International Journal of Food Microbiology, 10 (1990) 91-100 Elsevier

91

FOOD 80005

Analytical methods for Staphylococcus aureus Merlin S. Bergdoll Food Research Institute, University of Wisconsin, Madison, WI, U.S.A. Key words: Enterotoxin; Analytical methods; Staphylococcus spp.

Introduction

The staphylococcal enterotoxins are relatively low molecular weight proteins, 27000 to 29000 Da (Bergdoll, 1979). They are produced by the staphylococci, primarily Staphylococcus aureus, although the newer species, Staphylococcus intermedius and Staphylococcus hyicus have been reported to be enterotoxigenic (Adesiyun et al., 1984; Fukuda et al., 1984). The guideline followed for many years in the Food Research Institute to determine whether staphylococci might produce enterotoxin was to determine whether the particular strain produced either coagulase or thermonuclease (TNase). Formerly, both S. intermedius and S. hyicus would have been classified as S. aureus. Therefore, if we follow the current species classification, we cannot confine our testing of staphylococcal strains to S. aureus alone. To my knowledge, no one is currently working on the identification of additional enterotoxins although we do know that unidentified ones do exist. This is very difficult to accomplish because a specific biological test such as the monkey feeding test is necessary to identify the enterotoxin (Surgalla et al., 1953). Apparently, only about five percent of staphylococcal food poisoning outbreaks are due to unidentified enterotoxins. The enterotoxins that have been identified so far are classified as enterotoxins A (SEA), B (SEB), C 1 (SEC1), C~ (SEC2), C 3 (SEC3), D (SED) and E (SEE). The enterotoxin Cs are very closely related and can be identified by their cross-reactions with antibodies prepared against any one of the SECs. The other enterotoxins are identified by antibodies specific for each of the enterotoxins, although cross-reactions between SEB and the SECs (Lee et al., 1980) and SEA and SEE (Lee et al., 1978) do exist. Monoclonal antibodies have been prepared that are useful in the sandwich ELISA for the detection of SEB, SECs, SEA and SEE by one set of two antibodies for each group. Efforts to prepare monoclonal antibodies that cross-react with SEA, SED and SEE were generally unsuccessful, although an

Correspondence address: Food Research Institute, University of Wisconsin, 1925 Willow Drive. WI 53706, U.S.A. 0168-1605/90/$03.50 © 1990 Elsevier Science Publishers B.V.

92 TABLE 1 C o l l l m o n a m i n o acid sequence o f the e n t e r o t o x i n s Type of

A m i n o acid sequence

cnterotoxin

SEA

-Thr-A la-Cys-Met-Tyr-Gly-Gly-Val-Thr-Leu-His-Asp-Asn-Asn-Arg-Leu-Yhr-

SEB

-Lys-Thr-Cys-Met-Tyr-Gly-Gly-Val-Thr-Gln-Hi

SE(" I

-Lys-Thr-Cys-Met-Tyr-Gly-Gly-I le-Thr-Lys-Hi s-Glu-Gly-Asn-Hi s-Phe-Asp-

s-Gly-Asn-Asn-Glu-Leu-Asp-

antibody was prepared that reacts strongly with SEA and SEE but only weakly with SED. Attempts to use this antibody in the sandwich ELISA, coupled with specific antibodies to each of the enterotoxins were unsuccessful. Amino acid sequencing of SEA, SEB and SEC~ revealed that the enterotoxins have a similar structure, the cystine loop (Bergdoll and Robbins, 1973) and a common amino acid sequence (Bergdoll, 1985) (Table I). It has been speculated that this common sequence may be the toxic site in the enterotoxin molecule as it is the only area of homology between the enterotoxins (Huang et al., 1976). It was planned to synthesize a peptide containing this sequence and use it to prepare monoclonal antibodies to it; however, this was not accomplished because the individual responsible for the monoclonal work left the staphylococcal research group before this was undertaken. It was hoped that such an antibody could be used to detect and identify new enterotoxins. Attempts to detect unidentified enterotoxins with the cross-reacting monoclonal antibodies to SEB and the SECs, and SEA, SED, and SEE were unsuccessful.

Detection of enterotoxins

All of the methods for the detection of the enterotoxins are based on the use of the antibodies prepared against the enterotoxins. Essentially all of the antibodies in use have been prepared in rabbits using the individual purified enterotoxins (Robbins and Bergdoll, 1984). These polyclonal antibodies react with the enterotoxins in gels to give precipitin reactions which make them highly specific. The monoclonal antibodies cannot be used in gels because their reactions with the enterotoxins do not result in the formation of precipitates. Many types of gel reactions have been used in the detection of the enterotoxins, the most common ones being some form of the Ouchterlony gel plate or some form of the microslide (Ouchterlony, 1949, 1953; Crowle, 1958). These methods have been used widely in the determination of the enterotoxigenicity of staphylococcal strains. The modification of the Ouchterlony gel plate test that is used in the Food Research Institute and recommended to others is the optimum sensitivity plate (OSP) method (Robbins et al., 1974). It is easy to use and in conjunction with production of the enterotoxins by the membrane-over-agar method (Robbins et al.,

93

f IX

5×

@

IX

@

5×

IX

% 5×

Fig. 1. Enterotoxin analysis with Optimum Sensitivity Plates (OSP). A, B. C = SEA, SEB, SEC, respectively (4 ffg/ml), aA, aB, aC = anti-SEA, SEB, SEC, respectively. U nknow n UI contains approximately 0.5 p,g A and 6 /xg B per ml; unknown U2 contains no SEA, SEB, SEC; unknown U3 contains approximately 2 fig S E A / m l ; unknown U4 contains approximately 16 p,g C per ml. 1 x = unconcentrated unknowns; 5 X = 5 times concentrated unknowns.

1974) or the sac culture method (Donnelly et al., 1968) and is of adequate sensitivity to detect most enterotoxigenic staphylococci. The normal sensitivity is 0.5 ffg/ml but can be increased to 0.1 ~ g / m l by a 5-fold concentration of the staphylococcal culture supernatant fluids (Fig. 1). The microslide is used by some investigators but it is a rather difficult method to set up and make sure that good results will be obtained. Many things can go wrong with this method and experience is very important in using it successfully (Casman et al., 1969). More recently a question has arisen regarding the sensitivity of the gel diffusion methods for determining the enterotoxigenicity of staphylococcal strains. Igarashi et al. (1986) have reported that enterotoxin production of strains was observed by the reversed passive latex agglutination (RPLA) method that was not detectable by the OSP method, in the neighborhood of 10 to 20 n g / m l (Table II). This was confirmed by concentrating the culture supernatant fluid from five strains that tested positive for SEA by ELISA about 100-fold for testing by the OSP method. Positive results were obtained. The importance of this low production may be questioned; however, we have found strains that were implicated in food poisoning outbreaks to be negative by OSP but positive by ELISA. This resulted from the examination of strains by ELISA that were negative by OSP but positive by the monkey feeding test

94 TABLE II Staphylococcal strain testing by reverse phase latex agglutination (RPLA) method Strain

188 228 311 365 452 581 609 754 802 887 896 965

Enterotoxin RPLA

OSP a

SEA, SEA, SEA SEC SEA SEC SEA, SEA, SEB SEA SEA, SEA,

SEA, SED SEA

SED SED

SED SEB

SEB SED

SEA SEC SEA, SEB SEB SEA, SEA, SED

'~ Optimum sensitivity plate; modified Ouchterlony gel plate test.

(Kokan and Bergdoll, 1987). A number of these strains were positive for one or more of the identified enterotoxins by the ELISA method, particularly for SED (23 strains). Some of these strains had been isolated from food poisoning outbreaks which is significant because SED has been implicated as the second most important enterotoxin in food poisoning. It should be pointed out that SED is produced in the smallest amounts by strains of any of the enterotoxins (Robbins et al., 1974). It is interesting to note that only three of the strains produced low amounts of SEA, the enterotoxin implicated in 75% of staphylococcal food poisoning outbreaks (Wieneke and Gilbert, 1987). The production of 10 to 20 ng of enterotoxin/ml is probably of significance because only 100 to 200 ng of enterotoxin A was shown to be necessary to produce food poisoning (Evenson et al., 1987), with the amount present in the vehicle, 2% chocolate milk, being 0.50 to 0.75 n g / m l . Admittedly, the amount of enterotoxin produced by the membrane-over-agar method is 5 - 1 0 times that produced in shake flasks or even possibly in foods, yet if growth is sufficient, (10 s to 109 cells per ml), 1 to 2 ng of enterotoxin/g of food may be produced. This would be adequate to result in staphylococcal food poisoning in sensitive individuals. It is important, therefore, that standard procedures be developed for the examination of staphylococcal strains for enterotoxin production. The detection of enterotoxin in foods requires much more sensitive methods than those required for the determination of the enterotoxigenicity of strains. The quantity of enterotoxin present in foods involved in food poisoning outbreaks may vary considerably, from less than 1 n g / g to greater than 1 /~g/g. Usually, little difficulty is encountered in detecting the enterotoxin in foods involved in staphylococcal food poisoning outbreaks, however, outbreaks do occur in which the amount of enterotoxin is less than 1 n g / g , such as was the case with the 2% chocolate milk already mentioned. In such cases, the enterotoxin can be detected only by the most

