The purpose of this work was to explain how the caries-preventive agent xylitol interferes with the growth of Streptococcus mutans. It was found that the xylitol-sensitive strain of S. mutans 27352 (serotype g) and LG1 (serotype c) took up 14C-xylitol when the labelled pentitol was added to cells growing at the expense of glucose. Uptake of xylitol by growing cells of S. mutans 27352 XR and LG1 XR, two xylitol-insensitive spontaneous mutants, and of S. mutans GS5-2, which was also insensitive to xylitol, was practically inexistent under the same conditions. Alkaline phosphatase treatment followed by enzymatic analysis and thin-layer chromatography revealed that the accumulated product was xylitol phosphate. Intracellular concentrations of 5–7 mM for resting cells and of up to 60 mM for growing cells were calculated. Xylitol was phosphorylated at the expense of phosphoenolpyruvate by toluenized cells of S. mutans LG1, but not by toluenized cells of GS5–2 and S. mutans LG1 XR. The phosphorylation of xylitol was dependent on phosphoenolpyruvate and required the presence of both soluble and membrane cellular fractions in the reaction mixture. This indicated that xylitol was transported and phosphorylated by a phosphoenolpyruvate: sugar phosphotransferase system. The phosphoenolpyruvate-dependent phosphorylation by isolated membranes of S. mutans LG1 in the presence of the soluble fraction was inhibited by fructose but not by glucose, mannose or galactose. Measurement of phosphoenolpyruvate: phosphotransferase activities in isolated membrane revealed that strain 27352 and LG1 had activities for fructose and xylitol; membrane from 27352 XR and LG1 XR had very little activity for fructose and xylitol. It was concluded that xylitol was transported and phosphorylated by a constitutive phosphoenolpyruvate:fructose phosphotransferase system in S. mutans. The data suggested that xylitol toxicity in S. mutans is caused by the intracellular accumulation of xylitol phosphate.
Since the exposure of mutans streptococci to xylitol is known to select for xylitol-resistant (XR) natural mutants, the occurrence and long-term survival of such xylitol-resistant strains was evaluated in a cross-sectional sampling of participants of the Ylivieska xylitol study four years after the original two-year experimental period. Paraffin-stimulated whole saliva was first collected, and then plaque was collected and pooled. The salivary and dental plaque mutans streptococci were enumerated after growth on TSY20B agar. The proportion of XR strains was determined by autoradiography with 14C-xylitol. A strong and significant correlation (r = 0.645 and p = 0.005) between the number of mutans streptococci in saliva and in dental plaque was observed in non-consumers of xylitol. Such a correlation totally disappeared (r = 0.098 and p = 0.612) in xylitol-exposed consumers (habitual and former xylitol-consumers). The proportion of the salivary XR mutants (35%) in non-consumers (n = 16) was significantly lower than in the xylitol-exposed consumers (79%) (n = 27), (p = 0.0001) or in former consumers (75%) (n = 13), (p = 0.0008) or in the habitual consumers (83%) (n = 14), (p = 0.004). The proportion of XR mutants in dental plaque was, on the average, much lower than in the corresponding saliva. The proportion of XR in the plaque of xylitol non-consumers was half of that of the xylitol-exposed group, but the difference was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)
The effect of long-term consumption of refined xylitol on the natural populations of S. mutans in the human oral cavity has been investigated. Fifty-four S. mutans strains were isolated from adults and children who had been consuming commercial food products containing xylitol for a period of from 1 1/2 to 10 years. Twenty isolates were also obtained from control subjects who had never consumed xylitol-containing commercial food products. The inhibitory effect of xylitol on the isolated strains was determined by monitoring growth on glucose in the presence or absence of xylitol. This was used to define the sensitivity of each isolate to xylitol. Phosphoenolpyruvate:sugar phosphotransferase (PEP-PTS) activities were measured by means of the soluble and membrane fractions prepared from strains from both study populations. It was found that 87% of the fresh isolates from xylitol consumers were xylitol-resistant (XR), compared with only 10% of the strains isolated from the control subjects. The XR strains had low constitutive fructose PTS activity and very low xylitol-phosphorylating capacity. The xylitol-sensitive (XS) strains, however, had much higher levels of constitutive fructose PTS activity and phosphorylated xylitol 16 times more rapidly than did the XR strains. Evidence for the phosphorylation of xylitol by a fructose PEP-PTS in the XS strains was obtained. The growth inhibition by the intracellular accumulation of non-metabolizable toxic xylitol phosphate and its prevention by the presence of fructose are discussed.
We investigated the effect of xylitol on the growth of different oral bacteria in the presence of glucose. Xylitol inhibited the growth of all but one of ten strains of S. mutans and failed to inhibit the growth of the lactobacilli, actinomycetes, and other streptococci tested except S. sanguis 10556, which was slightly inhibited. However, the rate of acid production of the sensitive S. mutans strains was not equally affected by xylitol. These data, obtained with pure cultures of acidogenic oral bacteria, may explain the lack of an in vitro inhibitory effect of xylitol on dental plaque samples.
