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In-vitro acid production by the oral bacterium Streptococcus mutans 10449 in various concentrations of glucose, fructose and sucrose
Archs oral Bid. Vol. 30, No. 4, pp. 319-324, Printed in Great Britain. All rights reserved
1985 Copyright
0
0003-9969/85 $3.00 + 0.00 1985 Pergamon Press Ltd
IN-VITRO ACID PRODUCTION BY THE ORAL BACTERIUM STREPTOCOCCUS MUTANS 10449 IN VARIOUS CONCENTRATIONS OF GLUCOSE, FRUCTOSE AND SUCROSE R. Department
of Periodontology
DUCXJID
and Community Dentistry, University Scotland, U.K.
of Dundee,
Dundee
DDI
4HR,
Summary-At intermediate and high concentrations, the results with the sugars were similar, with lactic acid as the main end product. Over 4 h, the pH fell from approx. 7 to 4. At low monosaccharide concentrations (2 mM glucose, 2 and 5 mM fructose), after an initial pH drop and period of lactic-acid production, evidence of pH rise and lactic-acid consumption were noted. This did not happen when sucrose was added to the bacteria. There was evidence of a heterolactic-acid fermentation pattern at low-sugar concentrations, lactic, acetic and formic acids being produced in similar amounts. The results suggest that, when low-sugar concentrations are present in dental plaque, Strep. mutans is capable of co%ming previously-formed lactic acid.
MATERIALS
INTRODUCTION
The addition of glu’cose to dental plaque in uiuo results in an immed:late fall in pH followed by a slower recovery to the resting pH (Stephan, 1944). Stephan’s early work has been followed by numerous in -viva and in -vitro studies on the effects of sugars on dental plaque and dental plaque bacteria (Geddes, 1975; Kleinberg et d., 1982; Distler and Kriinke, 1983). It is now aclcepted that the main anionic product produced from sugars by dental plaque is lactate. The range of sugar concentrations used in various studies has been enormous, ranging from Stephan’s original 10 per cent glucose rinse (560mM), 580 mM-sucrose used by Geddes and McNee (1982) down to concentrations of 2.8 mM glucose used by Kleinberg et al. (1982). In the in-vitro studies especially, where there is no possibility of lactate and other products being lost through diffusion, this wide range of sugar concentrations could result in a wider range of lactate concentrations. Each mole of glucose consumed has the potential for producing 2 mol of la.ctate and each mole of sucrose the potential for producing 4mol of lactate. The ability of dental plaque to produce a subsequent rise in plaque pH may also be important in determining its cariogenicity (Kleinberg et al., 1982). Emphasis has been placed on the possible role of Veillonella in consuming lactic acid produced by Streptococcus mutllns (Mikx and van der Hoeven, 1975; Distler and Kronke, 1980) but there is also evidence that streptococci are able to consume lactic acid produced by their own glycolysis once the sugar substrate has been used up (Hu and Sandham, 1972). Cultures of oral bacteria are also able to reverse the initial pH fall seen afi:er sugar addition (Stephan and Hemmens, 1947); natural populations of dental plaque (Muntz, 1943) and salivary sediment (Sandham and Kleinberg, 1970) can do the same. This effect is especially marked in the presence of arginine, urea or the salivary .peptide sialin (Kleinberg et al., 1982). 319
AND METHODS
Bacteria Strep. mutans (NCTC 10449) was obtained from the National Collection of Type Cultures (Colindale, London, U.K.). Broth cultures were grown overnight in 400ml Todd-Hewitt broth (Difco, Detroit, U.S.A.). Bacterial suspension (350 ml) was harvested by centrifugation (MSE High Speed 18, M.S.E. Ltd, Crawley, U.K. used at 7000g for 20 min at 4°C) and the bacteria1 pellet resuspended in 10 ml phosphatebuffered saline (NaCl 8 g/l; Na,HPO, 1.15 g/l, KC1 0.2 g/l, KH,PO, 0.2 g/l) using a Tri-R, Stir-R homogenizer (Tri-R, Stir-R Instruments Inc., New York, U.S.A.). The resuspended pellet was recentrifuged, weighed (approx. 1.0 g wet wt) and resuspended in heart-infusion broth (Difco). Incubation
The bacterial suspension was pre-incubated for 30 min prior to the addition of the various sugar solutions dissolved in water so as to remove any traces of fermentable carbohydrate from the broth. To 30ml of the bacteria1 suspension 30 ml of the appropriate sugar solution pre-warmed to 37°C was then added. The diluted suspension was well mixed, left for 1 min to stabilize and the pH readings taken at various times over the next 4 h. The initial Strep. mutans concentration was 1.7 per cent for all experiments. Samples (1.2 ml) of the suspension were taken at timed intervals and immediately filtered through a 0.2 p membrane filter (Amicon, Lexington, U.S.A.) to remove bacteria and the filtrate was placed on ice before analysis of organic acids by isotachophoresis. Isotachophoresis
Lactic, acetic and formic acids were analysed using an LKB 2127 Tachophor (LKB Instruments, Bromma, Sweden) with a 5 mM HCI containing 0.2 per cent hydroxymethylcellulose and buffered to pH 3.9 with p-alanine as the leading electrolyte. The terminating electrolyte was 5 mM hexanoic
320
R.