95 sensitive methods. Another situation in which it is essential to use a very sensitive method is in determining the safety of a food for consumption. In such situations it is necessary to show that no enterotoxin is present by the most sensitive methods available. The initial methods for detecting enterotoxin in foods were extraction concentration methods with the microslide as the method for detection of the enterotoxin in the concentrated extract (Casman and Bennett, 1965; Reiser et al., 1974). The amount of food required was 100 g with concentration of the extract to 0.2 ml. Reaching this volume required removal of most of the extractable solutes which made considerable purification necessary. It was difficult to detect the enterotoxin at the n g / g level with these methods. The time required for completing these procedures was about 5 days. The first really sensitive method proposed for detection of enterotoxin in foods was the reversed passive hemagglutination (RPHA) method (Silverman et al., 1968). This appeared to be a promising method but it was difficult to eliminate the substances in some food extracts that produced non-specific hemagglutination. In addition, it was not always possible to couple the antibodies to the red blood cells. After much experimentation, research on this method was abandoned in favor of research on the radioimmunoassay (RIA). Considerable success was achieved with this method by treating the unknown sample with the specific antibodies followed by the radioactive enterotoxin (Miller et al., 1978). The enterotoxin-antibody complex was precipitated by use of protein A cells. The centrifuged precipitate which contained the antibody-enterotoxin complex was read in a scintillation counter to determine the amount of iodinated enterotoxin present. The amount of enterotoxin present in the unknown sample was indirectly related to the amount of radioactive enterotoxin present; the less the radioactivity, the greater the amount of enterotoxin in the food sample. It was possible to detect enterotoxin at the level of 1 n g / g of food using a simple extraction procedure without concentration of the extract. The method was used in the Food Research Institute for several years before development of the enzyme-linked immunosorbent assay (ELISA) methods. The basic requirements of handling radioactive materials along with the need for a scintillation counter and the purified enterotoxins limited the usage of this method to a few laboratories. The ELISA methods were applied to the detection of the enterotoxins in foods soon after they were originally developed for the detection of other proteins. Two types of ELISA procedures have been reported: (1) the competitive method in which the antibody reacts with the unknown sample before being treated with the enzyme-enterotoxin conjugate (Stiffler-Rosenberg and Fey, 1978) and (2) the sandwich method in which the antibody is treated with the unknown sample before the antibody-enterotoxin complex is treated with the enzyme-antibody conjugate (Saunders and Bartlett, 1977). The latter procedure is preferred because the amount of enzyme and, thus, color developed, is directly proportional to the amount of enterotoxin present in the unknown sample. This eliminates the need for the highly purified enterotoxins as crude or only partially purified enterotoxin is needed for preparation of a standard curve.

96 TABLE lII Detection of staphylococcal enterotoxins in foods by ELISA and RIA procedures Food

Enterotoxin

Amount of enterotoxin

Amount of enterotoxin detected ( n g / g )

added ( n g / g )

ELISA

RIA

Milk

SEA

0.63 1.25

0.63

0.54 0.95

Ham

SEA

0.63 1.25

0.34

0.72 1.14

Sausage

SEA

0.63 1.25

0.36

ND a 0.56

Cheese

SEA SED

0.63 0.63

0.59 0.15

Potato salad

SEB

0.63

0.18

" ND, not detectable.

The majority of users of the ELISA method use microtiter plates to which the antibodies are attached. The large number of wells in a microtiter plate provide for doing several samples at one time, although there may not be uniformity in all of the wells, particularly those around the edge of the plate. The use of a plate reader for recording the results which adds expense to the method, is necessary. An alternate procedure has been developed, and that is the use of polystyrene balls to which the antibodies are attached (Stiffler-Rosenberg and Fey, 1978; Freed et al., 1982; Fey et al., 1984). The ball method is more cumbersome because each ball must be handled separately. The main advantage is that a relatively large volume of the unknown sample can be used, and thus, increase the amount of enterotoxin adsorbed per sample. This makes the use of 1 ml volumes of substrate possible so that the color developed can be read in a simple colorimeter, an instrument that most laboratories would have available. The sensitivity of the ELISA methods are between 0.5 to 1.0 n g / g of food (Table III). Polyclonal antibodies prepared in rabbits have been and are used in the enterotoxin detection methods. The development of monoclonal antibodies to the enter° otoxins has made their use in enterotoxin analysis possible and currently they are being used in the development of an ELISA dip stick method for enterotoxin detection in foods. One kit that employs the ELISA ball method is now commercially available. Those who have used it have found it to be a very good method for detecting enterotoxin in foods (Fey and Pfister, 1983). A RPLA kit is available commercially that is advertised for use in the detection of enterotoxins in foods. The method is adequately sensitive for the detection of enterotoxin in solution, but the extraction method recommended for foods (9 ml b u f f e r / g food) is inadequate for the detection of the small amounts of enterotoxin that can be present to cause food poisoning in sensitive individuals (Igarashi et al.,

