OXOID

PubJis/1ed as a service to microbiology by Oxold Lllnl ted

Volume 8 No 1 April 1987

Reversed passive latex agglutination tests for the rapid and simple detection of bacterial toxins Hideo Igarashi and Junichi Sugiyama Department of Microbiology , Tokyo Metropolitan Research Laboratory of Public Health , Shinjuku-Ku , Tokyo 160 and Denka Seiken Co Ltd , Gosen-Shi, Niigata 959-16, Japan

Introduction A series of test kits for detecting bacterial toxins have recently been put on the market byOXOId Limited. These kits employ reversed pass­Ive tatex agglutination (RPLA) as the assay method and, in this article. the development, character­istics and value of the method for detecting bacterial toxins are briefly reviewed . Bacteria produce enzymes and toxins With vanous biological activities. Some of the products of their metabohsm such as toxins are closely associated With human diseases, sometimes acting as causative agents. It ;s tllerelore very useful to confirm the presence of tox ins in test samples for the diagnosis of certain diseases, How­ever. If the assay to detect such toxins involves expensive equip· ment. technically difficult or lengthy procedures, experience has shown that the test will not be widely used in the diagnOSIs of disease. Various techniques have been dev· eloped for the detection of bacterial

toxins. The easiest and most eco­nomical method which does not requi re specia l equipment IS that based upon the agglutination of preserved erythrocytes or latex particles as a reaction Indicator. RPLA was selected for the assay because of ItS excellenl sensitivity and specifiCity. It was originally developed as a method to detect staphylococcal enlerotoxln and later found sUitable for fun her applications In the detection of Vibno cho/erae enterotoxin. Esche­(fCh18 colt heat-labile enterotoxin and Clostridium perfrmgens enter· otOXIn. In the following part of this article these applications 01 RPLA for enterotoxin detection are described. Salmon and Tew 1 first described RPLA as a method to detect staph­ylococcal enterotoxins. The sensi­tivity of their method in detecting staphylococcal enterotoxin B was 1 ng in 1 ml 01 sample, a remarkably high sensilivity at that time. How­ever, the method remained widely unused because it could detect

enterotoxin B only whilst five immunologically different enlero­toxins A to E were known to exist. The low specificity of the sensitized tatex prepared by ditutlng specific antl·enterotoxin sera according to their method was another serious drawback. The practical applica­tion of RPLA for detecting bactenat toxins had to walt untilihe pUrlflca· lion of antl ·enterotoxln serum became feasible by affinity chrom­atography uSing staphylococal enterotoxlns as ligands.

tmprove ment of RPLA to p u r ify toxins and antisera The impmvement of techniques to purify enterotoxins and the availa­bitity of affinity chromatography for the purification of antisera have accelerated the practical applica­tion of RPLA for the detection of bacterial toxins. Conventional methods to purify staphylococcal enlerotoxlns reqUire five to six difficult processes to obtain satls· factory purity. The method de· veloped by Oda" USing Sp·

Table 1: Comparison of RPLA and APHA for recovery of experimentally added staphylococcal enterotoxins

Test Enterotoxins added Amounts recovered Recovery (,<g/g) (0/0)

Foods No. Type "gig RPLI), RPHA RPLA RPHA

1 " Sakura-mochi" A 0.1 0.08 0.08 80 80 2 ··Kusa·mochi· ' A 0.05 0.04 0.04 80 80

B 0.1 0.16 0.16 160 160 3 Omelet A 0.1 0.08 0.08 80 80

C 0.05 0.04 0.08 80 160 4 " Uzura-mame" A 0.1 0.08 0.08 80 80 5 'Uguisu-mame" C 0.05 0.04 0.04 80 80 6 Custard cream A 0.001 0 0

B 0.1 0.08 0.16 80 160 7 Eclair C 0.05 0.08 0.08 160 160 8 Meat ball A 0.05 0.04 0.04 80 80

A 0.001 0 0 9 Shu·rnai C 0.1 0.08 0.16 80 160

10 Salad B 0.05 0.08 0.08 160 160 11 "Inari-zushi" A 0.1 0.02 0.02 20 20

("Aburage") A 0.01 0.01 0.01 100 100 12 "Nori·maki" C 0.05 0.04 0.08 80 160 13 " Kinshi-tamago" A 0.1 0.04 0.04 40 40

