Prolonged expression and production of Staphylococcus aureus enterotoxin A in processed pork meat Nina Wallin-Carlquist a,1 , Dóra Márta a,b,1 , Elisabeth Borch c , Peter Rådström a, ⁎ a Applied Microbiology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden b Dept. of Microbiology and Biotechnology, Faculty of Food Science, Corvinus University of Budapest, H-1118, Budapest, Somlói út 14-16, Hungary c Swedish Institute for Food and Biotechnology, P.O. Box 5401, SE-402 29 Gothenburg, Sweden abstract article info Keywords: Enterotoxin A Staphylococcus aureus In vivo gene expression Virulence expression The bacteriophage-encoded staphylococcal enterotoxin A (SEA) is the toxin most frequently reported to be involved in staphylococcal food poisoning. In this study, sea expression and SEA formation were studied in four processed pork products: boiled ham, hot-smoked ham, Serrano ham (dry-cured Spanish ham) and black pepper salami. The products were selected because of their differences in intrinsic factors. As a reference, Staphylococcus aureus was cultivated under favorable planktonic growth conditions. Expression was mainly linked to bacterial growth for both meat products and broth cultures. In liquid broth, however, the relative level of sea mRNA peaked in the late exponential phase and then rapidly declined, while in the meat products allowing immediate growth, i.e. boiled and smoked ham, active sea expression occurred throughout the incubation period of seven days. Lower levels of sea mRNA and SEA were found in smoked ham compared to boiled ham, although viable counts of S. aureus on the two products were similar. Furthermore, the SEA concentration in the boiled ham reached a maximum after three days of incubation and then unpredictably decreased. In the Serrano ham, no increase in cell number was observed until day seven, and sea expression and extracellular SEA could only be detected on days five and seven. Finally, the black pepper salami with low pH and competing microbiota proved to be a difficult environment for the survival of S. aureus. The molecular mechanism behind the behaviour of S. aureus SEA expression is discussed in connection to the life-cycle of the SEA-encoding bacteriophage and the microbial communities in these pork products. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Staphylococcal food poisoning (SFP) was the fourth most common causative agent in food-borne illness within the EU in 2008 (EFSA, 2010). It is associated with food, generally protein-rich food, which requires extensive manual handling, often in combination with inadequate heating and/or inappropriate storage of the food (Le Loir et al., 2003; Smyth et al., 2004). In Korea, 30% of SFP incidents in 2001–2006 involved meat and meat products according to the Korean Food and Drug Administration and in the US, 36% of confirmed SFP outbreaks reported to CDC in 2007 involved pork meat products (CDC, 2007; Kim et al., 2009). Today, viable cell counts are performed to determine whether a food product is safe for human consumption. An investigation of SFP outbreaks in the UK in 1969–1990, revealed that the level of S. aureus in the incriminated food ranged from no detectable S. aureus to 1.5 × 10 10 colony forming units (CFU) per g food, with a median of 3×10 7 CFU/g (Wieneke et al., 1993). However, a more accurate way to determine the safety of food in case of SFP would be to measure the enterotoxin levels. In addition, it is a necessity to investigate and identify the conditions that prevent or stimulate enterotoxin expression and formation in food. At present, 21 staphylococcal enterotoxins or enterotoxin-like proteins have been identified (Schlievert and Case, 2007; Thomas et al., 2006), and the bacteriophage-encoded staphylococcal entero- toxin A (SEA) is the toxin most frequently reported to be involved in SFP (Cha et al., 2006; Kérouanton et al., 2007; Wieneke et al., 1993). Recently, it was proved that the sea expression is linked to the life- cycle of the SEA-encoding prophage to some extent (Sumby and Waldor, 2003), and previous reports have shown that the sea expression peaks in the late exponential growth phase (Borst and Betley, 1993; Czop and Bergdoll, 1974; Sumby and Waldor, 2003). To better understand the effects of environmental factors on the SEA formation, expression studies can be performed on model systems consisting of liquid cultures of pure bacteria, i.e. planktonic bacteria. However, these planktonic systems may be very different from the conditions in the actual food. In fact most bacteria in food appear to be associated with surfaces or tissues in various ways and International Journal of Food Microbiology 141 (2010) S69–S74 ⁎ Corresponding author. Tel.: + 46 46 222 3412; fax: + 46 46 222 4203. E-mail address: Peter.Radstrom@tmb.lth.se (P. Rådström). 1 These authors contributed equally to the paper. 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.03.028 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro