Antibacterial Effect of Lactoperoxidase and Myeloperoxidase Against Bacillus cereus

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1985, p /85/ $02.00/0 Copyright 1985, American Society for Microbiology Vol. 27, No. 1 Antibacterial Effect of Lactoperoxidase and Myeloperoxidase Against Bacillus cereus JORMA TENOVUO,1* KAUKO K. MAKINEN,' AND GUNNEL SIEVERS2 Department of Biochemistry, Institute of Dentistry, University of Turku, SF Turku,' and Department of Biochemistry, University of Helsinki, SF Helsinki,2 Finland Received 21 May 1984/Accepted 5 October 1984 An oral periodontopathic bacterium, Bacillus cereus, was inhibited both by lactoperoxidase (LP) and myeloperoxidase (MP) antimicrobial systems. With the LP-SCN--H202 system, the growth inhibition was directly proportional to the amount of OSCN- ions present. The OSCN-, which is the principal oxidation product of the LP (or MP)-SCN--H202 system at neutral ph, is a normal component of human saliva. The oxidation products of both peroxidase systems inhibited the growth of the bacteria. This inhibition was associated with reduced extracellular release of collagenase activity from the cells. With LP, the antimicrobial efficiency of the oxidizable substrates was SCN- > I-, and with MP, the efficiency was I- > Cl > SCN-, respectively. LP did not oxidize Cl1. Growing interest has recently been focused on innate, nonimmunoglobulin defense factors in the human mouth (14; I. D. Mandel and S. A. Ellison, in K. M. Pruitt and J. Tenovuo, ed., Chemistry and Biological Significance of the Lactoperoxidase System, in press) and on their possible association with dental caries (7, 17) or periodontal diseases (12, 13). However, because many innate (lysozyme, peroxidase, lactoferrin, agglutinins, etc.) and immune defense systems act in concert (Mandel and Ellison, in press), evaluation of the clinical significance of one particular factor is difficult. One of the major antimicrobial factors in human saliva is the peroxidase system (for review, see J. Tenovuo and K. M. Pruitt, J. Oral Pathol., in press). Human whole saliva contains peroxidase activity derived both from the salivary glands (28) and from the leukocytes of gingival crevices (12, 13). The substrates of the oral peroxidase system, thiocyanate ions (SCN-) and hydrogen peroxide, are also normal constituents of human saliva. Thiocyanate is derived from diet or tobacco smoke (40), and H202 is derived from bacteria (5), leukocytes (10) or from other host cells (22). Both the salivary peroxidase and the leukocyte myeloperoxidase can catalyze the oxidation of SCN- (9, 36), but myeloperoxidase is also able to oxidize Cl- that is not oxidized by the salivary peroxidase (20). At neutral ph, the principal antimicrobial product generated by the salivary peroxidase system is the hypothiocyanite (OSCN-) ion (36). The system is complex, but its net reaction is: peroxidase H202 + SCN- * OSCN + H20 The hypothiocyanite ion is a normal component of human parotid (22), submandibular (17), and whole saliva (34, 36) with normal values ranging from 10 to 250,uM (17, 22, 33, 34, 36). At low ph, the major product is hypothiocyanous acid (HOSCN) which is in an acid-base equilibrium with OSCN- (pk 5.3) (37). Due to its uncharged nature, HOSCN may penetrate microbial cell membranes more readily than OSCN- (37). This suggestion is supported by observations showing that the antibacterial effects of the peroxidase system are greater at low ph (9, 38). * Corresponding author. 