A 0.05 0.02 0.02 40 40 14 Cooked rice 8 0.01 0.01 0.Q1 100 100

C 0.01 0.01 0.01 100 100 15 Vienna sausage A 0.1 0.08 NS " 80 0 16 Bacon A 0.1 0.16 NS 160 0

8 0.025 0.02 NS 80 0 17 Grilled fish A 0.1 0.16 NS 160 0

Note: Recovery of enterotoxin was calculated on the base of the highest dilutions giving positive agglutination and minimum detectable dose of RPLA and RPHA. NOI detected

" NS: Nonspecific

Sephadex C-25 fo r purifying entero­tOXins A to E IS simpler to use and has produced purified enlerotoxins With high Yields. Rabbits were Immunized With each of these pUrified enteroloxins to prepare specifiC Immune-sera. The technique of affinity chromato­graphy, uSing very specific chemi· cal reactions such as enzyme­substrate reactions or antigen­antibody reactions, was developed, and the technique could be used for the purification of antisera with­out difficulty. Affinity chromato­graphy columns were prepared fixing each of the specificenterotox­ins A to 0 as lig~nds after allowing them to combine w ith CNBr­activated Sepharose 4B. The use of such columns produced specific Immunoglobulins to the enterotox­ins extracted from the immune sera. Senslttzed erythrocytes and latex particles were prepared by coating sheep erythrocytes or latex part· Icles with these specific immuno­globulins. Using these carriers the sensitivity and specificity of various

tests were examined. 5,6.7 The purification methods for V. cho/erae enterotoxin and8 CI. perfringens enterotox in were improved in parallel and resulted in the commercial availabili ty of these enterotoxin tests. Purified V. cholerae enterotox in was used to p repare rabbi t immune sera to the enterotoxin. The antibodies were passed through a ff inity chromat­ography columns, using the entero­toxin as ligand, to obtain speci fic immunoglobulins.

Preparation of sensitized latex Polystyrene latex particles 48GE (0.60-0.70,,) manufactured by Takeda Chemical Industries Ltd (Japan) were used. For sensitiza­tion purposes 25~glml of 0 .5 per cent latex suspension was mixed with the same volume of each type of specific staphylococcal entero: toxin immunoglobulin and incubat· ed in a 37 ° C water bath for 2 hours. Alte r sensitization. bovine

CONTINUED OVERLEAF

Table 2: Detection of enterotoxins in food responsible for staphylococcal food poisoning outbreaks using RPLA

Incident InCriminated Enterotoxin S. aureU$ No. food by RPL/I (clu/g)

Iype "gig

1 Rice ball (Salmon) A 0.04 3.0 x 108

2 Rice ball (Salmon) A 0.045 9.0 x 10' 3 Omelet A 0.004 1.2 x 105

4 Rice ball ("Okaka") A 0.008 D 0.016 6.6 x 109

5 " Kimishigure" A 0.16 1.1 x 10~

6 Rice ball (Salmon) A 0.032 4.3 x 108

7 Sandwich (Ham) A 0.032 D 0.04 3.2 x 108

8 Omelet A 0.064 4.7 x 109

9 Rice ball (Salmon) A 0.01 1.3 x 108

10 Rice ball A 0.Q4 4.4 x 108

tl Fish (boiled with soy sauce) A 0.08 9.4 x lOa 12 Lunch (scrambled eggs) A 0.08 2.9 x 109

13 Lunch (mixed foods) A 0.16 5.2 x 108

CFukiyose-Egg") A 1.28 2.0 x 109

14 Rice ball (Salmon) A 0.04 D 0.02 5.5 x 10'

15 Rice ball (" Takikomi") A 0.08 7.6 x 108

16 Lunch (Omelet) A 0.02 1.3 x 108

17 Lunch (Omelet) A 0.04 1.5 x 108

18 Rice ball (Salmon) A 0.128 1.5 x 109

19 Rice ball A 0.08 1.0 x 109

20 Rice ball C 0.04 7.0 x 10' 21 Lunch (Spaghetti) A 0.08

8 0.04 1.5 x 109

22 Lunch (Salmon) A 0.04 2.3 x 10' 23 Rice ball A 0.16 3.6 x 108

24 Rice bal l (Salmon) A 0.04 8.3 x 10~

25 Rice bal l ("Umeboshi") A 0.08 1.6 x 108

26 Boiled vegetable A 0.04 3.8 x 108

Printed by Surrey Fmc fl.lt Press Ltd Reclhlll SUlrey. England

f Table 3: Production of TS5T-l and enterotoxins by S. Bu,eus strains

Toxin(s)