DLJGUID
acid adjusted to pH 5.5 by the addition of solid tris(hydroxymethyl)methylamine. Separation was performed at a constant current of 40pA with the Tachophor set at 12°C. Background levels of organic acids
Following the pre-incubation period, detectable levels of lactic and acetic acids were found in the heart-infusion broth. The range for lactic acid at the start of the incubation was 2.2-3.7 mM and for acetic acid 0.1-0.3 mM. Formic acid was not detected in the broth prior to incubation. The concentrations given in Figs l-3 are those concentrations actually measured in the broth during the incubation. As in some experiments (i.e. addition of 2 and 5 mM fructose) the final lactate concentration was lower than at the start of the 4 h incubation, there must have been consumption of some of the lactate present at the start of the incubation period. Such consumption of lactate was also seen in control experiments where no glucose was added to the medium. Where values are given for the percentage ratio of organic acids produced by the bacteria, these are the ratios of the initial concentration subtracted from the final concentration for the appropriate acid. pH measurements
These were made using a combination gel-filled pH electrode (Orion Research, Cambridge, U.S.A.) and an Orion microprocessor ionalyser/901 calibrated for use at 37°C (slope: +61.5 mV/pH unit). RESULTS
Control experiments
A 4-h incubation of the Strep. mutans suspension in the absence of any added sugar caused a drop in pH of approx. 0.1 from 7.0 to 6.9. No lactic acid or other acid production by the bacteria could be detected during or by the end of the incubation. There was evidence of some lactate consumption but the reduction in the lactate concentration was not statistically significant. Effect of increasing glucose concentration
The effect on the pH and acid formation of adding 2, 5, 10, 50 and 500 mM glucose to the Strep. mutans suspension is shown in Fig. 1. The results are expressed as means f standard deviations of three experiments. As the glucose concentration increased, both the initial pH fall and the production of lactate increased. For example, in the first 40 min of incubation with 2.0 mM glucose, the pH fell from 7.1 to 6.0 and the lactate concentration rose from 3.7 to 5.2mM. This rise was followed by a decline in lactic-acid concentration to 4.6 mM which occurred as the pH rose from 6.0 to 6.3. This reduction in lactate concentration was not statistically significant. During the incubation, the acetate concentration rose from 0.2 to 1.8 mM and the formic-acid concentration from 0.0 to 1.5 mM so that at the end of 4 h the percentage ratio of acid products resulting from the fermentation of the glucose was lactic: acetic: formic, 26.8 per cent: 37.8 per cent: 35.4 per cent. Following the addition of 500 mM glucose, the pH fell continuously from a mean of pH 6.8 to 4.1 and
the lactic-acid concentration determined rose continuously from 3.7 to 22.9 mM, the acetic-acid concentration from 0.3 to 0.8 mM and formic acid from 0.0 to 0.9 mM. At the end of the incubation, the percentage ratio of products from the glucose fermentation was lactic: acetic: formic, 93.5 per cent: 2.2 per cent: 4.3 per cent. Effect of increasing fructose concentration
The effect on the pH and acid formation of adding 2, 5, 10, 50 and 500 mM fructose to the Strep. mutans cultures is shown in Fig. 2. At both 2 and 5 mM fructose, following the initial pH drop, there was a small rise in pH from 5.6 to 5.8 and from 5.1 to 5.3, respectively. This rise was associated in both instances by production of lactic acid followed by loss of lactic acid in the medium to levels below the initial one at the start of the incubation; thus, whilst there was a net production of acetate (1.3 mM in both cases) and formic acid (1.2 and 1.5 mM, respectively), there was an overall reduction in lactic-acid concentration from 2.6 to 2.0 mM in both instances over the 4 h. The reductions in lactic-acid concentration from its peak values was statistically significant (p < 0.05). At medium and higher concentrations of fructose (l&500 mM), the results were similar to those with glucose with a percentage ratio of the bacterial acid-products after 4 h of lactic: acetic: formic: 88.6 per cent: 3.3 per cent: 8.4 per cent at 500mM fructose. Effect of increasing sucrose concentrations
The effect on the pH and acid formation of adding 1, 2.5, 5, 25 and 250 mM sucrose to the Strep. mutans suspension is shown in Fig. 3. As sucrose is a disaccharide, concentrations half those of glucose and fructose were used. At 1 mM sucrose, the pH fell from 7.0 to 6.4 before rising to pH 6.6 by the end of the 4-h incubation. At the same time, the lactic-acid concentration in the broth rose from 3.1 to 4.0 mM and fell to 3.8 mM. After 4 h, the percentage ratio of the acids produced by the bacteria was lactic: acetic: formic, 30.3 per cent: 35.9 per cent: 35.0 per cent. Conversely, at the highest sucrose concentration (250mM), the pH fell from 6.8 to 4.2 and the lactic acid concentration rose from 2.2 to 22.8 mM over the 4-h incubation. The final ratio of acid produced by the bacteria was lactic: acetic: formic, 94.5 per cent: 3.0 per cent: 2.5 per cent. DISCUSSION
Several organic acids are now known to occur in dental plaque. Geddes (198 1) found acetic, propionic, lactic and succinic acids in all human dental plaque samples, with formate in 7.5 per cent and pyruvate in less than 50 per cent. Resting plaque or plaque 60 min after exposure to sucrose contained more non-volatile acids than lactic acid but, immediately after sugar exposure, lactic acid predominated (Geddes, 1975). Similar results were obtained by Vratsanos and Mande1 (1982) and Distler and Kriinke (1983) although the latter noted wide, unaccountable variations in the acid content of individual plaque samples. Using an improved HPLC method for the analysis of organic acids, Distler and Kriinke (1984) showed that formic
pH response
of Strep. mutansto sugar
concentration
321 25
@HI
2mM Glucose
(mM)
(pli
5mM Glucose
(mfvl) 20
e
6
1
1
(h)
2 25
(pH)
i
IOmM
Glucose
ImM)
fhl
2
7(PHI
25
50mM
Glucose
(mM) 20
20
6
0
1 (h)
2
4
0
1 (h)
- 25
(pH)
500mM
Glucose
.I_i . (mM)
- 20
E Formote
Acetate
-
15
Lactate
-
10
-5
1
Fig. 1. Acid production values for pH (0)
(h)
2
4
by Strep.mutans10449 in the presence of 2, 5, 10, 50 and 500 mM glucose. Mean and means f standard deviations for formate, acetate and lactate are given.
