days at 24 C is, within limits, proportional to

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1 IN VITRO STUDIES ON STAPHYLOCOCCAL ENTEROTOXIN PRODUCTION' H. SUGIYAMA, M. S. BERGDOLL, AND G. M. DACK Food Research Institute, University of Chicago, Chicago, Illinois Received for publication January 5, 1960 Staphylococcal enterotoxin is found in the culture supernatant after suitable incubation of food poisoning strains (Dack, 1956). Attempts to demonstrate the toxin within washed cells have not been successful. This extracellular nature of enterotoxin and the production of an antienterotoxin serum (Bergdoll, Surgalla, and Dack, 1959) suggested that the enterotoxigenicity of staphylococcal strains could be studied in vitro, using the formation of specific antigen-antibody precipitate lines in agar plates as the criterion of enterotoxin production. The present communication is concerned with (a) a simple in vitro method for demonstrating the production of enterotoxin, (b) the variation in enterotoxin production within a single strain of staphylococcus, and (c) additional evidence for the antigenic heterogeneity of enterotoxins produced by different strains. MATERIALS AND METHODS Staphylococcal strains were stock cultures maintained in this laboratory on veal infusion agar slants. Enterotoxigenicity of these strains was determined by monkey feeding tests. Most of the work was done with the S-6 strain from which the highly purified enterotoxin was prepared. Liquid culture medium used was amigen broth: 2 per cent amigen (pancreatic digest of casein containing amino acids and small peptides; Mead, Johnson and Company) supplemented with 10 mg/liter niacin and 0.5 mg/liter thiamine, ph 7.6. Amigen agar was prepared with this broth by adding 1.5 per cent agar (Difco) or 0.9 per centionagar S 2 (Consolidated Laboratories). Antiserum used in the serum plates was prepared by the immunization of rabbits with a purified S-6 (70 per cent purity) enterotoxin preparation (Bergdoll et at., 1959). 7-Globulins 1 This work was supported by contributions from various companies and associations of the food industries. were precipitated with alcohol and made to 8 times the original concentration by dissolving in saline containing 1:10,000 merthiolate. The antiserum produced two antigen-antibody precipitate bands with the crude S-6 enterotoxin. Adsorption with a lyophilized concentrate of culture supernatant of nonenterotoxigenic strain 305 resulted in an antiserum producing only one precipitation line with both crude and purified S-6 (70 per cent purity) enterotoxin preparations. Toxigenic Corynebacterium diphtheriae growing on agar containing a suitable concentration of diphtheria antitoxin are surrounded by halos of toxin-antitoxin precipitates (Ouchterlony, 1949; Groman, 1953). A comparable enterotoxin system was used to study the variation of enterotoxin production by the S-6 strain. The adsorbed antiserum was dialyzed for 3 days against saline to remove the merthiolate and sterilized by Millipore filter. A final concentration of 1:2 to 1:4 relative to the original antiserum concentration (e. g., 0.7 ml of 8X serum to 12 ml of agar) was added to the melted and cooled amigen agar immediately before pouring into petri plates. Quantitative differences in the amount of enterotoxin produced was studied by the simple diffusion technique of Oudin with the antiserum which contained only one antibody (lot 3 of Bergdoll et al., 1959). Actively growing cultures were inoculated into liter flasks containing 200 ml of amigen broth and incubated for 24 hr at 37 C on a shaking machine. The cells were removed by centrifugation and the merthiolated supernatant put on the Oudin tubes. The migration distance of the precipitate band in 7 days at 24 C is, within limits, proportional to the concentration of enterotoxin. In some instances the culture supernatant was concentrated. After dialysis for 3 days at 5 C with several changes of water, the material was lyophilized. Solutions of crude enterotoxin of known concentrations could be prepared by appropriate calculations from the dry weight and the volume of supernatant dialyzed. 265