97 TABLE IV Detection of staphylococcal enterotoxin (SET) in foods from outbreaks (from Wieneke and Gilbert, 1987) Food

Ham Vanilla slice Turkey & duck Lasagne. dried Salmon, canned Halloumi cheese Sheep milk cheese Corned beef Ham Smokey bacon spd. Salmon-mouse Ham rolls Beef rolls Pork Chicken Chicken chow mein Meat pies Corned beef Cold buffer

S. aureus

SET detected in food

count

SET by strain

1.5×10 9 1.3 × 10 9 3.0 × 10 8 2.0 × 10 8 3.5 × 10 6 ND ND 4.0 × 10 v 1.2×109 1.0 × 109 9 . 0 x 108 5.0 x 108 4.0 × 106 6.0 X 109 1.0 x 106 1.5 x 106 1.0 x 106 8.5 × 104 6.0×103

A A A A A

A, B A,D A, D A, D A, D A, D B C A A A A,B

Geldiff. A ND a ND ND A A,D A, D A, D B

ELISA plate

ELISA kit

RPLA kit

A A A A A A A A, B A¢ A~ Ac A~ Ac B

A A A A A A A A, B A,D A, D A, D A, D A B C ND ND ND ND

A A A A A A NSA b A, B Ac A, D A¢ A~ ND c B ND ND ND ND ND

ND ND ND ND

ND, not detected. b NSA, non-specific agglutination. Extract not tested for D.

1985). One paper has been published on the comparison of the various methods for the detection of enterotoxin in foods, including the R P L A method (Wieneke and Gilbert, 1987). In this work the same extraction procedure was used for all the methods, one that provides the sensitivity necessary for detection of enterotoxin in foods. In this comparison we see that the RPLA method is adequate in most situa, tions and could be used if the extraction procedures were those used in making this comparison (Table IV). The current procedures are adequate for detection of enterotoxin in foods and it is possible to do them in one day. There is always the request that the time for doing an analysis be shortened, but in reality, there is no great need to obtain results in less than the time now required. We have come a long way in improving the methods for enterotoxin detection in foods and it is not improbable that further improvements will be made in the future. References Adesiyun, A.A., Tatini, S.R. and Hoover, D.G. (1984) Production of enterotoxin(s) by Staphylococcus hyicus. Vet. Microbiol. 9, 487-495.

