Staphylococcus aureus Response to Lysostaphin in

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1 APPLIED MICROBIOLOGY, Jan. 1969, p Copyright ( 1969 American Society for Microbiology Vol. 17, No. 1 Printed in U.S.A. Staphylococcus aureus Response to Lysostaphin in Some Fermented Foods' ROBERT H. METCALF AND R. H. DEIBEL Department of Bacteriology and The Food Reseach Institute, College of Agriculture and Life Sciences, University of Wisconsin, Madison, Wisconsin Received for publication 9 October 1968 The addition of lysostaphin to starting materials for cheese and fermented sausage that were artificially contaminated with Staphylococcus aureus resulted in an initial decrease in the staphylococcal flora. In a simulated cheese process, lysostaphin remained with the curd after separation of the whey. In both cheese and fermented sausage samples that were produced experimentally in the laboratory, a significant S. aureus population ultimately developed, eveninthe presence of lysostaphin. Staphylococcal isolates from these treated products were not more resistant to the lytic enzyme than was the parent strain. Lysostaphin is unique in that it lyses only members of the genus Staphylococcus; thus, it would have no deleterious effect on the starter cultures used in cheese and sausage manufacture. Cheddar cheese and fermented sausage sometimes contain appreciable numbers of Staphylococcus aureus, and cheese has been implicated on occasion in food-poisoning outbreaks. We investigated the effect of adding the enzyme during the manufacture of cheese and sausage inoculated with S. aureus. MATERIALS AND METHODS Source and maintenance of strains. A typical S. aureus (strain 100) which produces enterotoxin A was obtained from Karl F. Weiss, Food Research Institute, University of Wisconsin. The strain was transferred in a medium containing tryptone (Difco), 10 g; yeast extract (Difco), 5 g; NaCl, 5 g; K2HPO4, 5 g; glucose, 5 g; and distilled water, 1 liter; the ph was adjusted to 7.2 (TYE medium). Experimental cheese was made with a mixed starter culture (Streptococcus lactis and Streptococcus cremoris) obtained from Edward Shannon, Department of Food Science and Industries, University of Wisconsin. This culture was transferred in litmus milk. The fermented sausage starter, Pediococcus cerevisiae, strain FPI, was transferred in APT Broth (Difco). All cultures were maintained by daily transfer. S. aureus and P. cerevisiae were incubated at 37 C, the mixed culture starter at 30 C. Experimental preparations of the various fermented foods were inoculated with 12- to 15- cultures. Buffers. Two buffer solutions were used in this study. One contained M/15 phosphate and 0.85% 1 Published with the approval of the Director of the Research Division, College of Agricultural and Life Sciences, University of Wisconsin. NaCl (PS buffer). The other consisted of 0.05 M tris(hydroxymethyl)aminomethane (Tris) buffer and 0.85% NaCl, ph 7.5. Lysostaphin. A lyophilized preparation of lysostaphin was generously supplied by P. A. Tavormina, Mead Johnson and Co., Evansville, Ind. Stock solutions of this lytic agent were prepared by dissolving it in the Tris buffer. To test for lysostaphin activity, a 12- culture of S. aureus 100 (grown in TYE broth) was chilled, centrifuged, washed twice, and resuspended in cold distilled water. A mixture of 0.5 ml of cell suspension, 9.0 ml of PS buffer, and 0.5 ml of lysostaphin (for test series) or 0.5 ml of Tris buffer (for control series) was dispensed into 18-mm matched test tubes. The initial optical density at 600 nm was generally about 0.8. After a zero reading was obtained, the tubes were tempered appropriately and incubated in a water bath at 37 C. Optical density determinations were made periodically. Cheese manufacture. A modification of the procedure to make small amounts of cheese in beakers was employed (2). Pasteurized whole milk (800 ml) was heated to 30 C in a 1-liter beaker and inoculated with 8 ml of the starter culture. Rennet extract (0.16 ml in 20 ml of water) was added with stirring 45 min after inoculation. After 30 min, the curd was cut, warmed gradually to 39.5 C over a 30-min period, and held at this temperature for an additional 1. The curd was pressed for approximately 2 ; then it was cut, salted to 1.5%, and pressed overnight at ambient temperature. To simulate Staphylococcus contamination, pasteurized market milk was heated to 30 C, inoculated, and held at this temperature for 20 min. The milk was then chilled to 6 C and held at this temperature overnight, prior to experimental cheese making. Sausage manufacture. Experimental laboratoryscale sausage was made by means of a previously de- 63

