Involvement of antigen I/II surface proteins in Streptococcus mutans and Streptococcus intermedius biofilm formation.

BACKGROUND/AIM: Dental diseases are caused by microorganisms organized in biofilms. Streptococcus mutans and Streptococcus intermedius are commensals of the human oral cavity. S. mutans is associated with caries, whereas S. intermedius is associated with purulent infections. Oral streptococci including S. mutants and S. intermedius express a family of surface proteins termed antigen I/II (Ag I/II). Ag I/II is implicated in adhesion; however, its role in biofilm formation has not yet been investigated. METHODS: By using isogenic Ag I/II-deficient mutants of S. mutans and S. intermedius we studied the influence of Ag I/II on in vitro biofilm formation. Biofilm was quantified in polystyrene microtiter plates and visualized by scanning electron microscopy. Ag I/II expression in planktonic and biofilm cells, as well as in the presence or absence of saliva was investigated by immunoblotting. RESULTS: In the presence of saliva, the Ag I/II-deficient mutants formed 65% less biofilm than the wild-types. In the absence of saliva, no difference was observed in S. mutans, whereas the S. intermedius Ag I/II mutant formed 41% less biofilm. Ag I/II expression was reduced in the presence of saliva. No differences in expression were observed between biofilm and planktonic cells. CONCLUSION: The results indicated that Ag I/II may be important during biofilm formation particularly in the presence of saliva. These findings may provide useful information regarding the importance of Ag I/II in biofilm formation and in the search of new strategies to control biofilm-mediated infections.

Cariogenic traits in xylitol-resistant and xylitol-sensitive mutans streptococci.

Long-term xylitol consumption leads to the emergence of xylitol-resistant (X-R) mutans streptococci. The aim of the present study was to compare cariogenic traits in X-R and xylitol-sensitive (X-S) strains. Six strains of mutans streptococci, three X-R and three X-S strains, were studied. Xylitol resistance and sensitivity were confirmed by growth in xylitol-supplemented media. Acid production from glucose or fructose or uptake of xylitol was initiated by adding (14)C-labelled glucose, fructose or xylitol to bacterial suspensions. The resultant metabolites were identified by HPLC. Lactate was the major metabolite from glucose, whether the bacteria were grown in the presence or the absence of xylitol. Lactate production per colony-forming unit was lower in X-S cells than in X-R cells. Fructose was metabolized by both X-R and X-S cells. Both X-R and X-S cells took up xylitol, but xylitol-5-P was detected in X-S cells only. Total polysaccharides were measured through production of C(14)-labelled ethanol-insoluble polymers from [U(14)-C]-sucrose. No difference in polysaccharide production was found between X-R and X-S cells. The present study thus does not support the contention that X-R are less cariogenic than X-S mutans streptococci.

Functional variation of the antigen I/II surface protein in Streptococcus mutans and Streptococcus intermedius.

Although Streptococcus intermedius and Streptococcus mutans are regarded as members of the commensal microflora of the body, S. intermedius is often associated with deep-seated purulent infections, whereas S. mutans is frequently associated with dental caries. In this study, we investigated the roles of the S. mutans and S. intermedius antigen I/II proteins in adhesion and modulation of cell surface characteristics. By using isogenic mutants, we show that the antigen I/II in S. mutans, but not in S. intermedius, was involved in adhesion to a salivary film under flowing conditions, as well as in binding to rat collagen type I. Binding to human fibronectin was a common function associated with the S. mutans and S. intermedius antigen I/II. Adhesion of S. mutans or S. intermedius to human collagen types I or IV was negligible. Hydrophobicity, as measured by water contact angles, and zeta potentials were unaltered in the S. intermedius mutant. The S. mutans isogenic mutants, on the other hand, exhibited more positive zeta potentials at physiological pH values than did the wild type. The results indicate common and species-specific roles for the antigen I/II in mediating the attachment of S. mutans and S. intermedius to host components and in determining cell surface properties.

Expression and functional properties of the Streptococcus intermedius surface protein antigen I/II.

Streptococcus intermedius is associated with deep-seated purulent infections. In this study, we investigated expression and functional activities of antigen I/II in S. intermedius. The S. intermedius antigen I/II appeared to be cell surface associated, with a molecular mass of approximately 160 kDa. Northern blotting indicated that the S. intermedius NCTC 11324 antigen I/II gene was transcribed as a monocistronic message. Maximum expression was seen during the early exponential phase. Insertional inactivation of the antigen I/II gene resulted in reduced hydrophobicity during early exponential phase, whereas no effect was detected during mid- and late exponential phases. Binding to human fibronectin and laminin was reduced in the isogenic mutant, whereas binding to human collagen types I and IV and to rat collagen type I was not significant for either the wild type or the mutant. Compared to the wild type, the capacity of the isogenic mutant to induce interleukin 8 (IL-8) release by THP-1 monocytic cells was significantly reduced. The results indicate that the S. intermedius antigen I/II is involved in adhesion to human receptors and in IL-8 induction.

