Factors Affecting the Resistance of Staphylococcus

APPLIED MICROBIOLOGY, Jan., 1967, p. 97-101 Copyright 1967 American Society for Microbiology Vol. 15, No. 1 Printed in U.S.A. Factors Affecting the Resistance of Staphylococcus aureus to Hydrogen Peroxide Treatments in Milk V. M. AMIN AND N. F. OLSON Department of Food Science and Industries, University of Wisconsin, Madison, Wisconsin Received for publication 12 August 1966 ABsTRAcr Staphylococcus aureus 196E was treated with 0.05% hydrogen peroxide in milk under varying conditions to determine the effects of treatment conditions and characteristics of the culture on bactericidal effectiveness of hydrogen peroxide. Time intervals required for 90 to 99.99% destruction of S. aureus decreased significantly as treatment temperatures increased from 37.8 to 57.2 C. Plots of survivor curves showed extended lags in destruction at 37.8 C, slight lags followed by logarithmic rates of destruction at 48.9 C, and logarithmic rates at 54.4 and 57.2 C except for trials in which there was very rapid initial destruction followed by logarithmic rates. S. aureus 196E was significantly more resistant to heat treatments at 54.4 C without added hydrogen peroxide than to treatment with 0.05% hydrogen peroxide at this temperature. Cultures grown at 37 C for 16 hr in milk were more resistant to hydrogen peroxide than were cultures grown at 35 C. Storage of cultures for 96 hr in milk at 4 C caused a decrease in the resistance of the culture. Numbers of staphylococci being treated had little effect on rates of destruction. Downloaded from http://aem.asm.org/ The first experimental use of hydrogen peroxide as a preservative in milk was reported in 1883 (11). Recently, hydrogen peroxide has been approved for treatment of milk for making Swiss and American types of cheese (Federal Register, Washington, D.C., 1962). In the latter use, hydrogen peroxide is added to milk at 37 to 54 C, allowed to act for 15 sec to 10 min, and then destroyed with catalase preparations before cheese making. The bactericidal effectiveness of hydrogen peroxide varies with the concentration used, the temperature and duration of treatment, and the types and numbers of microorganisms (1, 5-7, 10, 14). The resistance of Staphylococcus aureus to hydrogen peroxide has been reported to be less than that of aerobic sporeformers and greater than that of gram-negative bacteria (6, 7, 14). References in the literature on the factors affecting the destruction of S. aureus by hydrogen peroxide are limited. Olson and Price (Abstr. J. Dairy Sci. 45:852, 1960) found that the duration of treatment at 130 F (54.4 C) had a greater effect on destruction of S. aureus than increases in concentration of hydrogen peroxide from 0.02 to 0.12%. Asato and Olson (Abstr. J. Dairy Sci. 46:600, 1963) reported that mean D values (time 97 in minutes required for 90% destruction) were reduced from 5.17 to 1.51 min for S. aureus 72D when treatment temperatures were increased from 120 to 130 F (48.9 to 54.4 C). Walker and Harmon (13) found that 0.05% hydrogen peroxide destroyed greater numbers of S. aureus S1 and other microorganisms present in raw milk at 120 F (48.9 C) than could be attributed to the effect of heat alone. This added destructive effect of hydrogen peroxide was not observed at 130 F (54.4 C). In the present study, the effects of the following conditions on the resistance of S. aureus were determined: temperature of hydrogen peroxide treatment, storage of cultures at low temperatures in milk, growth temperature, and numbers of S. aureus being treated. MATERIALS AND METHODS Preparation of culture. Stock cultures of S. aureus 196E (ATCC 13565) were grown on Difco Standard Methods plate count agar (PCA) slants in screw-cap test tubes at 37 C for 16 hr. The tubes were stored at 4 C between monthly transfers. The stock cultures were washed from the PCA slants and activated for hydrogen peroxide treatments by four successive daily transfers in sterile reconstituted nonfat dry milk (NDM) containing 11% solids. on February 25, 2019 by guest