2 64 METCALF AND DEIBEL APPL. MICROBIOL. scribed method (1). Preparations which were to be inoculated with S. aureus (at the same time as starter culture inoculation) were first warmed to 25 C, inoculated, mixed, and held at this temperature for 20 min. Lysostaphin was then added to the test series which was mixed and dispensed into 100-ml beakers. The beakers were tempered in a 37 C water bath and incubated at this temperature for 24. Results from a previous study indicated that significant Staphylococcus growth occurs only in the aerobic or outermost areas of a fermented sausage. Under aerobic conditions, the staphylococci were observed to be more acid-tolerant (R. H. Deibel, unpublished data). Consequently, one beaker of a given test preparation was made for each sampling interval and only the uppermost 10 g of meat from each beaker was used for S. aureus plate counts. Plate counts. The first 10-fold dilution of milk or cheese was made in a 1.0% solution of sodium citrate with the aid of a mechanical blender. Subsequent dilutions were made in distilled water and 0.1-ml portions were surface-plated on Plate Count Agar (Difco) supplemented with 7.5% NaCl (SPCA). The cheesestarter organisms did not grow on this medium. Some of the colonies (5 to 10%) from the counted plates were isolated in Brain Heart Infusion Broth (Difco) for subsequent coagulase testing. The SPCA medium effectively suppressed other bacteria to the extent that most colonies on the plates were coagulasepositive staphylococci. Sausage (10 g) was blended with 90 mi of water for 3 min, and further dilutions were made in distilled water. Portions of this mixture (0.1 ml) were surfaceplated on Tellurite Glycine Agar (Difco) and incubated for 24 to 28 at 37 C. The SPCA medium did not inhibit the Pediococcus starter and contaminants in the sausage, necessitating use of the more selective medium. RESULTS Lysostaphin activity as a function of ph and temperature. At the ph of fresh whole milk, approximately 6.6, or, roughly, 1 ph unit below the optimum for lysis (3), it was observed that lysostaphin activity in a cell suspension of S. aureus in PS buffer was about 33% of that at ph 7.4 (Table 1). Under similar conditions, lysis at ph 5.8 (the approximate initial ph of the meat mix for sausage) was about 12% of that at ph 7.4. At 30 C, the enzyme was approximately 80% as active as it was at 37 C; at 5 C, it was about 10% as active as at 37 C (all ph values comparable). Thus, slight but measurable lysostaphin activity could be expected at raw-product holding temperatures. Lysostaphin activity in food. Various concentrations of lysostaphin were compared to determine the extent of lysis in Tris buffer (ph 7.5). Concentrations of 8, 5, and 4,ug/ml caused a 90% decrease in the optical density within 10 min, whereas concentrations of 3, 2, and 1 pg/ml TABLE 1. Effect of ph value and temperature on the susceptibility of S. aureus strain 100 to lysis by lysostaphin ph TemperatureTime to achieve value r 50% lysis activity Relative C min a Cells were washed once in respective buffers, and the optical density of the suspension was adjusted to approximately 0.8. effected decreases of 82, 75, and 50%, respectively, in the same period. Plate counts of S. aureus (approximate initial level of 106 cells/ml) incubated in pasteurized whole milk at 30 C for either 1 or 4 showed that 5,ug of lysostaphin per ml reduced the staphylococcal population fivefold as compared with the counts when 1 pg/ml was used. Concentrations of 10 and 25 pug of lysostaphin per ml were not significantly more effective in killing S. aureus in milk than was 5 Ag/ml. Therefore, a lysostaphin concentration of 5 pg/ml or per g of food was employed in subsequent experiments. Reliability of plate counts. Preliminary experiments indicated that lysostaphin remained active after the fermentation period, and the possibility of its carryover to the plating media existed. To determine the extent of this difficulty, dilutions of lysostaphin were prepared such that upon addition of 1 ml of S. aureus suspension, the concentrations of 5.0, 0.5, and 0.05,ug/ml would be obtained. These concentrations corresponded to the amount of lysostaphin in the first tee dilutions of a food product which contained 