Journal of Radiation Research and Applied Sciences

J. Rad. Res. Appl. Sci., Vol. 2, No.5, pp. 1003 - 1026 (2009)

Antimicrobial Effects of Sodium Fluoride, Xylitol and Metals Salts on in Vitro Growth Inhibition, Acid Production and Ultrastructure of Streptococcus mutans.
T. M. El-Mongy and A. B. Abd EI-Aziz.
Microbiology Department, National Center for Radiation Research and Technology, Atomic Energy Authority, Nasr City, Cairo, Egypt.
Email: abdelazezamany@gmail.com Received: 26/10/2009. Accepted: 21/12/2009.

ABSTRACT This study aimed to evaluate the effects of sodium fluoride (NaF), dietary sugars, sugar alcohols (xylitol and sorbitol) and different metals salts either separately or in combination, by different concentrations at different pH, on the growth inhibition, acid production and ultra structure of Streptococcus mutans. NaF was more effective at low pH, when NaF was added to actively growing Streptococcus mutans broth culture, the growth rate was unaffected by 75 ppm F-, slowed by 150 ppm F-, and immediately arrested by 300 or 600 ppm F-. On the other hand, the best effect of xylitol was at high pH. The effect of xylitol was more marked in the presence of NaF as the acid production was inhibited and the pH did not fall to 5.0. The response of Streptococcus mutans to metals salts was typical of this organism's response to a number of trace metals above optimum concentrations of which may be inhibitory. Synergistic effect observed by addition of metals salts by concentration ranged from 0.2 to 5.0mML-1, 300 ppm NaF and 5% xylitol. This formula can work at any pH value and causes no drop of the broth culture pH to below 5.0 which is the optimal pH for growth and multiplication of Streptococcus mutans, so this formula worked as pH buffer regulation and growth inhibition for S. mutans. Low concentration of this combined formula after 5 min only at 5.0 and 7.0 pH values caused effective complete destruction of the bacterial viable cells and this effect was observed clearly by Electron Microscope photo graph.
Key words: Sodium Fluoride, Xylitol, Metals Salts, Streptococcus mutans.

INTRODUCTION When the equilibrium of the oral environment changes, the proliferation of various bacterial species may hazardously increase and act together to initiate

1004 T. M. EL-MONGY& A. B. ABD EI-AZIZ /J. Rad. Res. Appl. Sci., Vol. 2, No.5(2009)
and further aggravate certain oral diseases, as periodontal diseases, dental caries(1), abnormal test acuity or halitosis with the growth of the dental plaque(2). Many investigative studies have documented the inhibition of plaque growth and the reduction of bacterial acid formation by the use of antibacterial agents added to mouthrinses or toothpaste preparations (2). According to their chemical characteristics, mouthrinses commercially available contain cationic, anionic and nonionic active ingredients which may, alter the bacterial membrane function. Certain metal ions may alter the function of the cells membrane and the enzymatic activity within the cell, impairing the production of acids during the glycolysis process (3). Among the cationic agents some divalent metal ions like Cu+2, Zn+2 and Sn+2, are most widely used. They are electro statically attracted by oral surfaces known to carry negatively charged groups, thus increasing the residence time of the active ingredient in the oral cavity (3). In the presence of xylitol and NaF, Streptococcus mutans (S. mutans), the oral organism nearly always used in such assays, is unable to acquire the nutrients necessary for its survival and reproduction (4). In order to be effective, the active ingredient (s) in a mouthrinse preparation must show a high substantivity (ability to maintain an effective concentration for prolonged periods of time) and to be able to interfere with the metabolism of targeted microorganisms. Additionally, its bactericidal effect would last as long as the agents active form is present at effective levels and should be harmless towards the oral mucosa and of low toxicity to humans, since certain volume of the substance may be swallowed during rinsing (1). The aim of this study was to evaluate in vitro the antimicrobial activity of sodium fluoride, xylitol combined with different metals salts at differing concentrations and initial pH on the growth, ultra structure and acid production by Streptococcus mutans and investigate whether this combination could result in an additive synergetic effects. MATERIALS AND METHODS Organism Streptococcus mutans an oral pathogen was obtained from the medical lab of the Department of Microbiology at Ain Shams University.

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Growth Medium The medium used was brain heart infusion (BHI) broth (Difco Laboratories). Streptococcus mutans was cultured in (BHI) broth containing 0.2% glucose, buffered with 0.36 % KH2PO4 and adjusted to pH 7.0 at 37C up to the exponential phase of growth (optical density [OD], 0.2 to 0.3). Three hundred micro liters of this suspension was transferred into 3 ml of test medium containing various sugar alcohols, dietary sugar or metal salts at different concentrations. The test tubes were incubated at 37C for 48 hrs. Each test was carried out in triplicate. All the medium components were autoclaved separately. All compounds included in the media as potential inhibitors were filter sterilized. Maintenance Medium The organism was maintained by weekly subculture on brain heart infusion agar plates. Analytical Determinations Growth measurement Absorbance at 650 nm against the standard medium was used as a measure of growth on a LW-V-200-RS spectrophotometer, with the measurements being performed every 1 to 2 hrs during the logarithmic phase of growth. The OD results were calculated as the means of three measurements. Measurement of acid production All pH measurements were performed by means of a glass electrode pH meter type with a Fisher pencil probe. Test materials comprised: 1. Commercial sugars (glucose, sucrose, maltose and fructose) and sugar alcohols (xylitol and sorbitol). 2. Aqueous sodium fluoride. 3. Pure metal salts of copper, magnesium, calcium, zinc and nitrite. Sugars or sugar alcohols Xylitol, sorbitol (Sigma Chemical Co., St. Louis, Mo.), fructose, glucose, maltose or sucrose (Merck, Darmstadt, Germany), was sterilized by filtration and separately added (5% wt/vol) or in mixture (5% tested sugar and 5% xylitol