TSST'1 (alone) TSST'1 + SEA TSST·l + SEB TSST·l + SEC TSST·l + SEA + SED TSST-l + SEA + SEa + SED SEA SEB SEA + SEB SEA + SED SEB + SEC + SED None

serum albumin (BSA) was added to the suspension at a final concentration of 0.5 per cent and the suspension was left undistur­bed at 4°C overnight. The latex suspension was then centrifuged at 3.000rpm lor 20 minules and dilu· led willi 0 .5 per cent BSA plus 0.075M phosphate buffered saline to a concentration of 0.16 per cent.6,]

Very similar methods were employ­ed for the sensitIzation of latex part­icles with V. cholerae enterotoxin, C/. perfringens enterotoxin and toxic shock syndrome toxin (TSST-l). "

RPLA as method to detect bacterial toxins When using RPLA to detect staph· ylococcal enlerotoxins in food sam pies 1 0 per cent or 50 per cent suspensions of homogenized food were centriluged at 14.000rpm lor 15 minutes at 4°e, Doubling dilutions of the supernatant were prepared in small test tubes and 25,.d volumes of each d ilution placed in rows of 5 wells. Where the culture isotate 01 Staphy· lococcus aureus was used as the sample. the isolate was shake· cu ltured in brain· heart in fusion broth lor t 8 hours and ten lold sequential dilutions of the supernat· an t were prepared in small test tubes. These dilutions as in the case 01 load samples were added in 25111 volumes to the weJJs. To these d iluted supernatants in the lour rows 01 wells one drop (25~1) of latex suspension sensitized with each of specific immu noglobul ins was then added. The lilth row 01 wells had 25~1 volumes 01 control latex suspension added, The plate was shaken well to mix Ihe contents and placed in a moisture box. It was then telt undisturbed lor 16·18

No. of TSS No. of Control strains strains

RPLA asp RPLA asp

0 4 1 3 11 9 1 0 0 0 1 0 3 3 2 1 2 0 1 0 1 0 0 0 1 0 0 1 0 0 1 3 0 0 3 1 1 0 4 2 0 0 1 0 1 4 6 10

hours and the results were read with the naked eye.6,7

To confi rm the specificity and sensitivity of the latex suspension sensitized with immunoglobulins speci fic for each type of enterotoxin A 10 0, doubling dilu tions 01 each type specific punfied en terotoxin (1 OOnglml) were prepared and the reaction of the sensitized latex was exam ined. Agglutinations were observed only With each latex sus· penSIOn and lIs respective entero· toxin shOWing that the speci ficity of the senSi tized latex suspension was adequate. The maximum dilution of staphylo· coccal en terotoxin to agglutinate the latex was 1 :64 lor all types. ind icating that the sensitivity of the sensitized latex was approximately 2ng per ml. The sensitl vily 01 the RPLA tests prepared in the same way lor detecting TSST· l . V. choterae enterotoxin, E. cotl heat·labile en terotoxin and Ct. perfringens enterotoxin were 1 ,1 2 1 ,10 110 and 4ng/ml respectively. The test procedures used to detect V. cholerae enterotoxin, E. coli heat-labile enterotoxin and CI perfrmgens enterotoxin differed in minor detai l from those used for staphylococcal enterotoxlns. 10 11

Application example of the test Table 1 shows assay data from load samples experimentally spiked with staphylococcal enterotoxin, com par ing rever sed passive haemagglutination (RPHA) using formalin-fixed sheep erythrocytes coated with speCific immunoglob· ulins to enterotoxins A to 0 with the RPLA test. " Food samples containing a lot of (a1. such as Vienna sausages, bacon and g rilled fish, developed non-specific reactions In RPHA demonstrating

Table 4: Comparison of detection of E.coli L T by RPLA test and by Y 1 cell assay

