Biocontrol of Salmonella Typhimurium in RTE foods with the virulent bacteriophage FO1-E2

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Abstract

Foodborne Salmonella infections are a major public health concern worldwide. Bacteriophages offer highly specific and effective biocontrol of such pathogens. We evaluated the broad host range, virulent phage FO1-E2 for reduction of Salmonella Typhimurium in different RTE foods. Samples were spiked with 1 × 103 Salmonella cells and treated with 3 × 108 pfu/g phage, and incubated for 6 days at 8 °C or 15 °C. At 8 °C, no viable cells remained following FO1-E2 application, corresponding to a more than 3 log10 unit reduction. At 15 °C, application of phage lowered S. Typhimurium counts by 5 log units on turkey deli meat and in chocolate milk, and by 3 logs on hot dogs and in seafood. In egg yolk, an effect was observed only after 2 days, but not after 6 days. Phage particles retained their infectivity, although they were readily immobilized by the food matrix, resulting in loss of their ability to diffuse and infect target cells. At the end of the incubation period, phage-resistant Salmonella strains appeared which, however, were not able to compensate for the initial killing effect. Altogether, our data show that virulent phages such as FO1-E2 offer an effective biocontrol measure for Salmonella in foods.

Highlights

►Salmonella biocontrol efficacy of bacteriophage FO1-E2 was evaluated. ►At 8 °C storage temperature, no viable bacteria were detected after treatment. ►At 15 °C storage temperature, Salmonella growth was suppressed by at least 2 and up to 5 log units on different foods. ►Phage particles were stable and retained infectivity. ►Bacteriophage FO1-E2 is an effective biocontrol agent for Salmonella.

Introduction

Salmonella Typhimurium (Salmonella enterica subspecies enterica, serovar Typhimurium) is a Gram-negative, rod shaped bacterium which belongs to the Enterobacteriaceae. It is a facultative intracellular human pathogen and the causative agent of non-typhoid salmonellosis. The disease is characterized by abdominal pain, diarrhea and nausea and is frequently transmitted via contaminated food or water. Salmonella pathogenicity and virulence factors have been reviewed and summarized in numerous articles (Coburn et al., 2007, Grassl and Finlay, 2008, Kingsley and Baumler, 2002, Parry et al., 2002, Santos et al., 2003). About 40,000 cases of non-typhoid salmonellosis per year are reported to the Centers for Disease Control and Prevention in the USA (Groseclose et al., 2004, Groseclose et al., 2002, Hopkins et al., 2003, Hopkins et al., 2005, Jajosky et al., 2006). However, taking into account the degree of under-reporting, approximately 1.4 million infections per year are estimated, resulting in 400–600 deaths (Mead et al., 1999, Voetsch et al., 2004). A recent outbreak in the US linked to eggs contaminated with Salmonella Enteritidis resulted in approximately 1500 cases of illness and a recall of more than 500 million eggs in 14 states (CDC, 2010). In the USA, more than 50% of salmonellosis is caused by only three serovars, namely Salmonella Typhimurium, Salmonella Enteritidis and Salmonella Newport (Jajosky et al., 2006). Salmonella Enteritidis and Salmonella Typhimurium also represent the predominant strains in Europe, and more than 100,000 confirmed cases of human Salmonellosis were reported in 2009 (European Food Safety Authority, 2011). Accordingly, non-typhoid salmonellosis is regarded as the second most common cause of foodborne zoonotic infection in the EU (Westrell et al., 2009). Therefore, the development and evaluation of new strategies for the control of Salmonella is urgently needed, especially in ready-to-eat (RTE) foods.

The concept of using phage application against spoilage bacteria and pathogens in foods has received increasing interest during the last years, and many studies support the value of this approach. Salmonella reduction after application of bacteriophages has been demonstrated for cheddar cheese (Modi et al., 2001), honeydew melon slices (Leverentz et al., 2001), mustard seeds (Pao et al., 2004), chicken frankfurters (Whichard et al., 2003), chicken skin (Pao et al., 2004), sprouts (Ye et al., 2010), and swine (Wall et al., 2010). It has also been demonstrated for other important foodborne pathogens such as Listeria monocytogenes (Carlton et al., 2005, Guenther et al., 2009), and several others.

