APPLIED MICROBIOLOGY, March, 1965Copyright © 1965 American Society for Microbiology

Vol. 13, No. 2Printed in U.S.A.

Detection of Staphylococcal Enterotoxin in FoodEZRA P. CASMAN AND REGINALD W. BENNETT

Division of Microbiology, Food and Drug Administration, Department of Health, Education, and Welfare,Washington, D.C.

Received for publication 24 September 1964

ABSTRACTCASMAN, EZRA P. (Food and Drug Administration, Washington, D.C.), AND REGINALD

W. Bennett. Detection of staphylococcal enterotoxin in food. Appl. Microbiol. 13:181-189. 1965.-Methods are described for the extraction and serological detection of trace

amounts of enterotoxins A and B in foods incriminated in outbreaks of staphylococcalfood poisoning. Evidence is presented for the probable applicability of the methods forthe detection of unidentified enterotoxins.

In 1947, a long-range program to developmethods for the detection of staphylococcalenterotoxin in foods was begun in the Divisionof Microbiology of the Food and Drug Adminis-tration. The biological tests which were availablefor the detection of enterotoxin were difficult toperform and of variable reliability. Of these, themonkey-feeding test was not sufficiently sensitiveand the procedure of injecting cats or kittens wasnot specific. The report by Dolman and Wilson(1938) of what appeared to be a specific antibodyfor the staphylococcal enterotoxin suggested thatthe problem might be solved by means of sero-logical procedures. It was necessary to demon-strate the antigenicity of the enterotoxin, todetermine the number of antigenic types ofenterotoxin that might be involved, to makeavailable for use the specific antitoxins, and todevelop methods for the serological detection ofthe enterotoxins in foods.We have reported studies (Casman, 1958,

1960) in which we demonstrated (i) the anti-genicity of enterotoxin, by conferring to cats apassive immunity to enterotoxin, with the use ofantiserum produced in rabbits; (ii) the occurrenceof two serological types of heat-resistant entero-toxin, of which only one appeared to be asso-ciated with food poisoning; (iii) the production bycertain staphylococci of other apparently heat-labile emetic substances, and (iv) the use of thegel double-diffusion test for the detection ofenterotoxin.

Since these studies were reported, one or moreadditional enterotoxins have been encounteredin food-poisoning incidents in England and, in afew instances, in this country. Only one of the twoserologically identified enterotoxins, however,continues to be associated with most outbreaksof food poisoning in the United States. This

enterotoxin is now designated enterotoxin A(Casman, Bergdoll, and Robinson, 1963). Strainsof food-poisoning origin which produce the "B"type of enterotoxin are rarely encountered.Fujiwara (1961) studied the production of entero-toxin B by strains incriminated in outbreaks offood poisoning in Japan. Enterotoxin B was pro-duced by 5 of 25 such strains. The 5 strains origi-nated from a common source and were laterfound by us to have identical phage patterns andto produce both "A" and "B" enterotoxins.Three strains isolated in 1961 in the state ofGeorgia are the only strains so far encounteredby us which are associated with food poisoningand produce the "B" and not the "A" type ofenterotoxin.Our development of a serological test for

enterotoxin A has made possible the detection ofthis common type of enterotoxin in foods. Wedemonstrated (Casman, McCoy, and Brandly,1963) the elaboration of enterotoxin A in meatafter relatively luxuriant growth of an entero-toxigenic staphylococcus. We now report thedetection of the small amounts of enterotoxinpresent in foods responsible for food-poisoningincidents, the nature of the problem, and ourpresent procedures and capabilities.Very small amounts of enterotoxin can be

expected to be present in such foods. The averageamount of enterotoxin A produced by staphylo-cocci in aerated Brain Heart Infusion broth is 2to 4 ,ug/ml of culture containing 15 to 20 billionorganisms. [This figure is based on the sensitivityof the slide gel diffusion test (Wadsworth, 1957;Crowle, 1958) which will detect enterotoxin whenpresent in a concentration of approximately 1Ag/ml.] In ham and in pastries responsible forfood-poisoning incidents, we have recently foundfrom 50 million to 200 million staphylococci per

181

on April 30, 2021 by guest

http://aem.asm

.org/D

ownloaded from

CASMAN AND BENNETT

gram. If we disregard the differences in abilitiesof various media to support the production ofenterotoxin and assume that there is a directcorrelation between growth and enterotoxinproduction, we could expect from 0.01 to 0.04 ,ugof enterotoxin per gram to be present in thesefoods. Such foods would have to be extractedand the extracts concentrated to an enterotoxincontent of 1 ,ug/ml to detect the enterotoxin bymeans of the slide gel diffusion test. Thus, todetect the presence of 1 ,ug of enterotoxin in 100 gof food, the extract would have to be concentratedto 1 ml. This degree of concentration of foodextractives results in the production of thickpastes which cannot be examined. It is neces-sary, therefore, to separate the enterotoxin notonly from the insoluble constituents of the foodsbut also from the soluble extractives.The procedure for examining foods for the

presence of enterotoxin may be divided into threesteps: (i) separation of the enterotoxin frominsoluble constituents, (ii) separation fromsoluble extractives, and (iii) concentration of theextracted and separated enterotoxin so that itmay be detected by means of the gel diffusiontest. We have examined three categories of foodsfor enterotoxin, using these procedures: (i) foodsto which measured amounts of enterotoxin wereadded, (ii) foods inoculated with enterotoxigenicstaphylococci, and (iii) samples of foods re-sponsible for food poisoning and containingenterotoxigenic staphylococci.

MATERIALS AND METHODSStaphylococcal strains. Strain 265-1 was isolated

in 1958 from cheese incriminated in an outbreak offood poisoning. It produces from 8 to 12 Ag ofenterotoxin A per ml of culture supernatant liquidwhen grown on semisolid Brain Heart InfusionAgar, pH 5.3 (Casman and Bennett, 1963). StrainD263 was received in 1957 from G. M. Dack, Uni-versity of Chicago. It was isolated from the stoolof a patient who developed enteritis during in-tensive antibiotic therapy. It produces 2 /Ag ofenterotoxin A and 20 to 30,g of enterotoxin B perml of culture supernatant when grown on semi-solid Brain Heart Infusion Agar, pH 5.3.

Strains 291, 293, and 294 were received in 1962from Betty Hobbs of London, England. They pro-duce enterotoxins which are neither enterotoxinA nor B. Strain 291 was isolated from cheese epi-demiologically responsible for three outbreaks offood poisoning in which 77 individuals were madeill. Strain 293 was isolated from chicken preparedin a small airport kitchen and was responsible forthe food poisoning of passengers aboard an air-craft. Strain 294 was isolated from the finger of afood handler associated with an outbreak of foodpoisoning caused by ham. Its phage pattern was

the same as that of staphylococci isolated fromthe ham.

Strain 315 is an enterotoxigenic staphylococcusproducing neither enterotoxin A nor B. It wasisolated in 1963 from cooked turkey epidemiolog-ically responsible for a food-poisoning incident inHouston, Tex. Six members of a family became illwith typical symptoms of staphylococcal foodpoisoning from 2 to 2.5 hr after eating the turkey.

Enterotoxins used in recovery experiments. Meas-ured amounts of enterotoxin were added to variousfoods in experiments designed to determine theefficiency of the extraction and concentration pro-cedures. The enterotoxins were produced by grow-ing enterotoxigenic staphylococci in sacs of cello-phane dialysis tubing lying on the surface of BrainHeart Infusion broth (Casman and Bennett, 1963).Strain D263 was employed for the production ofenterotoxins A and B. Two batches of this toxinwere used-one containing 40,g of enterotoxin Aand 800,g of enterotoxin B, and the other, 20,gof enterotoxin A and 2,000,ug of enterotoxin B perml.

In an experiment designed to determine the ap-plicability of our procedures to the detection ofenterotoxins which are not yet serologically identi-fied, similarly prepared enterotoxins were em-ployed, by use of strains 291, 293, 294, and 315.These enterotoxins were detected and very crudelymeasured by the cat test (Hammon, 1941) afterthe removal of interfering hemolysins by pan-creatic digestion (Casman and Bennett, 1963).

Foods used in recovery experiments. Measuredamounts of enterotoxins were added to feta cheese(prepared from goat's milk), to the coconut cus-tard portion of frozen coconut custard pie, tocooked and raw ground beef, and to cooked groundshrimp and turkey. The pie was baked as directedby the manufacturer. The beef, shrimp, and turkeywere cooked in flowing steam for 25 min.

Foods inoculated with strain 265-1. White turkeymeat, coconut custard, and shrimp were inocu-lated with strain 265-1. Cooked white turkey meat(150 g) was ground in a meat grinder and mixedwith 100 ml of distilled water in a Waring Blendorat high speed for 3 min. The resulting paste wasdistributed in petri dishes (diameter, 10 cm) at adepth of approximately 0.3 cm and autoclaved at15 lb of steam pressure for 10 min. Ground cookedshrimp (100 g) was mixed with 100 ml of distilledwater in the Waring Blendor at high speed for 3min, and the paste distributed in petri dishes andautoclaved in the same manner. The coconutcustard of a freshly baked frozen coconut custardpie was similarly distributed in petri dishes in ap-proximately 0.3 cm deep layers and autoclaved.An aqueous suspension containing approxi-

mately 3 X 108 staphylococci per milliliter wasprepared from a 24-hr nutrient agar slant cultureof strain 265-1, and 0.2 ml was spread over thesurface of the food in each petri dish. The plateswere incubated in air at 35 to 37 C for 48 hr. Thecultures of each of the foods were mixed and stored

182 APPL. MICROBIOL.

on April 30, 2021 by guest

http://aem.asm

.org/D

ownloaded from

STAPHYLOCOCCAL ENTEROTOXIN IN FOOD

at -18 C. Dilutions of the thoroughly mixed foodswere streaked on Staphylococcus 110 Agar (Difco)and on Plate Count Agar (Difco). The counts pergram of turkey paste, shrimp paste, and coconutcustard were 13.5, 15, and 2.5 billions, respectively.

Foods incriminated in outbreaks of food poison-ing. Foods which were epidemiologically responsi-ble for four outbreaks of food poisoning were alsoexamined for the presence of enterotoxin. In threeof the outbreaks involving cream pies, ham, andmacaroni salad, staphylococci which producedenterotoxin A were found in the incriminatedfoods. In another outbreak involving ham, onlynonenterotoxigenic staphylococci were detected.Banana cream pie and coconut cream pie, con-

sisting of flour, sugar, eggs, flavoring, and coconutor banana, were responsible for an outbreak of foodpoisoning in which seven individuals became ill4 hr after consuming the pies. The staphylococciisolated produced enterotoxin A. The coconutcream pie contained 200 million and the bananacream pie 72 to 73 million staphylococci per gram.