98 AMIN AND OLSON APPL. MICROBIOL. The reconstituted NDM was autoclaved at 121 C for 15 min. The cultures were incubated for 16 hr at 37 C with constant stirring by magnetic stirrers. In previous experiments, these incubation conditions produced cultures in the early maximal stationary growth phase. The cultures were stored for 8 hr at 4 C between incubation periods. The final culture in reconstituted NDM was diluted serially in sterile standard phosphate buffer to obtain approximately 10' cells per milliliter in the sterile reconstituted NDM (11% solids) used as the treatment medium. The first serial dilution was mixed for 3 min at high speed in a Waring Blendor to disperse clumps of cells. Microscopic examination indicated that this treatment was adequate. Hydrogen peroxide treatment. A 98-ml amount of the treatment medium was brought to the desired treatment temperature before 1.0 ml of the diluted culture was added. After 1 min, 1.0 ml of a 5.0% solution of hydrogen peroxide in redistilled water was added to adjust the hydrogen peroxide content to 0.05% in the treatment medium. The level of H20 in the stock solution was assayed by the cerate oxidimetric titration method. At specified time intervals after the hydrogen peroxide was added, 4.5-ml portions of the peroxide-culture mixture were removed and added to 0.5 ml of 0.0004% catalase (Armalase A 100; Armour Laboratories, Division of Armour and Co., Kankakee, Ill.) solution in 11% NDM at 32.2 C. The peroxide-culture-catalase mixture was held at 32.2 C for 20 min to destroy the hydrogen peroxide as indicated by tests with Peroxystyx (Miles Chemical Co., Division of Miles Laboratories, Inc., Elkhart, Ind.) The culturecatalase mixture was then cooled in an ice bath to prevent growth of surviving bacteria before plating. Enumeration of survivors. Numbers of survivors were counted on PCA after incubating at 37 C for 48 3 hr. The logarithms of counts of survivors were plotted against treatment times. Time intervals required for 90, 99, 99.9, and 99.99% destruction of the staphylococci were estimated from smooth curves based on the plots. Analyses of Variance, Duncan's New Multiple Range Test, and Dunnett's Multiple Comparisons Procedure were used to analyze statistically the time intervals for 99 and 99.9% destruction of the staphylococci by different treatment variations (12). RESULTS AND DIscussIoN eratures. Cultures of S. aureus 196E were treated at 54.4 and 60 C without added hydrogen peroxide, and at 37.8, 48.9, 54.4, and 57.2 C with 0.05% hydrogen peroxide. The maximal variation of temperature during treatment was + 0.2 C. The rates of destruction of S. aureus by heat treatments without added hydrogen peroxide are shown in Table 1. The time intervals required for 90% destruction were within the range of D values (90% destruction) reported for S. aureus by Bhatt and Bennett (Abstr. J. Dairy Sci. 47:666, 1964), Heinemann (3), Kadan et al. (4), Nevot et al. (8), Walker and Harmon (Abstr. J. Dairy Sci. 46:601, 1963), and the World Health Organization (14). The time intervals required for destruction of S. aureus with hydrogen peroxide are shown in Table 2. Extremely long treatment intervals were required at 37.8 C to attain even minimal 90% destruction, which would seem to eliminate this treatment temperature for commercial use. Increasing the treatment temperature between 37.8 and 54.4 C decreased the time for destruction, with the greatest effect occurring between 37.8 and 48.9 C. Only minor effects on rates of destruction occurred when treatment temperatures were increased from 54.4 to 57.2 C. Duncan's New Multiple Range Test (12) showed that destruction times at 37.8 C differed significantly from destruction times at the other treatment temperatures (Table 3). Differences between destruction times at 48.9, 54.4, and 57.2 C were not statistically significant, even though the time for 99.9% destruction at 54.4 C was less than half the time required at 48.9 C. The failure of Duncan's test to detect this difference was caused by heterogeneity of error resulting from greater variability, numerically, of destruction TABLE 1. Mean time intervals required for destruction of Staphylococcus aureus 196E in reconstituted nonfat dry milk at 54.5 and 60.0 Ca 90 99 99.9 99.99 C min min min min 54.4 22.4 46.1 67.5b -c 60.0 5.9 9.2 12.0 14.6 a Means of five replicate treatments. bextrapolated from survivor curve. c Experimental data not obtained. TABLE 2. Effect of temperature on time requiredfor destruction of Staphylococcus aureus 196E with hydrogen peroxide in reconstituted nonfat dry milka Treatment temp- 90.0 99.0 99.9 99.99 C m min min min 37.8 66.6 111.2 135.9b 154.4b 48.9 5.2 9.6 14.4 19.1 54.4 1.4 3.1 5.7 8.8 57.2 1.7 4.0 6.4 8.6 a Means of seven replicate treatments. b Extrapolated from survivor curves.