Culture fil trate Polymyxin B filtrate

Strain RPLA Y1 cell RPLA Yl cell

06- 21 2 128 64 512 06- 34 1 32 32 128 06- 38 2 128 128 4096 06- 44 06- 52 2 32 32 128 06- 54 2 64 64 256 06- 56 06- 60 2 64 128 512 06- 62 1 32 t28 2048 06- 69 t 32 8 64 06- 105 4 128 t28 2048 06- 108 06- 127 2 32 256 2048 06- ,149 06- 156 2 128 64 256 06- r65 8 64 32 128 06- 172 1 16 8 16 U- 154 2 32 128 512 U- 204 2 32 64 256 U- 208 8 256 2 8 U- 215 1 4 16 64 U-226 U- 443 4 64 8 512

The numbers indicate the highest dilu tion of the sample with a positive reaction.

Figure 1: Purification of anti sera by affin ity c hro m at ography.

that the meUlod IS Inadequate for use w ith these samples. RPLA. on the other hand. could detect the en terotoxins despite the presence 01 lat. Although RPLA requires a longer assay time than RPHA. the substantial superiority due to excellent specll ic lty 01 the RPLA suggests that it will become a more appropriate method for practical use. Table 2 11slS the type ol enterotoxin. concentration of the tOXin per gram of food , and the number of organ­Isms per gram 01 load lor 26 load samples tested by RPLA. Alilhese samples had caused staphy' lococcal food-poisoning. I/o Type A enterotoxin predominates over all other types in the number of cases investigated . Twenty-one strains of S. aureus Isolated from patients wi th toxic shock syndrome (TSS) and twenty strains of the same organism iso­lated from non·TSS patients were examined for enterotoxin and TSST' 1 production by RPLA and gel-diffusion (optimal sensitivity ptate. aSP) and the resutts are listed in Table 3 .12 RPLA is shown to be more sensitive than OSP since it recogn ises a greater number of entrotoxin or TS8T-1 producing strains. TSST' 1 assay procedures were the same as those described for the culture isolate of S. aureus. The influence of protein A on the tests todetectenterotoxlnsor TSST-l was negligible. tn Table 4 Ihe sensi livlly 01 RPLA and Yl cell assays in detecting E. coti heat· labile enterotoxin (L 1) are compared. 10 The quantity of L Tin the culture supernatant of E. coli was very small but treatment with polymyxin 8 caused an apparent increase of L T production by the organism. Tabte 5 lists the quantity 01 CI. perfringens enterotoxin appearing in the stools 01 patients al the acute and convalescen t stages of food poisoning.11

Conclusion The test kits introduced here are commercially available from Oxoid limited as SET·RPLA (S aureus enterotoxin A. B. C and 0) . TST· RPLA (staphylococcal toxic shock

Table 5: Detection of CI. perlringens enterotoxin from patient faeces

Slage Faecal specimens No. RPLA titer

Acute

Convalescent

N- t N- 2 N-3 N- 4 N- 5 N- 6 N- 7 N- 8 N- 9 N- 10 N-l1 N- 12

S- 1 S-2 S- 3 S- 4 S- 5 S- 6 S- 7 S- 8 S- 9 S- 10 S- 11 S- 12

2.560 10.240 40.960

640 10.240 40.960 10.240 40.960

1.280 10,240 40.960 40.960

320 2.560

5.120 10

1.280

20

10 2.560

The numbers indicate the highest dilution of the sample with a positive reaction.

syndrome toxin· 1) . VET·RPLA (V. choterae enterotoxin, E. coli heat· labile enterotoxin) and PET·RPLA (CI. perfringens enterotoxin). These are produced by a Japanese manufacturer, Denka Seiken Co Ltd under strict quality control and can be used for 12 months when stored at 2° to 8°C.