In another food-poisoning incident, three in-dividuals developed the symptoms of staphylococ-cal food poisoning 1 hr after consuming ham. Theham contained 50 million staphylococci per gram,and the isolated organisms produced entero-toxin A.

In a third outbreak of food poisoning, macaronisalad, consisting of cooked macaroni, salad dress-ing, canned milk, and sugar, was the common foodconsumed by 83 individuals who became ill 3 to 9 hrafter eating lunch in the cafeteria of an elementaryschool. The symptoms were nausea, vomiting, andheadache. The salad contained 1 million staphy-lococci per gram, and the isolated organisms pro-duced enterotoxin A.The fourth outbreak was apparently caused by

ham which was cooked, sampled without incident,wrapped in aluminum foil, and stored withoutrefrigeration for approximately 12 hr. Over aperiod of 5 days, seven individuals became ill withsymptoms typical of staphylococcal food poison-ing within 2 to 6 hr after eating the ham. The hamcontained approximately 3 billion coagulase-positive staphylococci per gram. Cultures wereprepared from eight isolated colonies and werefound to produce neither enterotoxin A nor B.Four of the eight cultures were tested for entero-toxigenicity, by use of the cat test, with negativeresults.

Separation of enterotoxin from insoluble foodconstituents. Foods were homogenized with diluentat high speed in a Waring Blendor. After centrifu-gation, the supernatant liquids were examined fortheir enterotoxin contents by the gel diffusion test,either directly or after further processing. Gen-erally, 20 g of food were blended for 3 min in 100 mlof 0.2 M NaCl and, after adjusting the mixture topH 7.4 to 7.5, centrifuged for 10 min at 32,800 X g.The supernatant liquid was decanted into abeaker. The sedimented food was resuspended byblending in 50 ml of 0.2 N NaCl at pH 7.4 to 7.5

and centrifuged again at 32,800 X g for 10 min.Floating particles of lipoidal material were re-moved. The pooled extracts were generally placedin cellophane dialysis tubing and immersed in 30%(w/w) polyethylene glycol 20,000 (PEG) (FisherScientific Co., Pittsburgh, Pa.) to reduce theirvolume.The presence of 0.2 M NaCl in the diluent dimin-

ished markedly the removal of enterotoxin duringcentrifugation. There appeared to be more reten-tion of the enterotoxin by the supernatant at pH7.3 to 7.5 than at lower or higher pH values. Thiswas especially evident when water, rather thansaline, was the suspending fluid.Amounts considerably larger than 20 g were ex-

amined when the presence of very small amountsof enterotoxin was indicated by the numbers ofstaphylococci detectable by plate count and mi-croscopic examination.

Separation of enterotoxin from soluble extractives.Two procedures used by others in purification ofstaphylococcal enterotoxin B were examined forthe separation of enterotoxin from food extrac-tives. Of these, adsorption onto carboxymethyl-cellulose (CM-cellulose; Schantz, 1964) was moreapplicable than filtration through Sephadex G-100(Frea, McCoy, and Strong, 1963).

CM-cellulose. Prior to the separation of entero-toxin with CM-cellulose, the extract was generallyconcentrated by dialysis against PEG to approxi-mately one-fourth its original volume. The dialysistubing was emptied and rinsed with two or three2-ml quantities of 0.2 M NaCl, and the pooledconcentrate and washings were adjusted to pH7.4 to 7.5 with 1.0 N NaOH. After centrifugationat 32,800 X g for 10 min to remove any precipitatethat appeared on concentration, the extract wasdiluted with 20 volumes each of distilled waterand 0.01 M sodium phosphate (pH 6.0). The extractwas adjusted to pH 6.0 with 0.01 M HSP04 andpassed through a column of CM-cellulose. A 1-gamount of CM-cellulose (Whatman Powder, CM70, manufactured by W. R. Balston, Ltd., 0.7meq/g capacity) was suspended in approximately100 ml of 0.01 M sodium phosphate (pH 6.0). Thesuspension was adjusted to pH 6.0 with 1.0 NNaOH. The column was packed by pouring thesuspended CM-cellulose over a plug of glass woolin a chromatographic tube 2 cm in diameter and40 cm long. The particles were allowed to settleunder flow conditions until the adsorbent reacheda height of approximately 2 cm. A loosely packedplug of glass wool was placed just above the CM-cellulose to prevent disturbing the column, andfines were removed by washing the column with100 to 200 ml 0.01 M sodium phosphate (pH 6.0).The column was then placed in the cold room,

and the diluted extract was allowed to passthrough it at a flow rate of approximately 1 to 2ml/min, mainly because it was convenient to dothis step overnight. When the last of the extractjust entered the adsorbent bed, the column was

washed with 100 ml of 0.01 M sodium phosphate

VOL. 13, 1965 183

on April 30, 2021 by guest

http://aem.asm

.org/D

ownloaded from

CASMAN AND BENNETT

(pH 6.0). When passage of the column was com-pleted prematurely, resulting in a drying of thecolumn, the latter was hydrated by passing 25 to50 ml of distilled water through it and then washedwith 100 ml of 0.01 M sodium phosphate (pH 6.0).The enterotoxin was eluted from the column with150 ml of 0.2 M sodium chloride in 0.2 M sodiumphosphate (pH 7.4).With some foods, excessive amounts of soluble

food constituents were adsorbed by the CM-cellulose at pH 6.0 so that the eluates could not beconcentrated to the degree possible with otherfoods.

