Behavior of Listeria monocytogenes in pH-modified chicken salad during refrigerated storage.

Introduction

Listeria monocytogenes is the causative agent of listeriosis in both humans and animals (Food and Drug Administration [FDA], 2001a). Although the incidence of listeriosis in the United States is relatively low (~2,500 cases per year), the ~20 percent fatality rate is the highest among the foodborne-pathogen diseases tracked by the Centers for Disease Control and Prevention (CDC) (1999). Other consequences of infection can include septicemia, meningitis, and stillbirth (FDA, 2001a). Populations that are most susceptible to listeriosis include pregnant women and individuals with a predisposing disease or diminished immune function (Farber & Peterkin, 2000). Because of the severity of infection, a zero tolerance (i.e., <1 CFU per two 25-g samples) has been established in the United States for L. monocytogenes in processed ready-to-eat (RTE) foods and for processed dairy and meat food products (Shank, Elliott, Wachsmuth, & Losikoff, 1996).

RTE foods, including prepared deli salads, may contain Listeria monocytogenes by way of contaminated ingredients, postprocess contamination, or both. A preservation strategy that combines temperature and acidulants can be used to control the survival and growth of L. monocytogenes in RTE foods. L. monocytogenes has been recovered from many RTE foods, including luncheon meats and coleslaw (CDC, 1999; Schlech et al., 1983). The presence of L. monocytogenes in such foods is often the result of postprocess contamination. Controlling the behavior of L. monocytogenes in foods is problematic because the microorganism is halotolerant, is ubiquitous in raw ingredients and the processing environment, is capable of survival or growth over a broad pH range (pH 4.4-9.6) and a broad temperature range (<1[degrees]C-45[degrees]C), and is capable of survival or growth in atmospheres of varied oxygen content (Farber & Peterkin, 2000). Since RTE foods usually are not heated before consumption, it is important that L. monocytogenes not be present, or if present, that it be incapable of growing to infective-dose levels.

The U.S. Department of Agriculture (USDA), FDA, and CDC have collaboratively produced an extensive risk assessment for L. monocytogenes that addresses the prevalence of this organism in foods, consumption patterns, the epidemiology of past listeriosis outbreaks and sporadic cases, and the doseresponse relationship (USDA, 2001). Although many classes of foods are discussed in the assessment, limited data were available for RTE deli salads such as chicken salad.

Predictions about the fate of L. monocytogenes obtained through the application of a proposed model from data derived from an actual food system may contribute to the development of guidelines for formulation and cold storage of RTE foods. This information may be particularly useful for retail food establishments that prepare and sell deli salads. In the study reported here, the objective was to determine the differences in growth and inactivation rates of L. monocytogenes in commercially produced RTE chicken salad modified to three pH levels (4.0, 4.6, and 5.2) when stored at three temperatures (5[degrees]C, 7.2[degrees]C, and 21.1[degrees]C) over the shelf life of the pasteurized salad (119 days).

Materials and Methods

Experimental-Condition Rationale

The temperatures evaluated in this study were determined by current FDA recommendations and some state and local requirements for cold storage of RTE foods in retail food establishments. The 2001 FDA Food Code (FDA, 2001b) currently recommends cold storage at 5[degrees]C (41[degrees]F), whereas many states or local jurisdictions require storage at 7.2[degrees]C (45[degrees]F). The Food Code also recommends that once the packaging has been opened, RTE foods should not be stored for more than seven days at 5[degrees]C (41[degrees]F) or four days at 7.2[degrees]C (45[degrees]F).

A survey of chicken salad purchased from 25 grocery stores and delicatessens located in mid-central Indiana in 2000 identified pH values ranging from pH 4.1 to 5.1 in 24 of the products, with the 25th sample having pH 5.4. In light of these findings, the present study evaluated a slightly broader pH range of 4.0-5.2. The pH values represented conditions that would potentially support inactivation (pH 4.0), survival (4.6), and growth (pH 5.2) of L. monocytogenes in food systems (FDA, 2001a).