1006 T. M. EL-MONGY& A. B. ABD EI-AZIZ /J. Rad. Res. Appl. Sci., Vol. 2, No.5(2009)
wt/vol) to the basic medium from a 50 % (wt/vol) stock solution. The inhibitory effect of sugars and sugars combined with xylitol was investigated by measurement of the acid production rate. Antimicrobial effectiveness was assessed by measuring reduction in optical density at 650 nm using a visible spectrophotometer. Aqueous sodium fluoride Freshly prepared aqueous solutions of NaF were sterilized by filtration and serially diluted in tubes with (BHI) broth to yield final fluoride concentrations of 0, 75, 150, 300, 600, 1200 or 1500 ppm and the broth pH was adjusted to values 7.0 and above with 1 N NaOH or to pH values below 7.0 with 1 N NaCl. Initial and terminal pH values after 24 and 48 hrs of incubation of agent-broth solutions were determined. In experiments to determine the effect of pH on fluoride inhibition, the buffer used was 1% KH2PO4 and the pH was adjusted as indicated in each experiment. OD of cell suspensions was determined during the time-course of the incubation. Metals Salts The antimicrobial efficacy of different metal salts against Streptococcus mutans was tested in (BHI) liquid medium. Zinc chloride, calcium phosphate, calcium carbonate, magnesium sulfate, cupper sulfate and sodium nitrite was added to the medium to final concentrations of 0, 0.2, 0.5, 1, and 5 mML-1 and incubated for 48 hours. The optical densities (OD) of cell suspensions were determined and the cultures pH was measured after 24 and 48hrs during the time-course of the incubations. Ultra structure Streptococcus mutans tested strain was grown in 5% xylitol, 5% glucose, 5% fructose, 5% sorbitol, control medium (BHI) and in the effective inhibition concentrations of NaF or tested metal salts (calcium, zinc and nitrite salts) for 24 hrs and the ultra structure of bacteria examined by electron microscope. S. mutans broth cultures were exposed to the combined formula of xylitol, NaF and effective metals salts at pH 5.0 and pH 7.0 for only 5 min and the bacterial ultra structure examined by electron microscope.

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Statistical Analysis Experimental data were subjected to analysis of variance (ANOVA) using SAS program 6.1., SAS institute, Cary, NC (5). One-way analysis of variance (ANOVA) was used to test the differences between the groups at each point in time. A significance level of =0.0001 was set for comparison of the groups. The data are the means of 3 independent experiments. Vertical bars indicate standard deviations. RESULTS In the present study, all pH and OD measurements were monitored to ensure that comparisons were made between cultures of comparable growth phase. The preliminary experiments indicated that the OD measurements gave good agreement with the direct count during the period of growth, so OD alone was used to estimate total cell numbers. The mean OD for the Streptococcus mutans after 10 hrs of cultivation in different broth media varies significantly (P< 0.0001) from one culture to another. It was 0.85 and 0.62 with 5% glucose and maltose respectively (Fig.1), whereas it was 0.86 in the control medium (BHI) and 0.75 in 5% sucrose (Fig.2). The mean OD values for the Streptococcus mutans remained less in xylitol-containing medium than in the control medium throughout the observation period for all media tested (P < 0.0001).Tested oral Streptococcus strain was inhibited by xylitol which exhibited a dose-related inhibition of Streptococcus mutans in the (BHI) medium. The difference was greater after 10 hrs of incubation, where the mean OD measurements for the S. mutans were 0. 46, 0.28 and 0.20 in 2.5, 5 and 10 % xylitol, i.e., it was 46.51%, 67.45% and 76.74% less in the xylitol-containing medium than the control culture (BHI), respectively (Fig. 2). The addition of glucose, maltose, or sucrose at concentration 5% did not alter the effect of xylitol, and growth inhibition was detected (Fig.1). The difference was greater after 8-15 hrs of the beginning of the cultivation. The mean OD measurements after 10 hrs for the Streptococcus mutans were 0.45 , 0.54 and 0.49 in 5% of glucose, maltose and sucrose combined with 5% xylitol respectively; i.e., it was 47.67, 37.20 and 43.02 % less in the xylitolcontaining medium than the control medium (R2 = 0.99, [ P < 0.0001]),

1008 T. M. EL-MONGY& A. B. ABD EI-AZIZ /J. Rad. Res. Appl. Sci., Vol. 2, No.5(2009)
(Figs.1and 2). Meanwhile the OD was 0.67 by using 5 % fructose. Xylitolinduced growth inhibition was prevented by the addition of 5 % fructose (OD, 0.62) (Fig.1). Sorbitol at concentration of 5% had no inhibition effect on the growth of Streptococcus mutans (OD, 0.51). Xylitol at a concentration of 5% was as effective alone as it was in combination with 5% sorbitol (Fig.2). The mean OD for the Streptococcus mutans after 10 hrs of cultivation was 0.30 in the medium containing both 5% sorbitol and xylitol. The OD values for the cultures grown on the experimental media containing different metal salts differed significantly (P < 0.0001) from those for cultures grown on the control medium. Growth of Streptococcus mutans, measured in terms of OD, in media containing (per Liter) 1.0 mM of ZnCl2, 5.0 mM of CaPO4, or 0.5 mM of NaNO2 over 48 hrs was compared with in control medium (BHI) at pH 7.0. Inhibition of 41.05 %, 77.32 % and 58.02 % (P<0.0001), were observed with ZnCl2, CaPO4, and NaNO2, respectively (Fig.3). MgSO4 at concentration 0.5 mM inhibited the growth of Streptococcus mutans by 37.79 %. Meanwhile, CaCO3 and CuSO4 at concentration 5.0 mM and 0.2 mM were having little effect on S. mutans activity (28.95% and 33.48 %, respectively) (Fig.3).
0.9 0.8 0.7 0.6 0.5
glucose+xy fructose+_xy suc+xy mal+xy gluc5% fruc5% suc5% Malt 5%