Gelfiltration. To separate enterotoxin from solu-ble extractives by gel filtration, the extract wasconcentrated by osmotically forced dialysis to 20ml or less, clarified by centrifugation at 32,800 X gfor 10 min and filtration through a membrane filter(average porosity, 0.5 A), and placed on a column(4 by 21 cm) prepared with 12 g of 140 to 400 meshSephadex G-100 (Pharmacia Laboratories, Inc.,New York, N.Y.) equilibrated with 0.2 M NaCl(pH 7.3 to 7.4). After the sample entered theSephadex, the column was eluted by gravity flowwith 0.2 M NaCl, maintaining a head of 15 to 20 cm.An elution pattern determined with a mixture ofenterotoxin A (20,g) and B (2,000,g) in 20 ml of0.2 M NaCl revealed that enterotoxin appeared inthe eluate at 95 ml and was no longer detectableat 210 ml. The eluates from 90 through 210 ml(total volume 120 ml) were combined and con-centrated.

Comparison of CM-cellulose and Sephadex G-100.In comparing adsorption to CM-cellulose to gelfiltration, 40 g of food, to which measured amountsof enterotoxins A and B had been added, wereextracted at pH 7.4 to 7.5 with 200 ml of 0.2 MNaCl. The extract was concentrated to at least40 ml by osmotically forced dialysis, centrifugedat 32,800 X g for 10 min, and filtered through amembrane filter (average porosity, 0.5 u). Thefiltrate was divided into two equal quantities.One portion was passed through CM-cellulose atpH 6.0, as described above, after dilution with 20volumes each of distilled water and 0.01 M sodiumphosphate, and the other was passed throughSephadex G-100.

Concentration of the eluates. Eluted toxin wasconcentrated by dialysis against PEG at 5 C. The150 ml of eluate from the CM-cellulose column orthe 120 ml of eluate from the Sephadex columnwere placed in 75 to 80 cm of cellophane dialysistubing (2.9 cm flat width) and immersed overnightin 100 ml of PEG. The concentrated contents werecollected in one end portion (about 10 cm) of thecellophane sac. The remaining 65- to 70-cm portionof tubing was rinsed with several 2- to 3-ml quan-tities of saline which were added to the smallersac. Concentration was continued by immersingthe end portion of the sac in fresh PEG until al-most complete dehydration was effected. The sacwas immersed briefly in saline to hydrate the con-tents. A Pasteur pipette was used to add the con-

centrate to the well in the plastic matrix of theslide gel diffusion assembly.

Gel diffusion test. A slide modification (Wads-worth, 1957; Crowle, 1958) of the double-diffusiontest of Ouchterlony was used to detect the entero-toxin. In this test, lines of precipitation develop ina thin layer of agar gel formed between a glassslide and a 0.3 cm thick square of plastic. Theplastic contains funnel-shaped holes in which thereactants are placed. Identification of the materialunder test is permitted by coalescence of its lineof precipitation with a reference line of precipita-tion formed by the interaction of antibody withknown enterotoxin. Results obtained with anti-gens, approximately 95% pure, indicate that thesensitivity of the test is such that an enterotoxinconcentration of approximately 1 pg/ml is requiredfor the formation of a line of precipitation. Thetest may be employed to determine, semiquantita-tively, the micrograms per milliliter of the ma-terial under examination, by multiplying its vol-ume by the reciprocal of the highest dilutiongiving a line of precipitation. Only 0.02 to 0.03 mlof antigen or serum is required to fill a well in theplastic matrix. The test was performed as de-scribed by Crowle (1958) except for the incorpora-tion of 0.04 M sodium barbital (pH 7.4), and 0.01%thimerosal (Merthiolate) in the agar gel. In theexamination of certain foods such as uncookedbeef, it was necessary to add 1 M glycine (Halbert,Swick, and Sonn, 1955) to the agar and to lowerthe sodium barbital concentration to 0.02 M to re-duce clouding which obscured the lines of precipi-tation.

RESULTS

Evaluation of CM-cellulose and Sephadex forthe separation of enterotoxin from food extractives.To determine the relative merits of adsorptiononto CM-cellulose and filtration through Sepha-dex for the separation of enterotoxin from solublefood constituents, approximately 20 Ag of entero-toxin A and 2,000 ,ug of enterotoxin B containedin 1 ml of sac culture toxin of strain D263 wereadded to 40-g quantities of feta cheese, coconutcustard, cooked beef, cooked ground shrimp, andcooked ground turkey. After blending and ex-tracting with 200 ml of 0.2 M NaCl (pH 7.4 to7.5), as described above, the extracts were con-centrated by dialysis against PEG to at least 40ml. They were clarified by centrifugation andfiltration through a membrane filter (averageporosity, 0.5 ,u) and divided into two equalquantities. One was subjected to gel filtration,and the other was diluted with 20 volumes eachof distilled water and 0.01 M sodium phosphate(pH 6.0) and passed through 1 g of CM-cellulose.The eluates were concentrated, when possible,to a final volume of approximately 0.1 ml. Be-cause of their viscidity, however, it was not