2 266 SUGIYAMA, BERGDOLL, AND DACK [VOL. 80) The identity with enterotoxin of the halo around the colonies was demonstrated by the agar diffusion technique similar to that used in the assay of the protective antigen of Bacillus anthracis (Thorne and Belton, 1957; Strange and Thorne, 1958). Plain amigen agar was poured into petri plates, 12 ml/9 cm diameter plates. After the surface of the agar was dry, the plate was inoculated in two rows 12 mm apart by lightly touching the surface with a straight inoculating needle. The inocula in each row were spaced 10 mm distance from each other. After 48 hr incubation at 37 C, reservoirs were dug in the agar with cork borers. Antigen wells of 7-mm diameter were placed between the developing colonies of each row so that the distance between the centers of the well and the adjacent colonies was 5 mm. A row of antiserum wells 5 mm in diameter and 7 mm apart was placed between the antigen rows so that the centers of an antiserum well and the two closest antigen sources of the same row represented the points of an equilateral triangle. The antigen wells were filled with the purified S-6 antigen, 50 or 100,ug/ml. The antiserum wells were filled with 4 X or 8 X concentrated, adsorbed antiserum in rnerthiolate saline. The plates were incubated at 25 C and read at 24 and 48 hr. Growth of the colonies could be arrested by flooding the plate with chloroform and drying before the wells were placed. RESULTS Typical halos are obtained around colonies of S-6 strain growing on antiserum containing agar. The halos around different colonies are due to the same antigen-antibody precipitate; when colonies are in close proximity the halos join, instead of developing independently (figure 1). Evidence that this halo is due to enterotoxin-antienterotoxin precipitate is presented below. When the S-6 culture was streaked on the antiserum agar, not all the colonies produced halos. This indicated that the culture was composed of cells of different enterotoxin producing capability. The absence of a halo would be due, most likely, to a total lack of enterotoxin production or to the production of an amount insufficient to attain the antigen-antibody ratio required for precipitation. The amount of enterotoxin produced by different clones of S-6 was studied. Depending on the size of the halo after 48-hr incubation, 3 types of colonies were differentiated on the antiserum agar plates: (a) no halo, (b) small halo, and (c) large halo. Colonies of each halo size were inoculated directly into flasks of amigen broth and the amount of enterotoxin produced was compared by the band migration in Oudin tubes. When no precipitation band was detectable, the concentrate of the supernatant was retested at 5 times the original supernatant concentration. Table 1 Figure 1. Halo of enterotoxin-antienterotoxin precipitate surrounding colonies of strain S-6 growing on antiserum agar. Not all colonies produce halo. Halo of two adjacent colonies join to indicate some antigen-antibody precipitate.

3 1960] VARIATION IN STAPHYLOCOCCAL ENTEROTOXIN PRODUCTION 267 TABLE 1 Enterotoxin production in broth using as inocula S-6 colonies having different halo sizes; Oudin tube readings, 7 days at 24 C Halo Size of Inoculum Colony Movement of Antigen-Antibody Band in Oudin Tubes, in Millimeters Individual colonies Average None 0.9 (4.6), 1.7 (4.4), (3.4) (5.5), 1.3 (5.8),0 (0), 0 (0), 0 (3.7)* Small 6.2, 7.2, 6.7, 5.8, 8.7, , 6.5 Large 13.5, 8.5, 11.0, 8.4, , 18.0,15.0, 17.1, 19.8, 18.5 Randomt 15.0, 9.7, 14.7, 6.3, , 12.2, 4.6 * Figures in parentheses are for 5X concentrates of culture supernatants. t Colonies picked at random from plain amigen agar plates. also includes the results when colonies chosen at random from plain amigen agar plates were used as the inoculums. The data indicate considerable variation in enterotoxin production when colonies of S-6 strain are selected at random for use as inocula in the production of enterotoxin. A separate experiment covering a period of about 4 months and involving 55 single colony isolates showed even greater variation. Colonies on blood agar plates were picked and three daily transfers made in amigen broth tubes before inoculating into flasks. The supernatants from these shaker flask cultures were measured for enterotoxin content by the simple diffusion technique of Oudin. No precipitate band was observed in 2 cultures even after 10X concentration; the specific precipitate appeared in 5 instances only