Experimental Design

The study was based on a full-factorial experimental design that evaluated the individual and combined effects of storage time (0 to 119 days), temperature (5.0[degrees]C, 7.2[degrees]C, and 21.1[degrees]C) and pH level (pH 4.0, 4.6, and 5.2). Nine combinations of pH and storage temperature were evaluated in three replicated experiments, for a total of 27 different experiments. In each of the three trials, three separate samples of chicken salad were analyzed at each sampling interval for each combination of temperature, pH, and storage time.

Description of the Chicken Salad

Three batches of commercially pasteurized chicken salad were provided by a commercial food processor. Each batch consisted of eight 5-lb containers of chicken salad shipped on dry ice by overnight courier within 24 hours of production and stored refrigerated. Within 48 hours of production, the microbial background of the chicken salad was assessed by an aerobic plate count (Maturin & Peeler, 2001) on tryptic soy agar (TSA) (Beckton, Dickinson and Co., Sparks, Maryland). Samples of the chicken salad were also tested for the presence of L. monocytogenes with the FDA isolation and enrichment procedures (Hitchins, 2001). To determine the initial pH of the chicken salad, the authors used a Model AR15 pH meter (Fisher Scientific, Pittsburgh, Pennsylvania) to measure three 50-g samples from each 5-lb container of chicken salad and averaged the readings. The shelf life of the chicken salad, as stated by the commercial producer, was 112 days for unopened packages.

Modification of the pH of the Food System

Within 48 hour of production, the authors modified the pH of the commercially pasteurized chicken salad by aseptically transferring 300 g of chicken salad to a sterile stomacher bag and then adding 50 mL of sterile distilled water (d[H.sub.2]O) and filter-sterilized solutions of either 2N acetic acid or 2M sodium acetate (Sigma Chemical Co., St. Louis, Missouri). All samples were blended in a Lab Stomacher (Seward Medical, London) for 30 seconds to ensure even distribution of the solutions throughout the chicken salad. Blending was done so that the particle size of the salad was only minimally affected. To prepare negative controls for pH modification, the authors added 50 mL d[H.sub.2]O to a 300-g batch of chicken salad. To determine any net change in available water resulting from the pH modification or blending, the water activity ([a.sub.w]) of representative samples was measured and compared with the [a.sub.w] of 300 g of chicken salad + 50 mL d[H.sub.2]O, as well as the [a.sub.w] of chicken salad samples that had not been modified or blended (Aqualab, Pullman, Washington, Model CX-2). The amount of acetic acid and sodium acetate added for pH modification is given in Table 1.

Preparation and Addition of the Inoculum 

Three strains of Listeria monocytogenes--Scott A, LCDC81-861, and F2365--were stored on TSA slants at 4[degrees]C. L. monocytogenes Scott A was provided by Dr. L.R. Beuchat (Center for Food Safety and Quality, University of Georgia, Athens, Georgia). Other strains were obtained from Dr. A. Bhunia (Purdue University, West Lafayette, Indiana). The strains used in this mixed culture were selected because of their resistance to environmental stress (Scott A) and acidic conditions (LCDC81-861 and F2365). To prepare broth cultures of each strain, a loopful of cells was transferred aseptically from the TSA slant into 50 mL tryptic soy broth (TSB) (Beckton, Dickinson and Co., Sparks, Maryland) and incubated at 37[degrees]C for 1 hour. A 1-mL aliquot of each broth culture was transferred to 100 mL of fresh TSB and incubated at 37[degrees]C for another 18 hours. To create acid-adapted cultures, 1-mL aliquots were transferred from each regular broth culture to 50 mL TSB + 0.1 percent acetic acid and incubated at 37[degrees]C for 72 hours. Aliquots of 1 mL were subsequently transferred to fresh TSB + 0.1 percent acetic acid and incubated at 37[degrees]C for 24 hours. This procedure was repeated until turbidity was consistently observed after incubation at 37[degrees]C for 18 hours. To increase the level of acid resistance, 1-mL aliquots were transferred to TSB + 0.2 percent acetic acid and incubated at 37[degrees]C for 48 hours. This procedure was repeated until growth was consistently observed after incubation at 37[degrees]C for 18 hours. The purpose of creating acid-adaptive stains was to adapt cells to a lower pH range and enhance the potential for cell survivability and growth within a food system that had a similar pH and acid-type environment. Cells grown in pH conditions that are optimum for growth (i.e., pH 7.0) and then subjected to a food system with a lowered pH can be "shocked," and growth and survivability can be more difficult.