184 APPL. MICROBIOL.

on April 30, 2021 by guest

http://aem.asm

.org/D

ownloaded from

STAPHYLOCOCCAL ENTEROTOXIN IN FOOD

possible to concentrate the eluates from theSephadex column to this degree.The average final volumes of the concentrates

and the titers and percentages of recovery of theenterotoxins of at least two experiments are pre-sented in Table 1.The use of Sephadex G-100 generally resulted

in the recovery of slightly more enterotoxin butdid not permit the degree of concentration pos-sible after adsorption to and elution from CM-cellulose. The latter procedure permitted thedetection of smaller amounts of enterotoxin andwas, therefore, selected for the separation ofenterotoxin from food extracts.

Recovery of enterotoxin added to foods. Sepaiate0.4-ml quantities of the sac culture toxin ofstrain D263, each containing approximately 16 ,ugof enterotoxin A and 320 ,ug of enterotoxin B,were added to 20-g quantities of coconut custardand cooked and raw ground lean beef. By use ofthe procedures described above (except for dilu-tion of the extracted foods with 10 volumes ratherthan 20 volumes each of distilled water and 0.01M sodium phosphate), 0.72 ml of cloudy, color-less end product was obtained from the mixtureof enterotoxin and coconut custard. It contained11 jig of enterotoxin A and 230 ,ug of enterotoxinB, representing recoveries of 69 and 72%, respec-tively. With cooked beef, 38% enterotoxin A and48%c enterotoxin B were recovered. When thesame procedure was applied to raw beef, solublemeat constituents were adsorbed by the CM-cellulose to such an extent that the eluates couldnot be concentrated to the degree possible withextracts of other foods. Furthermore, the extractscontained substances which diffused into andclouded the agar gel in the slide diffusion test,

obscuring the specific lines of precipitation. Theclouding was not prevented by the addition of1 M glycine to the agar or by lowering the sodiumbarbital content of the agar to 0.02 M. The sub-stances in uncooked beef which caused theclouding were not removed when the extract waspassed through a column of Sephadex G-100 priorto passage through CM-cellulose.

Because less of the extractives from raw beefwere adsorbed by the CM-cellulose at higher pHvalues, attempts were made to separate theenterotoxin by adsorption at pH 6.5 and also atpH 6.5 and pH 6.8 at reduced ionic strength. Thereduction in ionic strength was accomplished bydialyzing the 0.2 M NaCl extract of the meatagainst PEG until approximately one-fourth itsoriginal volume was reached, and then dilutingthe concentrated extract with 20 volumes each ofwater and 0.01 M sodium phosphate (pH 6.5 or6.8).The results of these studies (Table 2) reveal

that at pH 6.5, when the ionic strength was notreduced, there was a marked reduction in re-covery of enterotoxin. At pH 6.5 and reducedionic strength, there was only a slight reductionin recovery. At pH 6.8 and reduced ionic strength,the recovery was one-half that obtained at pH6.0. Since considerably less adsorption of meatextractives took place at higher pH values andespecially at pH 6.8, the loss of enterotoxin couldbe compensated for by greater reduction of thefinal volume. The loss of enterotoxin at higherpH values and the excessive amounts of meatextractives at pH 6.0 limit the effectiveness ofthe procedure to the detection of more thantrace amounts of enterotoxin in raw meat. Cloud-ing of the gel by extracts of raw meat obtained

TABLE 1. Recovery of enterotoxins A and B from food extracts by use of carboxymethylcelluloseand Sephadex G-100

Food extracted

Feta cheese....Coconut

custard.......Cooked beef....Cookedshrimp .......

Cookedturkey........

Carboxymethylcellulose

Concen-trate

ml

0.13

0.10.15

0.1

0.1

Enterotoxin A

Titer Recov-ered

1:37

1:321:33

1:23

1:48

/c43

3241

23

48

Enterotoxin B

Titer

1:2,500

1:1,8901:2,850

1:3,500

1:3,750

Sephadex G-100

Enterotoxin A Enterotoxin B

Recovered

30

1929

35

37.5

Concen-trate

ml

0.33

0.30.3

0.2

0.4

Titer

1:13

1:191:20

1:21

1:12

Recov-ered

35

4660

41

48

Titer

1:1,090

1:2,2001:1,600

1:1,890

1:1,640

Recovered

30

5048

37.5

55

VOL. 13, 1965 185

on April 30, 2021 by guest

http://aem.asm

.org/D

ownloaded from

CASMAN AND BENNETT

TABLE 2. Recovery of enterotoxins of strain D263after addition to foods

Food

Coconutcustard..

Cookedbeef ......

Rawbeef ......

Rawbeef ......

Rawbeef ......

Rawbeef ......

Adsorp- Itoni Cloud-reduced gel

6.0

6.0

6.0

6.5

6.5

6.8

+

* Containing 1 M glycinebarbital.

