The Antibacterial Action of the Various Components of the Lactoperoxidase System on a Cariogenic Strain of Streptococcus mutans JORMA TENOVUO and MATTI L. E. KNUUTTILA Institute of Dentistry, University of Turku, Turku, Finland

Physiological activity of lactoperoxidase and in vivo concentration of thiocyanate ions were shown to be inhibitory against a cariogenic strain of Streptococcus mutans. However, the amount of H202 in vivo may be too lowfor optimum inhibition by lactoperoxidase system. H202 alone also inhibited the growth of S mutans to some degree. J Dent Res 56(12): 1603-1607, December 1977. Lactoperoxidase, thiocyanate ions, and hydrogen peroxidase form an antibacterial system in saliva and milk.1"2 Only a few studies have dealt with the different components, the enzyme, thiocyanate, and hydrogen peroxide, of this antibacterial system. Morrison and Steele3 have observed that even very low concentrations (10-9 M) of milk lactoperoxidase inhibited the growth of lactobacilli and cocci and that the degree of inhibition was increased by increasing the concentration of the enzyme. They assumed that the role of thiocyanate ions in this system is simply to stabilize the enzyme in dilute solutions. Hoogendoorn4 has assumed that inadequate inhibition of streptococci in the mouth is the result of too low concentration of H202 produced by oral flora. Therefore, the system was activated by means of enzymes producing H202 and a significant reduction in the plaque index was observed.5 Conversely, H202 itself is antibacterial, e.g. for S cremoris at low concentrations.6 The purpose of the present study was to determine the effect of various concentrations of the components on the inhibition and to determine the optimum concentrations of these components. Differences in the degree of inhibition caused by H202 alone or by the lactoperoxidase Received for publication Septeinber 28, 1976. Accepted for publicationi Februars 1, 1977. This insestigation snas tlinancialls supported bs a grant from the Finnish Dental Societe. *BBL, Disisiol ot Bio Quest, Cockessitlle, Nid., U.S.A. tKlett-Summierssoni colorimeter (Iilter nio. 62). tSigma Cheinical Conipansv, St. Louis, Mo., U.S.A. §Analytical grade, BDH Chetnicals Lid., Poole, Eitgland.

system were studied. The knowledge of these concentrations is necessary for possible activation of this system in vivo.

Materials and Methods CULTIVATION OF S MUTANS. -S mutans (strain Ingbritt) was originally isolated from the human oral cavity and donated to this laboratory by Prof. Bo Krasse (University of Goethenburg, Sweden). The maintainance and the cultivation of the cells have been described earlier. 7 All cultivations were carried out in Trypticase-Phytone* based medium aerobically (without agitation) at 37 C. The turbidityt was followed and the pH was measured with calomel and glass electrodes at 25 C. CONCENTRATIONS OF VARIOUS COMPONENTS OF THE LACTOPEROXIDASE SYSTEM. - Commercial milk lactoperoxidase+ was used at concentrations of 2.50 x 10-9 M to 1.25 x 10-7 M, representing guaiacol units from 0.14 to 34. Peroxidase activity was determined according to the guaiacol§ method.8 Thiocyanate ions were used at concentrations of 0.31 to 3.1 mM (30 to 300 mg/l) and H202 at concentrations of 0.9 to 2.9 mM. The concentrations of iodide ions (added as KI) used in some experiments were 1, 10, and 100 ImM.

DETERMINATION OF ANTIBACTERIAL EFFECT OF THE LACTOPEROXIDASE SYSTEM. - A 15hour culture of S mutans was transferred to a

medium where the components of the lactoperoxidase system were simultaneously added. The turbidity and pH of the medium were measured. In one series of experiments, the enzyme, SCN- ions and/or H202 were added to the medium after 2-, 4- or 6-hour growth. All the combinations used in these experiments are presented in the figure legends. Results EFFECT OF LACTOPEROXIDASE CONCENTRATION ON GROWTH OF S MUTANS. - The results

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FIG 1. -The effect of lactoperoxidase (LPO) concentration on the growth of the cells of S mutans. The concentrations of H202 and KSCN were 2.4 mM and 0.93 mM, respectively. LPO concentrations: B, 0.14 U/ml; C, 1.8 U/ml; D, 5.2 U/ml; E, 11 U/ml; F, 34 U/ml. Curve A, H202 alone; curve G, control. The control culture contained none of the components.