[FIGURE 1 OMITTED]

In preparation for addition of the inoculum, 25-g samples of the pH-modified chicken salad and the negative-control chicken salad were prepared in sufficient numbers to accommodate enumeration through the 112-day shelf life. Each 25-g sample was added to a sterile polypropylene specimen cup (Fisher Scientific, Pittsburgh, Pennsylvania). The samples were tempered to a designated storage temperature (5.0[degrees]C, 7.2[degrees]C, or 21.1[degrees]C) for 4 hours before addition of the inoculum, which the authors prepared by combining 2-mL aliquots from each of the three standard broth cultures and each of the three acid-resistant broth cultures in a sterile tube (total volume: 12 mL). The cells were centrifuged, the supernatant was removed, and the cells were re-suspended into phosphate-buffered saline (Sigma Chemical Co., St. Louis, Missouri). The re-suspended cells were diluted to a level of approximately [10.sup.7] cells per mL in buffered peptone water (11 mL of the re-suspended cells were added to 99 mL buffered peptone water) (Beckton, Dickinson). The inoculum was enumerated by aerobic plate count (Maturin & Peeler, 2001) on TSA at 37[degrees]C for 48 hours.

The authors inoculated the individual 25-g samples of chicken salad by aseptically adding a 100-[micro]L aliquot of the inoculum to the surface of the chicken salad. The inoculum was quickly mixed into the chicken salad with the aid of a sterile pipette tip. The lids of the sample cups were replaced, and the samples were held at 5.0[degrees]C, 7.2[degrees]C, or 21.1[degrees]C for the duration of the study. The temperature of the storage incubator was continuously monitored by use of a data-logging thermocouple (Fisher Scientific, Pittsburgh, Pennsylvania).

Sample Randomization and Enumeration

During storage, samples were identified by placement on trays outfitted with numbered grids. On sampling days, a random-number generator (Hewlett-Packard 11c calculator) was used to produce a list of the grid number positions of the samples to be pulled. On each sampling day, one 25-g inoculated sample of each pH level (pH 4.0, 4.6, and 5.2), one 25-g negative-control un-inoculated sample for each pH level, one 25-g negative control of un-inoculated chicken salad + d[H.sub.2]O, and one 25-g sample of unmodified chicken salad were pulled for enumeration. Samples were enumerated with a five-tube most-probable-number (MPN) method, in a modified version (10 mL of stomached sample to 90 mL Modified University of Vermont broth [UVM]) (Beckton, Dickinson and Co., Sparks, Maryland) of the USDA/Food Safety and Inspection Service method for detection of L. monocytogenes (Ryser & Donnelly, 2001; Wang & Muriana, 1994). The MPN method with dilutions up to [10.sup.-8] was selected--after a brief experiment was conducted to compare the enumeration efficacy of MPN and direct plating methods on samples containing [10.sup.-3]-[10.sup.9] CFU/g--because it gave a more conservative estimate of survival and estimated levels lower than [10.sup.3] CFU/g.

Each 25-g sample was aseptically transferred to a sterile filter stomacher bag (Seward, London). The sample cup was rinsed twice with portions from a bottle containing 225 mL of sterile UVM broth. The rinse fluid was added to the stomacher bag along with the remaining UVM broth, and the contents were blended for 30 seconds in a lab stomacher. When very low numbers of L. monocytogenes were being enumerated, the authors created a [10.sup.-1] dilution series by transferring 20 mL of the blended sample homogenate to each of five empty sterile bottles. All MPN tubes were incubated at 30[degrees]C for 48 hours. After the 48-hour enrichment, 1-mL aliquots from each MPN bottle were transferred to tubes containing 10 mL Fraser broth (Beckton, Dickinson and Co., Sparks, MD) and incubated at 30[degrees]C for an additional 24 hours. All Fraser broth tubes that showed esculin hydrolysis (indicated by black color) were presumed positive for L. monocytogenes and were further confirmed by biochemical tests.

