Effect of fluoride on glucose incorporation and metabolism in biofilm cells of Streptococcus mutans.

The aim of this study was two-fold: firstly, to study the effect of high fluoride concentrations on carbohydrate metabolism in Streptococcus mutans present in biofilms on hydroxyapatite; and, secondly, to evaluate the effect of fluoride-bound hydroxyapatite on lactic acid formation in growing biofilms of Strep. mutans. Biofilms of a clinical strain of Strep. mutans on saliva-coated hydroxyapatite beads were incubated with sodium fluoride over a wide range of concentrations. At high fluoride concentrations (>10 mM) the incorporation of [14C]-labeled glucose decreased by 80-85%, at both pH 7.0 and 5.6. At lower fluoride concentrations, the effect of fluoride on the incorporation of labeled glucose was pH-dependent in both biofilm cells and in planktonic cells. At pH 7.0, fluoride at concentrations < 10 mM had little or no effect. Pretreatment of hydroxyapatite discs with fluoride varnish (Fluor Protector) or fluoride solutions caused a statistically significant reduction of lactic acid formation in associated, growing biofilms of Strep. mutans. Fluoride varnish and 0.2% (47.6 mM) sodium fluoride solution exhibited a statistically significant inhibitory effect on lactate production.

Analysis of sodium dodecyl sulfate-stable cell wall aminopeptidases in strains of viridans streptococci.

Cell wall extracts from nine strains of viridans streptococci representing five species were analyzed for aminopeptidases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with zymogram procedures revealed a common pattern of aminopeptidases in six strains, all having enzyme bands at 89 and 45 kDa. Three strains had an additional band at 200 kDa. Crossreactivity between aminopeptidases of all active strains was shown with crossed immunoelectrophoresis. Strains of Streptococcus mutans and S. sobrinus were without detectable cell wall aminopeptidases.

Effect of chlorhexidine gel treatment on the prevalence of mutans streptococci and lactobacilli in patients with impaired salivary secretion rate.

The potential pathogenic role of mutans streptococci in the etiology of dental caries is well-documented. Mutans streptococci are sensitive to chlorhexidine (CHX), and several methods for the clinical use of CHX have been described. An important target group for caries-preventive measures is patients with impaired salivary secretion due to the use of therapeutic drugs such as psychotropics. The aim of the present study was to compare two methods for antimicrobial treatment in such patients. Twenty-four volunteering patients at a hospital clinic were randomly divided into three groups: Group A, in which each participant was treated with 10% CHX gel in gel trays on two consecutive days, followed by application of a fluoride varnish; Group B, in which the patients were treated with the same CHX gel as above; and Group C, which was used as a control. After treatment, there was a reduction of mutans streptococci in the two groups treated with CHX. In group A, the sames collected one, three, and five weeks after the treatment were significantly lower than baseline values. No clearcut reduction of the number of mutans streptococci was observed in the subjects not treated with CHX. These data indicate that treatment with CHX gel in gel trays is superior to polishing the teeth with CHX gel. From a clinical point of view, our observations suggest that it is important to monitor the effect of antimicrobial treatment individually in order to optimize preventive programs in patients with impaired salivary secretion.

A study in vitro of threaded titanium pins used for retrograde obturation of root canals.

The microleakage of four retrograde filling materials was compared in vitro. Fifty-three single rooted teeth were instrumentated and root filled with resin chloroform and gutta-percha. The gutta-percha cones were left extruding from the access opening. All teeth were apicected and retrograde fillings placed. The materials used were a non gamma 2 amalgam (Amalcap), a glass ionomer cement (ChemFil II), threaded titanium pins cemented with a glass ionomer cement (ChemFil II) and identical titanium pins cemented with a silicone material (Adheseal). After removal of the gutta-percha with tweezers, a radioactive isotope solution was placed in the teeth. Extraradicular samples were taken at 3, 7, 28, 77 and 104 days. All retrograde fillings showed some microleakage. The group with titanium pins cemented with silicone showed the least leakage: significantly less than the teeth with glass ionomer cement (P < 0.01) and with amalgam (P < 0.01). No significant differences were found between other groups.

Purification and characterization of an aminopeptidase from Streptococcus mitis ATCC 903.

