lnternattonal Journal of Food Mtcrobiology. 12 (1991) 275-280 © 1991 Elsevier Science Publishers B.V. 0168-1605/91/$03.50

275

FOOD 00384

Short communication

Distribution of enterotoxigenic Staphylococcus aureus in ready-to-eat foods in eastern Nigeria T. Sokari Department of Bloloswal Sciences, Rwers State Umverslty of Science and TechnoloK*', Nkpolu, Port Harcourt. Ntgerta (Received 3 September 1990: accepted 19 November 1990)

Out of 880 samples of commonly consumed meat. fish and vegetable ready-to-eat foods from eastern Nigeria examined. 552 (62%) yielded coagulase-positlve Staphylococcus aureus, with 269 (48%) of the strains being enterotoxigemc. Two cowpea-based foods, akara and moin moin, whose methods of preparation and sale involve little or no post-processing hand contact, contained the smallest numbers of S. aureus. All the water samples from food preparation centres analysed, which had repeated hand contact during food preparation, yielded coagulase-posltive S. aureus. Generally, organisms producing enterotoxan A were the most frequently encountered, followed by producers of enterotoxm B. Among fish samples, however, the predominant strains were producers of enterotoxin B and enterotoxin A, in that order. The frequency of enterotoxans C- and D-producing stratus was about the same for the samples investigated. Key words: Enterotoxm; Staphylococcus aureus; Ready-to-eat foods

Introduction

Many outbreaks of staphylococcal food poisoning result from hand contact (Bryant et al., 1988). In some cases strains of Staphylococcus a u r e ~ recovered from food handlers have been found to be identical to those responsible for enterotoxin production in suspect foods (Eisenberg et al., 1975; Bryant et al., 1988). The predominant enterotoxin produced by strains o f S. aureus implicated in most cases of food poisoning is enterotoxin A (SEA) (Casman et al., 1967: Simkovicova and Gilbert. 1971, Terayama et al., 1972; Wieneke, 1974). Cases of staphylococcal food poisoning in which producers of enterotoxin B (SEB) were the predominant isolates have also been reported (Naidu, 1986). Studies have shown that the type of enterotoxin synthesized in foods by multiple enterotoxin producers could be influenced by pH, temperature and the type of oil present in the food (Gomez-Lucia et al., 1986b). It has also been shown that different foodstuffs and Correspondence address" T.G. Sokari, Department of Biological Sciences, Rivers State University of Science and Technology. Nkpohi, P.M.B. 5080. Port Harcourt. Nigeria.

276 laboratory media affect the types and amounts of enterotoxm produced bv multiple enterotoxin producers (Gomez-Lucia et al., 1986a). In the present study various ready-to-eat foods commonly consumed in eastern Nigeria as well as water from food preparation centres were examined for enterotoxigenic S. aureus. Attempts were made to determine whether differences existed in the enterotoxins most frequently produced by strains from different food sources.

Materials and Methods Foods

'Suya' - - thin slices of condiment-coated roasted beef on skewers - - were purchased at random from hawkers in traditional markets in Port Harcourt and Enugu, Nigeria. Three-hundred samples were tested. Fried fish and fried meat were purchased from randomly selected ' b e e r parlours' in Port Harcourt and Enugu. Twenty samples, 10 each of fish and meat, were obtained each day for 10 days. One-hundred dry (smoked) fish samples, 10 per day for 10 days, were purchased at random from traders in each of the markets from which suya were obtained. ' A k a r a ' - - balls of bean cake prepared by frying cowpea slurry until golden brown - - were purchased from local markets in Port Harcourt and Enugu, Nigeria. ' M o r n moin' - - cowpea pudding prepared by placing slurries, contained in small alluminium plates or wrapped in leaves, in water and boiling until the slurries set as firm gels - - were purchased from the same markets as akara. A total of 100 each of akara and moin moin samples were obtained. From time to time beer palours in Port Harcourt and Enugu run out of free-flowing tap water. As a result the operators of these food production centres keep basins of water which are used for washing plates from which customers would eat. A total of 40 samples of water used for primary washing and an equal number of samples of water used for final rinsing were collected for analysis. Detection o f S. aureus

All samples were analysed within one hour of collection or refrigerated and analysed as soon as possible afterwards. A 10-g amount of each food was homogenised in 10 ml of 0.1% sterile bacteriological peptone (Oxoid) containing 7.5% ( w / w ) NaCI and spread in duplicate on the surface of dried mannitol salt agar (MSA) (Oxoid CM 85) plates which were incubated at 3 7 ° C for 24-48 h. In addition 10 ml of 7.5% NaCI-peptone in 25-ml screw-capped bottles were heavily seeded with homogenate and incubated at 3 7 ° C for 48 h. These were then subcultured on MSA where direct culture failed to yield S. aureus. Suspected S. aureus colonies on MSA were transferred to nutrient agar (oxoid) slants and incubated at 37°C. Only coagulase positive stratus (Colhns and Lyne, 1976). were tested further in accordance with standard procedures (Collins and Lyne, 1976).

