1. Introduction

Since Escherichia coli O157:H7 was first identified as a foodborne pathogen in 1982, there has been a growing demand for methods to control contamination of this organism in the food industry (Riley et al., 1983). An estimated 73,480 cases of E. coli O157:H7-related illness are reported each year with approximately 85% due to foodborne transmission (Mead et al., 1999). Many food vehicles have been responsible for the transport of E. coli O157:H7, including apple cider (Besser et al., 1993), raw vegetables and sprouts ( Cieslak et al., 1993), and water ( Keene et al., 1994). Cattle are the primary reservoir of E. coli O157:H7 and the majority of outbreaks have stemmed from bovine products, primarily ground beef (Griffin and Tauxe, 1991).

Food-processing methods have been developed to interfere with bacterial homeostasis, prevent growth, or kill foodborne pathogens (Abee and Wouters, 1999). Some common hurdles used to control microbial growth are pH, sodium chloride, and storage temperature. Consumer demand for high-quality fresh or minimally processed products has resulted in many foods using a combination of acid, salt, and cold storage to prevent microbial growth. Within such foods, bacteria are exposed to sub-lethal stress conditions. Previous research has indicated that bacterial exposure to a sub-lethal stress can result in subsequent increased resistance to that particular stress or increased resistance to other stresses ( Jenkins et al (1988) and Jenkins et al (1990); Farber and Pagotto, 1992; Buncic and Avery, 1998; Leenanon and Drake, 2001).

E. coli O157:H7 is more acid tolerant than other E. coli and can survive in many acidic foods (Besser et al., 1993; Guraya et al., 1998; McIngvale et al., 2000). The acid tolerance of E. coli O157:H7 is believed to play a key role in pathogenesis and foodborne illness (Buchanan and Edelson, 1996). Three mechanisms for acid resistance have been proposed in E. coli O157:H7: an oxidative, an arginine-dependent, and a glutamate-dependent system (Lin et al., 1995). Monosodium glutamate (MSG) is by far the most common source of glutamate used as an additive in the food industry ( IFT, 1987). The use of MSG as a flavor enhancing ingredient in many foods such as oriental foods and beef jerky could pose an increased food safety risk as an additional source of glutamate for the glutamate-dependent acid resistance mechanism in E. coli O157:H7. While previous studies have shown the effects of different types of single sub-lethal stresses on E. coli O157:H7, this study was undertaken to determine the combined effects of cold, acid, sodium chloride, and MSG on the survival and subsequent acid resistance of E. coli O157:H7.

2. Materials and methods

2.1. Bacterial strains and culture conditions

E. coli O157:H7 ATCC 43895 (raw hamburger isolate) and E. coli O157:H7 ATCC 43890 (fecal isolate) were obtained from the culture collection in the Dept. Food Science, North Carolina State University (Raleigh, North Carolina). E. coli O157:H7 SEA 13B88 FDA was obtained from Dr. Pina Fratamico (USDA ARS, Wyndmoor, Pennsylvania). Stock cultures were stored in 30% (w/w) glycerol at ?80°C. Cultures were examined for purity and negative sorbitol fermentation monthly on sorbitol MacConkey agar (BBL, Cockeysville, Maryland). Virulence markers (slt, eae, and hly) were also confirmed monthly by PCR (Meyer et al., 1992; Gannon et al., 1997; Schmidt et al., 1995).

Cultures were activated prior to use by overnight growth in TSB, pH 7.2, at 37°C at least twice before each experiment. Static growth curves in TSB at 37°C were generated for each strain. Based on growth curves for each strain, stationary phase occurred after 18 h growth (10⁹ cfu/ml) and corresponded to OD₆₀₀ values of approximately 1.8 (Beckman, DU Series 500 spectrophotometer, Fullerton, California). For cell enumerations, serial dilutions were performed in sterile 0.1% peptone-water and pour plated in duplicate on tryptic soy agar. Plates were incubated at 37°C for 48 h prior to counting.

For cold storage studies, a three-strain cocktail was prepared. Each isolate was grown at 37°C for 18 h (approx. 10⁹ cfu/ml). Ten milliliters of each isolate was centrifuged at 10,000×g for 5 min. Supernatants were decanted and the pellets washed three times, each with 9 ml of sterile 0.1% peptone-water (Difco, Detroit, Michigan). Pellets were resuspended in 3.0 ml of sterile 0.1% peptone-water. The three resuspended pellets were pooled to a final volume of approx. 10 ml. One milliliter of this mixed strain cocktail was then used to inoculate each cold storage treatment to an initial concentration of 10⁶ cfu/ml.

2.2. Cold storage

Three pH values were studied: pH 7.2, 5.0, and 4.0. Sodium chloride and MSG were then studied within each pH in a factorial arrangement of treatments. The pH of TSB was adjusted with hydrochloric acid (35% w/w) (Seastar Chemical Inc., Pittsburgh, Pennsylvania) prior to autoclaving and pH confirmed following autoclaving. Sodium chloride (0, 2, or 4%) (Fisher Scientific, Fair Lawn, New Jersey) was incorporated into TSB at each pH prior to autoclaving. A 10% (w/v) stock solution of MSG (Sigma) was prepared in distilled deionized water. The solution was then filter-sterilized (0.22 ?m Millipore filter, Millipore Corporation, Bedford, Massachusetts) into a sterilized container. Appropriate volumes of this sterile stock solution were then added to autoclaved TSB pH/salt combinations to the final desired concentration (0, 0.5%, 1.0% w/v). TSB was then aseptically subdivided into 100 ml sterile glass bottles in 50 ml aliquots. MSG was added post-autoclaving to prevent heat degradation. Periodically a sample of the MSG stock solution was tested for contamination by inoculating a 1 ml volume into 9 ml TSB and examining for turbidity following incubation at 37°C for 24 h. Bottles were pre-chilled to 5°C prior to inoculation with the mixed strain cocktail. Cells were enumerated at time 0 and subsequently stored at 5°C. Cell numbers were subsequently enumerated after 7, 14, and 21 d storage.

error of survivors at each timepoint, were plotted for visual examination.