An aminopeptidase isolated from the cytoplasmic fraction of a cell extract of Streptococcus mitis ATCC 903 was purified 330-fold by ion-exchange chromatography, gel filtration, and hydroxyapatite chromatography. The partially purified enzyme had a broad substrate specificity. Twelve aminoacyl-beta-naphthylamide substrates were hydrolyzed and also several di-, tri-, tetra-, and pentapeptides and bradykinin. The enzyme hydrolyzed arginine-beta-naphthylamide at the highest rate. Optimal conditions for activity were at pH 7.0-7.2 and at 37-40 degrees C. The molecular weight of the enzyme was estimated to be 93,000. The enzyme was activated by Co2+ ions. Hg2+ inhibited the activity completely. SDS, EDTA, urea, and pCMB also inhibited activity. Inhibition by EDTA could be completely reversed by dialysis and addition of Co2+ ions. Reducing agents, sodium fluoride, and PMSF had no effect on the activity of the enzyme. The isoelectric point of the enzyme was at pH 4.3. High substrate concentrations inhibited activity. Substrate inhibition increased in the presence of high concentrations of Co2+ ions.

Quantitative radioactive analysis of microleakage of four different retrograde fillings.

Sealing properties of four different retrograde filling materials were investigated in vitro. Radioactive isotopes were applied in the root canal, and leakage into an extraradicular fluid was measured at regular intervals. The method permitted repeated observation of the specimens over prolonged periods of time. Forty single-rooted human teeth were biomechanically instrumented and obturated using calcium-hydroxide paste. Following obturation, an apicectomy was performed and retrograde cavities were filled with four different materials: group 1, non gamma 2 amalgam (Amalcap); group 2, glass ionomer cement (Ketac Silver); group 3, calcium-hydroxide-based root canal sealer (Sealapex); group 4, composite resin (Palfique Light-S). After removal of the calcium hydroxide, the teeth were immersed in a fluid. An isotope solution was then placed in the root canals. Samples were taken from the fluid at 0, 3, 7, 28, 56, 105, 210, 285 and 376 days to determine the radioactivity. It was found that Sealapex and Palfique Light-S showed significantly less leakage than amalgam and glass ionomer cement, which had the highest apical leakage.

Determination of non-immune binding of immunoglobulin G to Staphylococcus aureus by enzyme-linked immunosorbent assays.

Non-immune binding of human IgG to Staphylococcus aureus was studied by enzyme-linked immunosorbent assays using whole bacteria or bacterial cell walls as the solid phase. Two types of anti-human IgG peroxidase conjugates each with a low affinity for protein A, were used: F(ab')2-fragments of goat IgG and chicken IgY.

Arginine catabolism by strains of oral streptococci.

Arginine catabolism via the arginine deiminase pathway was found in Streptococcus sanguis 903. Citrulline and ornithine were released from resting cells incubated with arginine, arginine-containing peptides, or saliva. Maximum arginine catabolism by resting cells of S. sanguis 903 was found in the pH range 7-8 and at 45-48 degrees C. Arginine deiminase activity was found in the cytoplasm and in the cell-wall extract of this strain, while ornithine carbamoyltransferase activity was found in the cytoplasm and in extracts of cell walls and cytoplasmic membranes. Streptococcus mutans GS-5 and Streptococcus sobrinus strains OMZ 176 and 6715 could release arginine from salivary peptides but were incapable of significant arginine catabolism.

The influence of Streptococcus mutans on adhesion of Candida albicans to acrylic surfaces in vitro.

Adhesion of Candida albicans and Streptococcus mutans was studied by incubation of radiolabelled cells with acrylic test specimens in a chemically defined growth medium. Strep. mutans adhered firmly in the presence of sucrose, while C. albicans was only loosely attached to the acrylic in both glucose and sucrose media. Firm adhesion of C. albicans occurred when the yeast was incubated simultaneously with Strep. mutans, in the presence of sucrose. The adhesion of C. albicans was also stimulated by incubation with Strep. mutans culture supernatants. Adhesion was not affected by the presence of partially purified glucosyltransferase from Strep. mutans IB. Coaggregation between C. albicans and Strep. mutans upon growth in sucrose medium was observed by light and scanning electron microscopy. No coaggregation was observed in the presence of glucose.

Water-soluble glucans from Streptococcus mutans strain Ing-Britt as an energy source for bacterial growth.