OD
0.4 0.3 0.2 0.1 0 0 2 6 10 15 20 24 48

Time (hrs)

Time (hrs)

Fig. 1.

Fig (1): Growth of Streptococcus mutans in media contaning xylitol and different sugars measured in in media counts over 48hrs. Growth of Streptococcus mutansterms of ODcontaining different sugars and

xylitol measured in terms of OD counts over 48 hrs. [(-) Error bars represent standard deviation. (-) Means of data are significantly different (P < 0.0001)].

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1 0.9 0.8
BHI

0.7 0.6
OD

xylitol 2.5% xylitol 5% xylitol10% sorbitol 5% sorb+xy

0.5 0.4 0.3 0.2 0.1 0 0

10 Time (hrs) Time (hrs)

15

20

24

48

Fig. 2. Growth of Streptococcus mutans inmeasured containing different 48hrs. and Fig (2): Growth of Streptococcous mutans media in terms of OD counts ovr sugars xylitol measured in terms of OD counts over 48 hrs. [(-)Error bars represent standard deviation. (-)Means of data are significantly different (P < 0.0001)].
0.7
CaPO4 CaCO3 NaNO2 MgSO4

0.6

0.5

CuSO4 ZnCl2

0.4
OD

0.3

0.2

0.1

0 0 2 6 10 15 20 24 48

Fig (3): Effect of different metals salts on Streptococcus mutans growth measured Fig. 3. Effect of different metal salts in terms of OD. on Streptococcus mutans growth. [(-)Error

Time (hrs)

bars represent standard deviation. (-)Means of data are significantly different (P < 0.0001)].

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Table-1 reveals the effect of different treatments on the final pH of growth media. Xylitol was reported to inhibit the growth of Streptococcus mutans and the rate of acid production from glucose. These results were notable at pH 5.57.0. After 48 hrs of incubation demonstrated that maltose, glucose and fructose gave final pH values of 4.24, 4.15 and 4.92 compared with sucrose (final pH 3.95). Addition of 5% xylitol to the sorbitol cultures gave a final pH 6.27; whereas the final pH values with different tested xylitol concentrations ranged between 6.40 and 6.47); sorbitol-xylitol challenges gave less acid production than sorbitol (final pH 6.12); challenges alone (Table 1). It was concluded that the media which have high pH values were those containing xylitol alone or mixtures of xylitol and sorbitol. Xylitol was not able to inhibit the acid production from the easily fermented glucose (final pH of glucose/xylitol mixture 5.43); and fructose (final pH fructose/xylitol mixture 4.96).
Table 1. Effect of different treatments on the final pH of Streptococcus mutans growth media.
Treatment 10hrs BHI (control) Glucose 5% Glucose+xylitol (5%) Fructose 5% Fructose+xylitol (5%) Sucrose 5% Sucrose + xylitol (5%) Maltose 5% Maltose+xylitol (5%) Sorbitol 5% Sorbitol +xylitol (5%) Xylitol 2.5% Xylitol 5% Xylitol 10% ZnCl2 NaNO2 MgSO4 CuSO4 CaPO4 CaCO3 0.860 0.853 0.450 0.669 0.619 0.674 0.485 0.615 0.540 0.51 0.30 0.46 0.275 0.206 0.507 0.361 0.535 0.572 0.195 0.611 SD 0.028 0.015 0.007 0.009 0.050 0.009 0.007 0.006 0.014 0.021 0.032 0.042 0.021 0.005 0.003 0.021 0.007 0.006 0.007 0.002 OD 48hrs 0.483 0.645 0.305 0.403 0.383 0.481 0.225 0.325 0.117 0.213 0.176 0.119 0.103 0.092 0.471 0.300 0.361 0.455 0.099 0.523 SD 0.014 0.007 0.006 0.013 0.046 0.003 0.007 0.005 0.010 0.028 0.002 0.014 0.001 0.003 0.001 0.014 0.016 0.002 0.001 0.005 10hrs 4.81 5.67 5.74 5.65 5.69 5.72 5.61 5.55 5.33 5.95 6.08 6.10 6.17 6.22 4.97 6.31 5.26 5.82 6.17 5.00 pH 24hrs 4.72 4.75 5.51 5.09 5.08 4.16 5.19 4.60 5.67 6.05 6.20 6.38 6.40 6.43 4.62 6.44 4.78 6.31 6.57 4.54 48hrs 4.70 4.15 5.43 4.92 4.96 3.95 5.12 4.24 5.72 6.12 6.27 6.40 6.45 6.47 4.52 7.08 4.57 6.44 6.69 4.39

Initial pH 5.5. * Data are recorded as mean of 3 observations SD. * Significance change from control group at P< 0.0001.