References

1 Salmon. L.l and Tew, R W (1968). P,oc Soc Exp a'ol Med.. 129 . 539·542

2 Oda. T (1978) Jpn J Bac/errol. 33, 743·752 (In Japanese)

3 Oda. T (1980) Jpn. J Bactenol .. 35, 559·567 (In Japanese)

4 Terayama. T et al (1976) Ann Rep. Tokyo Melr Res Lab. PH. 27(1) . 5-9 (In Japanese)

5. Yamada. S .. IgarashI. Hand Terayama. T (1977). Mlcroblol Immunol.. 21 , 675-682.

6. Oda. T. el al (1979) Ann Rep Fukuoka Clly Hyg. Lab .. 4 . 33-37 (In Japanese)

7. Shmgakl. M. et al (1981) Ann Rep Tokyo Melr Res Lab PH , 32 . 129· 131 (In Japanese)

8. Finkelstein, RA and LoSpaltuto. J J (1970) . J. In fect. DIS .. 121 (Suppl) S63-S72

9. Sakaguchi, G .. Uemura. T and Riemann. H . P (1973) Appl Mlcroblol .. 26 . 762-767

10 SugIyama, J. (1983) . Yamaguchi Med J .. 32. 259·267 (In Japanese)

11 SugIyama, J. (1984) Yamaguchi Med J .. 33.227-233 (In Japanese)

12 IgarashI, H el al (1986) J Om Mlcroblol. 23. 509·512

13 Igarashi. H. and Shlngakl. M (1982) . Midea Circle, 27. 72·81 (In Japanese)

14. Igarashi, H et af (1985) , The StaphylococcI: Zbl. 8akt Suppl . 14 . 255·257

Bibliography

HarmOn, S.M. and Kauuer. D.A. (1986). J Food Protection, 49. 523-526.

8erry, P A .. Stringer. M.F. and Uemura, T. (1986). Lett. Appl. Microbia/., 2,101-102. Igarashi. H. el a/ (1986). J. Clin. Microbial" 23, 509·512. Park, C.E. and Szabo. R. (1986). Can. J. Microbial .. 32 . 723·727 . Cantonl, C .. Cattaneo. p. and BalzareUi, C. (1986). Arch. Velerm. ltaliano, 37, 87-88.

Staphylococcal enterotoxins in foods Merlin S Bergdoll, PhD, Food Research Institu te, Universi ty of Wisconsin , Madison, Wisconsin, USA.

Staphyloccocal food poisoning has undoubtedly existed for as long as the foods that are capable of supporting the growth of staphy­lococci . However. the first recard­ed cases of the typical symptoms of staphyloccocal food pOisoning were not reported until the 1800's. Although some illnesses were obviously related to the presence of staphylococci in tile implicated food. staphylococci as the cause of food poisoning was generally ignored. It was only after Dack et al l showed that a food poisoning outbreak in Chicago. in 1929 . was due to a tox in produced by the staphylococci Isolated Irom the sponge cakes involved , that staphylococcal food pOisoning be­came generally recognized . Interestingly . Barber ,2 in 1914, had related his illness to the growth of staphylococc i in milk from a mastitic cowan a farm he visi ted in the Philippines; however. this information was not generally avail­able to scientists In other parts of the world.

Toxin isolation and characterization Although Dack ef ai' found that staphylococcal food poisoning was due to a toxin produced by the staphylococcI , it was not until 1959 tllat Bergdoll el ai' reported the isolation of the tOXJn and HJbnlck and Bergdoll" reported Its characteri zation. The availability of a speCifiC anti­body to the toxln5 made detection of the toxin (antigen-antibody reac­tion in gels) much easier than using monkeys and/or cats. Soon it was discovered that staphylococcal food poisoning could be caused by more than one enterotoxin.5 To date, seven enterotoxins have been identified and purified ; namely, enterotoxins A-E (SEA-SEE) and one or more not yet identif ied other than by the monkey feeding tes!. Each enterotoxin is identified by ilS pattern of reaction with specific anti­bodies_ So far. three SEC's have been identified as (C ,. C, and C,). They react with the same major anti­body. but have different minor anti­genic reactions. All of the enlero, toxins have been characterized (Table 1) and studies of the amino aCid sequences of SEA. SEB and SEC have led authors to believe that [he area of homology of the three enterotoxins may be the toxic site (Table 2): especially since the biological activities of the entero­toxins in food poisoning appear to be ident ical,' One piece 01 evidence to support this theory was obtained when the substitution of the 5-6 histidine reSidues in SEA inactivated the emetic reaction in monkeys. ' The lact that SEA' and

Table 1: Properties of the enterotoxins.

Molecular weight: 26.000-29.000.

Isoelectric pOints: 7.0-8_6.

Absorbance: Peak at 277nm.

Enzyme resistance: Trypsin and pepsin.

Monkey emetic dose. Intragastric - 5-20jtg/anlmal. Intravenous - 0 . 02-0 . 5~ g/kg.