96 Numerous species of oral and enteric bacteria are inhibited by the oxidation products of the salivary peroxidase system. Among these are clinically important organisms such as lactobacilli (11), streptococci (21, 31, 32, 38), actinomyces (8), and salmonellae (24). However, gram-positive, catalase-negative bacteria are resistant to inhibition (8, 38). So far, Streptococcus mutans has been used as a test organism in most studies. Our study is the first to demonstrate the inhibition of growth and collagenase excretion of a periodontopathic bacterium, Bacillus cereus, by the peroxidase system. Because the human salivary peroxidase has never been successfully purified, we used bovine milk lactoperoxidase (LP) as a source of the enzyme activity. However, LP and human salivary peroxidase are catalytically very similar (32), albeit the enzymes are probably not identical (25). No difference exists in the nature of the antimicrobial oxidation products generated by these two enzymes (22, 32). On the other hand, leukocyte myeloperoxidase (MP) has a slightly different substrate specificity (10); therefore, the antimicrobial effect of purified human myeloperoxidase against B. cereus was also investigated. MATERIALS AND METHODS Reagents. Yeast extract, casein, and Casamino Acids (vitamin free) were obtained from Difco Laboratories (Detroit, Mich.). D-Glucose was from BDH Chemicals Ltd. (Poole, United Kingdom). 2-Mercaptoethanol, the collagenase substrate, and its cleavage product that was determined in the assay (see below) were purchased from Fluka (Buchs, Switzerland). The lactoperoxidase, prepared from bovine milk, was a product of Sigma Chemical Co., St. Louis, Mo. The purity index, A412/A280, of the preparation was Myeloperoxidase was purified from normal human leukocytes by the method of Bakkenist et al. (3). The purity index, A430/A280, of MP was ABTS [2,2'-azino-di(3-ethylbenzthiazoline- 6-sulfonic acid)] and catalase (from bovine liver) were products of Sigma. Hydrogen peroxide was purchased as a 30% solution (E. Merck AG, Darmstadt, Federal Republic of Germany) and stored at 4 C. Reduction of 5,5'-dithiobis(2- nitrobenzoic acid) (Aldrich Chemical Co., Milwaukee, Wis.) to 5-thio-2-nitrobenzoic acid with 2-mercaptoethanol was

2 VOL. 27, 1985 done as described previously (34). All reagents were of analytical grade. The water used was distilled and deionized. Growth conditions of B. cereus. The Bacillus sp. strain Soc 67, identified as B. cereus (29), was a kind gift of W. Loesche, University of Michigan. More detailed information on this strain was given in a previous paper from our laboratory (29). The strain was originally isolated from deep gingival pockets of a patient suffering from juvenile periodontitis. The bacteria were grown overnight in a complex medium [1 g of (NH4)2SO4, 5 g of NaCl, 3 g of KH2PO4, 7 g of K2HPO4 * 3H20, 100 mg of MgSO4 * 7H20, 1 g of Casamino Acids, 30 mg of tryptophan, 0.5% yeast extract; all in 1 liter of water] supplemented with 2% D-glucose (ph 6.8). For inhibition studies, a sample of the overnight culture was transferred to a fresh medium and incubated at 37 C in a shaking water bath (150 strokes per min). Antibacterial effect of the peroxidase systems on B. cereus. Immediately after inoculation of bacteria to the fresh medium, the medium was supplemented with various combinations of the LP or MP system components. Growth, ph, and extracellular collagenase activity were followed for 6 to 7 h at intervals of 1 h. The growth was measured as turbidity with a Klett-Summerson colorimeter (filter no. 62). In