5 Mug of lysostaphin per ml or per g of food. A 1-ml amount of S. aureus containing about 10,000 cells was added to each tube, mixed immediately, and 0.1 ml of this mixture was plated in triplicate on both SPCA and Tellurite Glycine Agar. The largest amount of sample that could be plated without significant carryover of lysostaphin was g. Therefore, numbers of S. aureus cells below 1,000/g could not be counted, and at least 30,000 cells/g were required for statistical accuracy. Growth in cheese containing lysostaphin. Lysostaphin was added to cheese before and after the overnight holding period to determine the effect of protracted exposure of the staphylococci to the

3 VOL. 17, 1969 S. AUREUS RESPONSE TO LYSOSTAPHIN IN FOODS 65 TABLE 2. Sequential S. aureus counts in experimentally prepared cheese with and without lysostaphin" Higher inoculum6 Lower inoculumb Stage of processing Total elapsed Lysostaphin added" Lysostaphin added" time No No lysostaphin Before After lysostaphin Before After added overnieh overnieh added overnigoeriht chillht chill chill Inoculated milk with S. aureus, chilled 20 min Overnight at 6 C 43 < <30 11 Warmed to 30 C < <30 <30 After rennet addition, curd formation < 30 < < 30 < 30 Curd after "cooking" ,700 <30 < <30 <30 Whey after "cooking" < 30 < < 30 <30 Curd before salting , <30 <30 Curd after overnight pressing , ,000 <30 <30 a Expressed as counts/g or ml X 1,000. b Counts represent the average of two trials. Concentration in initial milk was 5 jug/ml. enzyme during this period. The treatment was effective in reducing the staphylococcal count; however, depending upon the initial population level, the number in the final product was similar regardless at which of the two stages the enzyme was added (Table 2). The initial level of staphylococcal contamination was somewhat critical in determining the Staphylococcus level in the final product treated with lysostaphin. If the initial staphylococcal count was approximately 104/ml, or less, significant numbers of staphylococci were not detected in the cheese. However, if the initial count was slightly higher (approximately 5 X 104/ml), counts approaching 105/g were observed. Invariably, when the initial level of contamination was greater than 5 X 104, large staphylococcal counts were detected in the final product. In these instances, the staphylococcal count in the lysostaphin-containing cheese was generally 2 to 3 logs lower than in the controls without lysostaphin. Fate of lysostaphin during curd and whey separation. Whey samples were collected from experimental preparations made with and without lysostaphin. The control whey was divided into two portions and lysostaphin (5 ug/ml) was added to one. Each of these control whey preparations, as well as the test whey, was assayed for lytic activity with a cell suspension of S. aureus in Tris buffer. The activity of the whey from the test sample (lysostaphin added to milk) was about 7% of that of the control whey to which lysostaphin was added. No lytic response could be associated with the control whey without lysostaphin. The curd samples could not be tested in the same manner due to interference with optical density determinations. Testing higher dilutions of the curd would have yielded concentrations too low for accurate estimations of activity. Consequently, both whey and curd samples were tested for activity in another experiment by their introduction to a S. aureus suspension, preceded and followed by S. aureus counts. Again, the whey possessed minimal activity, whereas the curd evidenced considerable activity (Table 3). The addition of lysostaphin to the control whey resulted in a significant decrease in the staphylococcal count, thus indicating that the lysostaphin activity was not inhibited in the whey. These data indicate that most of the added lysostaphin remained in the curd during cheesemaking. Staphylococcal growth in sausage containing lysostaphin. A sausage mixture was prepared and inoculated with the sausage starter P. cerevisiae as described in Materials and Methods. To determine the effect of lysostaphin on S. aureus growth in sausage, the entire sausage mix was also inoculated with approximately 20,000 cells of S. aureus per g of raw sausage mix. After a sample was removed for an initial staphylococcal count, the entire mixture was divided into two equal portions, and sufficient lysostaphin was added to one portion to yield a final concentration of S pg/g of mixture.

