Invasiveness of Listeria monocytogenes strains of Caco-2 cells in response to a period of extreme salt stress reﬂecting salt-curing and rehydration of cod (Gadus morhua L.) Grete Lorentzen a, * , Saskia Mennen a , Ragnar L. Olsen b , Taran Skjerdal c a Noﬁma Marin, PO Box 6122, N-9291 Tromsø, Norway b Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway c National Veterinary Institute, PO Box 750, N-0106 Oslo, Norway article info Article history: Received 5 July 2010 Received in revised form 10 December 2010 Accepted 27 December 2010 Keywords: Listeria monocytogenes Extreme salt stress Invasiveness Caco-2 cells abstract Products like salt-cured ﬁsh contain approximately 15e21% NaCl and are rehydrated to 2e3% NaCl before preparation and consumption. These products are regarded as safe, but it has been shown that Listeria spp. is able to survive at extreme levels of salt and start to grow after rehydration. Thus, the ability of salt stressed Listeria monocytogenes to cause listeriosis, measured as its ability to invade Caco-2 cells was studied in this paper. Seven strains of L. monocytogenes and one Listeria innocua were cultivated in BHI to early and late stationary phase at 4  C. At both phases, the strains were exposed to either no salt or to salt stress comparable to that applied in the production of rehydrated salt-cured cod, i.e. 21% NaCl followed by dilution to 2% NaCl. In addition, the eight strains were cultivated in BHI with 2% NaCl, which is similar to the salt content as in rehydrated salt-cured cod and other ready-to-eat (RTE) products as well. The ability of non salt stressed L. monocytogenes strains to enter Caco-2 cells was signiﬁcant higher (p > 0.05) compared to the corresponding strains exposed to 21% NaCl for 96 h, followed by 2% NaCl for 48 h. On the other hand, L. monocytogenes cultivated in BHI with 2% NaCl showed a higher invasiveness of Caco-2 cells than both the other sample categories. As the ability to invade Caco-2 cells correlates with bacterial virulence, the results suggests that L. monocytogenes represent a lower food safety risk when exposed to salt-curing with extreme NaCl concentrations than exposure of a constant and moderate level of salt commonly used in RTE products. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Listeria monocytogenes can cause human listeriosis and it is recognized that nearly all cases are foodborne. Although listeriosis represents a small fraction of all illnesses due to known foodborne pathogens it is an important cause of severe illness. Invasive listeriosis accounts for approximately 4% of foodborne disease hospitalizations with a case-fatality rate in the range of 20e30% (Swaminathan & Gerner-Smidt, 2007). Invasive listeriosis is rare, however in Europe, increasing incidences of listeriosis is observed among persons more than 60 years (Goulet, Hedberg, Le Monnier, & de Valk, 2008) and public health ofﬁcials are concerned by the increasing number of cases since 1999 (EFSA, 2006). Epidemiological data indicate that certain products such as ready-to-eat (RTE) food are more likely to be associated with listeriosis than others (Lianou & Sofos, 2007). As L. monocytogenes is able to grow to high levels in many RTE products, including ﬁsh products containing 2e8% NaCl (Fernández-Segovia, Escriche, Gómez-Sintes, Fuentes & Serra, 2006; Gimenez & Dalgaard, 2004; Hwang, 2007; Lorentzen, Olsen, Bjørkevoll, Mikkelsen, & Skjerdal, 2010; Midelet-Buordin, Copin, Leleu & Malle, 2010; Pedro, Albuquerque, Nunes, & Bernardo, 2004), the risk of human listeri- osis increases with increasing storage time between preparation and consumption. Commercial production of RTE products from salt- cured ﬁsh, such as rehydrated salt-cured cod has increased. Salt-cured cod is processed by adding salt, i.e. by pickle salting or brining. A fully salt-cured product contains approximately 15 and 21% NaCl for salt- cured ﬁsh and klipﬁsh (dried salt-cured ﬁsh), respectively. After salt- curing, the product is rehydrated by soaking in water to lower the level of salt to about 2% NaCl. Despite extreme concentrations of salt in dried salt-cured ﬁsh, it has been found that Listeria spp. when added to fresh cod prior to salt-curing, was able to survive and start to grow after lowering the salt concentration to 2% NaCl (Lorentzen, Olsen, et al., 2010). Thus, assuming extreme concentrations of salt as a permanent food safety barrier has to be reconsidered and the food safety of RTE products of salt-cured cod needs to be studied. As far as * Corresponding author. Tel.: þ47 77 62 90 00; fax: þ47 77 62 91 00. E-mail address: grete.lorentzen@noﬁma.no (G. Lorentzen). Contents lists available at ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2010.12.013 Food Control 22 (2011) 1040e1045