acid is quantitatively the most important organic acid in resting plaque. The extent to whi’ch Strep. mutans contributes to acid production in dental plaque is not clear. van der Hoeven and Frank.en (1982) compared sucroseinduced acid produ’ction in rat plaques with and without Strep. mutans T2. The amount of lactic acid, 5 min after exposure to 10 per cent sucrose (292 mM), was significantly higher in the Strep. mutans-infected
plaque than in the Strep. mutans-free plaque, but significant differences at other times, or with formic or acetic acids were not noted. Analyses of acid production from Strep. mutans Ny341 mono-infected plaque taken from gnotobiotic rats showed equivalent amounts of formic, acetic and lactic acids in resting plaques where the free-sugar concentration was presumably low (van der Hoeven and Franken, 1984).
322
R. 9
@HI
DUGUID
25
2mM
Fructose
mM)
8
25
9
5mM
IpHI
20
E
15
7
10
6
Fructose
1
4
2
mM) 20
2
(h)
25
IOmM
4
(hl
1;
Fructose
:mM) 20
9
-
(pH)
50mM
Fructose
25
(mM)
6
-
20
I
1
2
4
(h)
(h) 25
(PHI
5OOmM
Fructose
cmM) 20
Iu
Formote Acetote Loctote
6
: . rnr Fig. 2. Acid production
A-
2
by Strep.
murans
in the presence of 2, details as in Fig. 1.
10449
In my experiments, formic, acetic and lactic acids were all produced by Strep. mutuns 10449. There was no evidence of propionic-acid production on the isotachophoresis traces; if propionic acid is produced by this strain it can only be in minute amounts. This is in accord with Distler and Kriinke (1980) for Strep. mutans 634 and T3/53. I did not analyse the incubation medium for ethanol which was reported by
5, 10, 50
and 500 mM fructose. Other
Carlsson and Griffith (1974) to be a product of Strep. mutans JC2.
The finding that Strep. mutans appears, under certain conditions, to be able to consume the lactate it has produced following sugar addition was also noted by Hu and Sandham (1972). However, Paulus (1983) found no evidence of lactate consumption in any of eight strains of Strep. mutans investigated.
pH response of Strep. mutans to sugar concentration -225
9
(PIi)
1 mM Sucrose
(mM)
8
323
9
(PHI
- 20
6
- 10
6
2.5mM
1CmM)
Sucrose
7
6
1
0
2
i
(h)
(h)
9
(PHI
5mM
Sucrcse
(mM) 20
8
6
i
i (h)
(h)
9
(pIi)
-
250mM
Swose
25
(mM)
8
- 20 Formote
7
6
0
1
(h)
Acetote
I
1 1 Loctote U
Fig. 3. Acid production by Strep. mutans 10449in the presence of 1, 2.5, 5,25 and 250 mM sucrose. Other details as in Fig. 1.