277

The wash water samples were centrifuged at 5000 rpm for 10 rain, and the resultant sediment was examined as described for the food homogenates. Production and detection o / e n t e r o t o x i n

The procedure of Jarvis and Lawrence (1970) was adopted in enterotoxin production by the strains of coagulase-positive S. aureus. Enterotoxin detection was by the method of Sokari and Anozie (1989). Reference enterotoxins A - D (SEA-SED) and their homologous antisera were kindly donated by Professor M.S. Bergdoll of the Food Research Institute, University of Wisconsin, U.S.A. The reagents were reconstituted in accordance with the instructions of the supplier.

Results and Discussion

The range of foods and related samples investigated is shown in Table 1. Out of 880 samples 552 (62.3~) yielded coagulase-positive S. aureus, 269 (48.7%) of which synthesized enterotoxin. The inability of the coagulase-positive strains from akara and moin moin, and from a substantial proportion of the other samples to produce enterotoxin (Table I) showed that there was no correlation between coagulase production and enterotoxigenicity. This is in agreement with the results of other workers (Bergdoll et al., 1967; Lachica et al., 1969, 1979). All the samples of water yielded coagulase-positive S. aureus strains (Table I). This might be a reflection of repeated hand contact during the washing of crockery, cutlery and other utensils.

TABLE I Enterotox,genicity among strains of Staphylococcus aureus isolated from ready-to-eat foods and w a t e r from food preparat,on centres Samples

Number tested

Number y,eld,ng Coagulasepositive

Number of stra,ns enterotoxigenic

S. aureus

(%)

(%) Suya Fried meat Dry (smoked) fish Fr,ed fish 'Akara" ' M o m moin" Wash water Rans¢ water

300 100 100 100 100 100 40 40

258 61 77 55 13 8 40 40

(86.0) (61.0) (77.0) (55.0) (13.0) (8.0) (100.0) (100.0)

153 (59.3) 28 (34.4) 38 (48.4) 32 (58.2) 0 0 10 (25.0) 8 (20.0)

Total

880

552 (62.3)

269 (49.9)

278 -

loo-

£NTE ROTOXINS

80-

A

B

C

D

GJ

--

60-

¢ L,0-

g o

20-

2 ~

L...........~-..~

~,..,,,....,..,v-..-~

TO (269)

SU (153)

~,-,,,,~...~",.,....-,.~',-.......-,r-...~

FM (28)

OF (38)

FF {32)

~--.,r

..-~

AK (0)

~

~

NO (0)

~,-

PW (10)

RW (8)

Enferofoxlgenic S.aureus m foods

Fig. 1. Enterotoxins produced by coagulase posiuve S. aureus recovered from ready-to-eat foods and assoc=ated samples. AK, "akara'; DF, dry (smoked) fish; FF, fried fish; FM. fried meat; MO, 'moin moin', PW, primary wash water; RW, nnse water; SU, 'suya'; TO, overall total; numbers in parentheszs. actual numbers of S. aureus tested.

The highest proportion of enterotoxigenic isolates was encountered among the strains from meat and fish products (Table I). This was probably because of repeated hand contact with these foods. In traditional Nigerian markets there is often an unacceptably high level of hand contact with foods; trader and potential customer each pick up the items on display as if to assess and reassess their value as they haggle over prices until a mutually agreed sum is arrived at. Enterotoxin A (SEA) was the most frequently encountered toxin among the strains of S. aureus from the meat products and water samples, the next in frequency was SEB. The most frequently encountered enterotoxin among isolates from fish products, however was SEB, followed by SEA. Producers of the other enterotoxins, SEC and SED, occurred in more or less equal proportions (Fig. 1). The reason for the dominance of SEA producers in meat products and SEB producers m fish products is not certain. It has been reported that pH, temperature, types of oils or other factors prevalent in foods could affect the types and amounts of enterotoxin synthesized by multiple enterotoxin producers (Gomez-Lucia eL al., 1986a,b). Whether, however, SEA producers grow better in meats while SEB producers grow better in fish can only be determined by further study.

