Growth inhibition of Streptococcus mutans and Leuconostoc mesenteroides by sodium fluoride and ionic tin.

K G Yost and P J VanDemark Appl. Environ. Microbiol. 1978, 35(5):920. Downloaded from http://aem.asm.org/ on December 29, 2011 by guest

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1978, p. 920-924

Vol. 35, No. 5

Printed in U.S.A.

0099-2240/78/0035-0920$02.00/0 Copyright 1978 American Society for Microbiology

Growth Inhibition of Streptococcus mutans and Leuconostoc mesenteroides by Sodium Fluoride and Ionic Tin
K. G. YOSTt* AND P. J. VANDEMARK Department of Microbiology, Cornell University, Ithaca, New York 14853 Received for publication 8 August 1977

Sodium fluoride caused inhibition of growth rate and growth levels of Streptococcus mutans with glucose as the primary energy and carbon source. Stannous fluoride increased growth lag and caused a much greater inhibition of growth rate than did sodium fluoride. Neither compound was found to be bactericidal when culture viability was measured after 6 days of incubation. Leuconostoc mesenteroides, which lacks a phosphotransferase system for sugar transport, showed less inhibition of growth rate with both inhibitors than did S. mutans, which possesses a phosphotransferase system. Metabolism of glucose or lactose which requires enolase activity showed sodium fluoride inhibition, whereas metabolism of arginine or pyruvate does not involve enolase activity and showed no inhibition of growth.

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Streptococcus mutans has received recogni- versity Medical College, Evanston, Ill. L. mesentertion as a primary agent of dental caries, being oides was obtained from the stock culture collection of the Department of Microbiology at found at most sites of caries (4). Although the sity, Ithaca, N.Y. Stock cultures were Cornell Univermaintained froharm the teeth, it is zen in heart infusion broth. organism itself does not capable of lowering the local pH below the solGrowth medium. The medium used was brain ubility limit of the teeth. Control of the acid heart infusion broth (Difco Laboratories) buffered produced would lead to a control of caries. The with 0.36% KH2PO4 and adjusted to pH 6.6. In expereffect of fluoride in caries control has been his- iments to determine the effect of pH on fluoride inhitorically attributed to hardening of the teeth, bition, the buffer was 1% KH2PO4 and the pH was but recently advanced theories have suggested adjusted as indicated. Media for S. mutans contained the effect may also be due to disruption of the 5 mM glucose, determined to be a limiting concentraecological balance in the mouth, through pre- tion, or other substrates in concentrations noted with mesenteroides contained 7.5 the vention of adsorption of salivary glycoproteins mMfigures. Media for L.comparable growth levels. All glucose to achieve to hydroxyapatite (15) or perhaps by inhibiting the medium components listed above were autoclaved growth of S. mutans directly. separately. All compounds included in the media as Enolase is inhibited by fluoride (20) and is the potential inhibitors were filter sterilized. An incubamost fluoride-sensitive enzyme in the glycolytic tion temperature of 35C was used. pathway (11). Fermentative growth dependent Growth measurement. Absorbance at 650 nm upon glycolysis is thus inhibited by fluoride and versus a water standard was used as a measure of blocks phosphoenolpyruvate (PEP) synthesis. S. growth on a Gilford Micro-Sample 300-N spectrophomutans possesses a PEP:phosphotransferase tometer. All pH measurements were performed on a system (PTS) which both phosphorylates and New Brunswick model pH-40 meter with a Fisher translocates glucose; phosphorylation is insen- pencil probe. sitive to fluoride to 10 mM (17). Leuconostoc RESULTS mesenteroides does not possess a PTS (A. H. Many elements have been examined for their Romano and J. D. Trifore, Abstr. Annu. Meet. Am. Soc. Microbiol. 1976, K103, p. 153). Since inhibitory effects upon caries (3), but relatively PEP is used for both energy and transport in S. few have shown significant results. More than mutans, but for only adenosine 5'-triphosphate any other element, fluorine has been the subject generation in L. mesenteroides, a decrease in of attention and investigation. A comparison of PEP production through inhibition of enolase growth inhibition of S. mutans by the sodiumshould be relatively more detrimental to growth halogen series showed the relative effectiveness of the ion in inhibiting growth (Fig. 1). for S. mutans. If the pathway used to metabolize a specific MATERIALS AND METHODS energy source does not pass through the enolase Organisms. S. mutans FAl was generously pro- conversion of phosphoglycerate to PEP, then an vided by Hutton D. Slade of the Northwestern Uni- increase in fluoride would not be expected to t Present address: 170 Tabor Rd., Morris Plains, NJ 07950. result in a marked decrease in growth with that
920