are seen in Figure 1. The 0.14 U/ml of lactoperoxidase did not reveal any inhibition besides that caused by H202. Enzyme concentrations above 5.2 U/ml inhibited the growth of the cells almost totally for 4 to 8 hours; after that the growth increased as in the control medium. There were no noticeable differences between 11 and 34 U/ml of lactoperoxidase in the antibacterial activity. EFFECT OF THIOCYANATE IONS ON GROWTH OF S MUTANS. -There were no remarkable differences between thiocyanate concentrations of 0.93 to 3.1 mM (90 to 300 mg/i) in the ability to inhibit the growth by the lactoperoxidase system (Fig 2). As little as 0.31 mM KSCN caused a slight inhibition. It was observed again that after the inhibitory period the growth increased very quickly as in the control medium. EFFECT OF H202 ON GROWTH OF S MUTANS. Figure 3 shows that H202 alone at concentrations above 1.8 mM inhibited the growth of the cells. The amount of H202 needed for effective antibacterial activity of the lactoperoxidase system was also above 1.8 mM. There was a clear difference between the inhibitory action of H202 alone and that of the lactoperoxidase system. H202 inhibited the growth to a lower extent than the lactoperoxidase system, but the inhibitory period was clearly longer.

EFFECT OF IODIDE IONS ON GROWTH OF S - The results of the present study indicate that iodide ions at the concentrations of 1.0 to 100 ftM did not replace thiocyanate ions in the lactoperoxidase system. In this assay, containing lactoperoxidase (5.2 U), iodide ions (1.0, 10, or 100 1M), and hydrogen peroxide (2.9 mM), the growth was inhibited only by H202. Nor did iodide ions potentiate or diminish the antibacterial action of the lactoperoxidase + SCN- + H202-system to any remarkable MUTANS.

extent.

CORRELATION BETWEEN GROWTH STAGES OF BACTERIA CELLS, LACTOPEROXIDASE SYSTEM AND H202. -Figure 4 shows that if H202 or the lactoperoxidase system was added to the growth media after 2 or 4 hours growth, the inhibition was remarkably lower when the cell numbers were high. The concentrations of lactoperoxidase, SCN- and H202 were the same at all addition points (0, 2, and 4 hours). The addition of a new sample of lactoperoxidase (5.2 U/ml), after 4 and 6 hours inhibitory period, to the growth medium which already contained the complete lactoperoxidase system, shortened the inhibitory period by approximately 2 hours when compared to the growth curve obtained with the lactoperoxidase system added to the medium at 0 hour (Fig 5).

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Discussion

The role of the auxiliary cofactors, SCN- and H202, in the lactoperoxidase-mediated antibacterial system has not yet been thoroughly clarified. Oram and Reiter9 observed that the oxidation of thiocyanate by lactoperoxidase in the presence of H202 produces as oxidation products, sulfate, CO2 and ammonia. Chung and Wood'0 suggested that intermediates, especially cyanate, could have a bactericidal effect. Evidence that the inhibition is due to an inhibitor formed by the oxidation of thiocyanate by lactoperoxidase and H202 is presented.4" Regardless of the role of thiocyanate in the lactoperoxidase system, it seems to be essential to the antibacterial activity, although even low

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concentrations (< 1 mM) are sufficient. The average thiocyanate concentration in human whole saliva, 0.9 to 1.7 mM (range 0.25 to 3 mM)4-'1 seems to be quite suitable for the antibacterial action of the lactoperoxidase system on S mutans. Several studies have provided evidence that smoking increases the concentration of SCN- in human whole saliva.'2'14 Courant'4 also observed that increased thiocyanate concentration led to an increased ability of the lactoperoxidase system to inhibit the growth of Lactobacillus acidophilus. In the present study, no such linearity was found on S mutans. The activity of lactoperoxidase needed for a strong inhibition of S mutans was about 10 U/ml. The average activity in human saliva is, according to our observations, 3 to 5 U/ml, the