[FIGURE 2 OMITTED]

A loopful of cells from Fraser-positive broth tubes were streaked onto modified Oxford (MOX) and PALCAM agars (Beckton, Dickinson), and were incubated at 35[degrees]C for 24 hours. Individual colonies showing esculin hydrolysis production were transferred into a tube of 10 mL TSB + 0.6 percent yeast extract (TSB-YE) (Beckton, Dickinson), streaked onto a plate of TSA + 0.6 percent yeast extract, and incubated at 35[degrees]C for 24 hours. A loopful of the culture from the TSBYE tube was transferred to each of four wells on a 96-well microtiter plate containing purple broth base (PB) + 0.5 percent rhamnose and PB + 0.5 percent mannitol (Beckton, Dickinson). The plate was incubated at 35[degrees]C for 24 hours. Single colonies from each TSA-YE plate were also stabbed onto 5 percent sheep blood agar (Remel, Lenexa, Kansas). The authors tested production of catalase by adding three drops of 3 percent hydrogen peroxide ([H.sub.2][O.sub.2]) (Sigma Chemical Co., St. Louis, Missouri) to the colonies present on the TSA-YE plate. The identification of L. monocytogenes was confirmed for those colonies demonstrating esculin hydrolysis (black color of colonies on MOX/PALCAM agars), rhamnose utilization with corresponding lack of utilization of mannitol, hemolysin production, and catalase production. The MPN was calculated from confirmed L. monocytogenes according to the method described by Swanson, Petran, and Hanlin (2001).

Determination of the Survival Curves

The average log of the MPN values at each sampling day for the three trials for each storage temperature was calculated and plotted against time to show the inactivation kinetics of L. monocytogenes in the chicken salad.

Statistical Analyses

Significance of the simple effects (time, temperature, and pH) and the interactions was determined by application of a two-way analysis of variance (ANOVA) to the experimental data (JMP[R], SAS Institute, Inc., Cary, North Carolina). The authors formulated a linear model of the behavior of L. monocytogenes in the food system by adding the parameter estimates for the three simple effects, the three two-factor interactions, and the three-factor interaction.

[FIGURE 3 OMITTED]

Results and Discussion

Microbial Growth and Inactivation

The nine different treatments evaluated in the experiments reported here included combinations of pH and temperature that could potentially support growth, survival, or inactivation of L. monocytogenes (Table 2). The food system did not, however, provide suitable conditions for growth or survival at any combination of pH and temperature, and L. monocytogenes was inactivated, albeit at different rates, over time in all nine treatments (Figure 1, Figure 2, Figure 3).

Inactivation occurred at all pH levels during storage at 5.0[degrees]C and 7.2[degrees]C (Figure 1, Figure 2); however, the rate of inactivation was greater at 5.0[degrees]C than at 7.2[degrees]C. The overall microbial reduction and inactivation rate was lower than other pH values, at pH 5.2, with a decrease of only 1.1 [log.sub.10] in 119 days at 7.2[degrees]C and 4.0 [log.sub.10] in 119 days at 5.0[degrees]C. Inactivation at pH 4.6 at 5.0[degrees]C and 7.2[degrees]C was comparable, with decreases of 3.8 and 3.3 [log.sub.10] seen after 119 days, respectively. Inactivation at pH 4.0 was more rapid at 5.0[degrees]C (>6 [log.sub.10] in 28 days) than at 7.2[degrees]C (>3 [log.sub.10] in 119 days).

Inactivation occurred at each pH level during storage at 21.1[degrees]C (Figure 3) and was most rapid at pH 4.0. Inactivation was almost immediate at 21.1[degrees]C/pH 4.0, but a lag phase of 10 to 12 days was evident before inactivation at pH 4.6 and pH 5.2. The inactivation rate observed at all three levels of pH was more rapid at the abuse temperature of 21.1[degrees]C (Figure 3) than at either 5.0[degrees]C or 7.2[degrees]C (Figure 1, Figure 2).

A two-way ANOVA was done on the experimental results to identify the significant factors and factor interactions (Table 3). At the 95 percent confidence level, statistical significance was identified in the simple effects of temperature (p < .0001) and time (p < .0001), and the interactive effects of temperature and time (p < .0001), and pH and time (p = .0103).