VOL. 35, 1978

energy source.
upon

INHIBITION OF S. MUTANS AND L. MESENTEROIDES

I1

921

The inhibition of growth based lactose, sodium pyruvate, and arginine hydrochloride for S. mutans was determined (Fig. 2). Lactose metabolism, like that of glucose, requires enolase and shows a similar inhibition pattern. Those pathways involved in the metabolism of arginine and pyruvate do not involve enolase activity, and growth on these substrates showed no inhibition. Growth was measured as a function of the initial medium pH for a range of concentrations of sodium fluoride (Fig. 3) and indicates that a significant increase in inhibition of growth by fluoride can be caused by more acidic conditions. A range of inorganic salts, some of which are shown in Table 1, was compared to common fluoride dentifrice agents for its inhibitory effects upon the growth of S. mutans. The most highly inhibitory compounds (Hg2Cl2, Na2AsO2) are also those most toxic to humans. The primary compounds studied for this work, sodium fluoride and stannous fluoride, were only moderately inhibitory. Surprisingly, sodium monofluorophosphate showed no inhibition at the lower values.

oD
c
0

S..
0
0

4%

Downloaded from http://aem.asm.org/ on December 29, 2011 by guest

4 3 NoF (mM)

FIG. 3. Effect of sodium fluoride on S. mutans growth at various initial pH levels, measured at 24 h.

TABLE 1. Inhibition of S. mutans growth by some inorganic salts

Inorganic salt

GrIoa
( g/g)

Maximum concn tested

O.T!A
0.6

_ l

NoCI, NaBr, Nal

.:

c 0.5
0

0
4

04
0.3

0.20.I

Halogn (mM) FIG. 1. S. mutans growth with sodium-halogen

QZ+yf
94S 4 h

1,000 480 NIc 240 NI RbCl2 100,000 3,000 NaMFPd 250 200 NaF 250 100 SnF2 aMinimum concentration causing at least 50% growth inhibition after 24 h. b only. Tested at 10, 100, and 1,000 NI, No inhibition within this range. d NaMFP, Sodium monofluorophosphate.
1,000
'

Na2WO4b Hg2Cl2b Na2AsO2b BaCl2b SrC12

1,000 10 10

1,000

1,000
1,000

Ag/g

0.7r
* o c 0.5 p

0.6- _
mM

lactose
0

U .0 04. O

,X0 .3
0.20
a um

Since the effect of fluoride inhibition of enolase is assumed to be due to the formation of fluoromagnesium phosphate complexes (21), thus depriving enolase of sufficient cofactor to act as a catalyst, addition of magnesium to the medium would be expected to reverse the inhibition. However, supplying magnesium chloride in a concentration series equivalent to that used for sodium fluoride had a slight stimnulatory ef-

~o

20 mM rginine

fect upon growth, but showed no reversal of

inhibition of 4.75 mM sodium fluoride (Fig. 4),

suggesting
that formation of such
may

complexes

lOmM pyruvote

not

be the point of inhibition.

* * * * * * 5 4 3 6 2 NoF (mM) FIG. 2. S. nrzutans growth with sodium fluoride and lactose, sodiuwmnpyruvate, and arginine hydrochloride as energy sobirces after 24 h. The observed growth
level with no Iadded energy source is 0.08.

Stannous fluoride has become a major source of fluoride in dentistry and toothpastes. Growth
of S. mutans in the presence of stannous fluoride and stannous chloride was found to be inhibited to a significantly greater degree than for sodium fluoride (Fig. 5). Magnesium and stannous ions both occur in the +2 oxidation state. If stannous

922

YOST AND VANDEMARK

without NoF

APPL. ENVIRON. MICROBIOL.

0.81.
0D .6
c

.0
.m 0.4
0

0.z*
1

76 mM NoF

whereas stannous fluoride did not inhibit growth at any concentration tested (Fig. 8). No detectable increase in the lag phase was observed for cultures inhibited by sodium fluoride; however, stannous fluoride caused a greatly increased lag period. In an experiment using an inoculum of growing cells, obtained from a steady-state culture in the chemostat, the lag at the highest concentrations of stannous fluoride was measured in hours (Fig. 9).

.--*

m.- -

.-0.4 r
2
3

Downloaded from http://aem.asm.org/ on December 29, 2011 by guest

6
0
E)
11

MgCI2 (mM)
FIG. 4. Effect of magnesium chloride on growth of S. mutans in the absence and presence of sodium fluoride after 24 h.
A

0.2

esNo

mesen?eoides,
L mesenteroides,

NoF

SnF2
S mulons,
-

.~0.1

wS mtons, SnF2

1\2 1

*
3

|* 4

(mM)
c
a

.o
D
0
U,

FIG. 6. Growth rate factor, ,u, for S. mutans and L. mesenteroides with sodium and stannous fluorides.
0

-0.2

-0.4
0

3-0.6

;07

1.6 1.2 0.6 Compound (mM) FIG. 5. S. mutans growth with stannous fluoride, stannous chloride, zinc chloride, and sodium fluoride after 24 h.