+*

_*

_*

_*

Per cent recovery

Entero-toxinA

69

38

30

9

25

15

Entero-toxin B

72

48

48

17.5

40

24

and 0.02 M sodium

TABLE 3. Staphylococci and enterotoxin A in foodsinoculated with strain 265-1

Food Staphylococci Enterotoxin A(billions/g) (jAg/g)

Coconut custard 2.5 0.2Shrimp paste 15.0 14Turkey paste 13.5 8-9

TABLE 4. Recovery of enterotoxin A in foods in-oculated with strain 265-1

Enterotoxin AStaphylo- Enterotoxin recovered

Food cocci/g A in 20 g(millions) of food Per

Amt cent

pg AgCoconut

custard..... 250 0.4 0.2 50Turkey....... 750 8-9 7 80Shrimp. 675 14 9 60

at pH 6.0 would also interfere with the detectionof lines of precipitation formed by traces ofenterotoxin.

Detection of enterotoxin A in foods inoculatedwith an enterotoxigenic staphylococcus. Foodswhich were inoculated with strain 265-1 andincubated at 35 to 37 C for 48 hr supported ex-cellent growth. Enterotoxin could be detected inthe 20% extracts of the turkey paste and shrimppaste without concentration.

Supernatant liquids from the 20% slurries,prepared in 0.2 M NaCl in a Waring Blendor andcentrifuged at pH 7.5 to 7.6 for 10 min at 32,800X g, were concentrated by dialysis against PEGand titrated by the gel diffusion test. The num-bers of staphylococci and amounts of enterotoxinA produced are presented in Table 3. It is evidentthat the turkey and shrimp are stuperior to coco-nut custard in support of growth of the staphylo-coccus and the production of enterotoxin A. Thenumbers of staphylococci per gram of the in-oculated foods are considerably greater than thosegenerally found in foods responsible for foodpoisoning.The inoculated foods were diluted with un-

inoculated food to approximate the numbers ofstaphylococci encountered in food-poisoningincidents. These diluted foods were examinedfor their enterotoxin contents. A 2-g amount ofinoculated coconut custard, containing approxi-mately 0.4 j,g of enterotoxin A, was diluted with18 g of coconut custard obtained from a freshlybaked frozen coconut custard pie. A 19-g amountof ground steamed turkey meat was mixed with1 g of turkey paste containing approximately 8 to

9 ,g of enterotoxin A; also, 19 g of groundsteamed shrimp were mixed with 1 g of shrimppaste containing approximately 14 ,ug of entero-toxin A. Slurries of the mixtures were preparedin 0.2 M NaCl and examined for their enterotoxincontents. The coconut custard slurry was proc-essed as described above (see Materials andMethods). The supernatant liquids obtained oncentrifuging the turkey and shrimp slurries weresimilarly processed except for dilution of thesupernatant liquids with 10 volumes rather than20 volumes each of distilled water and 0.01 Msodium phosphate before passage through theCM-cellulose. The final volumes after elutionand concentration were 0.1, 0.9, and 0.75 ml forthe custard, turkey, and shrimp, respectively.The approximate percentages of recovery ofenterotoxin A are presented in Table 4.

Detection of enterotoxin A in foods responsiblefor poisonings. The procedures which were em-ployed successfully for the detection of entero-toxin added to food and for the detection ofenterotoxin in foods which were inoculated withan enterotoxigenic staphylococcus were testedwith foods which were responsible for outbreaksof food poisoning. The types of foods (creampies, cooked ham, and macaroni salad) which wereavailable for these tests were such that CM-cellulose equilibrated at pH 6.0 was used toadsorb the enterotoxin from diluted extractsadjusted to pH 6.0. The numbers of staphylococciin the foods, as determined by the plating ofdilutions of their suspensions and by microscopicexamination, indicated that 20-g quantities of

186 APPL. MICROBIOL.

on April 30, 2021 by guest

http://aem.asm

.org/D

ownloaded from

STAPHYLOCOCCAL ENTEROTOXIN IN FOOD

the cream pies and ham samples could be ex-amined successfully.The number of staphylococci in the macaroni

salad was so small that the entire 360 g of samplewere examined as a 40% suspension in 0.2 M

NaCl (pH 7.5 to 7.6). After centrifugation at32,800 X g for 10 min, the large volume of super-

natant fluid was placed in dialysis tubing andconcentrated to 64 ml by dialysis against PEG.The concentrated supernatant was diluted with15 volumes of distilled water and 15 volumes of0.01 M sodium phosphate (pH 6.0), and passedthrough the CM-cellulose equilibrated to pH 6.0and eluted and concentrated as described above.The 0.15 ml of concentrated extract from 20 g

of the ham involved in the fourth food poisoningincident was only faintly positive for enterotoxinA. Concentration to a smaller volume was notattempted because the concentrate absorbedmoisture and overflowed its well in the plasticmatrix of the slide gel diffusion assembly.A definitely positive test was obtained, how-

ever, when the extract of an 80-g sample of theham was passed through Sephadex G-100 andthen through CM-cellulose. The 80-g portion wasextracted as a 40% slurry in 0.2 M NaCl, and wasconcentrated by osmotically forced dialysis. Theconcentrate was clarified by centrifugation andfiltration through a membrane filter and dividedinto two 14-ml quantities. These were separatelypassed through Sephadex G-100 (see Materialsand Methods), and the two eluates were combinedand concentrated to 26.8 ml. The 26.8 ml of con-centrate were diluted with 20 volumes each ofdistilled water and 0.01 M sodium phosphate(pH 6.0), passed through CM-cellulose, andeluted and concentrated as described above to0.2 ml of a brick-red suspension which did notabsorb moisture in the gel diffusion test.No attempt was made to measure the entero-

toxin in these foods. The undiluted concentratedextracts were placed in the wells of the plastic

matrix of the gel diffusion assembly. As indicatedin Table 5, positive results were obtained with allof the samples.When 20-g portions of the coconut cream pie

and banana cream pie were examined by gelfiltration on Sephadex G-100 (see above), thecolumn eluates became viscid on concentrationand could not be reduced in volume sufficientlyto permit detection of the enterotoxin.

