INFECTION AND IMMUNITY, Jan. 1982, p. 20-24 Vol. 35, No. 1 0019-9567/82/010020-05$02.00/0 Lactoperoxidase and Thiocyanate Protect Bacteria from Hydrogen Peroxide MICHAEL ADAMSON AND JAN CARLSSON* Department of Oral Microbiology, University of Umea', S-901 87 Umead, Sweden Received 22 June 1981/Accepted 2 September 1981 Lactoperoxidase and thiocyanate were shown to protect Escherichia coli and three oral streptococcal species from the bactericidal effect of hydrogen peroxide under aerobic conditions. Lactoperoxidase in the absence of thiocyanate was also protective for two of the bacterial species in a dilution medium but potentiated hydrogen peroxide toxicity for the other two under the same conditions. The products of the reaction between hydrogen peroxide and thiocyanate in the presence of lactoperoxidase were not bactericidal except in the case of E. coli, and then only under special conditions. The results demonstrate the effectiveness of lactoperoxidase and thiocyanate in protecting living cells from hydrogen peroxide toxicity. Although the effect on human cells was not examined in this study, extrapolation of these results to the cells of the oral mucosa would suggest an important protective role of lactoperoxidase and thiocyanate against the toxic effects of hydrogen peroxide in the oral cavity. Hydrogen peroxide is capable of killing a MATERIALS AND METHODS variety of life forms, including bacteria and Microorganisms. Streptococcus mutans strain mammalian cells (1, 6, 7, 12, 14, 20, 26), even NCTC 10449, S. salivarius strain NCTC 9759, S. though these cells may have highly developed sanguis strain ATCC 10556, and E. coli K-12 strain defense mechanisms against hydrogen peroxide AB1157 (3) were used as test strains. (8). For this reason, free hydrogen peroxide may Preparation of media. Trypticase-soy-yeast extract pose a threat to both human and bacterial cells (TSY) broth (pH 7.3) contained, per liter, 17 g of living in the human body. Among the putative Trypticase peptone (BBL Microbiology Systems, sites of hydrogen peroxide production in hu- Cockeysville, Md.), 3 g ofphytone peptone (BBL), 5 g mans are the surfaces of the oral cavity, where of yeast extract (Difco Laboratories, Detroit, Mich.), 5 hydrogen peroxide-forming bacteria constitute g of NaCI, 2.5 g of K2HPO4, and 2.5 g of glucose. The broth was autoclaved at 121°C for 20 min. The dilution up to 60% of the facultatively anaerobic flora solution was prepared by dissolving 4.3 g ofNaCI, 0.42 (18). Free hydrogen peroxide has not been de- g of Na2HPO4, 1.0 g of K2HPO4, and 10 g of sodium 1B- tected in the saliva, however (18), presumably glycerophosphate * 5H20 in 500 ml of water, pH 7.0 because it is quickly destroyed by the salivary (solution 1), and 0.24 g of CaCl2 and 0.1 g of enzymes lactoperoxidase and thiocyanate. MgC92 6H2O in 500 ml of water (solution 2) (25). Hydrogen peroxide is a substrate of lactoper- Solutions 1 and 2 were autoclaved separately, oxidase in oxidizing thiocyanate to hypothio- cooled, and pooled. Peptone-yeast extract-glucose cyanate (OSCN) (2, 15, 16). This product of the (PYG) agar medium was prepared from two solutions. reaction oxidizes bacterial sulfhydryl groups One solution contained, per liter, 20 g of tryptone (Difco), 10 g of yeast extract (Difco), and 20 g of NaCl. (23, 29), damages bacterial cell membranes (19, The other solution contained 28 g of agar (Difco) and 21), and causes a rapid bacteriostatic effect in 2 g of glucose. The two solutions were autoclaved both gram-positive and gram-negative bacteria separately, cooled to 50°C, mixed, and poured into (9; 17, 22, 24, 27; B. Reiter, A. Pickering, J. D. plates. Oram, and G. S. Pope, J. Gen. Microbiol. 33: The blood agar medium was prepared as described xii, 1963). A slower bactericidal effect has been by Holdeman et al. (13) and contained defibrinated reported for some gram-negative bacteria, in- horse blood (GIBCO, Bio-Cult Ltd., Paisley, Scot- cluding Escherichia coli (5, 28). Lactoperoxi- land) hemolyzed by freeze-thawing. dase and thiocyanate have also been shown to Chemicals. Hydrogen peroxide (30%o, wt/wt; Perhy- drol) was from E. Merck AG, Darmstadt, Germany. protect some oral bacteria from the effects of Lactoperoxidase (L 2005; from milk) was from Sigma bactericidal concentrations of hydrogen perox- Chemical Co., St. Louis, Mo. Potassium thiocyanate ide under anaerobic conditions (6). This study was from Riedel-De Haen AG, Seelze-Hannover, Ger- examines the effects oflactoperoxidase and thio- many. cyanate on the survival of bacteria treated with Exposure to lactoperoxidase-thiocyanate-hydrogen hydrogen peroxide under aerobic conditions. peroxide. The strains were grown at 37°C in the broth 20 VOL. 35, 1982 PROTECTION BY LACTOPEROXIDASE AND THIOCYANATE 21 under aerobic