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This study compared the effects of different concentrations of sodium fluoride on S. mutans growth (Fig.4).The addition of 300 ppm F- to actively broth cultures almost totally arrested the growth of the S. mutans (OD, 0.08) at pH 5.5. The addition of 1200 ppm F immediately terminated S. mutans growth (OD, 0.04). (R2 = 0.99, [P< 0.0001]). The inhibitory concentration of fluoride ions progressively diminished as the acidity increased, the inhibitory effect of fluoride was maximal at pH 5.5 and gradually decreased at pH values (6.0-7.5). The streptococci were inhibited by low fluoride concentrations (75 ppm) at pH 5.5 than (1500 ppm) at pH 7.5, where the OD was 0.13 and 0.31, respectively. The inhibitory effects of fluoride on acid production significantly increased (P< 0.0001) when the pH dropped from 7.0 to 5.5 (Fig. 4).
0.9 0.8 0.7 0.6 0.5
OD
control 75ppm 150ppm 300ppm 600ppm 1200ppm 1500ppm

0.4 0.3 0.2 0.1 0

5.5

6.5

7.5

pH various initial pH after 48 hrs.

pH

Fig (4): Effect of NaF different concentrations on Streptococcous mutans growth at Fig. 4. Effect of different NaF concentrations on Streptococcus mutans growth at various initial pH after 48 hrs.

At pH 7.0 there was significant decrease (P< 0.0001) in final pH with the increase of F- concentration. The final pH was 5.89 with 1500 ppm F-, where it was 5.66 with 1200 ppm F- and it decreased to reach 4.84 with 75 ppm F after 48hrs (Fig.5). The greatest effect of NaF was at pH 5.5 for all concentrations tested. As the pH decreased the inhibition effect of NaF increased and the smallest concentration used (75 ppm F-) gave OD 0.125, where it gave OD

1012 T. M. EL-MONGY& A. B. ABD EI-AZIZ /J. Rad. Res. Appl. Sci., Vol. 2, No.5(2009)
0.705 at pH 7.5. By increasing the concentration of NaF from 75 ppm to 300 ppm at pH 5.5 the OD was 0.08 and it was 0.046 with 1500 ppm F- after 48 hrs (Fig.5).
8 7

6 5

pH

4
BHI

F75ppm F150ppm

2 1 0 0

F300ppm F600ppm F1200ppm F1500ppm

10 time (hrs) Times (hrs)

14

20

24

48

Fig. 5. Effect of different Fig (5): Effect of NaF on growth media pH Streptococcus mutans NaF concentrations on pH values of growth media.

Three hundred ppm NaF was taken as the lowest effective concentration of F and its inhibitory effect in combination with others antimicrobial agents was tested. From Table 2. Fluoride (300 ppm) and xylitol 5% together have synergistic inhibitory effects on the mutans streptococci growth as the inhibition percentage increased by 87.14 more than NaF alone (300 ppm) as control medium at pH 7.0.
-

Metal ions were evaluated in combination with NaF as potential antimicrobial agents for growth inhibition of S. mutans. The inhibition percentage increased by 68.59, 84.53 and 53.13 with 1.0 mM of ZnCl2, 5.0 mM of CaPO4 and 5.0 mM of NaNO2, respectively. Meanwhile, MgSO4 and CuSO4 at concentration 0.5 mM and 0.2 mM with NaF having little inhibitory effect by 27.81% and 21.09%, respectively and the synergetic effect of 5.0 mM CaCO3 was 2.65% and can be neglected (Table 2). The final pH of tested bacteria broth culture in the presence of nitrite resulted in a higher pH (6.31) than was found in controls (pH 4.81) after 10 hrs. Presence of nitrite resulted in considerable reduction of acid production with the

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tested strain. With ZnCl2, CaPO4, and NaNO2, the pHs were pH 4.52, 6.69 and 7.08, respectively after 48 hrs of cultivation. Meanwhile, with MgSO4 and CuSO4 the final pH values were 4.57 and 6.44, respectively (Table1).
Table 2. Comparison of metal salts and xylitol inhibition effect after 48 hrs at pH 7.0 with (300 ppm NaF) as control medium.
Metals salts 1.0 mM ZnCl2 5.0 mM CaPO4 0.5 mM NaNO3 0.5 mM MgSO4 5.0 mM CaCO3 0.2 mM CuSO4 5% xylitol Inhibition % 68.59 84.53 53.13 27.81 2.65 21.09 87.14

*Significance change from control group at P< 0.0001.

Table 3. ANOVA Statistics

R-Square ANOVA SS

Mean Square

F-ratio

Mean

Root MSE

Treatment BHI (control) Glucose (5%) Glucose + xylitol (5%) Fructose 5% Fructose + xylitol (5%) Sucrose (5%) Sucrose + xylitol (5%) Maltose 5% Maltose+ xylitol (5%) Sorbitol (5%) Sorbitol + xylitol (5%) Xylitol (2.5%) Xylitol (5%) Xylitol (10%) ZnCl2 NaNO2 MgSO4 CuSO4 CaPO4 CaCO3

0.931 1.263 0.323 0.573 0.449 0.758 0.486 0.845 0.693 0.231 0.077 0.281 0.092 0.049 0.209 0.095 0.186 0.220 0.019 0.368

0.519 0.610 0.306 0.413 0.389 0.449 0.386 0.318 0.297 0.280 0.193 0.236 0.158 0.130 0.428 0.317 0.371 0.432 0.137 0.498

0.133 0.105 0.027 0.048 0.037 0.063 0.040 0.070 0.058 0.019 0.011 0.040 0.013 0.007 0.030 0.013 0.027 0.031 0.003 0.053