with the 1OX concentrate. Two cultures gave band migration of up to 5 mm with the unconcentrated supernatant, 8 cultures of 5 to 10 mm, 18 of 10 to 15 mm, 18 of 15 to 20 mm, and 2 cultures gave migration of 20 to 24 mm. A correlation exists between the enterotoxin produced and the size of the halo around the colonies used as the inocula. Least enterotoxin is found in the broth inoculated with colonies showing no halo, intermediate amounts with small haloed colonies, and most with large haloed colonies. Two of the 5 X concentrates prepared from nonhaloed colonies produced no demonstrable precipitate band; 10 X concentrates of the lyophilized preparations also showed no precipitation line. Thus, the S-6 culture is composed of cells which differ considerably in their ability to produce enterotoxin. This is probably not a phenomenon peculiar to this particular staphylococcal strain; when other strains producing the S-6 type of enterotoxin are streaked on antiserum agar, a similar range of halo sizes was observed. Large halo colonies obtained after successive selections may give higher and more consistent enterotoxin production as indicated in the following experiment. Part of a large haloed colony was suspended in saline and streaked on antiserum agar; the remainder of the colony was used to inoculate a flask of amigen broth. After 2 days incubation one of the colonies with the maximal halo size on the plate was suspended and restreaked. The process was repeated two times. Four of the largest haloed colonies from the last plate were used to inoculate flasks of broth. The supernatants of the flask cultures incubated 24 hr at 37 C were examined on Oudin tubes. The supernatant from the original colony had a migration of 17.5 mm; the four cultures after 3 successive selections of large haloed colonies gave 18.5, 20.2, 21.0, 19.7 mm. The halo size (enterotoxin production) around the colonies seems to be a function of the numerical proportion of at least two different types of cells constituting the colonies. When a large halo colony is suspended in saline and plated on antiserum agar, most or all of the colonies that develop are surrounded by large halos. When a nonhaloed colony is similarly propagated, very few of the developing colonies have halos; in some cases, none. Those colonies having minimal halo gave rise to 5 to 20 per cent haloed colonies. A partially purified S-6 enterotoxin was the antigen used in the preparation of the antiserum used in these plating experiments. After adsorption with a crude concentrate of a nonenterotoxic strain, only one band could be demonstrated in Oudin tubes with crude and purified S-6 enterotoxin preparations. Thus, the halo surrounding the colonies on antiserum agar is probably due to the specific precipitation of the S-6 type of enterotoxin. Additional evidence for this conclusion would be desirable in testing

4 268 SUGIYAMA, BERGDOLL, AND DACK [VOL. 80 the type of enterotoxin elaborated by strains other than S-6. The comparator cell technique (Surgalla, Bergdoll, and Dack, 1952) is not suitable for the study of large numbers of strains. Attempts to obtain the typical arrowhead line of precipitate between parallel streaks of an unknown and S-6 strains made at right angles to a strip of filter paper soaked in antiserum, as in the diphtheria virulence test in vitro (Elek, 1949; Freeman, 1951), were not satisfactory. The probable explanation is the low titer of one or both of the reactants. The double diffusion agar technique described previously proved excellent for the purpose. When the antigen reservoirs placed between the colonies were filled wvith the highly purified S-6 enterotoxin, a single line of precipitate was formed between the antigen and antiserum wells. When the adjacent colony produced enterotoxin of the S-6 type, a similar precipitation line formed and joined with that produced with the highly purified S-6 toxin. Heterologous precipitates would not join in these reactions of identity (Ouchterlony, 1953). Up to 6 strains could be tested on a single plate by appropriate placing of the colonies and the wells containing the S-6 enterotoxin; 10 colonies could be tested if the known enterotoxin was not used. Figure 2 is an Figure 2. Identity of immune precipitate band developed by colonies of staphylococcal strains