standard experiments, the medium contained enzyme (ca. 2,ug/ml), SCN- (1 mm), and various amounts of H202 (0.025 to 0.3 mm) to generate concomitantly various amounts of OSCN-. The possible inhibitory effect of the individual components of the peroxidase systems was tested separately. In all experiments, the medium inoculated with bacteria was first supplemented with peroxidase and SCN- (diluted in phosphate-buffered saline [ph 7.0]), followed by the addition of H202 (in distilled water) to activate the system. For assay of the total amount of OSCN- (or OCI-) generated, a sample was taken 1 min after the addition of H202 from an identical growth medium containing all the same ingredients except the cells. Chemical assays. Peroxidase activity was measured with ABTS as a donor (15). Enzyme activity calculations were based on the first 15 s of the reaction during which the A412 versus time curves were linear. One enzyme unit is equivalent to a change in A412 of 32.4 per min and corresponds to the amount of enzyme catalyzing the oxidation of 1 mmol of substrate per min under the assay conditions. This definition assumes an extinction coefficient of M-1 cm-' for ABTS at 412 nm. All spectrophotometric measurements were done with a Bausch & Lomb spectrophotometer. OSCN- and OC1- were assayed by reaction with 5-thio- 2-nitrobenzoic acid, the colored anionic monomer of 5,5'- dithiobis(2-nitrobenzoic acid), as originally described by Aune and Thomas (2). A slight modification of this method (34) was used in the present study. The reaction mixture was 64,uM 5,5'-dithiobis(2-nitrobenzoic acid) and 60,uM 2-mercaptoethanol in 2.0 ml of 0.1 M Tris-hydrochloride buffer (ph 8.0) and contained 50,ug of catalase per ml. The catalase was added to destroy possible residual H202 which would interfere with the assay. The concentration of 5-thio-2- nitrobenzoic acid was calculated assuming a molar extinction coefficient of at 412 nm (34). The extracellular collagenase activity of the cultures of B. cereus was tested in principle by the method of Wunsch and Heidrich (41) with a synthetic peptide derivative as the substrate (4-phenylazobenzyloxycarbonyl-L-prolyl-Lleucylglycyl-L-prolyl-D-arginine). In this method, the substrateis hydrolyzed, forming 4-phenylazobenzyloxycarbonyl- PEROXIDASES AND BACILLUS CEREUS 97 L-prolyl-L-leucine and a tripeptide (14). The spontaneous hydrolysis of the substrate was constantly followed and found to be negligible. Appropriate blanks were included in all determinations. At high concentrations (- 5 mm), SCNinterferes with this method, but these effects were corrected by including proper blanks. However, the SCN- levels that induced the formation of physiological amounts of the inhibitory oxidation products (notably OSCN-) did not exert any remarkable interference. The enzyme activities were expressed as A320. RESULTS Effect of LP-SCN--H202 system on bacterial growth. The lactoperoxidase enzyme (2,ug/ml) alone did not affect the bacteria in any way. Thiocyanate ions at high concentrations (30 mm) had a slight inhibitory effect on the growth (Fig. 1A), but the bacterial cells were clearly sensitive to H202 at concentrations higher than 0.5 mm (Fig. 1B). H202 at 10 mm completely inhibited the growth for 7 h A TIME (h) FIG. 1. Effect of SCN- and H202 on the growth of B. cereus. The calculated final concentrations of SCN- and H202 in the media are indicated. SCN- (in phosphate-buffered saline [ph 7.0]) and H202 (in distilled water) were added to the media 1 min before inoculation with bacteria.