Are sodium lauryl sulfate-containing toothpastes suitable vehicles for xylitol?

The hypothesis to be tested in this study was that toothpastes containing sodium lauryl sulfate (SLS) is unsuitable vehicles for xylitol. The bacteriostatic (and cariostatic) effect of xylitol is assumed to be caused by intracellular accumulation of xylitol-5-P in plaque bacteria. Experiments were designed to investigate whether presence of SLS would affect the uptake of xylitol by interacting with the bacterial membranes and thus inhibit xylitol-5-P formation. It was shown in an in vitro study that even very low concentrations of the strong anionic detergent SLS inhibited uptake of xylitol and xylitol-5-phosphate formation by dental plaque totally. The mild nonionic detergent ethoxylated stearyl alcohol (30x EO) had no such effect. In vivo experiments with toothpastes containing xylitol and either the strong or the mild detergent, showed that xylitol in toothpaste with SLS was not available for the plaque bacteria and gave no adaptation to xylitol, whereas in the presence of 30x EO it was available, and a xylitol adaptation was observed. Glucose metabolism, which was also studied for the plaque samples, was not significantly affected by presence of any of the 2 detergents, indicating that the amounts of xylitol in toothpastes were presumably too low to give clinical significant effects, even when mild detergents are used.

Xylitol fermentation by human dental plaque.

The aim of the present study was to examine xylitol metabolism by dental plaque collected immediately after the use of xylitol gum. Plaque was collected from 12 individuals immediately before and after xylitol exposure. The effect on xylitol metabolism by dental plaque of a 3 d discontinuation of the xylitol exposure was also examined. Xylitol metabolism by the plaque suspensions was initiated by adding [14C]xylitol and analyzed by HPLC. The results showed increased xylitol metabolism after 11 wk of chewing xylitol-containing gum. The ability to metabolize xylitol was rapidly reduced after the discontinuation of the xylitol exposure. It is suggested that an induction of enzymes in one or more of the species of plaque bacteria may have caused this effect. Glucose metabolism, which also was studied in the plaque samples, was decreased after xylitol exposure, but increased again 3 d after cessation of the xylitol exposure. It is suggested that the reduced glycolysis was caused by accumulation of intracellular xylitol-5-phosphate in some plaque bacteria during the xylitol exposure.

Effect of xylitol-containing chewing gum on sorbitol metabolism in dental plaque.

The aim of the present study was to examine whether a long-term use of chewing gum with xylitol as the only sweetener would affect sorbitol metabolism in dental plaque. Ten test subjects used xylitol-sweetened chewing gum for 12 weeks. Plaque was collected at three occasions; 1) Control plaque; 2) Test plaque I: plaque collected after 12 weeks of chewing xylitol-containing chewing gum; 3) Test plaque II: sucrose-stimulated plaque collected 2 d after Test plaque I was collected. Plaque suspensions were incubated with [14C]sorbitol, and uptake of sorbitol and production of sorbitol metabolites were determined by HPLC. Plaque formation and sorbitol uptake were significantly reduced.

Does the presence of xylitol in a sorbitol-containing chewing gum affect the adaptation to sorbitol by dental plaque?

It is known that xylitol inhibits sorbitol metabolism in some bacteria in vitro. The effect of xylitol/sorbitol-containing chewing gum on sorbitol adaptation of dental plaque was therefore examined. Ten subjects used this chewing gum for 12 wk, and plaque was collected before (control plaque) and after (test plaque) the exposure to sorbitol/xylitol. The metabolism of sorbitol by the plaque was examined with 14C-labeled sorbitol, and the radioactive metabolites were detected by high-performance liquid chromatography (HPLC). A considerable individual variation in acid formation was found. The mean values of total acids in the test plaque increased, as compared with the control plaque. An adaptation of dental plaque to sorbitol thus occurred in spite of the presence of xylitol in the chewing gum. The concentration of acetic acid predominated over other acids in both the control and test plaques. The proportions of acids expressed in percentage of total acids differed only slightly. Thus, long-term use of xylitol/sorbitol-containing chewing gum did not eliminate the adaptation of dental plaque to sorbitol.

