Ocurrence of Staphylococcus aureus and multiplex pcr detection of classic enterotoxin genes in cheese and meat products

Brazilian Journal of Microbiology (2009) 40:145-148 ISSN 1517-8382 OCURRENCE OF STAPHYLOCOCCUS AUREUS AND MULTIPLEX PCR DETECTION OF CLASSIC ENTEROTOXIN GENES IN CHEESE AND MEAT PRODUCTS Marcia Regina Pelisser1,2; Cátia Silene Klein3; Kelen Regina Ascoli3;Thaís Regina Zotti3; Ana Carolina Maisonnave Arisi1* Departamento de Ciência e Tecnologia de Alimentos, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, Florianópolis, SC, Brasil; 2Universidade do Contestado, Concórdia, SC, Brasil; 3EMBRAPA – Suínos e Aves, Concórdia, SC, Brasil. 1 Submitted: March 07, 2008; Returned to authors for corrections: June 29, 2008; Approved: November 02, 2008. ABSTRACT Multiplex PCR was used to investigate the presence of enterotoxins genes (sea, seb, sec, sed and see) and femA gene (specific for Staphylococcus aureus) in coagulase-positive staphylococci (CPS) isolated from cheese and meat products. From 102 CPS isolates, 91 were positive for femA, 10 for sea, 12 for sed and four for see. Key words: Staphylococcus aureus, enterotoxins, multiplex PCR Staphylococcus aureus is the predominant specie involved in staphylococcal food-poisoning outbreaks, although other coagulase-positive Staphylococci, such as S. intermedius and S. hyicus, may also be enterotoxigenic (5,34,35). S. aureus may produce a large variety of enterotoxins (A, B, C, D, E, G, H, I, J, K, L, M, N, O, P, Q, R and U), but 95% of poisoning outbreaks are caused by classical enterotoxins: A, B, C, D and E (24). Enterotoxin A is the most frequently produced (8,21). Staphylococcal enterotoxins are thermostable and also resistant to gastrointestinal proteases such as pepsin, explaining its ability in remaining active after ingestion (7,8,36). The amount of staphylococcal enterotoxins required for establishment of typical symptoms of food poisoning is very low, ranging from 20 ng to 1 μg (32). Which corresponds to approximately 105 staphylococci colony-forming units per gram of food (19). Several studies evaluated the capability of staphylococcal strains isolated from foods to produce the classical enterotoxins A, B, C, D and E (2,5,8,13,15,18,20,26,31,33). In Brazil, several studies reported counts of coagulase-positive Staphylococci above the maximum levels allowed by the Brazilian legislation (11) in sausages (colonial sausage), milk and milk products (1,3-6,10,12-14,23,25). The aim of the present work was to evaluate the presence of by coagulase-positive Staphylococci (CPS) in meat and milkderived products commercialized in Santa Catarina, SC, Brazil and to detect the presence of genes for classical staphylococcal enterotoxins A, B, C, D and E (sea, seb, sec, sed and see) and for gene femA, specific for S. aureus species, using multiplex PCR. A total of 72 food samples including mozzarella (15 samples), American cheese (15 samples), colonial cheese (15 samples), colonial sausage (18 samples) and salaminho (09 samples) were collected from markets in Alto Uruguai Catarinese region (AMAUC), in Santa Catarina state, Brazil, from 2005 to 2007. The mozzarella (A, B, C) and American cheese brands analyzed in this work are under Federal Inspection Service (SIF), while colonial cheese brands are under different inspection services: State Inspection (SIE) (G brand), Municipal Inspection (SIM) (H brand) and SIF (I brand). Five (A, B, C, D, E) out of six brands of colonial sausages are inspected by SIE, and G brand is inspected by SIM (Concórdia - SC). Salaminho brands (A, B, C) are inspected by SIF. For S. aureus enumeration (17), serial dilutions of food homogenates were plated on Baird Parker agar (Oxoid) with 5% egg yolk tellurite emulsion (Oxoid) and incubated at 35ºC for *Corresponding Author. Mailing address: Departamento de Ciência e Tecnologia de Alimentos, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina. Av. Admar Gonzaga, 1346 CEP 88034-001 Florianópolis - SC, Brasil. Tel.: 55 4837215382. E-mail: 145 Pelisser, M.R. et al. 48 h. After this period, typical colonies (circular, smooth, convex, gray to jet-black, frequently with light-colored (off-white) margin, surrounded by opaque zone and frequently with an outer clear zone) were counted and five colonies were transferred to MacConkey agar (Oxoid) and blood agar. Colonies that grew in blood agar but not in MacConkey were tested for Gram coloration, coagulase, catalase, oxidase, urease and maltose. Total DNA was extracted from 5 mL of a coagulase-positive staphylococcal culture grown at 35ºC (± 2ºC) for 16-24 h in Brain Heart Infusion (Merck) broth. DNA was isolated using the WizardÒ Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA) and lisozyme 10 mg.mL-1 (Sigma Aldrich). Enterotoxigenic S. aureus strains ATCC 13565 (sea), ATCC 14458 (seb), ATCC 19095 (sec), ATCC 23235 (sed) e ATCC 27664 (see) were used as positive controls and Staphylococcus xylosus ATCC 29971 as negative control. Detection of specific genes for S. aureus (femA) and for enterotoxins A, B, C, D, and E was carried out according to Mehrotra, Gehua, Johnson (27), with some modifications, yelding the expected amplicons: 102 bp for sea, 132 bp for femA, 164 bp for seb, 209 bp for see, 278 bp for sed, 451 bp for sec. For multiplex PCR detection of sec and femA genes, amplification reactions were performed in final volume of 25 mL containing PCR buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl), 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.4 mM of each primer, 1.25 U Taq DNA polymerase and 100-300 ng of template DNA. Reactions were carried out in MinicyclerTM (MJ Research, Inc. Watertown, MA) with the following program: initial denaturation at 94ºC for 5 min followed by 35 cycles of 94ºC for 2 min, 57ºC for 2 min and 72ºC for 60 s with a final extension at 72ºC for 7 min. For multiplex PCR detection of sea, seb, sed, see and femA genes, amplification reactions were performed in final volume of 50 mL containing PCR buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl), 3 mM MgCl2, 0.2 mM of each dNTP, 400 nM of each primer, 1.25 U Taq DNA polymerase and 100-300 ng of template DNA. Amplification profile was standardized in 94ºC for 5 min followed by 35 cycles of 94ºC for 2 min, 52ºC for 2 min and 72ºC for 3 min with a final extension at 72ºC for 7 min. PCR products were separated by electrophoresis at 80 V for 70 min in 2% agarose gel and stained with ethidium bromide. Gels were visualized in a UV transilluminator and images were digitalized with a digital camera (CANON Powershot A70). Presence of coagulase-positive staphylococci (CPS) was detected in 33 out of 72 analyzed samples. In American and colonial cheeses, from 30 samples analyzed, 19 presented contamination by CPS. However, presence of CPS was not detected in the 15 mozzarella samples. 28.8% of samples contaminated with CPS presented counts with levels above 103 CFU.g-1- which is the upper limit established by RDC nº 12 of the Brazilian National Sanitary Control Agency (11). Counts in samples inspected by municipal, st (...truncated)