3 98 TENOVUO, MAKINEN, AND SIEVERS 150 A620 OSCN- (NM) CONTROL TIME (h) FIG. 2. Effect of OSCN- ions on the growth of B. cereus. The media were supplemented with LP (2 plg/ml) and SCN- (1 mm). Various amounts of H202 (calculated final concentrations, to 0.3 mm) were added 1 min before the addition of bacteria. The amount of OSCN- was assayed from the growth medium just before the bacteria were added. Based on the effects of the individual components of the LP system, the amounts of LP and SCN- selected for standard experiments were 2,ug/ml and 1 mm, respectively. The amount of H202 was varied to generate various amounts of the inhibitor, the OSCN- ion. The inhibition of bacterial growth increased with increasing OSCN- concentration in the growth medium (Fig. 2). When the bacterial growth was plotted against the initial concentration of OSCN- (measured in the absence of bacteria) at 1-h intervals after the cultivation was started, regression lines with high correlation coefficients (0.750 to 0.937) were obtained (Fig. 3). The intercepts on the x-axis indicated that the amounts of OSCN - required for complete inhibition for 2, 3, 4, 5, and 6 h were 116, 127, 138, 150, and 191,uM, respectively. This suggests that during the log phase, an OSCN- concentration ca. 10,uM higher was required for each additional 1-h growth delay. When high concentrations of OSCN- (356 pm) were generated during the mid-log phase (2 h after inoculation of bacteria, A620 = 40), OSCN- could completely stop the growth for several hours (not shown). Even with these high concentrations of OSCN-, all OSCN- disappeared from the medium in less than 1 h. When OSCN- was generated in the presence of the bacterial cells (H202 was added slowly to the medium which was supplemented with bacteria, enzyme, and SCN-), the growth delay was about 30 min longer than with the same amount of OSCN- generated before the cells were added. The effect of ph on bacterial growth was also studied. In standard experiments, the ph of the medium (6.8) decreased markedly, to ca. ph 5.5, as the growth increased. However, the drop in ph did not affect the growth since no difference was observed between cultures in which ph was kept constant (ph 6.5) with small additions of solid sodium carbonate and those in which ph was allowed to drop. Addition of a reducing agent, 2-mercaptoethanol, to the cultures did not restore the growth which was inhibited by OSCN-. This was studied with 2-mercaptoethanol concentrations of 0.01 to 0.5 mm. The addition of 2-mercaptoethanol was made 3 or 5 h after inoculation to the cultures which were inhibited by OSCN- (148,uM). Effect of the LP-SCN--H202 system on extracellular collagenase. In control experiments, the extracellular collagenase activity reached its maximum after 4 h of growth. Thereafter, the activity decreased, although the bacterial growth still continued. Inhibition of growth by the LP system caused a respective delay in the extracellular release of bacterial collagenase. Effect of MP-halide (or SCN-)-H202 system on bacteria. The inhibitory effect of the MP system against B. cereus was studied with SCN-, Cl-, and I- as oxidizing substrates. MP alone or any of the substrates per se did not affect the bacterial growth at the concentrations used. In all experiments, the amount of MP was comparable to that of LP in previous experiments; also, the activities of these two enzymes were similar (about 250 mu/ml) with ABTS as a substrate. The growth medium contained 0.09 M NaCl. When the medium was supplemented with MP and H202 (final concentration, 0.1 mm) immediately before inoculation with bacteria, only a small yield of OCl- (6.5 pum) was observed. This caused, however, ca. 1 h of growth delay when compared to the control experiment. When MP was replaced by LP, no OCl- could be detected. Higher concentrations of Cl- (0.2 M) and H202 (0.3 mm) in the medium did not remarkably enhance either OCl- generation (maximum, 10 pim) or growth inhibition. The MP-1--H202 system seemed to be a more potent inhibitor of B. cereus than was the LP-1--H202 combination (Fig. 4). Identical concentrations of I- and H202 caused 120 A [OSCN] rm ANTIMICROB. AGENTS CHEMOTHER FIG. 3. Regression lines of bacterial growth (A620) versus initial concentration of OSCN- (measured in the absence of bacteria) in the medium after 2, 3, 4, 5, and 6 h of growth. The number of individual measurements for each slope varied from 43 to 50, the results being obtained from 10 to 12 different experiments. The correlation coefficients for various times were as follows: 2 h, ; 3 h, ; 4 h, ; 5 h, ; and 6 h, The A620 without bacteria is subtracted from values on the y axis.