0.018 0.018 0.012 0.012 0.021 0.011 0.023 0.009 0.020 0.012 0.016 0.020 0.011 0.006 0.004 0.013 0.009 0.006 0.006 0.012

404.84 309.69 194.60 332.33 84.59 527.71 78.46 869.10 148.80 141.94 41.54 95.19 111.74 177.91 201.51 83.91 326.48 743.13 84.11 346.79

0.997 0.996 0.994 0.996 0.987 0.997 0.986 0.998 0.992 0.992 0.976 0.989 0.991 0.994 0.999 0.988 0.996 0.998 0.988 0.997

3.49 3.02 3.83 2.90 5.40 2.43 5.88 2.83 6.63 4.15 8.46 8.70 6.89 4.85 0.89 4.00 2.43 1.50 4.14 2.47

CV

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Ultra structure Examination of S. mutans exposed to xylitol, glucose, fructose and sorbitol at concentration 5%, NaF (300 ppm) and different metals salts which gave high synergetic effect with NaF (calcium, zinc and nitrite) or control medium (BHI) for 24 hrs by electron microscopy (EM photos,1-8) indicated that a xylitol-containing reaction mixture caused distinct alterations in bacterial ultra structure without notable effect on the total viability of the strain and the cell wall of S. mutans became more diffuse, the proportion of damaged S. mutans increased. Degrading cells, autolysis, and intracellular vacuoles were frequently seen in the reaction mixtures after exposure to xylitol for 24 hrs, but not after exposure to other sugars or control medium. The Streptococcus mutans in the medium containing xylitol and sorbitol remained viable until the end of the test. The autolysis of Streptococcus mutans was seen in BHI and in the medium containing 5% sorbitol, which is a typical phenomenon in streptococci cultures after rapid logarithmic growth and which is thought to be mediated by autolytic enzymes. The bacteria in the media containing an extra carbon source for growth, i.e., fructose, glucose, or sucrose at concentration of 5%, had high OD values at the end of the experiment without autolysis. Fluoride ion (F-) was more effective in growth inhibition than the non xylitol(X), non (X)Non F-. XF- was more effective than F- alone, and combination of metals salts with F- X was the most effective in the inhibition of Streptococcus mutans growth and the rate of acid production. Examination of S. mutans exposed to F- X- metal salts mixture for 5min, at pH 5.0 and 7.0, by electron microscopy indicated complete damaged of the microbial cells.

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1-Effect of glucose on Streptococcus mutans

2-Effect of BHI on Streptococcus mutans

3-Effect of CaPO4 on Streptococcus mutans 4-Effect of ZnCl2 on Streptococcus mutans

5-Effect of Sorbitol on Streptococcus mutans

6-Effect of Fructose on Streptococcus mutans

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7-Effect of NaNO3 on Streptococcus mutans

8-Effect of xylitol on Streptococcus mutans

9-Effect of NaF (300 ppm F) at pH 7.0 on Streptococcus mutans

10-Effect of NaF (300 ppm F) at pH 5.0 on Streptococcus mutans

11-Effect of combined formula at pH 5.0 on Streptococcus mutans

12-Effect of combined formula at pH 7.0 on Streptococcus mutans

Effect of different treatments on Streptococcus mutans Ultra structure. (Electron microscope photos, 1-12).

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DISCUSSION Sugars and other fermentable carbohydrates, after being hydrolyzed by salivary amylase, provide substrate for the action of oral bacteria, which in turn lower the plaque and salivary pH. The resultant action is the beginning of tooth demineralization (6). Our study indicates that xylitol causes marked inhibition of S. mutants growth while, glucose, sucrose, and maltose had no effect on this inhibition effect of xylitol which was eliminated by fructose. Sorbitol as sugar alcohol has no effect on the bacterial growth in this study (Fig.1). Acid production by S. mutants was increased by tested dietary sugars, and reduced by tested sugars alcohols (Table-1). Also, the present data revealed that glucose and maltose give similar falls in pH, while fructose had only slightly smaller effects compared with sucrose. In other study pure culture of S. mutants was found to produce a moderate to large decrease in pH with glucose meanwhile, galactose produces a significantly smaller decrease in pH than does glucose (7). In another study the sugar alcohols, sorbitol and mannitol reported to slowly fermented to acid by oral bacterial, and xylitol is virtually nonfermentable (8). From Table-1, it is clear that sorbitol gave a small pH drop whereas xylitol caused a negligible decrease in pH and addition of xylitol to the bacterial suspension caused inhibition of acid production from sorbitol by S. mutans. Our present data are in agreement with previous study, where Streptococcus mutans cells grown on a medium containing xylitol and the mixture of sorbitol and xylitol formed less acid from glucose(9). In addition, from the present data in Table-1, it was noticed that the only mixtures which increased the pH values of the suspensions were those containing xylitol alone or mixtures of xylitol and sorbitol. Xylitol was not able to inhibit the acid production from the easily fermented glucose and fructose. Addition of small amounts of xylitol to the sorbitol cultures gave a growth below that of cultures with no extra carbon (10). The first step of xylitol metabolism in mutans group streptococci is entry of xylitol into the bacterial cell via the fructose phosphotransferase system and xylitol does not cause growth inhibition in the presence of fructose(8). Xylitol