Lactic-acid consumption by plaque was noted by Muntz (1943) and in the mixed bacteria of salivary sediment by Sandham and Kleinberg (1970). In my experiments, lactic-acid consumption was a consistent finding at low concentrations of glucose and, especially, fructose, though not with sucrose. At some of these low monosaccharide concentrations, all the lactic acid originally produced was lost by the end of
the 4-h incubation. Although the rise in pH was not large (0.2-0.3 pH unit), it does represent a considerable reduction in the amount of acid present (N 50 per cent reduction in [H+]). Rise in pH due to base formation has been studied in a number of species of oral bacteria, following the addition of arginine or arginine-peptide by Kleinberg et nl. (1982) though they listed Strep. mutans 10449 as what they called a
324
R. DUGUID
non-pH rise organism with arginine or argininepeptide. I did not investigate the effect of arginine or arginine-peptide on acid production by Strep. mutans 10449 but the glucose concentration (2.8 mM) used by Kleinberg et al. (1982) is close to the glucose/fructose concentrations where evidence of lactate consumption was seen (2 mM). The pathway by which lactic acid is lost from the incubation medium is not clear and requires further investigation. Neither is it clear why this effect is more evident with fructose than with glucose, though it may be relevant that fructose-1,6-diphosphate is a positive effector of lactate dehydrogenase (Wittenburger et al., 1971). Carlsson and Griffith (1974) showed that with Streptococcus sanguis and Strep. mutans and to a lesser extent with Streptococcus salivarius and Streptococcus bovis, when glucose is limiting, the fermentation pattern is heterolactic rather than homolactic. Similar findings were reported by Thomas, Ellwood and Longyear (1979) for Streptococcus lactis. Both groups of workers used chemostat cultures. I found similar results when Strep. mutans 10449 was grown in batch culture. I conclude that considerable care has to be taken when attempting to relate the carbohydrate metabolism of Strep. mutans and, in all likelihood, other streptococci to Stephan-type pH changes. At high sugar concentrations, Strep. mutans is predominantly a homolactic fermenter. At lower sugar concentrations, pathways of both heterolactic fermentation and the further metabolism of lactate operate. Thus, it seems likely that, when low fermentable-sugar concentrations prevail in dental plaque, Strep. mutans is capable of metabolizing its own previously-formed lactic acid and that diffusion out of plaque and the metabolic activity of other oral bacteria (such as Veillonella) need not necessarily be involved. This potential ability of Strep. mutans to metabolize lactic acid further at low sugar concentrations is another variable which may affect the cariogenicity of different strains of this organism and the dental plaques they inhabit. Acknowledgement--I am very grateful to Mrs Fran. ston for her skillful technical assistance.
John-
REFERENCES Carisson J. and Griffiths D. J. (1974) Fermentation products and bacterial yields in glucose-limited and nitrogenlimited cultures of streptococci. Archs oral Biol. 19, 1103-l 109.
Distler W. and Kriinke A. (1980) Acid formation by mixed cultures of cariogenic strains of Streptococcus mutans and Veillonella alcalescens. Archs oral Biol. 25, 655-658. Distler W. and Kriinke A. (1983) The acid pattern in human dental plaque. J. dent. Res. 62, 87-91. Distler W. and Kriinke A. (1984) Acids in single-site human resting plaque. Caries Res. 18, 160-I 61. Geddes D. A. M. (1975) Acids produced by human dental plaque metabolism in siru. Caries Res. 9, 98-109. Geddes D. A. M. (1981) Studies on metabolism of dental plaque: diffusion and acid production of human dental plaque. In: Frontiers of Oral Physiology: The Environment of the Teeth. Karger, Base]. Geddes D. A. M. and McNee S. G. (1982) The effects of 0.2 per cent (48 mM) NaF rinses daily on human plaque acidogenicity in situ (Stephan curve) and fluoride content. Archs oral Biol. 27, 765-769. Hoeven J. S. van der and Franken H. C. M. (1982) Production of acids in rat dental plaque with or without Streptococcus mutans. Caries Res. 16, 375-382. Hoeven J. S. van der and Franken H. C. M. (1984) Effect of fluoride on growth and acid production by Srreptococcus mutans in dental plaque. Infect. Immun. 45, 356359. Hu G. and Sandham H. J. (1972) Streptococcal utilization of lactic acid and its effect on pH. Archs oral Biol. 17, 729-743. Kleinberg I., Jenkins G. N., Chatterjee R. and Wijeyeweera L. (1982) The antimonv UH electrode and its role in the assessment and interpretation of dental plaque pH. J. dent. Res. 61, 1139-1147. Mikx F. H. M. and Hoeven J. S. van der (1975) ,_Symbiosis of Streptococcus mutans and Veillonella alcalescens in mixed continuous cultures. Archs oral Biol. 20, 407-410. Muntz J. A. (1943) Production of acids from glucose by dental plaque material. J. biol. Chem. 148, 225-236. Paulus M. (1983) Zur FIhigkeit kariogener Streptokokken, Glucose iiber Lactat aerob vollstiindig abzubauen. Med. Diss., Erlangen, West Germany. Sandham H. J. and Kleinberg I. (1970) Contribution of lactic and other acids to the pH of a human salivary sediment system during glucose catabolism. Archs oral Biol. 15, 1263-1283. Stephan R. M. (1944) Intra-oral hydrogen-ion concentrations associated with dental caries activity. J. dent. Res. 23, 257-266. Stephan R. M. and Hemmens E. S. (1947) Studies of changes in pH produced by pure cultures or oral microorganisms. J. denr. Res. 26, 15-41. Thomas T. D., Ellwood D. C. and Longyear V. M. C. (1979) Change from homo- to hetero-lactic fermentation by Streptococcus lacris resulting from glucose limitation in anaerobic chemostat cultures. J. Bact. 138, 109-I 17. Wittenberger C. L., Palumbo M. P., Bridges R. B. and Brown A. T. (1971) Mechanisms for regulating the activity constituent destructive pathways in Streptococcusfbecalis J. dent. Res. 50, 109~1113. Vratsanos S. M. and Mandel I. D. (1982) Comparative plaque acidogenesis of caries-resistant vs. cariessusceptible adults. J. denf. Res. 61, 465-468.