4 VOL. 27, 1985 longer growth delay with MP than with LP. On the other hand, when SCN- was used as a substrate, LP was much more effective in producing OSCN- than was MP (Table 1). However, even a small yield of OSCN- by the MP system resulted in a relatively strong inhibition. This inhibition could not result from unreacted H202, since 0.2 mm peroxide caused only about 15% inhibition of growth at mid-log phase. In accordance with the results obtained with the LP system, in experiments with MP, the extracellular release of collagenase was also directly proportional to the degree of growth inhibition. DISCUSSION Metabolic activities and growth of numerous gram-positive and gram-negative bacteria are inhibited by the LP- SCN--H202 or the MP-halide-H202 systems (for review, see reference 10 and K. M. Pruitt and B. Reiter, in K. M. Pruitt and J. Tenovuo, ed., The Lactoperoxidase System: Chemistry and Biological Significance, in press, and J. Tenovuo and K. M. Pruitt, J. Oral Pathol., in press). In general, bacteria grown anaerobically are more susceptible to inhibition than are those grown aerobically (5). Resting cells or cells in the stationary phase are less susceptible to killing or inhibition than are growing cells (24). Several studies have shown that gram-negative bacteria are less sensitive to the inhibitory effects of the peroxidase systems than are grampositive bacteria. However, gram-negative bacteria can also be killed by the LP system (19, 35; Pruitt and Reiter, in press), especially when the permeability of the bacterial cell envelope is increased (35; Pruitt and Reiter, in press). Clearly, increased permeability is associated with increased TIME (h) FIG. 4. The growth of B. cereus (A620) as affected by LP-1-- H202 and MP-I--H202 systems. The activities of LP and MP added to the medium were comparable (250 mu/ml). The added concentration of 1- was 1 mm in all experiments. The bacteria were added 1 min after the peroxidase systems were activated by H202. PEROXIDASES AND BACILLUS CEREUS 99 TABLE 1. Effect of LP-SCN--H202 and MP-SCN--H202 systems on the growth of B. cereus System' H202 (mm)b OSCN- (um)c % Inhibition' LP-SCN MP-SCN 'The level of SCN- was 1 mm (calculated final concentration in the medium), and the activities of LP and MP added to the medium were about the same (250 mu/ml). b Added slowly to prevent possible toxicity of H202. The amounts are calculated final concentrations in the medium. c Assayed 1 min after addition of H202 and immediately before the bacteria were added. dgrowth inhibition compared to that of the control culture (with no peroxidase system components) at mid-log phase (after 4 h of growth). susceptibility to both LP systems (24, 35) and MP systems (27). Our results with a gram-positive bacterium, B. cereus, are in accordance with earlier results obtained with other oral pathogenic bacteria (Pruitt and Reiter, in press). The amount of OSCN- required for complete growth inhibition for 6 h was about 200,uM. In human saliva, the levels of OSCN- range from 10 to 250 p.m (17, 22, 33, 34, 36) with an average of ca. 40,uM (34). Results of recent studies indicate that it is possible to increase the amount of OSCNin vivo above 100,uM with the aid of a mouthrinse containing low amounts of SCN- and H202 (18). Thus, at least in theory, it seems possible to prevent the metabolism, growth, or both of many oral pathogens, including B. cereus, in human whole saliva with a simple mouthrinse technique. Hypothiocyanite levels higher than 100,uM are also very effective in preventing glucose-stimulated acid production by dental plaque (33). However, it may be argued that a periodontopathic bacterium preferably multiplying in subgingival areas is never exposed to the inhibitory action of OSCN- ions present in saliva. Therefore, we studied also the effect of the leukocyte MP-halide-H202 system on this bacterium. Human gingival crevice is the major source of oral leukocytes and the gingival exudate contains MP (13), halides, and SCN- (1). Orogranulocyte peroxidase activity (MP) and the severity of periodontal