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FIG 3. -The effect of H202 concentration on the growth of the cells of S mutans. The concentrations of LPO and KSCN were 5.2 U/ml and 0.93 mM, respectively. H202 concentrations: A, 2.9 mM; B, 1.8 mM; C, 1.2 mM; D, 0.9 mM. E, control curve. Symbols: H202 alone, * [email protected]; LPO + KSCN +

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specific activity being 5.91 + 1.45 U/mg protein in subjects with low caries incidence. 15 An antibacterial system which is composed of myeloperoxidase, iodide ions, and H202 has been observed in leukocytes. 16 Myeloperoxidase can replace lactoperoxidase in the salivary lactoperoxidase system.2 Also thiocyanate ions could be substituted by iodide ions without loss of antibacterial activity when tested on L. acidophilus.2 The concentration of 1- in these experiments was 2 gtM. The human salivary iodide ion concentration is approximately 10 gM.'2 Iodide ions at concentrations of 1 to 100 jaM in the present study were not able to substitute for thiocyanate ions in the lactoperoxidase system tested on S mutans. Nor did these iodide concentrations diminish the antibacterial activity of the system, although it is known that there is a negative correlation between the amount of iodide and thiocyanate ions in human saliva. 12 H202 alone inhibited the growth of S mutans. The difference between the inhibitory action of H202 alone and the complete lactoperoxidase system was evidently due to the differences in H202 concentrations during the assay period. In the lactoperoxidase system, the enzyme seems to need H202 for its antibacterial action, obviously for the oxidation of thiocyanate, and thus uses up all of the H202 available in a few hours, whereafter the growth increases as in the control medium. Conversely, H202 alone inhibits the growth to a minor extent and the growth increases slowly, obviously due to slow decomposition of H202 during the cultivation. The strain of S mutans used in this study does not produce H202 itself. The amount of H202 in the oral cavity in vivo has been a matter of controversy. Kraus et al'7 assumed that the low concentration (< 1 jag/ml) in saliva is a result of destructive agencies, e.g. catalases and peroxidases, present in saliva. However, about 58% of aerobic oral bacteria produce H202. 17 The concentrations of H202 used in the present study may not be physiological, but were chosen because such amounts may occur locally in dental plaque in persons with aerobic oral flora and because reduction in the amount of dental plaque by means of enzymes producing such amounts of H202 has been achieved.4 Additionally, these in vitro tests are not possible to perform with very low concentrations of H202. The inhibitory action of the lactoperoxidase system becomes weaker as the cell number increases. Morrison and Steele3 have observed the same phenomen on S cremoris 972.

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(A) and H202 (B) on the growth of the cells of S mutans. The components of the LPO-system were added after 0 (A-A), 2 ( --) or 4 (V-V) hours growth to the medium. The respective symbols for H202: 0 (A-A), 2 (A-A) or 4 (O--) hours. The control culture, O -, presented in A contained none of the components. The concentrations of LPO, KSCN and H202 were 5.2 UJml, 0.93 mM and 2.4 mM, respec-

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FIG 5. - The effect of LPO additions during the cultivation on the growth of the cells of S mutans. Symbols: LPO + KSCN + H202 (added at 0 hour), A -A; LPO + KSCN + H202 (the components added at 0 hour and a new sample of LPO, 5.2 U/ml, after 4 and 6 hours growth), 0- *; H202, A-A; control, O -0. The concentrations of the components were the same as in Figure 4.

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The present study clearly showed that in vivo activation of the human salivary lactoperoxidase system against S mutans could be made possible by elevating the concentration of H202 to its optimum level (above 1.8 mM). The production of H202 by oral flora might frequently be too low for the necessary concentration.4

5. KOCH, G.; EDLUND, K.; and HOOGENDOORN, H.: Lactoperoxidase in the Prevention of Plaque Accumulation, Gingivitis and Dental Caries. II, OdontolRevy 24:367-372, 1973. 6. ANDERS, R. F.; HOGG, D. M.; and JAGO, G. R.: Formation of Hydrogen Peroxide by Group N

However, it is known that H202 at concentrations higher than 3 mM inactivates lactoperoxidase.4 The present study also showed that the enzyme concentration for a strong inhibition ought to be approximately 10 U/ml, i.e. 2 to 3 times higher than that normally found in human whole saliva.