Several factors affecting the amount of fluoride ingested during toothbrushing by 2- to 7-year-old children were investigated. The specific purpose of this study was to determine the contribution of age, the amount of dentifrice used, and rinsing after brushing to the variation in the ingestion of fluoride dentifrice. Four hundred and five children brushed their teeth in front of a portable sink. The tubes of dentifrice in gel (0.24% NaF) were weighed before and after use to determine the amount of toothpaste used. The fluoride content of the collected liquids was determined with a fluoride-ion-specific electrode. The amount of fluoride ingested was derived by determining the difference between the amounts used and recovered. The amount of dentifrice used, the age, and the rinsing habits, entered in a multiple regression model, explained up to 66 percent of the total variation in the amount of fluoride ingested. The amount of dentifrice used accounted by itself for 60 percent of the total variation. Therefore, these results indicate that the quantity of dentifrice used was the most important factor affecting the ingestion of fluoride through toothbrushing by young children.
Xylitol reduces plaque but the reduction mechanism is largely unknown. The main aim of the present study was to determine whether the xylitol-induced reduction in the amount of plaque and the number of mutans streptococci could be demonstrated in subjects with (presumably) high levels of xylitol-resistant (XR; not inhibited by xylitol) mutans streptococci acquired following previous xylitol consumptions. 37 healthy dental students participated in the double-blind study. All subjects had been uncontrolled, habitual consumers of xylitol-containing products for at least 1 yr before the study. A 1-month washout period was followed by a 2-week test period during which either xylitol, xylitol-sorbitol or unsweetened chewing gum base was chewed 3-5 x a day. Plaque and saliva samples were collected at baseline and at the 2-week point for determination of the amount of plaque, microbiological variables, and hydrolytic enzymes. Mixtures of xylitol and sorbitol seemed to perform equally well with respect to reduction in the amount of plaque but not the number of mutans streptococci. Thus, polyols were the active ingredients of chewing gums able to modulate the amount of plaque and its microbial composition. Xylitol reduced plaque with a mechanism which appeared not to be associated with the study-induced changes in the proportion (%) of mutans streptococci in plaque, the number of salivary mutans streptococci, the proportion of XR strains in plaque or saliva, or the hydrolytic enzyme activities of plaque.
The parental strain Streptococcus sobrinus (Streptococcus mutans ATCC 27352), which is known to transport, phosphorylate and accumulate xylitol intracellularly as nonmetabolizable xylitol-phosphate (xylitol-sensitive (XS) strain) and its xylitol-resistant (XR) spontaneous mutant were used to further investigate the inhibitory action of xylitol on oral streptococci. Fructose-grown XR cells did not accumulate xylitol-phosphate, indicating that the inducible fructose PTS is incapable of transporting the pentitol. The intracellularly accumulated pentitol-phosphate by the XS cells did not prevent the subsequent uptake and degradation of glucose or fructose, despite a drop in the PEP pool and a 50% inhibition of the glucose but not the fructose catabolism. Intracellular dephosphorylation of the pentitol-phosphate and release of xylitol in the extracellular medium resulted in a rapid decrease of the intracellular level of this nonmetabolizable product. A Mg(++)- or Mn(++)-independent sugar-phosphate hydrolysing activity capable of splitting xylitol-phosphate was demonstrated in both XS and XR strains. Preincubation in the presence of N1-ethylmaleimide (NEM) and xylitol or NEM and fructose resulted in the subsequent inhibition of both xylitol uptake and efflux. The efflux kinetic at various temperatures is compatible with a facilitated diffusion by the phosphotransferase system EIIfru without, however, excluding the existence of an additional exit route, but it excludes a simple diffusion exit process. The results are consistent with the existence of a xylitol futile cycle contributing to the growth inhibition of S. sobrinus by the pentitol without excluding a toxic effect of xylitol-phosphate. Discrepancies in the literature on the action of xylitol on S. mutans could be explained in the light of the evidence presented.
The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is made of several proteins. Two of them are designated general proteins because they are required for the transport and phosphorylation of all sugars of the PTS. These two proteins are found in the soluble fraction of cellular extracts and are termed HPr and enzyme I (EI). We reported in this work the purification and the characterization of these two proteins from Streptococcus salivarius ATCC 25975. HPr was purified by DEAE-cellulose chromatography, molecular sieving on Ultrogel AcA44, and carboxymethylcellulose chromatography. Sodium dodecyl sulfate electrophoresis in the presence of urea revealed a single band with a molecular weight of 6700. The protein contained no tryptophan and had a pI of 4.8. The purification scheme of EI was as follows: DEAE-cellulose chromatography, hydroxylapatite chromatography, DEAE-Sephadex A-50 chromatography, preparative electrophoresis, and molecular sieving on Ultrogel AcA34. The five-step purification for EI produced a 199-fold purified preparation with a specific activity of 530 mumol of HPr phosphorylated per minute per milligram of protein at 37 degrees C. The fraction obtained after filtration on Ultrogel AcA34 gave one band (68 000) on sodium dodecyl sulfate - polyacrylamide gel electrophoresis. The molecular weight of the native enzyme determined by gel filtration at 4 degrees C was 135 000, suggesting that it was a dimer. Enzyme I had a pI of 4.2, a pH optimum of 6.7, a Km for HPr of about 27 microM, a Km for phosphoenolpyruvate of 0.48 mM, and kinetics that were consistent with a Ping-Pong mechanism. Evidence had been obtained which indicated that S. salivarius enzyme I was antigenically very similar to enzyme I from various strains of Streptococcus mutans, but not to the enzyme from Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis, and Escherichia coli.
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