98 Bergdoll, M.S. (1979) Staphylococcal intoxications. In: H. Reimann and F.L. Bryan (Eds.), Food-borne Infections and Intoxications, 2nd Edn, Academic Press, New York, pp. 443-494. Bergdoll, M.S. (1985) The staphylococcal enterotoxins--an update, In: J. Jeljaszewicz (Ed.), The Staphylococci, Zbl. Bakteriol. Suppl. 14, Gustav Fischer Verlag, Stuttgart, New York, pp.2 47-254. Bergdoll, M.S. and Robbins, R.N. (1973) Characterization of types of staphylococcal enterotoxins. J. Milk Food Technol. 36, 610-612. Casman, E.P. and Bennett, R.W. (1965) Detection of staphylococcal enterotoxin in food. Appl. Microbiol. 13, 181-189. Casman, E.P., Bennett, R.W., Dorsey, A.E. and Stone, J.E. (1969) The micro-slide gel double diffusion test for the detection and assay of staphylococcal enterotoxins. Health Lab. Sci. 6, 185-198. Crowle, A.J. (1958) A simplified micro double-diffusion agar precipitation technique. J. Lab. Clin. Med. 52, 784-787. Donnelly, C.B., Leslie, J.E. and Black, L.A. (1968) Production of enterotoxin A in milk. Appl. Microbiol. 16, 917-924. Evenson, M.L., Hinds, W.M., Bernstein, R.S. and Bergdoll, M.S. (1988) Estimation of human dose of staphylococcal enterotoxin A from a large outbreak of food poisoning involving chocolate milk. Int. J. Food Microbiol. 7, 311-316. Fey, H. and Pfister, H. (1983) A diagnostic kit for the detection of staphylococcal enterotoxins (SET) A, B, C and D (SEA, SEB, SEC, SED). In: S. Avrameas et al. (Eds.), Immunoenzymatic Techniques. Elsevier, Amsterdam, pp. 345 348. Fey, H., Pfister, H. and Riiegg, O. (1984) Comparative evaluation of enzyme-linked immunosorbent assay systems for the detection of staphylococcal enterotoxins A, B, C and D. J. Clin. Microbiol. 19, 34 38. Freed. R.C., Evenson, M.L., Reiser, R.F. and Bergdoll, M.S. (1982) Enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxins in foods. Appl. Environ. Microbiol. 44, 1349-1355. Fukuda, S., Tokuna, H., Ogawa, O., Sasaki, M., Kishimoto, T., Kawano, J., Shimizu, A. and Kimura, S. (1984) Enterotoxigenicity of Staphylococcus intermedius strains isolated from dogs. Zbl. Bakteriol. Hyg. A 258, 360-367. Huang, I.Y., Schantz, E.J. and Bergdoll, M.S. (1976) The amino acid sequence of the staphylococcal enterotoxins. Jpn. J. Med. Sci. Biol. 28, 73-75. lgarashi, H., Shingaki, M., Fujikawa, H., Ushioda, H. and Terama, T. (1985) Detection of staphylococcal enterotoxins in food poisoning outbreaks by reversed passive latex agglutination, In: J. Jeljaszwicz (Ed.) The Staphylococci, Zbl. Bakteriol. Suppl. 14, Gustav Fischer Verlag, Stuttgart, New York, pp. 255 257. Igarashi, H., Fujikawa, H., Shingaki, M. and Bergdoll, M.S. (1986) Latex agglutination test for staphylococcal toxic shock syndrome toxin 1. J. Clin. Microbiol. 23, 509 512. Kokan, N. and Bergdoll, M.S. (1987) Detection of low enterotoxin-producing Staphylococcus aureus strains. Appl. Environ. Microbiol. 53, 2675-2676. Lee, A.C.-M., Robbins, R.N. and Bergdoll, M.S. (1978) Isolation of specific and common antibodies to staphylococcal enterotoxins A and E by affinity chromatography, Infect. Immun. 21, 387-391. Lee, A.C.-M., Robbins, R.N. and Bergdoll, M.S. (1980) Isolation of specific and common antibodies to staphylococcal enterotoxins B, C 1 and C z. Infect. Immun. 27, 432-434. Miller, B.A., Reiser, R.F. and Bergdoll, M.S. (1978) Detection of staphylococcal enterotoxins A, B, C, D, and E in foods by radioimmuno-assay, using staphylococcal cells containing protein A as immunosorbent. Appl. Environ. Microbiol. 36, 421-426. Ouchterlony, O. (1949) Antigen-antibody reactions in gels. Acta Pathol. Microbiol. Scand. 26, 507-515. Ouchterlony, O. (1953) Antigen-antibody reactions in gels. IV. Types of reactions in coordinated systems of diffusion. Acta Pathol. Microbiol. Stand. 29, 231-240. Reiser, R.F., Conaway, D. and Bergdoll, M.S. (1984) Detection of staphylococcal enterotoxin in foods. Appl. Microbiol. 27, 83-85. Robbins, R.N. and Bergdoll, M.S. (1984) Production of rabbit antisera to the staphylococcal enterotoxins. J. Food Protect. 47, 172-176. Robbins, R., Gould, S. and Bergdoll, M. (1974) Detecting the enterotoxigenicity of Staphylococcus aureus strains. Appl. Microbiol. 28, 946 950.

99 Saunders, G.C. and Bartlett, M.L. (1977) Double-antibody solid-phase enzyme immunoassay for the detection of staphylococcal enterotoxin A. Appl. Environ. Microbiol. 34, 518-522. Silverman, S.J., Knott, A.R. and Howard, M. (1968) Rapid, sensitive assay for staphylococcal enterotoxin and a comparison of serological methods. Appl. Microbiol. 16, 1019-1023. Stiffler-Rosenberg, G. and Fey, H. (1978) Simple assay for staphylococcal enterotoxins A, B, and C: modification of enzyme-linked immunosorbent assay. J. Clin. Microbiol. 8, 473-479. Surgalla, M.J., Bergdoll, M.S. and Dack, G.M. (1953) Some observations of the assay of staphylococcal enterotoxin by the monkey-feeding test. J. Lab. Clin. Med. 41, 782-788. Wieneke, A.A. and Gilbert, R.J. (1987) Comparison of four methods for the detection of staphylococcal enterotoxin in foods from outbreaks of food poisoning. Int. J. Food Microbiol. 4, 135 143.