0.4

-0.8
-

Uloo..~~~ .*'
1
2
3
4
5

1.0

-1.2
6
7

8
so-

DAYS

inhibits by competing with magnesium for a cofactor position, zinc, as another +2 oxidation state transition metal, might be expected to show inhibition similar to that of stannous ions. Zinc, however, enhanced rather than inhibited growth. Growth was measured as a function of time and of inhibitor concentration. The growth rate factor was determnined as the slope of the semilogarithmic plot of growth versus time during the exponential phase. Inhibition of the growth rate as a function of concentration for S. mutans and L. mesenteroides is shown in Fig. 6. Growth rate inhibition is not the only effect of the inhibitors. After reaching a maximum, turbidity stabilized at a lower value for the rest of the incubation period (Fig. 7). At the highest concentration, sodium fluoride inhibited growth,

FIG. 7. Long-term growth of S. mutans with dium fluoride. Symbols: 0, 0 mM; O, 1.19 mM; 2.38 mM; *, 3.57 mM; *, 4.76 mM; *, 5.95 mM.
0

O,

-0.2
-

-0.4

-J

0, -0.6 -0.8
-1.0
-1.2

Ii
0

p a

4 5 DAYS

FIG. 8. Long-term growth of S. mutans with stanSymbols: 0, 0 mM; O, 0.32 mM; 0.64 mM; *, 0.96 mM; *, 1.28 mM; *, 1.60 mM.
nous fluoride.
0,

VOL. 35, 1978

40

INHIBITION OF S. MUTANS AND L. MESENTEROIDES

923

The effects of both sodium and stannous fluorides on L. mesenteroides are less dramatic than those shown by S. mutans. This may be / due to their relative energy yielding systems. If _as fluoride were to inhibit only at the site of PEP synthesis, both organisms would show net zero .J I5 it adenosine 5'-triphosphate synthesis (6). If fluoride were to act only at translocation, L. mes.a enteroides would show no inhibition since it 0.4 1.6 12 lacks a PTS (Romano and Trifore, Abstr. Annu. SaF3 (mM) Meet. Am. Soc. Microbiol. 1976, K103, p. 153). FIG. 9. Growth initiation lag for S. mutans with S. mutans would show a decreased growth rate, stannous fluoride. Inoculum from chemostat. but would reach equivalent final growth levels due to diffusion of glucose. Since none of these DISCUSSION situations was shown to develop, a more complex S. mutans has been implicated as the cause of form of inhibition is suggested. cavities and has always been found at the site of The marked effect of stannous fluoride on the smooth surface caries (10). Caries may be con- growth rate with essentially no effect upon trolled by eliminating the causative agents. Al- growth levels suggests a different mode of action though fluoride is not bactericidal at concentra- than for sodium fluoride, perhaps upon the PTS tion as high as 24 mM (2), it may stop or reduce in vivo. The delay in growth inhibition caused caries without eliminating S. mutans (14). How- by stannous fluoride is too rapid for mutation ever, at these concentrations it does affect (Fig. 9) and may represent an adaptation of the growth of this organism. cell machinery to the unfavorable concentraWarburg and Christian (21) showed the sen- tions of inhibitor present. sitivity of enolase to fluoride and suggested the Fluoride dentifrices contain 0.1 to 0.2% fluomechanism was due to the formation of a fluo- ride, which acts as only 5 to 10 yg/g during rophosphate complex with magnesium, the en- brushing (13), yet they can still effect a decrease olase cofactor. The results of Wang and Himoe in caries (1). Use of 0.5% fluoride gels can be (20) show cooperative binding of fluoride and even more effective (8,9). Plaque has been found phosphate to enolase in an enolase-magnesium- to accumulate fluoride to levels of 9.5 umol/g phosphate-fluoride complex. In this tightly (net wt) even among people from low-fluoride bound phosphate-fluoride complex, enzyme ac- areas (7). Higher concentrations are found in tivity is a function of fluoride concentration. plaque exposed to fluoridated water (5). AlGrowth in the presence of inhibitor was found though only 7 to 10% of this is in the ionic form to be independent of the magnesium concentra- (19), a relation with caries control exists. This tion (Fig. 4). Such a result would be expected if ionic fluoride plus the reservoir of fluoride bound the binding of the complex to the enzyme were in the plaque and teeth may act in the type of irreversible. growth indicated above to control caries. A decreased production of PEP through inhibition of enolase would more severely affect LMRATURE CITED the facultatively anaerobic bacteria due to their 1. Bartelstone, H. J., I. D. Mandel, and N. W. Chilton. reliance on the PEP-powered PTS and lower 1961. Critical evaluation of clinical studies with stanyield of adenosine 5'-triphosphate from glucose nous fluoride dentifrices, p. 135-142. In H. R. Muhle(16) than strictly aerobic organisms which genmann and K. G. Konig (ed.), Caries Symposium Zurich. erate ample adenosine 5'-triphosphate through Hans Huber, Berne, Switzerland. 2. Bibby, B. G., and M. VanKesteren. 1940. The effect of respiration.
35 30
**
-