Detection of enterotoxins other than "A" and"B." The applicability of the above proceduresto the detection of enterotoxins which have notbeen serologically identified was determined.Such enterotoxins were produced with strains291, 293, 294, and 315 by use of the sac culturetechnique. They were studied with respect totheir adsorption to and elution from CM-celluloseand their detection when mixed with meat bymeans of the procedures developed for the de-tection of enterotoxins A and B. The intravenousinjection of cats was used for their detection andassay.To determine the approximate cat-vomiting

doses (Casman, 1958) of the toxins, portionswere digested with pancreatin to remove hemoly-sins and injected intravenously. From 2 to 4cat-vomiting doses of each toxin were dilutedwith 20 volumes each of distilled water and 0.01M sodium phosphate (pH 6.0), and passed through1 g of CM-cellulose equilibrated at pH 6.0. TheCM-cellulose columns were washed, and eachwas eluted with 150 ml of 0.2 M NaCl in 0.2 Msodium phosphate (pH 7.4 to 7.5). The eluateswere reduced in volume by dialysis against PEG,digested with pancreatin, and injected intra-venously into cats. The results of the toxin assaysand the emetic responses to the digested eluatesfrom the CM-cellulose columns are presented inTable 6.Having determined that the enterotoxins

were adsorbed to and eluted from CM-cellulose,we blended 5 ml of each toxin with 20 g of cooked

TABLE 5. Detection of enterotoxin A in foods responsible for food poisonings

Group | Food (mSyloi/g Amt examined Final vol of extract EnteGroup Food ~~~~~~~~~(millions)toiA9 ini~~~~~~txi

I Coconut cream pie 200 20 0.05-0.1 +Banana cream pie 72.5 20 0.05-0.1 +

II Ham 50 20 0.1 +

III Macaroni salad 1.0 360 0.1 +

IV Ham 3 billion* 20 0.15 ?3 billion* 80 0.2 +

* Nonenterotoxigenic.

VOL. 13, 1965 187

on April 30, 2021 by guest

http://aem.asm

.org/D

ownloaded from

CASMAN AND BENNETT

TABLE 6. Adsorption of unidentified enterotoxins toCM-cellulose

Emetic Toxinresponses* to passed Per cent Emetic

Strain toxins thirough eluate responses*CM- per cat to eluates

0.5 ml 1.0 ml cellulose

ml

291 1/2 1/2 2 100 1/1293 1/1 2/3 2 100 1/1294 1/2 1/2 2 100 1/1315 0/2 2/3 4 50 1/2

* Number of catsinjected.

vomited/number of cats

ground beef and 100 ml of 0.2 M NaCl and at-tempted to demonstrate the presence of the toxinsby use of the separation and concentration tech-niques described above for the detection of entero-toxins A and B. The eluates from the CM-cellulose columns were concentrated and digestedwith pancreatin, and one-half of each was in-jected intravenously into cats.The eluates obtained on processing beef to

which enterotoxins produced by strains 291, 293,294, 315, and 304 had been added producedemetic responses at 136, more than 300, 36, 47,and 39 min, respectively. Two 20-g quantitiesof the cooked beef were processed in the samemanner without the addition of enterotoxin.Neither of two cats gave emetic responses on

injection of the entire amount of each eluate.

DISCUSSiONIn previously reported studies (Casman et al.,

1963) on staphylococcal growth and enterotoxinproduction in meats, we used less efficient pro-

cedures for extracting, separating, and concen-

trating enterotoxin. The use of water instead of0.2 M NaCl resulted in extraction of less entero-toxin but was responsible also for extraction ofless of the meat constituents. The use of CM-cellulose at pH 6.8, rather than pH 6.0, similarlyresulted in the adsorption of smaller amounts ofboth enterotoxin and meat extractives. Finally,because concentration of the eluates by means ofthe LKB ultrafilter was used, there was an ap-preciable loss of enterotoxin. We have foundthat approximately 25% of crude enterotoxinpasses through the ultrafilter. The amounts re-

covered were much smaller than amounts one

would expect to be present as a result of the ex-

cellent growth of the staphylococcus with whichthe meats were inoculated.