SEB9 have been cloned makes it possi ble to do specific amino acid residue substitutions in the mole­cule, thus enabling H1e toxic site to be determined . The type of enterotoxin most fre­quently involved in staphylococcal food poisoning IS SEA. It is found in about 75 per cent of outbreaks. SED is the second most important cause of food poisoning outbreaks: the others are of less importance. Studies have revealed an associa­tion between the enterotoxin type. certain foods, and the sou rce of the staphylococci. For example. baked ham is tl1e food item most fre­quently involved in outbreaks in the United States: the enterotoxin generally involved is SEA. Human staphylococcI are primarily SEA producers and in these outbreaks the source of the staphylococci was th e food hand ler . SEA appears to be associated with other meats as well. such as canned corned beef. A number of ou tbreaks occurred in England. in 1979. Irom imported corned beef and the enterotoxin Involved was SEA. Enterotoxin B IS seldom Involved In staphylococcal food pOisoning but appears to be uniquely associated With food pOisoning from balled eggs. namety Easter eggs Three small outbreaks occurred In

WJsconSJn and were followed by one involVing several hundred children In Catifornla ~n 1983) after an Easter egg hunt.1 In all cases. the enterotoxin produced by the staphylococci involved and the one detected in the eggs was SEB. The source of the contamination of the eggs involved in the large outbreak was from an infection on the hand of the individual prepari ng the eggs. The source of the WisconSin outbreaks was not ascertained, but may have been human as well. The enterotoxin most frequently associated with bOVine and oVlne mastitis is SEC. If the contamination carries through to the finished produc t. then the cause of any Illnesses would be SEC or SED. However, the staphylococci may be destroyed during the process­Ing of the raw matenal and if food poisoning results in this case, it would be due to post -processing contamination.

Staphylococcal species The major staphylococcus specie to produce enterotox in is Staphy­lococcus aureus. Its two major characteristics are coagulase and thermonuclease (TNase) produc­tion (Table 3) and it is easily distinguished from the other major species, S. epidermidis which produces neither coagulase nor TNase. In recent years. two new species have been placed be­tween S. aureus and S . epider­midis, namely S. intermedius and S. hyicus. both of which may produce coagulase and TNase (Table 3) . Both S. in/ermedius" and S. hyicus12 have been shown to produce enterotoxin. In general, the coagulase-negative species such as S. epidermidis do nof produce enterotoxin, however, at least one outbreak was reported to be due to this species, 13 and because of this. coagulase­negative staphylococci cannot be ignored if they are present in large numbers in food. and should be

Table 2: Common amino acid sequence of the enterotoxins.

SEA: -'fhr-Ala-Cys-Met-Tyr-Gly-Gly-Val-Thr-Leu-His-Asp-Asn-Asn-Arg-Leu-Thr-

SEB: -Lys-Thr-Cys·Met-Tyr-Gly-Gly·Val·Thr-Leu-His-Gly-Asn-Asn·Glu-Leu-Asp-

SEC,: -Lys-Thr-Cys-Met-Tyr-Gty-Gly-tte-Thr-Lys-His-Gtu-Gly-Asn-His-Phe-Asp-

Table 3: Characteristics of Staphylococcus species.

Property S. aureus

Pigment

Coagulase

DNase

Haemolysis

Mannitol (an)

Acetoin

Clumping

Hyaluronidase

Lysostaphin

• = Over 900k

checked for enterotoxin produc­tion. Fortunately, not all coagulase­poslhvestaphylococci can produce enterotOXin, or otherWise there would be many more staphylo­coccal food pOisoning outbreaks_ The staphylOCOCCI are easily Isolated by plattng on Batrd-Parker agar ThiS medium was developed by Dr Tony Batrd-Parker of Unllever several years ago and has become the medium of cholce,14 It contains Ingredients such as li th ium ch londe which Inhibits the growth of other contaminating organisms and potassium telJurite which is reduced by S. aureus to form black colonies. To confi rm that the black colonies produced are S. aureus, several are tested for coagulase production. The plate gel-diffUSion method known as the optimum senSi tivity plate (OSP) method along with production by the membrane-over­agar method was conSidered ade· quate for detecti ng the enterotoxi­geniclty of staphylococcal strains. 15 The OSP method has a sensitivity of about 100ng/ml and the membrane-over-agar production method yields about 5-10 times more enterotoxin than the shake­flask method.' 5 However. when more sensitive methods such as