1018 T. M. EL-MONGY& A. B. ABD EI-AZIZ /J. Rad. Res. Appl. Sci., Vol. 2, No.5(2009)
retarded the growth of mutans streptococci in the presence of glucose, galactose, maltose, lactose (L) or sucrose as an energy source. The addition of sorbitol at concentrations of 1, 2.5 or 5% to the growth medium did not affect the growth of Streptococcus pneumoniae and neither inhibited nor enhanced the xylitol induced growth impairment (9). Xylitol inhibited acid production by washed cells of Streptococcus mutans from glucose, galactose, maltose, lactose or sucrose (12-83% inhibition). However, in the presence of Fr, no inhibition of acid production was observed (10). Xylitol was reported to inhibit the glycolysis and growth of S. mutans (direct inhibition) and inhibit the rate of acid production from glucose, with a decrease in the intracellular level of fructose 1, 6-bisphosphate and an intracellular accumulation of xylitol 5-phosphate (X5P) (indirect inhibition) (11). Many elements have been examined for their inhibitory effects upon caries, but relatively few have shown significant results. More than any other element, fluorine has been the subject of attention and investigation. This study compared the effect of different concentrations of NaF at different initial pH and the present data reveled that, as the acidity of the growth culture increased the effect of low concentrations of F- increased (Figs. 4, 5). In comparison, the inhibitory effect of NaF, SnF2, and SnC12 on the growth of Streptococcus mutans was studied in vitro and the results showed that, sodium fluoride arrested the growth at concentrations 300ppm and 600 ppm F- and it showed some bactericidal activity at 150 and 300 ppm F-, and at 600 ppm F- was totally bactericidal (12). Previous report has shown that the cell membrane is impermeable to the fluoride anion and that intracellular accumulation of fluoride depends on translocation of hydrogen fluoride across the membrane (13). Also, it was investigated that the inhibitory effects of fluoride on acid production increased when the pH dropped from 5.5 to 4.0. A concentration of 5 ppm fluoride inhibited the acid production activity of Streptococcus mutants at pH 5 and below. These results suggest that fluoride in dental plaque may affect acid production below pH 5.5 (14). In another study 1 mML-1 fluoride, used with the glucose or provided continuously, reduced both the rate of change and the degree of fall in pH, and in doing so prevented the enrichment of S. mutans in the culture (15).

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This study indicates that fluoride and xylitol together have synergistic inhibitory effects on the acid production of S. mutans and xylitol has the potential to enhance the inhibitory effects of low concentrations of fluoride (Table-2). It is clear that xylitol effect was most marked in the presence of fluoride. Under these conditions, the rate of lactate production was reduced at least 3fold, 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 metabolic effects on S. mutans of low levels of fluoride (16). In a previous study, demineralization is inhibited and remineralization is accelerated when 5% xylitol is used with (500 ppmF-) fluoride, compared to toothpastes containing 500 ppm F- only in vitro. Toothpaste containing 500 ppm F- and 5% xylitol might be beneficial, both with respect to its caries inhibiting effect and decrease in the risk of dental fluorosis (17). In another study, the combination of fluoride and xylitol (fluoride (0-6.4 mM) and/or xylitol (60 mM)) inhibited acid production more effectively than fluoride or xylitol alone. Analyses of intracellular glycolytic intermediates revealed that xylitol inhibited the upper part of the glycolytic pathway, while fluoride inhibited the lower part. This study indicates that fluoride and xylitol together have synergistic inhibitory effects on the acid production of mutans streptococci and suggests that xylitol has the potential to enhance inhibitory effects of low concentrations of fluoride (18). Inhibition of the formation and metabolism of dental plaque by zinc salts has been well documented (3, 19). The mechanisms remain unclear, but studies suggest that the inhibition of acid production is correlated with adsorption of zinc on the bacterial cell wall (19). Factors other than concentration may affect on zinc action such as the difference in zinc salts applied and methods of application. Zinc may also reduce plaque bulk by inhibiting extra cellular polysaccharide-producing enzymes of plaque organisms (20). The effect of various metallic salts upon growth and acid production has been investigated. In the present study, inhibition was high with CaPO4, and NaNO2, respectively, moderate with ZnCl2 and MgSO4 and low with CaCO3 and CuSO4 (Fig.3). Other study reported that copper, nickel; gold, silver, and mercury salts exerted the greatest inhibitory action on acid production.

1020 T. M. EL-MONGY& A. B. ABD EI-AZIZ /J. Rad. Res. Appl. Sci., Vol. 2, No.5(2009)
Magnesium, cobalt, manganese, aluminum, iron, and chromic salts showed little or no activity. Copper at a concentration of 0.25 mg. per 100 ml. of saliva containing sucrose definitely inhibited acid formation while a concentration of 3 to 4 mg per 100 ml of saliva caused complete inhibition of acid production (21). In the present study, the final pH of tested bacteria broth culture in the presence of nitrite resulted in a higher pH (6.31) than was found in controls (pH 4.81) after 10 hrs (Table-1). It has been reported that nitrite at acidic pH has been shown to be antibacterial particularly against the highly cariogenic species Streptococcus mutans (22). At an acid pH, nitrite is converted to nitrous acid and hence to nitric oxide (NO) and nitrogen dioxide (NO 2) radicals which are harmful to bacteria S. mutans (23). The NO radical can react with various other substances, e.g. super oxide (O2), iron and thiols. All of these interactions prove damaging to various cellular targets. NO2 is also able to interact with DNA causing deamination or cross-linking (24). At acidity levels below pH 5.0, low concentrations of nitrite (0.2 mM) caused effective complete killing of the periodontal bacteria (25). Calcium has been implicated in cariostasis by the formation of labile reaction products with fluoride enhancing its uptake (26). The acidic calcium phosphate (containing 0. 7 M Ca, 1.9 M PO4) treatment markedly enhanced the ability of the enamel to acquire fluoride and their protection effect could be conferred by direct and indirect effects and can exert antimicrobial effects, including glycolysis inhibition (27). Mg2+ and phosphate also were important for inhibition and they formulated a modulus for predicting inhibition based on the concentrations of three key agents, F-, Mg2+, and phosphate. In a previous study, inhibition has been found to occur at 5 mM inorganic phosphate and 2 mM Mg2+ in the presence of 16-54 mM fluoride (28). It has been investigated that xylitol, NaF and ZnCl2 in combination inhibited the growth of S. sobrinus OMZ 176 when added to Brain Heart Infusion broth. Fluoride was bactericidal only at pH 4.0 (29). However, the combination of zinc plus fluoride was strongly bactericidal at all pH values that were tested. 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