Two fractions of water-soluble glucans with different molecular weight were produced by Streptococcus mutans IB. The larger glucan had a molecular weight of about 40,000. The molecular weight of the small glucan was estimated to be 4,100 by gel filtration chromatography and by biochemical methods. These glucans were tested for their ability to initiate and support growth of S. mutans IB and Streptococcus sanguis 903. S. sanguis 903 could grow with the low molecular weight glucan at a reduced rate compared with glucose. S. mutans IB could not utilize any of the glucans. No endo- or exo-glucanase activities could be detected in culture supernatants of any of the strains. In addition to maltose S. sanguis 903 could also utilize maltotriose, maltopentaose and maltoheptaose for growth while S. mutans IB could not grow with maltopentaose or maltoheptaose.

Adhesion of Streptococcus rattus and Streptococcus mutans to metal surfaces.

The adhesion of Streptococcus rattus BHT and Streptococcus mutans IB to metal specimens of amalgam, silver, tin and copper was studied using (6-3H)thymidine labeled cells. In the standard assay the metal specimens were suspended by a nylon thread in an adhesion solution containing a chemically defined bacterial growth medium (FMC), sucrose, and radiolabeled bacteria. Maximum amounts of adhering bacteria were obtained after about 100 min of incubation. Saturation of the metal specimens with bacteria was not observed. Both strains also adhered in the absence of sucrose, indicating that glucan formation was not necessary for adhesion. However, in the presence of glucose, adhesion was only 26-45% of that observed in the presence of equimolar sucrose. Sucrose-dependent stimulation of adhesion seemed to be due to increased cell-to-cell adhesion capacity. Isolated radiolabeled water-insoluble and water-soluble polysaccharides produced from sucrose by S. rattus BHT were not adsorbed to the metal surfaces.

Peptide utilization by oral streptococci.

Streptococcus mitis ATCC 903 and Streptococcus sanguis 804 could utilize large peptides present in proteose peptone as their sole source of nitrogen but grew poorly in amino acid medium. By contrast, Streptococcus mutans BHT and S. mutans IB grew in amino acid medium but could not utilize large peptides. Streptococcus sp. strain Bravato had requirements for both peptides and amino acids. Peptide size did not seem important for the utilization of peptides by S. mitis except that dipeptides were not utilized. Large peptides were essential for depressed hyaluronidase synthesis in nongrowing S. mitis. Intracellular aminopeptidase activities were high in S. mitis ATCC 903 and Streptococcus sp. strain Bravato, intermediate in S. sanguis 804, and low in S. mutans BHT and S. mutans IB.

Protoplast formation and localization of enzymes in Streptococcus mitis.

Cells of Streptococcus mitis ATCC 903 were converted to stable protoplasts by the cell wall-degrading M-1 enzyme of the mutanolysin complex isolated from Streptomyces globisporus. Over 90% of total glucokinase (EC 2.7.1.2), aminopeptidase (EC 3.4.11.1), and dextranglucosidase (EC 3.2.1.70) was recovered in the cytoplasmic fraction, whereas over 20% of total invertase (beta-fructofuranosidase: EC 3.2.1.26) was released during protoplast formation. ATPase (EC 3.6.1.3). chymotrypsin-like protease (EC 3.4.21.1), arginine aminopeptidase (EC 3.4.11.6), and lactate dehydrogenase (EC 1.1.1.27) were detected in Triton X-100 extracts of the cytoplasmic membrane fraction by crossed immunoelectrophoresis in combination with enzyme-staining procedures. By these methods, NADH dehydrogenase (EC 1.6.99.3), aminopeptidase, and lactate dehydrogenase were detected in the cytoplasmic fraction. Aminopeptidases in the cytoplasmic fraction differed from this activity in the membrane fractions in electrophoretic mobility and substrate specificity.

Regulation of synthesis of beta-fructofuranosidase (invertase) in Streptococcus mitis.

beta-Fructofuranosidase activity was found to be cell-bound in Streptococcus mitis ATCC 903. The following evidence suggests that induction functions as a regulatory mechanism for beta-fructofuranosidase in S. mitis: (1) on transfer of glucose-grown exponential phase bacteria to sucrose medium, the specific activity of beta-fructofuranosidase increased fourfold in the course of one generation; (2) other sugars had no stimulatory effect on the rate of synthesis of beta-fructofuranosidase; (3) the effect of sucrose on the rate of synthesis of beta-fructofuranosidase could be measured within a few minutes. Glucose, fructose and mannose repressed beta-fructofuranosidase. The addition of glucose to bacteria growing on sucrose repressed beta-fructofuranosidase for about one generation. The intracellular concentration of glucose was considerably increased during repression, while the intracellular concentration of glycolytic intermediates did not vary significantly.