Detection of the Genes for Staphylococcus aureus Enterotoxins by PCR

Tkáãiková a. , A. Tesfaye, I . Mikula: Detection of the Genes for Staphylococcus aureus Enterotoxins by PCR. Acta Vet. Brno 2003, 72: 627-630. The aim of this study was to determine the presence of genes for staphylococcal enterotoxins in group of 32 isolates of Staphylococcus aureus from 75 samples (sheep, cow and goat raw milk) collected from sub-clinically and clinically sick sheep, goats and cows raised in different agricultural farms in the Eastern region of Slovakia and also from 12 samples of sheep cheeses. Fifteen out of 32 isolates of S. aureus carried gene for enterotoxin C (SEC): 12 from sheep raw milk and one from each of goat and cow raw milk and sheep cheese, too. Fifteen out of 32 isolates carried gene for enterotoxin D (SED): 12 from sheep raw milk, two from cow raw milk and one from sheep cheese. Two out of 32 isolates carried genes for enterotoxin A (SEA) and B (SEB): one positive isolate for SEA from sheep raw milk and the other SEB from sheep cheese. Cheese, cow, goat, raw milk, sheep Staphylococcus aureus produces different extra-cellular protein toxins and virulence factors, which stipulate its pathogenity due to their enterotoxins. The enterotoxins pass from the digestive tract to the blood circulation and consecutively activate the nerve centre of the emetic reflex, causing nausea, vomiting, abdominal cramps and diarrhea, which can lead to dehydration and the collapse of the organism (Gouloumes et al. 1996). There are several types of staphylococcal enterotoxins: SEA, SEB, SEC1, SEC2, SEC3, SED and SEE. It was found that some strains of Staphylococcus aureus produce more than one type of enterotoxins (Halpin-Dohnálek and Marth 1989). Therefore the presence of Staphylococcus aureus in the food can be potential risk for health, particularly, if the food is not stored at the prescribed temperature. There are several procedures to detect staphylococcal enterotoxins. The production of enterotoxin needs long incubation time (20 h). Some factors, which affect the incubation period, are the pH, the water activity and the used substrates (Sharma et al. 2000). Numerous methods are based on the evidence of the enterotoxins directly in the food (ELISA, reversal passive latex agglutination and others), with a possibility to detect nanogram amounts of enterotoxins in one gram or in one mili-liter of food (Burdová et al. 1994; Gouloumes et al. 1996; Strachan et al. 1997). The advantage of these methods is that enterotoxins are detected even if the producer Staphylococcus aureus should not be identified by the classical bacteriological procedure, because it is usually devitalized by temperature. DNA amplification methods (polymerase chain reaction, PCR) can show the presence of enterotoxigenic strains of Staphylococcus aureus before the expression of enterotoxins on the base of specific gene sequences and in this way detect the potential source of contamination. The advantage of the PCR methods is that it is able to detect genes which code the production of staphylococcal enterotoxins also from heat treatment of food, because the DNA remains unchanged (Tsen and Chen 1992; Holeãková et al. 2000). ACTA VET. BRNO 2003, 72: 627–630 Address for correspondence: Doc. MVDr. a. Tkáãiková, PhD. Department of Microbiology and Immunology University of Veterinary Medicine Komenského 73, 041 81 Ko‰ice, Slovakia Phone: +421 556 336 084 Fax: +421 556 335 641 E-mail: mikula@uvm.sk http://www.vfu. cz/acta-vet/actavet.htm Sharma et al. (2000) published multiplex PCR for the detection of all staphylococcus enterotoxins. They used one universal and five specific primers in one reaction. The result showed that this type of one-step PCR is very useful for detection of different staphylococcal enterotoxin genes. The aim of this work was to detect enterotoxins in sets of Staphylococcus aureus, isolated from the cow, sheep and goat raw milk and also sheep cheese. Materials and Methods Bacter ia l s t ra ins Samples of Staphylococcus aureus were isolated by standard bacteriological guideline (EN ISO 6888) from the cow, sheep and goat raw milk and sheep cheese and stored at -20 °C in 50% (vol/vol) glycerol, 50% Brain heart infusion (BHI; Oxoid Ltd., England). Bacterial isolates were grown at 37 °C in BHI broth before the extraction of the DNA. DNA isolat ion Sediment obtained after centrifugation (3 min, 3500 x g) of 1.5 ml of broth culture (24 h, 37 °C) was washed with 100 l 0.5% Triton X-100 (Koch-Light Lab. Ltd., England). Sediment was resuspended in 100 l 0.5% Triton X-100. The isolates were incubated at 95 °C for 10 min and centrifuged at 3500 x g for 5 min. The supernatant was used as a source of DNA. Oligonucleot ide pr imers Oligonucleotide primers were used for PCR reaction according to Sharma et al. (2000). DNA amplif icat ion PCR reaction was done according to Sharma et al. (2000). Detect ion of amplif ied DNA For detection, 10 l of PCR reaction mixture was electrophoresed on a 2% w/v agarose gel (Gibco BRL, USA), diluted in 1xTAE buffer (Sambrook at al. 1989), stained with ethidium bromide (Amresco, USA) in concentration 0.1 μg/ml and viewed under ultraviolet light. The 100 bp DNA Ladder (New England BioLabs, England) was used as a standard of molecular weight.