Effects of xylitol/sorbitol combinations on bacterial growth and metabolism in Streptococcus sobrinus OMZ 176.

Combinations of the non-cariogenic sugar alcohols xylitol and sorbitol are widely used as sucrose substitutes in lozenges and chewing-gums. In S. sobrinus OMZ 176 xylitol inhibits the metabolism of sorbitol, and the xylitol-induced bacterial growth inhibition of S. sobrinus OMZ 176 has been shown to be enhanced by sorbitol. In combination with xylitol the sorbitol transport changes from a pts- to a non-pts transport, while the transport of xylitol is unaffected by the presence of sorbitol. It would be of interest to know how much xylitol has to be added to a sorbitol-containing medium to inhibit the metabolism of this sugar alcohol. In the present study no optimal inhibitory concentrations of xylitol/sorbitol combinations were found. Growth and acid production in S. sobrinus OMZ 176 are essentially the same for xylitol: sorbitol ratios in the range from 1:7 to 7:1. The presence of intracellular xylitol metabolites seems to be essential.

Adaptation of dental plaque to sorbitol after 3 months' exposure to chewing gum.

Five subjects used sorbitol-containing chewing gum for a period of 12 wk. Plaque was collected before and after the sorbitol exposure and also 12 wk after the termination of the exposure. The individual plaque samples were incubated with 14C-labeled sorbitol, and the medium was examined by HPLC. It was found that plaque samples from all subjects catabolized more sorbitol after the exposure. The flora adapted to sorbitol was persistent, and all the subjects had a higher capacity for metabolizing sorbitol even 12 wk after the end of the sorbitol exposure than before it. Formate, acetate, ethanol, and lactate were the major catabolites of sorbitol. Small amounts of succinate and propionate were found in some samples.

Xylitol 5-P formation by dental plaque after 12 weeks' exposure to a xylitol/sorbitol containing chewing gum.

Five subjects used xylitol-containing chewing gum for 12 wk. Dental plaque was collected before and after the exposure to xylitol. The plaque samples were examined for their capacity to form xylitol 5-P by incubation with 14C labeled xylitol, extraction and separation on HPLC. It was found that the capacity of the plaque to form xylitol 5-P was not reduced during the xylitol exposure in any of the subjects. No other xylitol-derived metabolites were observed. The inhibitory capacity of xylitol thus appears to be maintained after 12 wk exposure to xylitol.

Ultrastructural changes in Streptococcus sobrinus induced by xylitol, NaF and ZnCl2.

The effect of xylitol, NaF, or ZnCl2 on the ultrastructure of Streptococcus sobrinus OMZ 176 was studied. The bacteria were grown statically in air for 8 h in brain-heart infusion broth supplemented with 33 mM Xylitol, 2.5 mM NaF, 0.5 mM ZnCl2, separately or in combination. The bacteria were washed briefly and prepared for scanning and transmission electron microscopy. All three agents and their combinations induced distinct ultrastructural alterations. The alterations were related to various aspects of cell morphology and varied in nature and extent among the agents. The most pronounced ultrastructural alterations were seen in bacteria grown in the presence of zinc ions. At the concentrations used, fluoride appeared to induce the least effect.

Combined effect of xylitol, NaF and ZnCl2 on growth and metabolism of Streptococcus sobrinus OMZ 176.

Xylitol, NaF and ZnCl2 in combination inhibited the growth of S. Sobrinus OMZ 176 when added to Brain Heart Infusion broth. Thin-layer chromatography, followed by autoradiography of cell extracts, was used to study the inhibiting mechanism. Glucose uptake was reduced, the glycolysis inhibited at the glucose 6-phosphate- and fructose- 1.6-diphosphate level and the accumulation of xylitol metabolites was increased. These effects in combination probably accounted for the inhibition of growth.

Sorbitol increases the growth inhibition of xylitol on Strep. mutans OMZ 176.