disease seem to correlate positively (12). It is also known that human dental plaque is able to release MP from polymorphonuclear leukocytes (30). Therefore, it is likely that the MP-halide (or -SCN-)-H202 system is operative in gingival crevices and possibly also within dental plaque. The SCN- levels in gingival exudate (1) and plaque fluid (7) are relatively low (30 to 50,uM), and so far no reports on the presence of OSCN- in these fluids exist. However, because most crevicular leukocytes are active in phagocytosis (26), the subsequent extracellular microbicidal activity due to the presence of the MP system is likely to occur (6). The significance of crevicular leukocytes in protection from periodontal diseases is indisputable (26), and the MP system contributes in a major way to the protective, antimicrobial function of human polymorphonuclear leukocytes (6, 10) İn our study, MP was remarkably less effective than LP in catalyzing the oxidation of SCN-. This is in contrast to the results of studies by Wever et al. (39), who found no

5 100 TENOVUO, MAKINEN, AND SIEVERS difference between LP and MP in their ability to catalyze the oxidation of SCN- to OSCN-. However, these results are not comparable, since in our study the reactions were carried out in a complex growth medium instead of buffer. It should also be remembered that the medium contained Cl-, and further addition of SCN- obviously resulted in the simultaneous generation of OSCN- and OCI-. On the other hand, the method used to detect the oxidation products does not differentiate between OSCN- and OC1-. OC1- is a more potent oxidizing agent than OSCN- (20), which may explain the strong inhibition obtained by low amounts of oxidizing equivalents in the medium (Table 1). With the LP-SCN-- H202 system, the only product was OSCN-, since LP did not oxidize Cl-. In contrast to Cl-, the MP-1--H202 system was more effective against B. cereus than was the LP-I-- H202 system. Although no definitive conclusions can be made on the basis of this study, it seems that with LP and MP, the order of efficiency of the substrates was SCN- > I- and 1- > C1- > SCN-, respectively. B. cereus is a very potent producer of an extracellular collagenase (29) which is most likely a clinically significant virulence factor. The production, excretion, or both of the collagenase reaches its maximum after 4 h growth, i.e., during the log phase, and is strongly dependent on the composition of the medium (29). The extracellular collagenase is not, however, a direct measure of bacterial growth rate since during the late-log phase the amount of enzyme decreases even if the cell number is still increasing. The mechanism responsible for the decrease of the collagenase activity is unknown. It may be explained either as autodegradation of the enzyme molecule or formation of an irreversible enzyme-inhibitor complex. The decrease of activity was not linked to the changes in ph of the medium during the growth. According to the results of the present study, the inhibitory effects of the peroxidase systems on extracellular collagenase are nonspecific; i.e., they are due to the prevention of the start of the log phase rather than to specific inhibition of the enzyme itself. Although sufficient amounts of OSCN- are clearly antimicrobial, some studies have proven the whole peroxidase system to be even more effective than OSCN- alone (4, 32). These findings suggest that in addition to OSCN-, some other short-lived antimicrobial oxidation products are generated by peroxidase and SCN-. In fact, kinetic and polarographic studies have indicated that OSCN- can be further oxidized by LP and H202 to yield other products, possibly 02SCN-, 03SCN-, or both (23). These other products may account for the observed difference between the antimicrobial properties of the peroxidase system compared to those of nonenzymatically produced OSCN-. Surprisingly, B. cereus is also a good producer of extracellular acid. Although the metabolic significance of this acid production for B. cereus is not clear, it was inhibited by the peroxidase