In the present studies, extraction with 0.2 M

NaCl followed by adsorption to CM-cellulose and

concentration of the eluate by dialysis againstPEG permitted the serological detection of 38 to69% of the enterotoxin A and 48 to 72% of theenterotoxin B which was added to selected foods.The method was used successfully to detect verysmall amounts of enterotoxin added to custard,cooked turkey, and shrimp, as well as the entero-toxin in foods responsible for food poisoning.The presence of extractives which clouded thegel of the gel diffusion test for enterotoxin andlimited the degree of concentration of the eluatesreduced the applicability of the procedure forthe detection of small amounts of enterotoxin inraw beef. This deficiency may not be importantfrom a practical point of view, however, sinceraw meats are generally not involved in staphylo-coccal food poisoning.As indicated by our study of the macaroni

salad which was responsible for an outbreak offood poisoning, amounts of food considerablygreater than 20 g may be examined. This pro-cedure is especially applicable when the fooddoes not contain extractives which are adsorbedby and eluted from the CM-cellulose.When excessive amounts of these interfering

extractives are present in a food, the applica-bility of the CM-cellulose method is limited toextracts of samples considerably larger than 20 gwhich are first passed through Sephadex G-100or to samples containing relatively large numbersof enterotoxigenic staphylococci. For example,the ham which was incriminated in an outbreakof food poisoning, and which contained 50million staphylococci per gram, contained justenough enterotoxin A to permit its detectionwhen the extract from 20 g was concentrated to0.1 ml. Further concentration was not possiblebecause of the presence of other extractives, sothat the presence of fewer, or less enterotoxigenic,staphylococci could have resulted in a negativetest for enterotoxin. Under such circumstances,the availability of a serological test more sensitivethan the slide double-diffusion test would be verydesirable. Preliminary trials with the hemag-glutination-inhibition test in which erythrocyteswere sensitized with partially purified entero-toxin, indicate that an approximately 10-foldincrease in sensitivity may be expected, and thatits application for the detection of enterotoxinin foods will depend on the availability of anti-genically pure enterotoxin.The methods developed for the detection of

enterotoxins A and B may be applicable to thedetection of staphylococcal enterotoxins not yetidentified serologically. Generalization, however,is not possible at this time, because we know

188 APPL. MICROBIOL.

on April 30, 2021 by guest

http://aem.asm

.org/D

ownloaded from

STAPHYLOCOCCAL ENTEROTOXIN IN FOOD

neither the number of types represented by thetoxins included in this study, nor the numberinvolved in food poisoning.The slide double-diffusion test of Crowle

(1958) and Wadsworth (1957) is especiallyvaluable for the detection of enterotoxin in foods.It is technically simple and requires very smallamounts (0.02 to 0.03 ml) of reagents. Entero-toxin is detected at concentrations of 1 ,ug/ml, asensitivity which is the same as that of the tubedouble-diffusion test of Oakley and Fulthorpe(1953). The lines of precipitation are easilyidentified through their coalescence with linesformed by reference toxins-a property which isvaluable in the examination of food extractswhich may produce lines of precipitation notcaused by specific reaction with antibody. Suchprecipitation would present a considerableproblem in the single-diffusion test of Oudin(1952) or the double-diffusion test of Oakley andFulthorpe (1953).

LITERATURE CITEDCASMAN, E. P. 1958. Serological studies of staphy-

lococcal enterotoxin. Public Health Rept. (U.S.)73:599-609.

CASMAN, E. P. 1960. Further serological studies ofstaphylococcal enterotoxin. J. Bacteriol. 79:849-856.

CASMAN, E. P., AND R. W. BENNETT. 1963. Culturemedium for the production of staphylococcalenterotoxin A. J. Bacteriol. 86:18-23.

CASMAN, E. P., M. S. BERGDOLL, AND J. ROBINSON.

1963. Designation of staphylococcal entero-toxins. J. Bacteriol. 85:715-716.

CASMAN, E. P., D. W. McCoy, AND P. J. BRANDLY.1963. Staphylococcal growth and enterotoxinproduction in meat. Appl. Microbiol. 11:498-500.

CROWLE, A. J. 1958. A simplified micro double-diffusion agar precipitin technique. J. Lab.Clin. Med. 52:784-787.

DOLMAN, C. E., AND R. J. WILSON. 1938. Experi-ments with staphylococcal enterotoxin. J.Immunol. 35:13-30.

FREA, J. I., E. McCoY, AND F. M. STRONG. 1963.Purification of type B staphylococcal entero-toxin. J. Bacteriol. 86:1308-1313.

FUJIWARA, K. 1961. Serological problems in staph-ylococcal food poisoning. Am. Rept. Inst. FoodMicrobiol., Chiba Univ., Japan 14:1-8.

HALBERT, S. P., L. SWICK, AND C. SONN. 1955. Theuse of precipitin analysis in agar for the studyof human streptococcal infections. J. Exp. Med.101:557-576.

HAMMON, W. McD. 1941. Staphylococcus entero-toxin: an improved cat test, chemical and im-munological studies. Am. J. Public Health31:1191-1198.

OAKLEY, C. L., AND A. J. FULTHORPE. 1953. Anti-genic analysis by diffusion. J. Pathol. Bacteriol.65:49-0.

OUDIN, J. 1952. Specific precipitation in gels.Methods Med. Res. 5:335-378.

SCHANTZ, E. J. 1964. Purification and charac-terization of staphylococcal enterotoxin B. Proc.Army Science Conference. West Point, N.Y.

WADSWORTH, C. 1957. A slide microtechnique forthe analysis of immune precipitates in gel.Int. Arch. Allergy Appl. Immunol. 10:355-360.

VOL. 13, 1965 189

on April 30, 2021 by guest

http://aem.asm

.org/D

ownloaded from