It was observed in a previous study that the growth of Streptococcus mutans strain OMZ 176 on sorbitol was inhibited by xylitol. The aim of the present study was to investigate the mechanisms involved in this inhibition. It was shown that the uptake of 14C-sorbitol was delayed when the cells had been pre-exposed to xylitol, and that the only labelled substance found intracellularly was sorbitol; no further metabolization occurred. This is in contrast with untreated normal cells, where sorbitol is taken up by a specific phosphotransferase system (pts). The 14C-xylitol metabolism of the cells was qualitatively unchanged in the presence of sorbitol; an intracellular accumulation of 14C-xylitol-phosphate (xylitol-P) and 14C-xylulose-phosphate (xylulose-P) was observed. However, a reduced uptake of xylitol was observed in the presence of sorbitol. Xylitol thus appears to change the pathway by which sorbitol is taken up by the cells. An inducible permease may replace the normal sorbitol pts when xylitol is present. No further metabolization of this intracellular sorbitol seemed to occur in the resting cell suspensions. It was furthermore observed that the presence of sorbitol enhanced the inhibitory potential of xylitol. The accumulation of intracellular sorbitol coincided with markedly increased xylulose-P/xylitol-P ratio. It may be speculated that, if xylulose-P were the major inhibitor of the glycolysis instead of xylitol-P, as previously assumed, an increased concentration of xylulose-P induced by sorbitol could explain that sorbitol enhances the inhibition potential of xylitol. It is not evident, however, how intracellular sorbitol could affect the xylulose-P/xylitol-P ratio.

Xylitol metabolism in xylitol-sensitive and xylitol-resistant strains of streptococci.

The metabolism of xylitol in xylitol-sensitive strains (strains whose growth is inhibited by xylitol) and xylitol-resistant strains (growth not inhibited) of oral streptococci was compared. Both xylitol-sensitive and xylitol-resistant strains took up xylitol. In the sensitive cells, the xylitol was probably transported via a phosphotransferase system. This resulted in intracellular accumulation of xylitol-5-phosphate and xylulose-5-phosphate. These metabolites were not detected in the xylitol-resistant strains, which probably transported xylitol via a permease system. It appeared that the resistant strains were able to utilize xylitol as carbon and energy source in the absence of other carbohydrates.

Further studies on the growth inhibition of Streptococcus mutans OMZ 176 by xylitol.

Thin-layer chromatography (TLC) of extracts of cells which had been exposed to 14C-xylitol indicated that xylulose-phosphate is produced by the cells in addition to the previously reported xylitol-phosphate. Resting cell cultures of Streptococcus mutans OMZ 176 pretreated with xylitol were exposed to 14C-glucose and glycolytic metabolites identified by TLC of boiling water extracts of the cells. The developed TLC-sheets showed an accumulation of 14C-hexose-6-phosphates in the xylitol-treated bacteria. This could indicate that a xylitol metabolite, or metabolites, compete with fructose-6-phosphate for the phosphofructokinase, since the glycolysis is inhibited at this step. It was also shown that after accumulation of xylitol-5-phosphate, the bacteria were able to expel xylitol, presumably after and intracellular dephosphorylation of xylitol-5-phosphate; a "futile cycle" is thus present in these cells. Xylitol is taken up and phosphorylated, and at a later step dephosphorylated and expelled. The most important inhibition mechanism was judged to be the competitive inhibition of the glycolysis at the fructose-6-phosphate level.

Metabolism of xylitol in dental plaque.

It has been reported previously that xylitol added to glucose used to challenge dental plaque in vivo caused a reduced acid formation. The aim of the present study was to approach the mechanism by which xylitol may affect glucose catabolism in plaque bacteria. Suspensions of freshly collected 4-day-old plaque bacteria were incubated, one batch with labeled xylitol, one with labeled glucose, in vitro at 37 degrees C. Samples of cells were taken out at time intervals, collected on paper discs and subjected to scintillation counting. It was observed that the plaque bacteria took up xylitol, the uptake increasing with incubation of more than 3-4 h, whereas the same cells took up glucose immediately. Cells which had taken up xylitol were extracted with boiling water, extracts concentrated and applied on thin-layer chromatography sheets. A radioactive component with mobility like xylitol-5-phosphate was isolated from the cell extracts, and also a component where labeled xylitol was associated with macromolecules. It is suggested that the accumulation of the metabolities within the cells inhibits glycolysis.

Addition of xylitol to the growth medium of Streptococcus mutans OMZ 176--effect on the synthesis of extractable glycerol-phosphate polymers.

Addition of 5% xylitol to growing cultures of Streptococcus mutans OMZ 176 reduced the amount of extractable glycerol-phosphate polymers in these cells compared to S. mutans cultures grown in medium with 5% glucose added. The glycerol-phosphate polymers were extracted from the cells by hot phenol-water-extraction, and separated by column chromatography. Lipoteichoic acid (LTA) was identified by indirect haemagglutination tests and lipid analysis. The amount of LTA extracted from the cells or present in the medium was not significantly different. It is suggested that the accumulation of intracellular xylitol-phosphate observed in a previous study causes an effect similar to glucose starvation, which is known to affect the composition of the cell wall.