systems. The inhibition of acid production has been frequently used as a reliable indicator of the antimicrobial action of the LP system on streptococci (38; Pruitt and Reiter, in press). In accordance with results obtained with S. typhimurium (24), the acid production (and growth) of B. cereus was not reversed by the addition of excess reducing agent (2-mercaptoethanol). This suggests that the actual mechanism of inhibition differs from species to species since acid production by streptococci is easily restored by 2-mercaptoethanol. Indeed, several different inhibitory mechanisms have been found (Pruitt and Reiter, in press). Some of these mechanisms are not reversed by sulfhydryl compounds (35). ANTIMICROB. AGENTS CHEMOTHER. In conclusion, our results show that a periodontopathic bacterium, B. cereus, is sensitive to the inhibitory action of both LP and MP systems. Both of these systems are present in the human mouth and thus contribute to the overall antimicrobial capacity of saliva and gingival exudate. Therefore, individual differences in these systems may alter host response against pathogenic bacteria. ACKNOWLEDGMENTS This study was supported by the Paulo Foundation and by the Academy of Finland. LITERATURE CITED 1. Anttonen, T., and J. Tenovuo Crevicular thiocyanate and iodide ions: cofactors of the antimicrobial peroxidase system in leucocytes. Proc. Finn. Dent. Soc. 77: Aune, T. M., and E. L. Thomas Oxidation of protein sulfhydryls by products of peroxidase-catalyzed oxidation of thiocyanate ion. Biochemistry 17: Bakkenist, A. R. J., R. Wever, T. Vulsma, H. Plat, and B. F. Van Gelder Isolation procedure and some properties of myeloperoxidase from human leucocytes. Biochim. Biophys. Acta 524: Bjorck, L., and 0. Claesson Correlation between concentration of hypothiocyanite and antibacterial effect of the lactoperoxidase system against Escherichia coli. J. Dairy Sci. 63: Carlsson, J., Y. Iwami, and T. Yamada Hydrogen peroxide excretion by oral streptococci and effect of lactoperoxidasethiocyanate-hydrogen peroxide. Infect. Immun. 40: Clark, R. A Extracellular effects of the myeloperoxidase-hydrogen peroxide-halide system. Adv. Inflammation Res. 5: Cole, M. F., S. D. Hsu, B. J. Baum, W. H. Bowen, L. I. Sierra, M. Aquirre, and G. Gillespie Specific and nonspecific immune factors in dental plaque fluid and saliva from young and old populations. Infect. Immun. 31: Germaine, G. R., and L. M. Tellefson Effect of human saliva on glucose uptake by Streptococcus mutans and other oral microorganisms. Infect. Immun. 31: Kersten, H. W., W. R. Moorer, and R. Wever Thiocyanate as a cofactor in myeloperoxidase activity against Streptococcus mutans. J. Dent. Res. 60: ' 10. Klebanoff, S. J., and R. A. Clark The neutrophil: function and clinical disorders. North-Holland Publishing Co., Amsterdam. 11. Klebanoff, S. J., and R. G. Luebke The antilactobacillus system of saliva. Role of salivary peroxidase. Proc. Soc. Exp. Biol. Med. 118: Klinkhamer, J. M., and M. D. Mitchell Orogranulocyte peroxidase activity as a measure of inflammatory periodontal disease. J. Dent. Res. 58: Kowolik, M. J., and M. Grant Myeloperoxidase activity in human gingival crevicular neutrophils. Arch. Oral Biol. 28: Makinen, K. K Characteristics of the hydrolysis of 4-phenylazobenzyl-oxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-arginine (a collagenase substrate) by enzyme preparations derived from carious dentine. Acta Odontol. Scand. 28: Makinen, K. K., and J. Tenovuo Observations on the use of guaiacol and 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid) as peroxidase substrates. Anal. Biochem. 126: Mandel, I. D In defense of the oral cavity, p In I. Kleinberg, S. A. Ellison, and I. D. Mandel (ed.), Proceedings: Saliva and Dental Caries (a special supplement to Microbiology Abstracts). Information Retrieval, Inc., Washington, D.C. 17. Mandel, I. D., J. Behrman, R. Levy, and D. Weinstein The salivary lactoperoxidase system in caries-resistant and -susceptible adults. J. Dent. Res. 62: MAnsson-Rahemtulla, B., K. M. Pruitt, J. Tenovuo, and T. M.

