Oral Microbiol Imtnutwl 1992: 7: 124-126
Effects of xylitol and fluoride on the response to glucose pulses of Streptococcus tnutatis T8 growing in continuous culture
Short communication A. H. Rogers, A. G. Bert Microbiology Laboratory, Department of Dentistry, University of Adeiaide, South Austraiia
Rogers AH. Bert AG. Effects of xylitol atidfluorideon the re.s'pon.se to glucose pulses of Streptococcus mutans T8 growing in contitutous culture. Oral Micfobiol Itntnunol 1992: 7: 124-126. Streptocuccus tnutans T8 was grown glucose-limited at pH 7.0 in a chemostat and pulsed, under pH free-fall conditions, with glucose, xylitol or a mixture of the two. Experiments were conducted in the absence or continual presence of low levels (1 mmol-1'') of fluoride. Culture filtrates of samples taken at frequent intervals were assayed for carbohydrate and fennentation end-products. Fluoride had little effect on the organism's response to glucose until the culture pH fell to ca. 5.0, at which point the rate of lactate production was reduced some 3-fold. Xylitol affected the response to glucose but its effect was most marked in the presence of fluoride. Under these conditions, the rate of lactate production was reduced at least 3-fold, the pH did not fall to 5.0 and only about 50% of the added glucose was consumed. This suggests that xylitol can augment the tnetabolic effects on S. tnutans of low levels of fluoride.
Fluoride has many adverse effects on oral bacteria (8). For example, at low levels it reduces the acid tolerance, and therefore cariogenic potential, of Streptococcus tnutans by disturbing the normal proton currents across the cell membrane (13). However, the contribution of such antimicrobial activities to the overall anti-caries effect of fluoride is unclear. Recent studies by Marsh et al., using a defined mixed-culture chemostat system, have clarified this issue. By pulsing the stable consortium of 9 oral species with glucose, and in the absence of pH control, they demonstrated that the low pH generated by sugar fermentation, rather than carbohydrate availability per se, selectively enriched potentially cariogenic species such as S. mutans and Lactobacillus casei (2). At low environtnental pH but not at a controlled pH of 7.0, the presence of sub-MIC (tninimal inhibitory concentration) levels of fluoride (1 mtnol 1"') decreased the rate of acid production and degree of fall in pH, and prevented enrichment of S. mutans. Furthermore, the pH-mediated inhibition of other members of the consortium was minimized (3). It was con-
cluded that such low, but biologically relevant, levels of fluoride could, therefore, contribute to caries prevention (3). Xylitol is an effective anti-caries agent (II). Small daily doses can produce a significant reduction in the salivary levels and plaque proportions of S. mutans (10). Xylitol uptake by these organisms leads to an energy-consuming futile cycle (15, 20, 21), which probably accounts for this observed ecological disadvantage that they subsequently suffer. Indeed, we have recently found that transient exposure to xylitol of a mixed continuous culture of S. nmtans and Streptococcus tndleri, growing glucose-limited, resulted in the selective depression of the former. In contrast, pulsing with other sugars, including sorbitol, gave 5. mutans an ecological advantage (18). Using ecologically relevant continuous culture conditions (2, 3), the purpose of this study was to examine the interactive effects of xylitol and tluoride on a strain of S. mutans. As a prelude to this investigation (see below), a number of preliminary experiments (data not shown) were done, in which a glucose-limited chemostat culture of 5. mu-
Key words: xyiitoi; fiuoride; Streptococcus mutans; continuous cuiture; giucose puise Dr. A. H. Rogers, Microbiology Laboratory, Department of Dentistry, University of Adeiaide, GPO Box 498, Adelaide 5001, South Austraiia Accepted for pubiication July 27, 1991