Acknowledgements The author wishes to express his gratitude to Professor M.S. Bergdoll of the Food Research Institute, University of Wisconsin, Madison, WI, U.S.A., for the kind donation of the reference enterotoxins ( A - D ) and their specific antisera used in this

279 study, and to S.O.U. Ejim of the Department of Biological Sciences, Rivers State University of Science and Technology, Nkpolu, Port Harcourt. Nigeria, for typing the manuscript.

References Bergdoil. M.S., Weiss, K.F. and Muster, M.J. (1967) The producuon of staphylococcal enterotoxm by a coagulase-negative microorganisms. Bact. Proc. p, 12. Bryant, R.G., Jarvis. J. and Guibert0 G. (1988) Selecttve enterotoxin production by a Staphylococcus aureus strain implicated in a foodborne outbreak, J. Food Protect. 51, 130-131. Casman, E.P., Bennett, R.W., Dorsey, A.E. and lssa. J.A. (1967) Identification of a fourth staphylococcal enterotoxm, enterotoxin D. J. Bacteriol. 94, 1875-1882. Collins, C.H. and Lyne, C.M. (1976) Microbiological Methods. Butterworths, London, pp. 403-408. Eisenberg, M.S., Gaarslev, K., Brown, W., Horwitz, M. and Hill, D. (1975) Staphylococcal food poisoning abroad a commercial aircraft. Lancet ii, 595-600. Gomez-Lucia, E., Bianco. J.L., Dominguez, L., Valle, J. and Suarez. G. (1986a) Translocation of the synthesis of enterotoxins A and B by Staphylococcus aureus $6 in cheese. 2nd World Congress Foodborne Infections and Intoxications. 26-30 May 1986. Abstracts, p. 95. Gomez-Lucm, E., Fernandez, J., Vandilio, S., Vazqucz, J.A. and L. Domingue,z, 1986b. Influence of pH and type of oil used for mayonnaise elaboration m the development of Staphylococcal aureus and enterotoxin production. 2nd World Congress Foodborne Infections and lntoxicatsons 26-30 May 1986, Abstracts, p. 153. Jarvas. A.W. and Lawrence, R.C. (1970) Production of high titres of enterotoxins for the routine testing of staphylococci. Appl. Microbiol. 19, 698-699. Lachica0 R.V.F., Weiss, K.F. and Deibel, R.H. (1969) Relauonships among coagulase, enterotoxin and heat-stable deoxyribonuclease production by Staphylococcus aureus. Appl. Microbiol. 18, 126-127. Lacluca. R.V.F., Genigeorgis, C. and Hoeprich, P.D. (1971) Meta-chromatic agar diffusion methods for detecting staphylococcal nucleas¢ activity. Appl. Environ. Microbiol. 21, 823-826. Naidu, A.S. (1986) Enterotoxieological and epidenuological investigation of staphylococcal food poisonmg outbreaks. 2nd World Congress Foodborne Infections and lntoyacattons. 26-30 May 1986, Abstracts, p. 52. S~mkovacova, M. and Gilbert, R.J. (1971) Serological detection of enterotoxin from food potsoning stratus of Staphylococcus aureus. J. Med. Microbiol. 4. 19-30. Sokan, T.G. and Anozie, S.O. (1989) Modified single radial immunodiffusion method for screening staphylococcal isolates for enterotoxin. Food Microbiol. 6. 45-48. Terayama, T.. lgarashi. H., Ushioda. H. and Zen-Yogi, H. (1972) studies on Staphylococcal food potsoning. III. Enterotoxin productsvity and coagulase types of stratus of Staphylococcus aureus originated from food poisonm S incidents, healthy subjects and commercml foods. J. Food Hyg. Soc. Jpn. 13, 549-554. Wteneke, A.A. (1974) Enterotoxin production by strains of Staphylococcus aureus tsolated from food and human bemgs. J. Hyg. Camb. 73, 255-262.

Distribution of enterotoxigenic Staphylococcus aureus in ready-to-eat foods in eastern Nigeria.

Out of 880 samples of commonly consumed meat, fish and vegetable ready-to-eat foods from eastern Nigeria examined, 552 (62%) yielded coagulase-positiv...
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