with that produced by highly purified S-6 enterotoxin. Difference in antigenic specificity of toxins. Left to right, top row: S-6 colony, purified S-6 enterotoxin, 291 strain; middle row: S-6 antiserum wells; bottom row: 196E colony (enterotoxigenic but not S-6 type), purified S-6 enterotoxin, 269 colony (nonenterotoxigenic by monkey feeding test). example. The identity of the precipitate line formed by the purified S-6 enterotoxin with those formed by the S-6 and 291 colonies is shown by the continuous wavy line; no precipitate line is formed by strains 196E (enterotoxigenic, but antigenically different from S-6) and 269 (nonenterotoxigenic by monkey feeding tests). A total of 29 strains of staphylococci were tested by this method. None of 8 strains which were nonenterotoxigenic by the monkey feeding test produced a precipitate line. Of 21 other strains whose culture supernatants elicited emesis in monkeys, 10 showed the reaction of identity with the purified S-6 enterotoxin, the remaining 11 showed no precipitation line, even on repeated testing. All of the strains which produced the S-6 precipitate band produced distinct halos when plated on antiserum agar. DISCUSSION The quantitative difference in enterotoxin produced by different single colony isolates of a given strain has practical importance. For purification purposes there is obvious advantage in starting with crude enterotoxin of maximal possible titer. In the examination of an unknown strain for enterotoxigenicity, false negative results could be obtained if only a single colony picked at random is studied. Human beings are more responsive to enterotoxin than monkeys (Dack, 1956; Wilson, 1959) and even "new" monkeys vary considerably in their sensitivity. Subcultures producing the greatest amount of enterotoxin are desirable when using monkeys to determine the enterotoxigenicity of suspected strains especially when the toxigenicity is low. The selection of the largest haloed colonies as inocula should contribute toward producing toxins of highest potency. At least two explanations are possible for the difference in halo size, or the amount of enterotoxin, produced by different subcultures of the same S-6 strain. (a) The cells of a given colony may represent a homogeneous population, the cells of different haloed sized colonies producing different amounts of enterotoxin. (b) The cells of a given colony may represent a mixture of two types of cells, the difference in halo size of the colonies being dependent on the ratio of the number of cells producing none to minimal amounts of enterotoxin and those producing large amounts. The large halo colonies would be those wn-ith a preponderance of good enterotoxin

5 1960] VARIATION IN STAPHYLOCOCCAL ENTEROTOXIN PRODUCTION producing cells while the nonhaloed colonies would be those with a population dominated by the poor enterotoxin producers. The results presented would favor the latter explanation. The result with enterotoxin is similar to that obtained in the study of staphylococcal hemolysins (Elek and Levy, 1954). This does not mean that the various strains could not differ in the maximal amount of enterotoxin produced. Whether the "pure" nonhaloed colonies produce no enterotoxin or in amounts insufficient to be detected by the method was not established. Most of the broth cultures inoculated with nonhaloed colonies showed a precipitate band in the Oudin tubes. These could be explained by the presence in the colony, or the appearance in broth, of toxin producing cells. Several amigen broth cultures inoculated with nonhaloed colonies showed no evidence of enterotoxin, even at 10 times the concentration of the original supernatant. On a previous occasion a variant (labeled strain S-6-R) of the S-6 has been obtained by chance. Monkey feeding tests showed that this strain did produce some enterotoxin, but evidence obtained by the Oudin technique indicated it was not of the S-6 type (Bergdoll et al., 1959). According to Casman (personal communication) the S-6-R produces enterotoxin of the 196-E type. Whether the "pure" nonhaloed colony is similar to S-6-R or is devoid of enterotoxin producing capability needs further investigation. The genetics of enterotoxin production was not the purpose of this study. However, it would seem that the subcultures of S-6 