conditions. In exponential growth, they 4 were diluted in aerated dilution solution to a density of E. coli K-12 AB1157 about 3 x 104 organisms per ml. At this dilution of the growing culture, no components of the broth inter- fered with hydrogen peroxide toxicity or substituted for thiocyanate in its reaction with lactoperoxidase. A 0.2-ml sample ofbacterial suspension was added to 1.8 ml ofthe dilution solution containing various combina- *_4--_-4_44 4 tions of lactoperoxidase and thiocyanate. Hydrogen peroxide was added to the bacteria 2 min after the start 90 0 of the dilution procedure. Samples (0.1 ml) were taken Co v 2 at regular time intervals from the reaction mixtures _-J and were spread over the surface of agar plates. When 0 -J3 the plates were incubated anaerobically, they were w stored at 37°C in an anaerobic box with an atmosphere 0 of 1OTo H2 and 5% CO2 in nitrogen (31). The numbers w of viable cells were determined. -J In one series of experiments, a 0.2-ml sample of strain AB1157 in exponential growth in broth was co LL added to 1.8 ml of aerated dilution solution giving 0 about 107 organisms per ml. All other conditions were a: the same as above. Only under these conditions could w a bactericidal effect of the hydrogen peroxide-lacto- peroxidase-thiocyanate combination be observed. z 1 RESULTS 2 When E. coli at a density ofabout 3 x 103 cells per ml of dilution solution was exposed to 1 mM hydrogen peroxide, lactoperoxidase protected I I I Il from the bactericidal effect of hydrogen perox- 0 1 2 3 4 ide in both the presence and absence of thiocya- TIME (h) nate (Fig. 1). Similar results were obtained with FIG. 1. Killing ofE. coli in dilution solution at 37°C S. salivarius. When S. mutans was tested under when exposed under aerobic conditions to various similar conditions, lactoperoxidase protected combinations of lactoperoxidase, thiocyanate, and hy- only in the presence of thiocyanate. In the drogen peroxide. The number of viable cells deter- absence ofthiocyanate, lactoperoxidase potenti- mined on blood agar incubated anaerobically after ated the bactericidal effect of hydrogen peroxide various time intervals in the presence of (1) 1 mM on S. mutans (Fig. 2). Results with S. sanguis H202; (2)1 mM H202 plus lactoperoxidase (25 ,ug) plus 2 mM thiocyanate; (3) 1 mM H202 plus lactoperoxi- were similar to those obtained with S. mutans. dase (25 ,ug/ml); (4) no addition. Similar viable counts A bactericidal effect of lactoperoxidase-thio- were obtained on PYG agar incubated aerobically. cyanate-hydrogen peroxide greater than that of hydrogen peroxide itself was demonstrated only in E. coli and then only under very specific conditions (Fig. 3). An exponentially growing culture of E. coli K-12 strain AB1157 in broth nate under these conditions (Fig. 3) did not was diluted 10-fold in dilution solution giving a protect from hydrogen peroxide toxicity, as in density of about 1 organisms per ml and ex- the experiments illustrated in Fig. 1. However, posed for more than 1 h to lactoperoxidase- cells exposed to hydrogen peroxide in the pres- thiocyanate-hydrogen peroxide. Many of the ence of lactoperoxidase did recover much faster cells were damaged to such an extent that they than cells exposed to hydrogen peroxide in the could not recover when incubated on PYG agar absence of lactoperoxidase. The former cells or blood agar aerobically. When incubated an- started to grow rapidly within 1 h, whereas the aerobically on blood agar, however, most of the others did not (Fig. 3). cells recovered even after a 3-h exposure. A pronounced bacteriostatic effect of lactoperoxi- DISCUSSION dase-thiocyanate-hydrogen peroxide on E. coli In the presence of thiocyanate, lactoperoxi- was found under these conditions (Fig. 3), and dase has been shown to protect microorganisms readily visible colonies did not usually develop from the bactericidal effect of hydrogen perox- on the agar plates until after more than 24 h of ide under anaerobic conditions (6). The present incubation. The surviving cells under all other study demonstrated that this protection against conditions (Fig. 3) formed large colonies within hydrogen peroxide toxicity was also effective 24 h. Lactoperoxidase in the absence of thiocya- under aerobic conditions. For a variety oforgan- 22 ADAMSON AND CARLSSON INFECT. IMMUN. 4 9[ S. mutans NCTC 10449 E. coli K-12 AB1157 9 , 4 v 2 90 cm 8 -J 7 co cn -J w 0 0 w -J C') -J -J U- w 0 3 w wm-) CID -J 1 z LA. 0 2 w o 2 1 z TIME (h) 4 FIG. 2. Killing of S. mutans in dilution solution at 37°C when exposed under aerobic conditions to vari- ous combinations oflactoperoxidase, thiocyanate, and hydrogen peroxide. See legend to Fig. 1. Similar viable counts were obtained on blood agar incubated anaero- bically and on PYG agar incubated aerobically.