Phages offer a number of desired properties: (i) they are designed to kill their host cells, (ii) they are usually highly specific and do not cross species or genus barriers, (iii) they are self-replicating and self-limiting, and (iv) they are ubiquitously distributed in nature (and consequently also in food), representing the most abundant replicating unit on earth (Rohwer and Edwards, 2002). Phages are commensals of humans and animals (Greer, 2005, Marks and Sharp, 2000), and they have been isolated from a wide range of foods like ground beef (Hsu et al., 2002, Kennedy et al., 1986, Whitman and Marshall, 1970, Whitman and Marshall, 1971), pork, chicken and other meat products (Hsu et al., 2002, Kennedy et al., 1986), chilled and frozen crabmeat (DiGirolamo et al., 1972), fermented dairy products like cheese and yogurt (Kilic et al., 1996), and from lettuce and mushrooms (Hernandez et al., 1997, Kennedy and Bitton, 1987). Hence, phages can be considered a part of the natural microflora of foods.

It is essential to use virulent, i.e., non-integrating, obligately lytic, non-transducing bacteriophages for biocontrol of pathogens (Hagens and Loessner, 2010). The prototype virulent Salmonella phage Felix-O1 (FO1) was first described by Felix and Callow in the 1930s (Felix and Callow, 1943). It is a member of the Myoviridae and features an 86.2 kb dsDNA genome (Whichard et al., 2010). FO1 offers a broad host range within the genus Salmonella (Cherry et al., 1954). Some Escherichia coli strains have also been claimed to be sensitive to FO1 (Hirsh and Martin, 1983, Welkos et al., 1974), although this issue may require further clarification. We use the newly isolated phage strain FO1-E2, which is related to FO1 but features an approximately 2 kb deletion in a genetic region of unknown function (unpublished data), and shows an even broader host range. The major aim of this study was to evaluate the potential and efficacy of FO1-E2 for control of Salmonella Typhimurium in different RTE foods often contaminated with Salmonella. We found that FO1-E2 is able to reduce Salmonella counts in foods at 15 °C storage temperature by up to 5 log units and can suppress Salmonella counts below the detection limit in food stored at 8 °C.

Section snippets

Bacteria, phage, and culture conditions

Salmonella and E. coli were cultured in Luria Bertani (LB) broth (Oxoid, Hampshire, UK), at 37 °C. To specifically determine the colony forming units (cfu) of Salmonella Typhimurium re-isolated from the spiked foods, drug resistant strains DB 7155 stmR and DB 7155 camR were employed. Strain DB 7155 stmR is a derivative of DB 7155 obtained by repeated passage of the wildtype on media containing streptomycin, which resulted in resistance to 500 μg/ml streptomycin. Strain DB 7155 camR was

Results

A prerequisite for recovery of the target bacteria from artificially contaminated (spiked) foods is the use of selectable host strains featuring a stable marker such as antibiotic resistance. Analysis of the stability of drug resistance in DB 7155 stmR and DB 7155 camR indicated that both strains were able to stably maintain the marker under non-selective conditions, in either LB broth or on sterilized hot dog samples, over a 6 days period (Table 1). Comparison of the growth rates of DB7155 stmR

Discussion

Our data demonstrate the suitability of the virulent and broad host range bacteriophage FO1-E2 for control of Salmonella Typhimurium in several ready-to-eat foods. Phage application at 8 °C significantly reduced Salmonella viable counts below the detection limit without enrichment. An overall reduction by at least 2 logs was observed in all tested foods except egg yolk (equals a killing efficiency of at least 99%). This has strong impact on the growth of the organism, and represents a major

Acknowledgments

We are grateful to Stefan Miller for providing bacteriophage FO1-E2, and to Monique Herensperger and the late Ursula Schuler for their excellent technical assistance.

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    Present address: Institute for Food and Beverage Innovation, ZHAW Zurich, 8820 Wädenswil, Switzerland.

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