1985 Copyright
0
0003-9969/85 $3.00 + 0.00 1985 Pergamon Press Ltd
IN-VITRO ACID PRODUCTION BY THE ORAL BACTERIUM STREPTOCOCCUS MUTANS 10449 IN VARIOUS CONCENTRATIONS OF GLUCOSE, FRUCTOSE AND SUCROSE R. Department
of Periodontology
DUCXJID
and Community Dentistry, University Scotland, U.K.
of Dundee,
Dundee
DDI
4HR,
Summary-At intermediate and high concentrations, the results with the sugars were similar, with lactic acid as the main end product. Over 4 h, the pH fell from approx. 7 to 4. At low monosaccharide concentrations (2 mM glucose, 2 and 5 mM fructose), after an initial pH drop and period of lactic-acid production, evidence of pH rise and lactic-acid consumption were noted. This did not happen when sucrose was added to the bacteria. There was evidence of a heterolactic-acid fermentation pattern at low-sugar concentrations, lactic, acetic and formic acids being produced in similar amounts. The results suggest that, when low-sugar concentrations are present in dental plaque, Strep. mutans is capable of co%ming previously-formed lactic acid.
MATERIALS
INTRODUCTION
The addition of glu’cose to dental plaque in uiuo results in an immed:late fall in pH followed by a slower recovery to the resting pH (Stephan, 1944). Stephan’s early work has been followed by numerous in -viva and in -vitro studies on the effects of sugars on dental plaque and dental plaque bacteria (Geddes, 1975; Kleinberg et d., 1982; Distler and Kriinke, 1983). It is now aclcepted that the main anionic product produced from sugars by dental plaque is lactate. The range of sugar concentrations used in various studies has been enormous, ranging from Stephan’s original 10 per cent glucose rinse (560mM), 580 mM-sucrose used by Geddes and McNee (1982) down to concentrations of 2.8 mM glucose used by Kleinberg et al. (1982). In the in-vitro studies especially, where there is no possibility of lactate and other products being lost through diffusion, this wide range of sugar concentrations could result in a wider range of lactate concentrations. Each mole of glucose consumed has the potential for producing 2 mol of la.ctate and each mole of sucrose the potential for producing 4mol of lactate. The ability of dental plaque to produce a subsequent rise in plaque pH may also be important in determining its cariogenicity (Kleinberg et al., 1982). Emphasis has been placed on the possible role of Veillonella in consuming lactic acid produced by Streptococcus mutllns (Mikx and van der Hoeven, 1975; Distler and Kronke, 1980) but there is also evidence that streptococci are able to consume lactic acid produced by their own glycolysis once the sugar substrate has been used up (Hu and Sandham, 1972). Cultures of oral bacteria are also able to reverse the initial pH fall seen afi:er sugar addition (Stephan and Hemmens, 1947); natural populations of dental plaque (Muntz, 1943) and salivary sediment (Sandham and Kleinberg, 1970) can do the same. This effect is especially marked in the presence of arginine, urea or the salivary .peptide sialin (Kleinberg et al., 1982). 319
AND METHODS
Bacteria Strep. mutans (NCTC 10449) was obtained from the National Collection of Type Cultures (Colindale, London, U.K.). Broth cultures were grown overnight in 400ml Todd-Hewitt broth (Difco, Detroit, U.S.A.). Bacterial suspension (350 ml) was harvested by centrifugation (MSE High Speed 18, M.S.E. Ltd, Crawley, U.K. used at 7000g for 20 min at 4°C) and the bacteria1 pellet resuspended in 10 ml phosphatebuffered saline (NaCl 8 g/l; Na,HPO, 1.15 g/l, KC1 0.2 g/l, KH,PO, 0.2 g/l) using a Tri-R, Stir-R homogenizer (Tri-R, Stir-R Instruments Inc., New York, U.S.A.). The resuspended pellet was recentrifuged, weighed (approx. 1.0 g wet wt) and resuspended in heart-infusion broth (Difco). Incubation
The bacterial suspension was pre-incubated for 30 min prior to the addition of the various sugar solutions dissolved in water so as to remove any traces of fermentable carbohydrate from the broth. To 30ml of the bacteria1 suspension 30 ml of the appropriate sugar solution pre-warmed to 37°C was then added. The diluted suspension was well mixed, left for 1 min to stabilize and the pH readings taken at various times over the next 4 h. The initial Strep. mutans concentration was 1.7 per cent for all experiments. Samples (1.2 ml) of the suspension were taken at timed intervals and immediately filtered through a 0.2 p membrane filter (Amicon, Lexington, U.S.A.) to remove bacteria and the filtrate was placed on ice before analysis of organic acids by isotachophoresis. Isotachophoresis
Lactic, acetic and formic acids were analysed using an LKB 2127 Tachophor (LKB Instruments, Bromma, Sweden) with a 5 mM HCI containing 0.2 per cent hydroxymethylcellulose and buffered to pH 3.9 with p-alanine as the leading electrolyte. The terminating electrolyte was 5 mM hexanoic
320
R.