+ +

+

+ +

+

+

+

HS

S. intermedius

+

+

+

+

HS

S_ hyicus S. epidermidis

+1-

+/-

+/-

+ +1-

+

HS SS

Figure 1: Examples of food products associated with staphylococcal food CONTINUED OVERLEAF poisoning , cream and milk (top), processed meats and eggs (bottom)_

Toxin Detection Oxoid have launched a range of kits for the detection of bacterial toxins in food. faeces, and cultural isolates. A reversed passive latex agglutination (RPLA) technique is employed. The four kits detect staphylococcal enterotoxins A.B. C. and D (SET-RPLA: Code DR 900) . Vibrio cholerae enterotoxinlE. coli heat labile enterotoxin (VET-RPLA: Code DR 920) ; Clostridium perfringens enterotoxin (PET-RPLA: Code 930): and staphy­lococcal toxin shock syndrome toxin (TST-RPLA: Code DR 940). The use 01 highly purified specific antibodies ensu res a sensitivity as low as 1-2ng of tox in per mt. Thesimplicityofthemethod , coupled with the remarkable sensitivity, permits the detection of these important toxins by almost any laboratory.

~-~- ~----------

radioimmunoassay (RIA), enzyme~ l inked immunosorbent assay (ELISA) and reversed passive latex agglulinalion (RPLA) were devel~ oped, it was discovered that some strains produced so little toxin (1 Ongl ml) Ihal il was nol deleclable by Ihe OSP method~ This was demonstraled by Igarashi et al." who used the RPLA method and compared their results to those obtained with the same strains by Ihe OSP mel hod (Table 4). The RPLA mel hod can be used lor test· in9 st ra ins production.

for ente ro toxin

illness in sensitive individuals. This was shown when 12 one· half pin t cartons (236m I) of 2 percent choc· olate milk were examined quanti· tatively for SEA alter an outbreak among school children in 1985. The amount present was between 0.50 and 0.75ng/ml of milk. Many of those who became ill drank only one carton.

Confirming the diagnosis of staphy lococca l food poisoning The symploms of staphylococcal food pOisoning are quite character­IstiC. that IS. vomiting and/or diarrhoea developing 1-6 hours after the ingestion of food contain­Ing enlerotoxln .. The confirmation of staphylococcallood poisoning was done by the Isolallon of large numbers 01 staphylococci (approxi~ mately 10') from the implicated food. However, since methods for detecting enterotoxin In foods became avai lable. demonstrating the presence of enterotoxin in the food provides better confirmation. It has been shown that 100 to 200ng 01 SEA is su llicient toresul tin

Up until recently, the methods avail~ able to most laboratories for the detection of enterotoxin in foods involved a rather lengthy extraction and concentration process. The enterotoxin was detected by the microslide method,' I but it was diff icult to detect the enterotoxin at the concen tration considered necessary (1 nglg) wi lhout consid~ erable experience: even then not all operators were able to achieve thiS sensitivity. Newer and more senSitive methods such as RIA and ELISA were developed but the purified enterotoxin needed for RIA and Ihe amounts of antibody needed for ELISA were not gen· erally available.

Figure 2: RPLA test results showing 'smooth button ' negatives and 'granular dispersed' positives.

In the last two years. kits for use In

detecting the enteroloxlns In foods have become available commercI­ally. One of these kits makes use of the RPLA assay developed by Igarashi el a/18 for the detection of enleroloxlns. Unfortunately. the

Staphylase Test" (DR595)

food extraction procedure recom­mended by the Japanese scientISts is Inadequate for deteCting entero· tOXin concentrations of 1 ng/g of food. The sensitivity achievable with Ihe RPLA may be adequate for detecting enterotoxin in foods Implicated in most staphylococcal

Simple Rapid Reliable Cost -effective

Now a test for the identification of StaRh. aureus is available from your leading supplier of microbiological products

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"

