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

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.

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 milk-derived 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 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, state and federal inspection services differed significantly, being those in SIF inspected samples significantly lower ( mean of 1.4x103CFU.g-1) than the samples inspected by the other two services (1.5x105 and 1.7x105 CFU.g-1, for SIE and SIM, respectively). In 6 samples of colonial cheese, CPS counts were above 105 CFU.g-1. According to FDA (19), in foods with such high counts of CPS the presence of staphylococcal enterotoxin is likely. In 9 out of 18 samples of colonial sausages, concentration of CPS was above the limit established by the Brazilian legislation (11), and in 6 samples, the counts were above than 105 CFU.g-1. All salaminho samples were negative for CPS.

From the 200 Staphylococcus spp. isolates, 116 originated from American and colonial cheeses and 84 from colonial sausage. 102 (51%) strains were characterized as coagulase-positive staphylococci (CPS), being 67 from colonial sausage and 35 from American and colonial cheeses. The remaining 98 isolates (49%) were coagulase-negative staphylococci (Table 1).

Table 1

Prevalence of femA, sea, seb, sec, sed and see genes in coagulase-positive Staphylococci strains isolated from foods.

Type of Food	Number of Isolates	CPS	CPS femA positive	CPS sea positive	CPS se bpositive	CPS sec positive	CPS sed positive	CPS see positive
Colonial and American Cheeses	116	35	35	0	0	0	11	1
Colonial Sausage	84	67	56	10	0	0	1	3*
Total	200	102	91	10	0	0	12	4

These strains were also positive for the presence of sea gene.

CPS = Coagulase-positive Staphylococci.

CPS isolates were analyzed by multiplex PCR in order to detect femA, sea, seb, sec, sed and see genes (Fig. 1). 91 out of 102 isolates were positive for femA gene. All femA gene negative CPS isolates (11 isolates) have been isolated from colonial sausage. The sed, sea and see genes were detected in 12, 10 and 4 isolates, respectively (Table 1). None of the isolates was positive for seb or sec genes.

Figure 1

Multiplex PCR of CPS strains isolated from A) cheese, B) colonial sausage. A) lane 1: Ladder 50bp (Promega); lane 2: water; lane 3: negative control (Staphylococcus xylosus ATCC 29971); lane 4: positive control (mix of four ATCC strains – 13565 sea, 14458 seb, 23235 sed and 27664 see); lanes 5 – 14: S. aureus strains isolated from cheese. B) Lane 1: Ladder 50 bp (Promega); lane 2: positive control (mix of four ATCC strains – 13565 sea, 14458 seb, 23235 sed and 27664 see); lanes 3 – 13: S. aureus isolated from colonial sausage; lane 14: negative control (Staphylococcus xylosus ATCC 29971).

The femA gene was detected in all 35 CPS isolates from cheeses, confirming the biochemical characterization. From cheese samples, sed was detected in ten isolates and see was detected in one isolate (Tab.1). In sed gene positive isolates, amplification of fragments with 600 and 700 bp was also observed (Fig.1A). The presence of these two extra fragments was observed only when the sed fragment was also present. Other authors have also reported the presence of unexpected fragments in some dairy products evaluated by multiplex PCR for the presence of enterotoxin genes (16).

Among the 56 CPS isolates from colonial sausage that were positive for femA, seven were also positive for sea and one for sed (Fig. 1B, Table 1). Three isolates amplified the enterotoxin genes sea and see simultaneously.

Several authors have also reported that enterotoxin genes sea and sed are the most common in staphylococci isolated from foodstuffs (5,7,8,30,33). In Brazil, Carmo et al. investigated the presence of staphylococci in minas fresh cheese and raw milk and observed that the isolates were able to produce enterotoxins A, B and C (13). Cheese produced in Serra da Canastra, MG, also contained S. aureus isolates able to produce enterotoxins B and C (9).

Detection of the enterotoxin genes in the CPS isolates coming from a food is not an indication that the toxins are effectively present in this food. Morandi et al. (28) evaluated 107 CPS isolates from dairy products, and observed that enterotoxin genes were detected in 67% isolates, but only 52% of the isolates were capable to produce enterotoxins. These authors reported that the results obtained by multiplex PRC and immunoassay tests were in agreement when tested for the presence of enteroxins A, C and D (16).

Results of the present work confirm that multiplex PCR is a rapid and sensitive method for screening of enterotoxigenic coagulase-positive Staphylococci, being highly specific. Results also indicate that better hygiene practices and better inspection are required in the production of the foods included in the study.