DLJGUID
acid adjusted to pH 5.5 by the addition of solid tris(hydroxymethyl)methylamine. Separation was performed at a constant current of 40pA with the Tachophor set at 12°C. Background levels of organic acids
Following the pre-incubation period, detectable levels of lactic and acetic acids were found in the heart-infusion broth. The range for lactic acid at the start of the incubation was 2.2-3.7 mM and for acetic acid 0.1-0.3 mM. Formic acid was not detected in the broth prior to incubation. The concentrations given in Figs l-3 are those concentrations actually measured in the broth during the incubation. As in some experiments (i.e. addition of 2 and 5 mM fructose) the final lactate concentration was lower than at the start of the 4 h incubation, there must have been consumption of some of the lactate present at the start of the incubation period. Such consumption of lactate was also seen in control experiments where no glucose was added to the medium. Where values are given for the percentage ratio of organic acids produced by the bacteria, these are the ratios of the initial concentration subtracted from the final concentration for the appropriate acid. pH measurements
These were made using a combination gel-filled pH electrode (Orion Research, Cambridge, U.S.A.) and an Orion microprocessor ionalyser/901 calibrated for use at 37°C (slope: +61.5 mV/pH unit). RESULTS
Control experiments
A 4-h incubation of the Strep. mutans suspension in the absence of any added sugar caused a drop in pH of approx. 0.1 from 7.0 to 6.9. No lactic acid or other acid production by the bacteria could be detected during or by the end of the incubation. There was evidence of some lactate consumption but the reduction in the lactate concentration was not statistically significant. Effect of increasing glucose concentration
The effect on the pH and acid formation of adding 2, 5, 10, 50 and 500 mM glucose to the Strep. mutans suspension is shown in Fig. 1. The results are expressed as means f standard deviations of three experiments. As the glucose concentration increased, both the initial pH fall and the production of lactate increased. For example, in the first 40 min of incubation with 2.0 mM glucose, the pH fell from 7.1 to 6.0 and the lactate concentration rose from 3.7 to 5.2mM. This rise was followed by a decline in lactic-acid concentration to 4.6 mM which occurred as the pH rose from 6.0 to 6.3. This reduction in lactate concentration was not statistically significant. During the incubation, the acetate concentration rose from 0.2 to 1.8 mM and the formic-acid concentration from 0.0 to 1.5 mM so that at the end of 4 h the percentage ratio of acid products resulting from the fermentation of the glucose was lactic: acetic: formic, 26.8 per cent: 37.8 per cent: 35.4 per cent. Following the addition of 500 mM glucose, the pH fell continuously from a mean of pH 6.8 to 4.1 and
the lactic-acid concentration determined rose continuously from 3.7 to 22.9 mM, the acetic-acid concentration from 0.3 to 0.8 mM and formic acid from 0.0 to 0.9 mM. At the end of the incubation, the percentage ratio of products from the glucose fermentation was lactic: acetic: formic, 93.5 per cent: 2.2 per cent: 4.3 per cent. Effect of increasing fructose concentration
The effect on the pH and acid formation of adding 2, 5, 10, 50 and 500 mM fructose to the Strep. mutans cultures is shown in Fig. 2. At both 2 and 5 mM fructose, following the initial pH drop, there was a small rise in pH from 5.6 to 5.8 and from 5.1 to 5.3, respectively. This rise was associated in both instances by production of lactic acid followed by loss of lactic acid in the medium to levels below the initial one at the start of the incubation; thus, whilst there was a net production of acetate (1.3 mM in both cases) and formic acid (1.2 and 1.5 mM, respectively), there was an overall reduction in lactic-acid concentration from 2.6 to 2.0 mM in both instances over the 4 h. The reductions in lactic-acid concentration from its peak values was statistically significant (p < 0.05). At medium and higher concentrations of fructose (l&500 mM), the results were similar to those with glucose with a percentage ratio of the bacterial acid-products after 4 h of lactic: acetic: formic: 88.6 per cent: 3.3 per cent: 8.4 per cent at 500mM fructose. Effect of increasing sucrose concentrations
The effect on the pH and acid formation of adding 1, 2.5, 5, 25 and 250 mM sucrose to the Strep. mutans suspension is shown in Fig. 3. As sucrose is a disaccharide, concentrations half those of glucose and fructose were used. At 1 mM sucrose, the pH fell from 7.0 to 6.4 before rising to pH 6.6 by the end of the 4-h incubation. At the same time, the lactic-acid concentration in the broth rose from 3.1 to 4.0 mM and fell to 3.8 mM. After 4 h, the percentage ratio of the acids produced by the bacteria was lactic: acetic: formic, 30.3 per cent: 35.9 per cent: 35.0 per cent. Conversely, at the highest sucrose concentration (250mM), the pH fell from 6.8 to 4.2 and the lactic acid concentration rose from 2.2 to 22.8 mM over the 4-h incubation. The final ratio of acid produced by the bacteria was lactic: acetic: formic, 94.5 per cent: 3.0 per cent: 2.5 per cent. DISCUSSION
Several organic acids are now known to occur in dental plaque. Geddes (198 1) found acetic, propionic, lactic and succinic acids in all human dental plaque samples, with formate in 7.5 per cent and pyruvate in less than 50 per cent. Resting plaque or plaque 60 min after exposure to sucrose contained more non-volatile acids than lactic acid but, immediately after sugar exposure, lactic acid predominated (Geddes, 1975). Similar results were obtained by Vratsanos and Mande1 (1982) and Distler and Kriinke (1983) although the latter noted wide, unaccountable variations in the acid content of individual plaque samples. Using an improved HPLC method for the analysis of organic acids, Distler and Kriinke (1984) showed that formic
pH response
of Strep. mutansto sugar
concentration
321 25
@HI
2mM Glucose
(mM)
(pli
5mM Glucose
(mfvl) 20
e
6
1
1
(h)
2 25
(pH)
i
IOmM
Glucose
ImM)
fhl
2
7(PHI
25
50mM
Glucose
(mM) 20
20
6
0
1 (h)
2
4
0
1 (h)
- 25
(pH)
500mM
Glucose
.I_i . (mM)
- 20
E Formote
Acetate
-
15
Lactate
-
10
-5
1
Fig. 1. Acid production values for pH (0)
(h)
2
4
by Strep.mutans10449 in the presence of 2, 5, 10, 50 and 500 mM glucose. Mean and means f standard deviations for formate, acetate and lactate are given.