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food poisoning outbreaks but is in· adequate lor determining the satety of a suspected food be· cause of a possible processing problem. The test is advertised to detect 1 ~2ng/ml, bul one should be aware that the actual sensitivity is determined by the amount of fluid/g used to extract the lood which should not be more than 1 ~ 2mllg and not the 9mllg suggested. It the kit is to be useful for thiS purpose. an improved extracllon procedure must be work­ed out and shown to have the necessary sensitivity. that is, 1 nglg of food. Until this is done. some precaution should be added about uSing the method for determining the safety 01 a food. The second kit that is commercially available IS one produced in SWitzerland Dr H Fey ot the University of Bern was Instrumental In ItS development and it is based on Ihe sandwich ELISA" tech· nlque using polystyrene balls as the antibody carrier. The use of dillerent colour~coded balls lor each enterotoxin makes it possible to examine a food for aU of the enterotoxlns simultaneously. The sensitivity of the test enables the detection ot less than 1 nglg of lood, primarily because of the large volume of extract used in the method. However. It IS cumber· some to handle the balls separate~ Iy. particularly If several tests need to be run at the same time.

Summary The advent of the enterotoxin test kit 1$ making it possible for almost any laboratory to lest loads lor the

presence of enterotoxin. This will also aid research Into the mechan· ism of enterotoxin production in dif­ferent foods under a variety of condi tions.

References

Dack. G.M el al (1930). J Prevent. Med .. 4, 167· 175

2. Barber. M.A. (1914) Phillppme J Sci .. sect B 9, 515·519.

3. Bergdoll. M.S. el al (1959) ArCh. Blochem 8Iophys., 85, 62-69

4. Hmlck, H.E. and Bergdoll. M.S (1959). Arch 810chem BlOphys .. 85, 70·73.

5 Bergdoll . M.S e/ 81 (1959) J Immunol .. 83, 334·338

6. Huang. 1 Y. e/ al (1976) Antmal. Plant, Mlcrobtol Toxms. 2, 131·135

7. Stelma. G.N and Bergdoll . M S (1982) . Blochem. Blophys Res Commun. . lOS, 121 -126

8 Belley. M J elal{l984) Proc NaIl Acad. SCI USA. . 81 , 5179·5183

9 Ranelll, D.M. el al (1985). Proc. Nail Acad. SCI USA. , 82, 5850

10. Memll. G.A. el 8/ (1984). JAm. Med. Assoc .. 252, 1019·1022.

t 1. Fukuda. A. el a/ (1984) . Zbl. Bakl Hyg. A. . 258, 360·367

12. Adeslyun. A.A. el al (1984) . Vet MlcroblOI. . 9, 487·495

13 Brecklnndge. J.C. and Bergdoll .

14

15

16

MS. (1971) . N. Eng/ J. Moo .. 284 , 541·543. BaIrd-Parker , A.C (1962) J Appl Baclenol .. 25, 12· 19 RobbIns. R N. and Bergdoll . M.S (1984) . J Food Protect . 47 , 172-176 IgarashI. H. et 81 (1986). J Mlcroblol. 23,509·512

Clm

17 Casman. E.? and Bennet\. R W (1965) Appl. MicroblOl . 13 , 18 1·189.

18. IgarashI. H. e/al(t985) Zbl 8akl. Mlkroblol. Hyg. I. Abt. Suppl .. 14, 255·257.

19. Fey. H. el al (1984) . J . Clm. Mlcroblol .. 19, 34·38.

Table 4 : Staphylococca l strain testing by RPLA method

Strain Enteroto xin

RPLA OSP t88 SEA. SED SEA, SED 228 SEA. SED 3t 1 SEA SEA 365 SEC 452 SEA SEA 581 SEC SEC 609 SEA, SED 754 SEA, SEB SEA, SEB 802 SEB SEB 887 SEA 896 SEA, SEB SEA, -965 SEA, SED SEA, SED

Note A reader has noticed that the wrong reference was cited for elastase production in Table 1 of Max Atkinson 's article on the genus Aeromonas (CUlture 1986 7(2)). Thus lor data referring to elastase production please consult: Popoff, M. and Veron, M. (1976). J . Gen. Microbiol., 94 11 ·22.

Culture is provided as a service to microbiology by Oxoid Limited, Wad e Road, Basingstokp.. Hampshire RG24 OPW. England. The opinions expressed In thIS publication are those ollhe aulhors concerned and do not necessarily tepresentthe vIews 01 the publishers. Media Medica. or the sponsors. Oxoid LImited. © 1987