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the inhibition of growth and it suggested that xylitol can augment the metabolic effects on S. mutans of low levels of fluoride (30). In the present study the ultra structure of S. mutans bacteria revealed that, xylitol has a harmful effect on the bacterial cell wall and the proportion of damage increased after exposure to xylitol for 2 hrs, as compared with other sugars or control medium S. mutans exposed to 5% xylitol for 2 hrs failed to form the longer chains comprising 4 to 6 cocci at a time that were seen in the control media. The ultra structure of the bacteria was not damaged after exposure to 5% glucose or 5% fructose, but after sorbitol exposure the cell wall structure became slightly more diffuse as compared with xylitol and control media (BHI). Previous reports have shown that the cell wall of five strains of pneumococci exposed to 0.5%-5% xylitol and glucose, fructose and sorbitol at concentration 5% or control medium (BHI) for 0.5-2 hrs became more diffuse and less well defined; the main diameter of the polysaccharide capsule became ragged and small as compared with the control media after exposure to xylitol for 2 h, but not after exposure to other sugars or control medium. The phenotype of all pneumococcal strains was opaque before xylitol exposure and became almost transparent both in xylitol and in control medium during the experiment (31). In another study, a xylitol-containing reaction mixture caused distinct alterations in bacterial ultra structure without notable effect on the total viability of the strain. Incubations in media containing 50 mg/ml of glucose, fructose, sucrose, lactose, sorbitol or mannitol as the primary carbon source did not affect bacterial ultra structure. Xylitol degrading cells and autolysis, intracellular vacuoles and lamellate formations in the cytoplasmic membrane were frequently seen independent of the concentration of xylitol in the reaction mixtures. Despite the alterations in ultra structure of the xylitol-incubated bacteria, there was no difference in their viability when compared to the controls (32). CONCLUSION The experimental results in our study support the combined effectiveness of the ingredients that the literature suggests. Calcium phosphate reduces demineralization of teeth and plaque formation through its bactericidal effect on

1022 T. M. EL-MONGY& A. B. ABD EI-AZIZ /J. Rad. Res. Appl. Sci., Vol. 2, No.5(2009)
anaerobic microorganisms and work as remineralizing agent. Zinc chloride reduces plaque and decalcification via its extra- and intra-cellular attachment, disrupting the metabolic activity of the microbiota responsible for plaque. Xylitol inhibits bacteriological growth. Zinc chloride enhances the activity of the sodium fluoride and adds its own antimicrobial properties, and sodium nitrite serves as a desensitizer that also complexes the metals, enhancing their activity on the enzymes of the microbiota. The five ingredients work together to reduce etiological factors, microbiota and their by-products, plaque and decalcification. In this way, they combine to aid in the maintenance of a normal host/parasite balance. REFERENCES 1. Burguera-Pascu, M., Rodrguez-Archilla, A., Burguera, J. L., Burguera, M., Rondon, C. and Carrero, P. (2007) Flow injection online dilution for zinc determination in human saliva with electro thermal atomic absorption spectrometry detection. In: Tenth flow injection analysis international conference, Porto, Portugal, 38 September.204-210. 2. Nakajoa, K., Imazatob, S., Takahashib, K., Kibab, W., Ebisub, S. and Takahashia, N. (2009) Fluoride released from glass-ionomer cement is responsible to inhibit the acid production of caries-related oral Streptococci. Dental., 1428: 412-418. 3. Giertsen, E. (2004) Effects of mouth rinses with triclosan, zinc ions, copolymer and sodium lauryl sulphate combined with fluoride on acid formation by dental plaque in vivo. Caries Res., 38:430-5. 4. Watanabe, M., Asatsuma, M., Ikui, A., Ikeda, M., Yamada, Y. and Nomura, S. (2005) Measurements of several metallic elements and matrix metalloproteinases (MMPs) in saliva from patients with taste disorder. Chem. Senses., 30:121-5. 5. SAS program 6.1., SAS institute, Cary, NC (1998) One-way analysis of variance (ANOVA) using SAS program 6.1. 6. Touger-Decker, R. and van Loveren, C. (2003) Sugars and dental caries. Am. J. Clin. Nutr., 78: S881-92. 7. Sheiham, A. (2001) Dietary effects on dental diseases. Public Health Nutr., 4: 569-91.