acid is quantitatively the most important organic acid in resting plaque. The extent to whi’ch Strep. mutans contributes to acid production in dental plaque is not clear. van der Hoeven and Frank.en (1982) compared sucroseinduced acid produ’ction in rat plaques with and without Strep. mutans T2. The amount of lactic acid, 5 min after exposure to 10 per cent sucrose (292 mM), was significantly higher in the Strep. mutans-infected
plaque than in the Strep. mutans-free plaque, but significant differences at other times, or with formic or acetic acids were not noted. Analyses of acid production from Strep. mutans Ny341 mono-infected plaque taken from gnotobiotic rats showed equivalent amounts of formic, acetic and lactic acids in resting plaques where the free-sugar concentration was presumably low (van der Hoeven and Franken, 1984).
322
R. 9
@HI
DUGUID
25
2mM
Fructose
mM)
8
25
9
5mM
IpHI
20
E
15
7
10
6
Fructose
1
4
2
mM) 20
2
(h)
25
IOmM
4
(hl
1;
Fructose
:mM) 20
9
-
(pH)
50mM
Fructose
25
(mM)
6
-
20
I
1
2
4
(h)
(h) 25
(PHI
5OOmM
Fructose
cmM) 20
Iu
Formote Acetote Loctote
6
: . rnr Fig. 2. Acid production
A-
2
by Strep.
murans
in the presence of 2, details as in Fig. 1.
10449
In my experiments, formic, acetic and lactic acids were all produced by Strep. mutuns 10449. There was no evidence of propionic-acid production on the isotachophoresis traces; if propionic acid is produced by this strain it can only be in minute amounts. This is in accord with Distler and Kriinke (1980) for Strep. mutans 634 and T3/53. I did not analyse the incubation medium for ethanol which was reported by
5, 10, 50
and 500 mM fructose. Other
Carlsson and Griffith (1974) to be a product of Strep. mutans JC2.
The finding that Strep. mutans appears, under certain conditions, to be able to consume the lactate it has produced following sugar addition was also noted by Hu and Sandham (1972). However, Paulus (1983) found no evidence of lactate consumption in any of eight strains of Strep. mutans investigated.
pH response of Strep. mutans to sugar concentration -225
9
(PIi)
1 mM Sucrose
(mM)
8
323
9
(PHI
- 20
6
- 10
6
2.5mM
1CmM)
Sucrose
7
6
1
0
2
i
(h)
(h)
9
(PHI
5mM
Sucrcse
(mM) 20
8
6
i
i (h)
(h)
9
(pIi)
-
250mM
Swose
25
(mM)
8
- 20 Formote
7
6
0
1
(h)
Acetote
I
1 1 Loctote U
Fig. 3. Acid production by Strep. mutans 10449in the presence of 1, 2.5, 5,25 and 250 mM sucrose. Other details as in Fig. 1.
