitory Concentration, BIC] was calculated by colony counts from surface scratching of glass slides in the bacterial media that supplied with 1% sucrose. Glucosyltransferase expression was detected by 15% SDS poly acrylamide gel electrophoresis. Concentration of 1mg/ml of ethyl acetate honey beeswax extract was inhibited completely biofilm and it was prevented the production of glucosyltransferase enzyme. The concentration of formation 6 mg/ml of the extract had bacteriostatic effect and 30 mg/ml concentration of this extract had bacteridicidal for S. mutans [P<0.01]. Thu sub-bacterial concentration honey beeswax extract was able to block the major enzyme that contributes to S. mutans biofilm formation

Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans

Streptococcus mutans membrane-bound P- and F-type ATPases are responsible for H+ extrusion from the cytoplasm thus keeping intracellular pH appropriate for cell metabolism. Toluene-permeabilized bacterial cells have long been used to study total membrane-bound ATPase activity, and to compare the properties of ATPase in situ with those in membrane-rich fractions. The aim of the present research was to determine if toluene permeabilization can significantly modify the activity of membrane-bound ATPase of both F-type and P-type. ATPase activity was assayed discontinuously by measuring phosphate release from ATP as substrate. Treatment of S. mutans membrane fractions with toluene reduced total ATPase activity by approximately 80 percent and did not allow differentiation between F- and P-type ATPase activities by use of the standard inhibitors vanadate (3 µM) and oligomycin (4 µg/mL). Transmission electron microscopy shows that, after S. mutans cells permeabilization with toluene, bacterial cell wall and plasma membrane are severely injured, causing cytoplasmic leakage. As a consequence, loss of cell viability and disruption of H+ extrusion were observed. These data suggest that treatment of S. mutans with toluene is an efficient method for cell disruption, but care should be taken in the interpretation of ATPase activity when toluene-permeabilized cells are used, because results may not reflect the real P- and F-type ATPase activities present in intact cell membranes. The mild conditions used for the preparation of membrane fractions may be more suitable to study specific ATPase activity in the presence of biological agents, since this method preserves ATPase selectivity for standard inhibitors.

Genetic engineering and dental caries.

Dental caries, a multifactorial disease requires four principle factor: the host, the microflora, the substrate & time for its occurrence and can be prevented or managed by elimination/modification of either of the above factors. The conventional preventive measure being followed for long time for the dental caries are not successful to the desirable extent due to their non avaibailaballity in the rural areas, lack of awareness & inaccessibility of dental services. Therefore, the focus has now been shifted to submicroscopic level to ensure that these measures can be reached to the farthest areas & each & every member of the population is benefitted. Few of the measures taken are. i) Genetically modifying the S. Mutans: ii) Searching The antagonist peptides to work against the specific enzyme system (Glucosyltransferase) of S. Mutans. iii) Changing the oral environment by those Genetically modified organisms that will produce bases (instead of acids) & these bases provides a milieu favoring remineralization. This paper discusses various ways in which genetically modified strains of microogranisms or genetically modified strains of microogranisms of genetically modified foods can help in the prevention of caries.

Fatores enzimáticos de agressão produzidos por bactérias aeróbicas da cavidade bucal / Production of histolytic enzymes by aerobic oral bacteria

Verificou-se a produção de hialuronidase, condroitin sulfatase, lecitinase e gelatinase em diferentes amostras bacterianas, isoladas da cavidade bucal. Hialuronidase foi produzida por 20 das 30 amostras de S. aureus, 18 das 21 amostras de S. pyogenes e todas as 4 amostras de A. viscosus. Nenhuma amostra produziu condrotin sulfatase e gelatinase; todas as 30 amostras de S. aureus e todas as amostras de A. viscosus produziram lecitinase