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8. Edgar, W. M. and Dodds, M. W. (1985) The effect of sweeteners on acid production in plaque. Int. Dent. J., 35 (1): 18-22. 9. Tapiainen, T., Kontiokari, T., Sammalkivi, L., Sammalkivi, L. Ikaheimo, I., Koskela, I. and Uhari, M. (2001) Effect of xylitol on growth of Streptococcus pneumoniae in the presence of fructose and sorbitol. Antimicrobial Agents and Chemotherapy, 45: 1669. 10. Kakuta, H., Iwami, Y., Mayanagi, H. and Takahashi, N. (2003) Xylitol inhibition of acid production and growth of mutans Streptococci in the presence of various dietary sugars under strictly anaerobic conditions. Caries Res., 37 (6): 404-9. 11. Miyasawa, H., Aizawz, S. and Takahashi, N. (2006) Difference in xylitol sensitivity of acid production among Streptococcus mutans strains, and its biochemical mechanism. Oral Microbiol. Immunol., 21 (4): 201-5. 12. Marquis, R. E., Sarah, A, C. and Mota-Meira, M. (2003) Fluoride and organic weak acids as modulators of microbial physiology. FEMS Microbiology Reviews., 26(493): 530-535. 13. Germaine, G, R. and Tellefson, L, M. (1986) Role of the cell membrane in pH-dependent fluoride inhibition of glucose uptake by Streptococcus mutans. Antimicrobial Agents and Chemotherapy, 29 (1): 58-61. 14. Okuda, K. and Frostell, G. (1982) The effect of fluoride on the acid production of Streptococcus mutans and other oral Streptococci. Swed. Dent J., 6 (1):29-36. 15. Bradshaw, D. J., Marsh, P. D., Hodgson, R. J. and Visser, J. M. (2003) Effects of glucose and fluoride on competition and metabolism. FEMS Microbiology Reviews, 26 (493): 507- 510. 16. Rogers, A. H. and Bert, A. G. (1992) Effects of xylitol and fluoride on the response to glucose pulses of Streptococcus mutans T8 growing in continuous culture. Oral Microbiol. Immunol., 7 (2):124-6. 17. Sano, H., Nakashima, S., Songpaisan, Y. and Phantumvanit, P. (2007) Effect of a xylitol and fluoride containing toothpaste on the remineralization of human enamel in vitro. J. Oral Sci., 49 (1):67. 18. Maehara, H., Iwami, Y., Mayanagi, H. and Takahashi, N. (2005) Synergistic inhibition by combination of fluoride and xylitol on glycolysis by mutans Streptococci and its biochemical mechanism. Caries Res., 39:5218.

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19. He, G., Pearce, E. I. F. and Sissons, C. H. (2002) Inhibitory effect of ZnCl2 on glycolysis in human oral microbes. Arch. Oral Biol., 47:117129. 20. Burguera-Pascu, M., Guez-Archilla, A.R. and Pilar, B. (2007) Substantivity of zinc salts used as rinsing solutions and their effect on the inhibition of Streptococcus mutans. Journal of Trace Elements in Medicine and Biology., 21: 92101. 21. Aranha, H., Strachan, C., Arceneaux, J. E. and Byers, B. R. (1982) Effect of trace metals on growth of Streptococcus mutans in Teflon chemostat. Infect. Immun., 35:45660. 22. Xia, D. S., Liu, Y., Zhang, C. M., Yang, S. H. and Wang, S. L. (2003) Antimicrobial effect of acidified nitrate and nitrite on six common oral pathogens in vitro. J. Dent., 31(5):367-70. 23. Doel, J. J., Hector, M. P., Amirtham, C. V., Al-Anzan, L. A., Benjamin, N. and Allaker, R. P. (2004) Protective effect of salivary nitrate and microbial nitrate reductase activity against caries. Eur. J. Oral Sci., 112 (5):424-8. 24. Li, H., Thompson, I., Carter, P., Whiteley, A., Bailey, M., Leifert, C. and Killham, K.(2007) Salivary nitrate-an ecological factor in reducing oral acidity. Oral Microbiol. Immunol., 22 (1):67-71. 25. 25- Radcliffe, C.E., Akram, N., Charlotte, E., Anthony, S. and Hurrell, F. (2003) Effects of nitrite and nitrate on the growth and acidogenicity of Streptococcus mutans. J. Dent., 53:436-53. 26. Antonucci, J. M. and Skrtic, D. (2005) Matrix resin effects on selected physicochemical properties of amorphous calcium phosphate composites. J. Bioact. Compat. Polym., 20:29-49. 27. Itthagarun, A., King, N., M., Yiu, C. and Dawes, C. (2005) The effect of chewing gums containing calcium phosphates on the remineralization of artificial caries-like lesions in situ. Caries Res., 39:251-254. 28. Watson, G. K., Cummins, D. and van der Ouderaa, F. J. G. (1991) Inhibition of acid production by Streptoccocus mutans NCTC 10449 by zinc and the effect of metal speciation. Caries Res., 25, 431437. 29. Izaguirre-Fernandez, E. J., Eisenberg, A. D. and Curzon, M. E. (1989) Interactions of zinc with fluoride on growth, glycolysis and survival of Streptococcus mutans. Caries Res., 23(1):18-25.

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30. Scheie, A. A., Assev, S. and Rolla, G. (1988) Combined effect of xylitol, NaF and ZnCl2 on growth and metabolism of Streptococcus sobrinus OMZ176. APMIS., 96 (9):761-7. 31. Tapiainen, T., Sormunen, R., Kaijalainen, T., Kontiokari, T., Ikaheimo, I., and Uhari, M. (2004) Ultrastructure of Streptococcus pneumoniae. Journal of Antimicrobial Chemotherapy, 54:225228. 32. Tuompo, H., Meurman, J. H., Lounatmaa, K., and Linkola, J. (1983) Effect of xylitol and other carbon sources on the cell wall of Streptococcus mutans. Scand. J. Dent. Res., 91(1):17-25.


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