Lactic-acid consumption by plaque was noted by Muntz (1943) and in the mixed bacteria of salivary sediment by Sandham and Kleinberg (1970). In my experiments, lactic-acid consumption was a consistent finding at low concentrations of glucose and, especially, fructose, though not with sucrose. At some of these low monosaccharide concentrations, all the lactic acid originally produced was lost by the end of
the 4-h incubation. Although the rise in pH was not large (0.2-0.3 pH unit), it does represent a considerable reduction in the amount of acid present (N 50 per cent reduction in [H+]). Rise in pH due to base formation has been studied in a number of species of oral bacteria, following the addition of arginine or arginine-peptide by Kleinberg et nl. (1982) though they listed Strep. mutans 10449 as what they called a
324
R. DUGUID
non-pH rise organism with arginine or argininepeptide. I did not investigate the effect of arginine or arginine-peptide on acid production by Strep. mutans 10449 but the glucose concentration (2.8 mM) used by Kleinberg et al. (1982) is close to the glucose/fructose concentrations where evidence of lactate consumption was seen (2 mM). The pathway by which lactic acid is lost from the incubation medium is not clear and requires further investigation. Neither is it clear why this effect is more evident with fructose than with glucose, though it may be relevant that fructose-1,6-diphosphate is a positive effector of lactate dehydrogenase (Wittenburger et al., 1971). Carlsson and Griffith (1974) showed that with Streptococcus sanguis and Strep. mutans and to a lesser extent with Streptococcus salivarius and Streptococcus bovis, when glucose is limiting, the fermentation pattern is heterolactic rather than homolactic. Similar findings were reported by Thomas, Ellwood and Longyear (1979) for Streptococcus lactis. Both groups of workers used chemostat cultures. I found similar results when Strep. mutans 10449 was grown in batch culture. I conclude that considerable care has to be taken when attempting to relate the carbohydrate metabolism of Strep. mutans and, in all likelihood, other streptococci to Stephan-type pH changes. At high sugar concentrations, Strep. mutans is predominantly a homolactic fermenter. At lower sugar concentrations, pathways of both heterolactic fermentation and the further metabolism of lactate operate. Thus, it seems likely that, when low fermentable-sugar concentrations prevail in dental plaque, Strep. mutans is capable of metabolizing its own previously-formed lactic acid and that diffusion out of plaque and the metabolic activity of other oral bacteria (such as Veillonella) need not necessarily be involved. This potential ability of Strep. mutans to metabolize lactic acid further at low sugar concentrations is another variable which may affect the cariogenicity of different strains of this organism and the dental plaques they inhabit. Acknowledgement--I am very grateful to Mrs Fran. ston for her skillful technical assistance.
John-
REFERENCES Carisson J. and Griffiths D. J. (1974) Fermentation products and bacterial yields in glucose-limited and nitrogenlimited cultures of streptococci. Archs oral Biol. 19, 1103-l 109.
Distler W. and Kriinke A. (1980) Acid formation by mixed cultures of cariogenic strains of Streptococcus mutans and Veillonella alcalescens. Archs oral Biol. 25, 655-658. Distler W. and Kriinke A. (1983) The acid pattern in human dental plaque. J. dent. Res. 62, 87-91. Distler W. and Kriinke A. (1984) Acids in single-site human resting plaque. Caries Res. 18, 160-I 61. Geddes D. A. M. (1975) Acids produced by human dental plaque metabolism in siru. Caries Res. 9, 98-109. Geddes D. A. M. (1981) Studies on metabolism of dental plaque: diffusion and acid production of human dental plaque. In: Frontiers of Oral Physiology: The Environment of the Teeth. Karger, Base]. Geddes D. A. M. and McNee S. G. (1982) The effects of 0.2 per cent (48 mM) NaF rinses daily on human plaque acidogenicity in situ (Stephan curve) and fluoride content. Archs oral Biol. 27, 765-769. Hoeven J. S. van der and Franken H. C. M. (1982) Production of acids in rat dental plaque with or without Streptococcus mutans. Caries Res. 16, 375-382. Hoeven J. S. van der and Franken H. C. M. (1984) Effect of fluoride on growth and acid production by Srreptococcus mutans in dental plaque. Infect. Immun. 45, 356359. Hu G. and Sandham H. J. (1972) Streptococcal utilization of lactic acid and its effect on pH. Archs oral Biol. 17, 729-743. Kleinberg I., Jenkins G. N., Chatterjee R. and Wijeyeweera L. (1982) The antimonv UH electrode and its role in the assessment and interpretation of dental plaque pH. J. dent. Res. 61, 1139-1147. Mikx F. H. M. and Hoeven J. S. van der (1975) ,_Symbiosis of Streptococcus mutans and Veillonella alcalescens in mixed continuous cultures. Archs oral Biol. 20, 407-410. Muntz J. A. (1943) Production of acids from glucose by dental plaque material. J. biol. Chem. 148, 225-236. Paulus M. (1983) Zur FIhigkeit kariogener Streptokokken, Glucose iiber Lactat aerob vollstiindig abzubauen. Med. Diss., Erlangen, West Germany. Sandham H. J. and Kleinberg I. (1970) Contribution of lactic and other acids to the pH of a human salivary sediment system during glucose catabolism. Archs oral Biol. 15, 1263-1283. Stephan R. M. (1944) Intra-oral hydrogen-ion concentrations associated with dental caries activity. J. dent. Res. 23, 257-266. Stephan R. M. and Hemmens E. S. (1947) Studies of changes in pH produced by pure cultures or oral microorganisms. J. denr. Res. 26, 15-41. Thomas T. D., Ellwood D. C. and Longyear V. M. C. (1979) Change from homo- to hetero-lactic fermentation by Streptococcus lacris resulting from glucose limitation in anaerobic chemostat cultures. J. Bact. 138, 109-I 17. Wittenberger C. L., Palumbo M. P., Bridges R. B. and Brown A. T. (1971) Mechanisms for regulating the activity constituent destructive pathways in Streptococcusfbecalis J. dent. Res. 50, 109~1113. Vratsanos S. M. and Mandel I. D. (1982) Comparative plaque acidogenesis of caries-resistant vs. cariessusceptible adults. J. denf. Res. 61, 465-468.