which showed no enterotoxin even with 10 X concentrates of amigen culture supernatant are stable. The other types of subcultures seem capable of reversion. A suspension of non- and large-haloed colonies gives rise to a few colonies of the opposite type when sufficient numbers of colonies are examined. A large haloed colony selected through several transfers always produced a predominance of large haloed colonies, a nonhaloed colony gave mostly nonhaloed colonies. When the opposite type of colony appeared, they could be maintained in this state with occasional reversions to the original type. It is possible that different cell lines vary in the frequency of reversion to the opposite type. Growth in amigen broth did not seem to have significant selectivity for either of the types since the amount of enterotoxin produced was correlated with the size of the halo around the colonies used for the inocula. 269 The reaction of identity method should facilitate the survey of staphylococcal strains as to their enterotoxin type. The evidence presented in this paper gives additional proof of the existence of at least two different serological types of enterotoxin (Thatcher and Matheson, 1955; Bergdoll et al., 1959; Casman, 1959). A suitable antiserum against the 196E enterotoxin type has not yet been obtained in our laboratory, but the preparation of such an antiserum has been indicated by Casman (1959) who adsorbed out the extraneous antibodies from antiserum prepared against relatively crude 196E enterotoxin. When this type of antiserum becomes available, screening for other antigenic types of enterotoxin will be possible. SUMMARY Quantitative difference in enterotoxin production by different subcultures of a single strain of staphylococcus has been demonstrated. The size of the zone of enterotoxin-antienterotoxin precipitate around the colonies developing on antiserum agar could be used as a guide to the selection of inocula for the best production of enterotoxin. The size of the precipitate halo is determined by the proportion in the colony of cells which produce and those that do not produce enterotoxin. A simplified method for the direct demonstration of the antigenic type of enterotoxin is described. REFERENCES BERGDOLL, M. S., SURGALLA, M. J., AND DACK, G. M Staphylococcal enterotoxin: identification of a specific precipitating antibody with enterotoxin-neutralizing property. J. Immunol., 83, CASMAN, E. P Further serological studies on staphylococcal enterotoxin. Bacteriol. Proc., 1959, 60. DACK, G. M Food poisoning, 3rd ed. Univ. Chicago Press, Chicago. ELEK, S. D The plate virulence test for diphtheria. J. Clin. Pathol., 2, ELEK, S. D. AND LEvY, E The nature of discrepancies between hemolysin in culture filtrates and plate hemolysin patterns of Staphylococci. J. Pathol. Bacteriol., 68, FREEMAN, V. J Studies on the virulence of bacteriophage-infected strains of Corynebacterium diphtheriae. J. Bacteriol., 61,

6 270 SUGIYAMA, BERGDOLL, AND DACK [VOL. 80 GROMAN, N. B Evidence for the induced nature of the change from nontoxigenicity to toxigenicity in Corynebacterium diphtheriae as a result of exposure to specific bacteriophage. J. Bacteriol., 66, OUCHTERLONY, Antigen antibody reactions in gels. Acta Pathol. Microbiol. Scand., 26, OUCHTERLONY, Antigen-antibody reactions in gels. IV. Types of reactions in coordinated systems of diffusion. Acta Pathol. Microbiol. Scand., 32, STRANGE, R. E. AND THORNE, C. B Further purification studies on the protective antigen of Bacillus anthracis produced in vitro. J. Bacteriol., 76, SURGALLA, M. J., BERGDOLL, M. S., AND DACK, G. M Use of antigen-antibody reactions in agar to follow the progress of fractionation of antigenic mixtures: application to purification of staphylococcal enterotoxin. J. Immunol., 69, THATCHER, F. S. AND MATHESON, G. H Studies with staphylococcal toxins. II. The specificity of enterotoxin. Can. J. Microbiol., 1, THORNE, C. B. AND BELTON, F. C An agar diffusion method for titrating Bacillus anthracis immunizing antigen and its application to a study of antigen production. J. Gen. Microbiol., 17, WILSON, B. J Comparative susceptibility of chimpanzees and Macaca mulatta monkeys to oral administration of partially purified Staphylococcal enterotoxin. J. Bacteriol, 78, Downloaded from on January 26, 2019 by guest