7. KNUUTTILA, M. L. E., and MXKINEN, K. K.:

Conclusions The study suggests that the efficiency of lactoperoxidase system to inhibit the growth of S mutans in vivo is related mostly to the amount of H202 available. Physiological concentrations of SCN- ions and activity of salivary peroxidases are quite suitable for the antibacterial action. Iodide ions at physiological concentrations were not able to replace SCN- ions in lactoperoxidase system against S mutans although such observations have been made with lactobacilli. It seems possible that in the oral cavity the inhibition of the growth of lactobacilli and cocci can be accounted for by both lactoperoxidase system and for H202 alone. The skilled technical assistance of Mlrs. Aila Lahteeniaki and Miss Rauni Suominen is gralefull) acknoswledged.

Streptococci and Its Effect on Their Growth and Metabolism, ApplMicrobiol 19:608-612, 1970.

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References 1. REITER, B.; PICKERING, A.; and ORAM, J. D.: in Microbial Inhibitors in Food, MOLIN, N. (ed),

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Almqvist and Wiksell, Stockholm, p 297. 2. KLEBANOFF, S. J., and LUEBKE, R. G.: The Antilactobacillus System of Saliva. Role of Salivary Peroxidase, Proc Soc Exp Biol Med 118:483-486, 1965. 3. MORRISON, M., and STEELE, W. F.: Lactoperoxidase, the Peroxidase in the Salivary Gland, in Biology of the Mouth, PERSON, P. (ed), Washington, 1968, pp 89-1 10. The Effect of H.: 4. HOOGENDOORN, Lactoperoxidase-Thiocyanate-Hydrogen Peroxide on the Metabolism of Cariogenic Microorganisms In Vitro and In the Oral Cavity, Academic dissertation, Haag, Holland, 1974.

15. 16.

17.

Purification and Characterization of a Phosphatase Specifically Hydrolyzing p-Nitrophenyl Phosphate from an Oral Strain of Streptococcus mutans, Arch Biochem Biophys 152:685-701, 1972. CHANCE, B., and MAEHLY, A. C.: in Methods in Enzymology, Vol II, Academic Press, New York, 1964, p 764. ORAM, J. D., and REITER, B.: The Inhibition of Streptococci by Lactoperoxidase, Thiocyanate and Hydrogen Peroxide. The Effect of the Inhibitory System on Susceptible and Resistant Strains of Group N Streptococci, J Biochem 100:373-381, 1966. CHUNG, J., and WOOD, J. L.: Oxidation of Thiocyanate to Cyanide and Sulfate by the Lactoperoxidase-Hydrogen Peroxide System, Arch Biochem Biophys 141:73-78, 1970. HOGG, D. McC., and JAGO, G. R.: The Antibacterial Action of Lactoperoxidase. The Nature of the Bacterial Inhibitor, Biochem J 117:779-790, 1970. TENOVUO, J., and MXKINEN, K. K.: Concentration of Thiocyanate and Ionizable Iodine in the Saliva of Smokers and Non-smokers,J Dent Res 55:661-663, 1976. ARMENIO, G.; LAFORGIA, P. D.; and BUONSANTO, M.: Variation in Concentration of Thiocyanates in Relation to Tobacco Smoke, Chem A bstr47:8867, 1953. COURANT, P.: The Effect of Smoking on the Antilactobacillus System in Saliva, Odontol Revy 18:251-261, 1967. TENOVUO, J.: The Variation of Salivary Peroxidase Activities in Persons of Different Oral Health, A cta Odont Scand 34:163 -168, 1976. KLEBANOFF, S. J.: Myeloperoxidase-Mediated Antimicrobial Systems and Their Role in Leukocyte Function, in Biochemistry of the Phagocytic Process, SCHULTZ, J. (ed), North-Holland, 1970, pp 89-110. KRAUS, F. W.; NICKERSON, J. F.; PERRY, W. I.; and WALKER, A. P.: Peroxide and Peroxidogenic Bacteria in Human Saliva, J Bacteriol 73:727-735, 1957.

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The antibacterial action of the various components of the lactoperoxidase system on a cariogenic strain of Streptococcus mutans.

The Antibacterial Action of the Various Components of the Lactoperoxidase System on a Cariogenic Strain of Streptococcus mutans JORMA TENOVUO and MATT...
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