4 66 METCALF AND DEIBEL APPL. MICROBIOL. An initial reduction in the number of viable S. aureus cells was observed in the sausage preparations containing lysostaphin. At 10, when maximum counts were obtained in the control sausage, there was more than a 3-log difference in counts in the two sausages, but this difference diminished to approximately 1 log after 24 of incubation at 37 C (Table 4). This experiment was performed on tee separate occasions, and comparable results were obtained. Lysostapliin-resistant mutants of S. aureus. No naturally occurring S. aureus isolate has been reported which is completely resistant to lysostaphin. Some strains, however, are more resistant than others, and it is possible to select for more resistant mutants by inoculating a large number of cells into a medium which contains a high level of lysostaphin. By use of the 5-tube, most-probable-number technique and 10 jig of lysostaphin per ml in TYE TABLE 3. Effect ofcurd and whey from cheese made from lysostaphin-containing milk on the plate counts of S. aureusa Substance Incubation Controlb TestO period Whey 0 5,300 5,500 Id 13,000 3,300 2 <30 Curd 0 6,300 5,800 ld 17,000 < a Expressed as count per g X 1,000. bno lysostaphin was added. c Lysostaphin concentration in milk was 5 jug/ml. d Lysostaphin was added to control series (final concentration 1 Ag/ml) immediately after 1- sample was removed. TABLE 4. Effect of lysostaphin on growth of S. aureus in fermented sausagea Incubation period Controlb Testc 0 17b <30 4 1,300 < ,000 < , aresults represent the average of tee trials and are expressed as counts per g X 1,000. b No lysostaphin was added. Lysostaphin concentration was 5,g/g. medium containing 0.5% Tris and 0.05% K2HPO4 (ph 7.5), it was observed that the mutation rate of S. aureus to lysostaphin resistance, determined by growth in the presence of lysostaphin within 7 days, was one mutant cell for every 4.65 X 106 cells. These mutants were not completely resistant to lysostaphin, and they were lysed eventually in cell suspension at approximately 3 % of the rate of the parent strain. Many S. aureus colonies were isolated from the cheese and sausage samples which contained lysostaphin. Each of these isolates was tested and found to be as sensitive to lysostaphin as was the parent strain. DISCUSSION Our experiments were conducted to determine the feasibility of controlling Staphylococcus growth in two fermented products which have a history of occasional staphylococcal contamination. Neither of these food products was conducive to optimal lysostaphin activity which could be associated with decreased ph values as well as the presence of organic material. Despite the reduced activity, the enzyme effected an initial reduction of the added S. aureus cells in the manufacture of both cheese and sausage. Cheese, unlike sausage, could have lost the enzyme activity during whey and curd separation; however, sufficient data were obtained to indicate that most of the activity remained with the curd. As the ph value of the product continued to decrease, lysostaphin activity also decreased. It appeared that the beneficial activity of lysostaphin was restricted to the initial fermentation period when the ph value of the menstruum was compatible with lysis. The development of a large Staphylococcus population took place upon continued incubation, even in the acid environment of sausage (final ph 4.3). In the manufacture of cheddar cheese, a good, active starter culture will produce enough acid to suppress the production of enterotoxin. The possibility of S. aureus enterotoxin production in cheddar cheese arises mainly when there is subnormal acid development (4). In the experiments reported, the ph of the cheese after overnight pressing was quite high (average 5.8). This represented the type of poor quality cheese in which enterotoxin production could be a problem. Again, however, addition of the lytic agent did not avert the ultimate growth of a significant staphylococcal population. It would appear that complete control of staphylococcal growth in cheese and sausage tough the agency of this highly specific lytic enzyme is not feasible under the conditions we

5 VOL. 17, 1969 S. AUREUS RESPONSE TO LYSOSTAPHIN IN FOODS 67 employed. However, some reduction in S. aureus counts was effected by lysostaphin, and its usefulness against lower levels of initial S. aureus contamination remains to be investigated. ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance of Vicki Wesen. This investigation was supported by Public Health Service grant Al 7691 from the National Institute of Allergy and Infectious Diseases and by contributions from the food industry to the Food Research Institute at the University of Wisconsin. LITERATURE CITED 1. Deibel, R. H., G. D. Wilson, and C. F. Niven, Jr Microbiology of meat curing. IV. A lyophilized Pediococcus cerevisiae starter culture for fermented sausage. Appl. Microbiol. 9g Sandine, W. E., P. R. Elliker, G. H. Wilster, R. L. Stein, and A. W. Anderson A beaker test for estimating gasproducing tendency of lactic acid starter cultures. J. Dairy Sci. 40: Schindler, C. A., and V. T. Schuhardt Purification and properties of lysostaphin-a lytic agent for Staphylococcus aureus. Biochim. Biophys. Acta 97: Zeen, V. L., and V. F. Zeen Relation ofacid development during cheesemaking to development of staphylococcal enterotoxin A. J. Dairy Sci. 51: Downloaded from on September 11, 2018 by guest