.7-0
0.6

A,~

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The presence of sodium fluoride and stannous fluoride decrease the growth rate of S. mutans 0.05 h-1 per 1 mM and 0.2 h-' per 1 mM, respectively (Fig. 6). Since the acid produced by the bacteria is the actual cause of cavities (12), the reduction of acid production as a part of metabolism becomes important. The estimated growth in the mouth is two or three cell divisions per day (18), as compared to a 2-h generation time observed in this study. Decreasing the generation time and the acid production through the addition of inhibitor might then be dramatic.

fluorine on mouth bacteria. J. Dent. Res. 19:391-402. 3. Bourne, G. H. (ed.). 1967. World review of nutrition and dietetics, vol. 7. Hafner Publishing Co., Inc., New York. 4. Bowen, W. H. 1974. Microbiological aspects of dental caries. Adv. Caries Res. 2:xxv-xxxii. 5. Dawes, C., G. N. Jenkins, J. L. Hardwick, and S. A. Leach. 1965. The relations between the fluoride concentration in the dental plaque and in drinking water. Br. Dent. J. 119:164. 6. Doelle, H. W. 1975. Bacterial metabolism. Academic Press Inc., New York. 7. Hardwick, J. L., and S. A. Leach. 1963. The fluoride content of the dental plaque. Arch. Oral. 8 (Suppl.): 151. 8. Horowitz, H. S. 1973. Fluoride: research on clinical and public health applications. J. Am. Dent. Assoc. 87:1013.

924

YOST AND VANDEMARK

APPL. ENVIRON. MICROBIOL.

15. Rolla, G., and B. Melsen. 1975. Desorption of protein and bacteria from hydroxyapatite by fluoride and monofluorophosphate. Caries Res. 9:66-73. 16. Roseman, S. 1969. The transport of carbohydrates by a bacterial phosphotransferase system. J. Gen. Physiol. 54: 138s-184s. 17. Schachtele, C. F., and J. A. Mayo. 1973. Phosphoenolpyruvate-dependent glucose transport in oral streptococci. J. Dent. Res. 52:1209-1215. 18. Scherp, H. W. 1971. Dental caries: prospects for prevention. Science 173:1199-1205. 19. Singer, L., B. A. Jarvey, P. Venkateswarlu, and W. Armstrong. 1970. Fluoride in plaque. J. Dent. Res. 49:455. 20. Wang, T., and A. Himoe. 1974. Kinetics of the rabbit muscle enolase-catalyzed dehydration of 2-phosphoglycerate. Fluoride and phosphate inhibition. J. Biol. Chem. 249:3895-3902. 21. Warburg, O., and W. Christian. 1941. Isolation and crystallization of enolase. Naturwissenschaften 29:589-590.

9. Horowitz, H. W., and S. B. Heifetz. 1972. The current state of topical fluorides in preventive dentistry, p. 34. In E. Newbrun (ed.), Fluorides and dental caries. Charles C Thomas, Publisher, Springfield, Ill. 10. Ikeda, T., H. J. Sandham, and E. L. Bradley, Jr. 1973. Changes in Streptococcus mutans and lactobacilli in plaque in relation to the initiation of dental caries in Negro children. Arch. Oral Biol. 18:555-566. 11. Kanapka, J. A., and I. R. Hamilton. 1971. Fluoride inhibition of enolase activity in vivo and its relationship to the inhibition of glucose-6-P formation in Streptococcus salivarius. Arch. Biochem. Biophys. 146:167-174. 12. Keyes, P. H. 1968, Research in dental caries. J. Am. Dent. Assoc. 76:1357-1374. 13. Loesche, W. J. 1976. Chemotherapy of dental plaque infections. Oral Sci. Rev. 9:65-107. 14. Loesche, W. P., S. A. Syed, R. J. Murray, and J. R. Mellberg. 1975. Effect of topical acidulated phosphate fluoride on percentage of Streptococcus mutans and Streptococcus sanguis in plaque. Caries Res. 9:139-155.

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