VOL. 15, 1967 RESISTANCE OF S. AUREUS TO HYDROGEN PEROXIDE 99 times caused by treatment at 37.8 C than by treatment at the other temperatures. To eliminate this heterogeneity, the data for the treatment at 37.8 C were eliminated from the analysis (12). When this was done, significant differences were found between the destruction times at 48.9 C and the two higher treatment temperatures (Table 4). Significant differences between destruction rates at 48.9 and 54.4 C were found also in trials where only these two temperatures were compared. Plots of survivor curves showed an extended lag in destruction during treatment at 37.8 C. Destruction rates were logarithmic after the lag. A slight lag followed by logarithmic destruction or wholly logarithmic destruction rates occurred during treatment at 48.9 C. Logarithmic destruction rates were obtained at 54.4 and 57.2 C, with the exception of trials in which numbers of survivors decreased very rapidly during initial stages of treatment and then at a logarithmic rate. The variations in rates of destruction may have been caused by the combined effects of heat and hydrogen peroxide. At 37.8 C, the effects of both agents would be minimal, causing a lag in destruction. As the temperature increased, the bactericidal effectiveness of both heat and hydrogen peroxide would increase, giving logarithmic or more rapid destruction rates. Hansen and Rieman (2) reported lags in destruction of bacteria during exposure to weak concentrations of disinfectants. They attributed the lag to the time required for diffusion of the disinfectant to the lethal site of the cell. Pulay and Toth (9) observed nonlogarithmic death rates of Escherichia coli at 37 C or lower in the presence of hydrogen peroxide (0.42 g per liter) in phosphate buffer (ph 6.7). At 47 and 57 C the death rates appeared to be logarithmic without the significant lag in destruction observed at 37 C. The data in Tables 1 and 2 indicate that S. aureus 196E was more resistant to heat treatments TABLE 3. Ranking of the mean time intervals for 99 and 99.9% destruction ofstaphylococcus aureus from data of Table 2a Destruction 37.8 C 48.9 C 57.2 C 54.5 C % min min min min 99.0 111.21 9.61 4.02 3.10 99.9 135.93 14.37 6.44 5.72 a Any two means not underscored by the same line are significantly different (P < 0.01). TABLE 4. Ranking of the mean time intervals for 99 and 99.9% destruction ofstaphylococcus aureus from data of Table 2a Destruction 48.9 C 57.2 C 54.5 C % mint min min 99 9.61 4.02 3.10 99.9 14.37 6.44 5.71 a Any two means not underscored by the same line are statistically different (P < 0.01). at 54.4 and 60 C than to hydrogen peroxide treatments at 54.4 C. The added destructive effects of hydrogen peroxide at these higher temperatures were not observed by Walker and Harmon using S. aureus S1 (13). It is possible that other strains of S. aureus would also show this variability in resistance to heat and heat plus hydrogen peroxide. Storage effects. Cultures of S. aureus 196E were grown at 37 C for 16 hr in sterile NDM and subdivided. One portion was treated with 0.05 % hydrogen peroxide at 37.8, 48.9, and 54.4 C. The second portion of the culture was stored at 4 C for 96 hr and then treated in the same way as the original culture. Storage of cultures reduced their resistance to hydrogen peroxide (Table 5). This reduction was most apparent when cultures were treated at 37.8 C. The lower resistance after storage was evident also with treatments at 48.9 C, but not at 54.4 C. The effects of treatment temperature and storage were found to be statistically significant. Interaction between temperature and storage was also significant, indicating that effects of storage on resistance of cultures varied with the treatment temperature used. Dunnett's statistical procedures (12) indicated that resistance of cultures was significantly lower after storage when cultures were treated at 37.8 C. Effects of storage on the resistance of cultures to treatment with hydrogen peroxide at 48.9 and 54.4 C were not statistically significant, even though time intervals for destruction at 48.9 C were reduced by 35 to 50% after storage. The lack of sensitivity of the statistical analyses for treatments at 48.9 C may have been caused again by the large differences in destruction times at 37.8 C before and after storage. Growth temperature. Cultures of S. aureus 196E were grown at 35 and 37 C for 16 hr with constant stirring, and then were treated with 0.05% hydrogen peroxide at 48.9 and 54.4 C. Cultures