tatis was pulsed with glucose, xylitol or glucose plus xylitol in the absence or continuous presence of low levels of fiuoride (1 mmoll"') at a controlled pH of 7.0. An identical series of experiments was conducted at a controlled pH of 5.5. The relevant findings were that (a) the response of the glucose-limited culture to xylitol pulses was much the same under all conditions tested - that is, the culture O.D. fell slightly, as did acid production, and low levels of glucose appeared in culture filtrates about 4 h post-pulsing; (b) at pH 7.0, in the presence or absence of fluoride, xylitol had no effect on the response of the culture to glucose when the two sugars were added simultaneously; and (c) at the constant pH of 5.5, fluoride slowed the response of the culture to a glucose pulse. This effect was even more marked when xylitol was added concurrently with the glucose. Under these conditions, glycolysis appeared to be shut down for the first 4 h, following which the culture did respond, albeit poorly, to the added glucose. S. tnutans T8, a local isolate (16), was grown in a BioFlo, model C-30 chemostat (New Brunswick Scientific, NJ)
Xylitol plus fluoride affeets S. mutans under conditions described previously (14). Briefly, the organism was grown at an imposed dilution rate of D = 0.1 h ' (/j = 6.9 h) in a filter-sterilized, chemically defined medium containing 10 mmoll"' glucose to give glucoselimiting conditions (9). The temperature was controlled at 37 "C and both the culture vessel and medium reservoir were continually gassed with a N^/COn (95;5) tnixture. In one series of experiments the pH was controlled at 7.0 and, after steady-state conditions had been achieved, the culture was pulsed with glucose (final concentration, 20 mmol • I"') or a mixture of glucose plus xylitol, the latter at a final concentration of 100 mtnol-1"'. Imtnediately after a pulse, the pH was allowed to free-fall, as would occur in vivo in plaque (3), and the pH control set so that the pH would not fall below 5.0. From an identical set of experiments, in which Ouoride (as KF) was added to the medium reservoir at a final concentration of 1 mtnol-1"' (3), we were able to tnonitor the effect of its continual presence. In all experimetits, following each pulse, satnples were withdrawn frotn the culture vessel at frequent intervals and the following parameters determined; O.D.560 „„,, pH and the levels of carbohydrate, lactate, formate and acetate in culture filtrates assayed using high-pressure liquid chrotnatography techniques based on those of Guerrant et al. (6). For each pulse, the above procedure was repeated 18-24 h later when steady-state conditions had been re-established. Since, in each case, the results for the second pulse were essentially the same as for the first, only the data for the latter are shown (Fig. 1). Following a glucose pulse (Fig. la, control), the culture pH fell to 5.0 in 1-1.5 h, whereafter base was consumed to maintain the pH at that level. Complete consumption of the added glucose took 4-5 h and the O.D. doubled within 1-2 h. Lactate increased markedly; indeed, its rate of production over the first 2 h was ca. 600 nmol mg dry wt"' min ' and averaged ca. 300 nmol mg dry wt ' min~' over the 6-h period. The gradual fall in the levels of acetate and formate over the 6-h period is consistent with the cessation of production of these 2 metabolites and their subsequent washout. This indicates that, as expected (4), fermentation rapidly switches from hetero- to homo- in response to carbohydrate excess. This phenomenon also occurred under other test conditions
I
a) Glue
r8
r1.4
125
40-1
Effects of xylitol and fluoride on the response to glucose pulses of Streptococcus tnutatis T8 growing in continuous culture
Short communication A. H. Rogers, A. G. Bert Microbiology Laboratory, Department of Dentistry, University of Adeiaide, South Austraiia
Rogers AH. Bert AG. Effects of xylitol atidfluorideon the re.s'pon.se to glucose pulses of Streptococcus mutans T8 growing in contitutous culture. Oral Micfobiol Itntnunol 1992: 7: 124-126. Streptocuccus tnutans T8 was grown glucose-limited at pH 7.0 in a chemostat and pulsed, under pH free-fall conditions, with glucose, xylitol or a mixture of the two. Experiments were conducted in the absence or continual presence of low levels (1 mmol-1'') of fluoride. Culture filtrates of samples taken at frequent intervals were assayed for carbohydrate and fennentation end-products. Fluoride had little effect on the organism's response to glucose until the culture pH fell to ca. 5.0, at which point the rate of lactate production was reduced some 3-fold. Xylitol affected the response to glucose but its effect was most marked in the presence of fluoride. Under these conditions, the rate of lactate production was reduced at least 3-fold, the pH did not fall to 5.0 and only about 50% of the added glucose was consumed. This suggests that xylitol can augment the tnetabolic effects on S. tnutans of low levels of fluoride.