6 VOL. 27, 1985 Le A mouth rinse which optimizes in vivo generation of hypothiocyanite. J. Dent. Res. 62: Marshall, V., and B. Reiter Comparison of the antibacterial activity of the hypothiocyanite anion towards Streptococcus lactis and Escherichia coli. J. Gen. Microbiol. 120: Morrison, M., and G. Schonbaum Peroxidase-catalyzed halogenation. Annu. Rev. Biochem. 45: Pruitt, K. M., M. Adamson, and R. Arnold Lactoperoxidase binding to streptococci. Infect. Immun. 25: Pruitt, K. M., B. MAnsson-Rahemtulla, and J. Tenovuo Detection of the hypothiocyanite (OSCN-) iop in human parotid saliva and the effect of ph on OSCN- generation by the salivary peroxidase antimicrobial system. Arch. Oral. Biol. 28: Pruitt, K. M., J. Tenovuo, R. W. Andrews, and T. McKane Lactoperoxidase-catalyzed oxidation of thiocyanate: polarographic study of the oxidation products. Biochemistry 21: Purdy, M. A., J. Tenovuo, K. M. Pruitt, and W. E. White, Jr Effect of growth phase and cell envelope structure on susceptibility of Salmonella typhimurium to the lactoperoxidasethiocyanate-hydrogen peroxide system. Infect. Immun. 39: Revis, G. J Immunoelectrophoretic identification of peroxidase in parotid saliva. Arch. Oral. Biol. 22: Scully, C Phagocytic and killing activity of human blood, gingival crevicular and salivary polymorphonuclear leukocytes for oral streptococci. J. Dent. Res. 61: Sips, H. J., and M. N. Hamers Mechanisms of the bactericidal action of myeloperoxidase: increased permeability of the Escherichia coli cell envelope. Infect. Immun. 31: Slowey, R. R., S. Eidelman, and S. J. Klebanoff Antibacterial activity of the purified peroxidase from human parotid saliva. J. Bacteriol. 96: Soderling, E., and K. Paunio Conditions of production and properties of the collagenolytic enzymes by two Bacillus strains from dental plaque. J. Periodontal Res. 16: Taichman, N. S., C. C. Tsai, P. C. Baehni, N. Stoller, and W. P. PEROXIDASES AND BACILLUS CEREUS 101 McArthur Interaction of inflammatory cells and oral microorganisms. IV. In vitro release of lysosomal constituents from polymorphonuclear leukocytes exposed to supragingival and subgingival bacterial plaque. Infect. Immun. 16: Tenovuo, J., and M. L. E. Knuuttila Antibacterial activity of salivary peroxidases on cariogenic strain of Streptococcus mutans. J. Dent. Res. 56: Tenovuo, J Formation of the bacterial inhibitor, hypothiocyanite ion, by cell-bound lactoperoxidase. Caries Res. 13: Tenovuo, J., B. MAnsson-Rahemtulla, K. M. Pruitt, and R. Arnold Inhibition of dental plaque acid production by the salivary lactoperoxidase antimicrobial system. Infect. Immun. 34: Tenovuo, J., K. M. Pruitt, and E. L. Thomas Peroxidase antimicrobial system of human saliva: hypothiocyanite levels in resting and stimulated saliva. J. Dent. Res. 61: Thomas, E. L., and T. M. Aune Susceptibility of Escherichia coli to bactericidal action of lactoperoxidase, peroxide, and iodide or thiocyanate. Antimicrob. Agents Chemother. 13: Thomas, E. L., K. P. Bates and M. M. Jefferson Hypothiocyanite ion: detection of the antimicrobial agent in human saliva. J. Dent. Res. 59: Thomas, E. L Lactoperoxidase-catalyzed oxidation of thiocyanate: the equilibria between oxidized forms of thiocyanate. Biochemistry 20: Thomas, E. L., K. A. Pera, K. W. Smith, and A, K. Chwang Inhibition of Streptococcus mutans by the lactoperoxidase antimicrobial system. Infect. Immun. 39: Wever, R., W. M. Kast, J. H. Kasinoedin, and R. Boelens The peroxidation of thiocyanate catalysed by myeloperoxidase and lactoperoxidase. Biochim. Biophys. Acta 709: Wood, J. L Biochemistry, p In A. A. Newman (ed.), Chemistry and biochemistry of thiocyanic acid and its derivatives, Academic Press, Ltd., London. 41. Wunsch, E., and H.-G. Heidrich Zur quantitative Bestimmung der Kollagenase. Z. Phys. Chem. 333: Downloaded from on October 16, 2018 by guest