100 AMIN AND OLSON APPL. MICROBIOL. TABLE 5. Time required for destruction of Staphylococcus aureus 196E with hydrogen peroxide at various temperatures before and after storage at 4 C for 96 hr in reconstituted NDM Storage time Replicates 90.0 99.0 99.9 99.99 hr C mtn min mitt min 0 96 37.8 4 5 52.4 43.1 101.6 70.6 126.8a 103.1 145.4a 125.7a 0 48.9 5 4.8 9.0 13.7 18.2 96 5 3.0 5.9 9.1 12.2 0 54.4 5 1.7 3.5 5.8 8.6 96 5 0.75 2.6 4.8 8.1 a Extrapolated from survivor curves. TABLE 6. Effect of growth temperature on the resistance of Staphylococcus aureus 196E to hydrogen peroxide in reconstituted nonfat dry milk at 48.9 and 54.4 Ca Growth temp Treatment temp 90 99.0 99.9 99.99 C C min min min min 35 48.9 2.8 5.0 8.1 12.2 37 5.8 10.0 14.3 18.7 35 54.4 0.5 1.2 2.1 3.3 37 1.9 4.1 5.9 7.6 a Means of four replicate treatments. grown at 35 C were less resistant than cultures grown at 37 C (Table 6). The differences in resistance were statistically significant at both treatment temperatures. Numbers of bacteria determined by plate count were lower in cultures grown for 16 hr at 35 C than in cultures grown at 37 C. This difference might have been caused by slower growth or unfavorable conditions for growth. Either condition would have lowered the resistance of the culture. Number of cells. Cultures of S. aureus 196E containing approximately 1010 cells per milliliter after incubation for 16 hr were serially diluted to give 103, 104, 105, and 106 cells per milliliter in the treatment media. The four diluted cultures were treated at 54.4 C with 0.05% hydrogen peroxide. Adjustment of the number of cells to 103, 104, 105, and 106 per milliliter had only slight effects on destruction rates during hydrogen peroxide treatments (Table 7). The time intervals required for 90 to 99.99% destruction of cultures containing 103 cells per milliliter were shorter than with the higher levels of cells. This difference was caused by a very fast destruction rate during the initial stages of treatment, since destruction rates TABLE 7. Effect of numbers of Staphylococcus aureus cells on rates of destruction by hydrogen peroxide treatments at 54.4 Ca No. of cells/ml prior to treatment 90.0 99.0 99.9 99.99 min min min min 103 2.9 6.1 10.8 104 4.8 9.5 12.4 15.1 105 4.8 8.7 12.7 15.2 106 4.8 9.0 11.5 13.5 a Means of six replicate treatments. for this culture were similar to the other three cultures during later stages of treatment. The data in this study illustrate the importance of some of the treatment conditions and characteristics of S. aureus which should be considered in developing methods of treating milk with hydrogen peroxide. Possible guidelines for treatment of milk are suggested by the data. However, the possible differences in resistance between strains of S. aureus must be taken into account before the results from this study can be used to delineate treatment conditions. ACKNOWLEDGMENT This investigation was supported by Public Health Service research grant EF-00204 from the Bureau of State Services, Division of Environmental Engineering and Food Protection. LITERATURE CITED 1. CURRAN, H. R., F. R. EVANS, AND A. LEvITON. 1940. The sporicidal action of hydrogen peroxide and the use of crystalline catalase to dissipate residual peroxide. J. Bacteriol. 40:423-434. 2. HANSEN, N. H., AND H. RIEMAN. 1963. Factors affecting the heat resistance of non-sporing organisms. J. Appl. Bacteriol. 26:314-333.

VOL. 15, 1967 RESISTANCE OF S. AUREUS TO HYDROGEN PEROXIDE 101 3. HEINEMANN, B. 1957. Growth and thermal destruction of Micrococcus pyogenes var aureus in heated and raw milk. J. Dairy Sci. 40:1585-1589. 4. KADAN, R. S., W. H. MARTIN, AND R. MICKELSEN. 1963. Effects of ingredients used in condensed and frozen dairy products on thermal resistance of potentially pathogenic staphylococci. Appl. Microbiol. 11:45-49. 5. LUCK, H. 1956. The use of hydrogen peroxide as a dairy preservative. Dairy Sci. Abstr. 18:364-385. 6. NAMBUDRIPAD, V. K. N., H. LAXMINARAYANA, AND K. K. IYA. 1949. Bactericidal efficiency of hydrogen peroxide. I. Influence of different concentrations on the rate and extent of destruction of some bacteria of dairy importance. Indian J. Dairy Sci. 2:65-69. 7. NAMBUDRIPAD, V. K. N., AND K. K. IYA. 1951. Bactericidal efficiency of hydrogen peroxide. II. Influence of different concentrations of peroxide on the rate and extent of destruction of some more bacteria of dairy importance. Indian J. Dairy Sci. 4:38-44. 8. NEvOT, A., P. LAFoNT, AND Y. LAFONT. 1958. De la destruction des bacteries par la chaleur; etude de l'efficacite de la pasteurisation du lait. Inst. Natl. Hyg. (Paris) Monograph No. 18. 9. PULAY, G., AND S. T6TH. 1964. The germicidal effect of hydrogen peroxide with special reference to sterilization of cheese milk. Tejipari Kut. Koze 7:3-16. 10. ROUNDY, Z. D. 1958. Treatment of milk for cheese with hydrogen peroxide. J. Dairy Sci. 41:1460-1465. 11. SCHRODT, M. 1883. Ein neues Konservierungsmittel fur Milch und Butter. Milch-Ztg. 13:785. 12. STEEL, R. G. D., AND J. H. ToRmI. 1960. Principles and procedures of statistics, p. 99-156, 232-249. McGraw-Hill Book Co., Inc., New York. 13. WALKER, G. C., Am L. G. HARMON. 1965. Hydrogen peroxide as a bactericide for staphylococci in cheese milk. J. Milk Food Technol. 28:36-40. 14. WORLD HEALTH ORGANIZATION. 1962. Milk hygiene, p. 17-18, 423-447. World Health Organization, Geneva, Switzerland. Downloaded from http://aem.asm.org/ on February 25, 2019 by guest