Fluoride has many adverse effects on oral bacteria (8). For example, at low levels it reduces the acid tolerance, and therefore cariogenic potential, of Streptococcus tnutans by disturbing the normal proton currents across the cell membrane (13). However, the contribution of such antimicrobial activities to the overall anti-caries effect of fluoride is unclear. Recent studies by Marsh et al., using a defined mixed-culture chemostat system, have clarified this issue. By pulsing the stable consortium of 9 oral species with glucose, and in the absence of pH control, they demonstrated that the low pH generated by sugar fermentation, rather than carbohydrate availability per se, selectively enriched potentially cariogenic species such as S. mutans and Lactobacillus casei (2). At low environtnental pH but not at a controlled pH of 7.0, the presence of sub-MIC (tninimal inhibitory concentration) levels of fluoride (1 mtnol 1"') decreased the rate of acid production and degree of fall in pH, and prevented enrichment of S. mutans. Furthermore, the pH-mediated inhibition of other members of the consortium was minimized (3). It was con-
cluded that such low, but biologically relevant, levels of fluoride could, therefore, contribute to caries prevention (3). Xylitol is an effective anti-caries agent (II). Small daily doses can produce a significant reduction in the salivary levels and plaque proportions of S. mutans (10). Xylitol uptake by these organisms leads to an energy-consuming futile cycle (15, 20, 21), which probably accounts for this observed ecological disadvantage that they subsequently suffer. Indeed, we have recently found that transient exposure to xylitol of a mixed continuous culture of S. nmtans and Streptococcus tndleri, growing glucose-limited, resulted in the selective depression of the former. In contrast, pulsing with other sugars, including sorbitol, gave 5. mutans an ecological advantage (18). Using ecologically relevant continuous culture conditions (2, 3), the purpose of this study was to examine the interactive effects of xylitol and tluoride on a strain of S. mutans. As a prelude to this investigation (see below), a number of preliminary experiments (data not shown) were done, in which a glucose-limited chemostat culture of 5. mu-
Key words: xyiitoi; fiuoride; Streptococcus mutans; continuous cuiture; giucose puise Dr. A. H. Rogers, Microbiology Laboratory, Department of Dentistry, University of Adeiaide, GPO Box 498, Adelaide 5001, South Austraiia Accepted for pubiication July 27, 1991
tatis was pulsed with glucose, xylitol or glucose plus xylitol in the absence or continuous presence of low levels of fiuoride (1 mmoll"') at a controlled pH of 7.0. An identical series of experiments was conducted at a controlled pH of 5.5. The relevant findings were that (a) the response of the glucose-limited culture to xylitol pulses was much the same under all conditions tested - that is, the culture O.D. fell slightly, as did acid production, and low levels of glucose appeared in culture filtrates about 4 h post-pulsing; (b) at pH 7.0, in the presence or absence of fluoride, xylitol had no effect on the response of the culture to glucose when the two sugars were added simultaneously; and (c) at the constant pH of 5.5, fluoride slowed the response of the culture to a glucose pulse. This effect was even more marked when xylitol was added concurrently with the glucose. Under these conditions, glycolysis appeared to be shut down for the first 4 h, following which the culture did respond, albeit poorly, to the added glucose. S. tnutans T8, a local isolate (16), was grown in a BioFlo, model C-30 chemostat (New Brunswick Scientific, NJ)
Xylitol plus fluoride affeets S. mutans under conditions described previously (14). Briefly, the organism was grown at an imposed dilution rate of D = 0.1 h ' (/j = 6.9 h) in a filter-sterilized, chemically defined medium containing 10 mmoll"' glucose to give glucoselimiting conditions (9). The temperature was controlled at 37 "C and both the culture vessel and medium reservoir were continually gassed with a N^/COn (95;5) tnixture. In one series of experiments the pH was controlled at 7.0 and, after steady-state conditions had been achieved, the culture was pulsed with glucose (final concentration, 20 mmol • I"') or a mixture of glucose plus xylitol, the latter at a final concentration of 100 mtnol-1"'. Imtnediately after a pulse, the pH was allowed to free-fall, as would occur in vivo in plaque (3), and the pH control set so that the pH would not fall below 5.0. From an identical set of experiments, in which Ouoride (as KF) was added to the medium reservoir at a final concentration of 1 mtnol-1"' (3), we were able to tnonitor the effect of its continual presence. In all experimetits, following each pulse, satnples were withdrawn frotn the culture vessel at frequent intervals and the following parameters determined; O.D.560 „„,, pH and the levels of carbohydrate, lactate, formate and acetate in culture filtrates assayed using high-pressure liquid chrotnatography techniques based on those of Guerrant et al. (6). For each pulse, the above procedure was repeated 18-24 h later when steady-state conditions had been re-established. Since, in each case, the results for the second pulse were essentially the same as for the first, only the data for the latter are shown (Fig. 1). Following a glucose pulse (Fig. la, control), the culture pH fell to 5.0 in 1-1.5 h, whereafter base was consumed to maintain the pH at that level. Complete consumption of the added glucose took 4-5 h and the O.D. doubled within 1-2 h. Lactate increased markedly; indeed, its rate of production over the first 2 h was ca. 600 nmol mg dry wt"' min ' and averaged ca. 300 nmol mg dry wt ' min~' over the 6-h period. The gradual fall in the levels of acetate and formate over the 6-h period is consistent with the cessation of production of these 2 metabolites and their subsequent washout. This indicates that, as expected (4), fermentation rapidly switches from hetero- to homo- in response to carbohydrate excess. This phenomenon also occurred under other test conditions
I
a) Glue
r8
r1.4
125
40-1