In the study reported here, the faster rate of inactivation and greater reduction in surviving cells seen during storage at 5.0[degrees]C and pH 4.0 may have been affected by the storage temperature, as other investigators have reported generation times for L. monocytogenes nearly twice as long at 4[degrees]C as at 8[degrees]C (Rosenow & Marth, 1987). In such a dynamic population, a generation time greater than the rate of inactivation would result in a net decrease in population. This behavior could be the result of the potential existence of two subpopulations created by the combined use of both standard and acid-adapted strains in the inoculum. The acid-adapted strains may have a slower rate of inactivation than the standard strains. Further experiments are required to confirm this hypothesis, however.

Since growth was not observed in any of the conditions used in the pH-modified chicken salad, the study reported here does not provide scientific support for the preferred use of 5.0[degrees]C cold storage, as opposed to 7.2[degrees]C cold storage, specifically for the inhibition of growth of L. monocytogenes in pH-modified chicken salad. Additional studies should be completed on other RTE foods to determine if this observation is consistent with results for other foods or if it is unique to pH-modified chicken salad.

The inactivation of L. monocytogenes in chicken salad observed in the study reported here cannot be explained solely by the use of an acidulant--in this case, acetic acid. Further identification and evaluation of the individual and combined effects of the specific ingredients, and the dispersion and physical positioning of components in the chicken salad could provide a better explanation for the inactivation seen at pH levels and temperatures that should normally support growth of L. monocytogenes.

Significance of the Research to Public Health

Within the past decade, the retail food industry and the food-processing/food-manufacturing industry have been increasingly concerned with the presence and growth of L. monocytogenes in ready-to-eat foods. Storage and display of ready-to-eat foods such as protein-based salads present a unique challenge to operators of retail food establishments. Risk assessment studies have been done; however, limited information is currently available about prevalence, growth, survival, and inactivation of L. monocytogenes in protein-based salads. This circumstance, together with the high reported mortality rates (20 percent) for L. monocytogenes infections from food, has also generated considerable interest on the part of USDA and FDA.

A wide variety of resources, including the FDA Bad Bug Book (FDA, 2001a), suggest that L. monocytogenes is capable of growing within the pH and temperature environment selected in this study. The USDA Pathogen Modeling Program (USDA, 2004) also suggests that growth would occur under many of the conditions studied (Table 4). With this approach, growth was predicted at all temperatures studied and at a pH of 4.6 and 5.2. Inactivation was predicted only at the lower pH (4.0). Although these resources are invaluable tools for the food industry to use, it is important to consider studying the specific food system of interest. The study reported here, for example, did not result in microbial growth as predicted by these resources.

The microbial inactivation of L. monocytogenes observed in the present study correlates with some aspects of previous studies on the behavior of this pathogen in mayonnaise products and mayonnaise-based deli salads. George and Levett (1990) studied the growth of L. monocytogenes in pH-modified commercial coleslaw (pH 4.0, 5.0, 6.0, and 7.0) stored at temperatures ranging from 5[degrees]C to 25[degrees]C. In this research, a synergistic effect of temperature and pH, which inhibited growth at any storage temperature in samples with pH [less than or equal to] 5.0, and eventual inactivation or "erratic growth responses" in viable populations of L. monocytogenes were observed. Erickson and Jenkins (1991) assessed the fate of [10.sup.3] cells/g of non-acid adapted L. monocytogenes in home-style salads prepared with commercial mayonnaise and determined survival during storage at 4[degrees]C for up to 10 days and at 12.8[degrees]C for two days. The fate of L. monocytogenes in the home-style salads was complex and dependent on salad formulation; however, growth to a level >5 [log.sub.10] CFU/g was observed in the home-style chicken salad held for 10 days at 4[degrees]C. The chicken salad used in those studies contained only six ingredients (i.e., diced cooked chicken breast, mayonnaise, chopped green onion, table salt, lemon juice, and black pepper) and was not intended to represent a commercial product, such as the commercially prepared chicken salad used in the study reported here. The growth observed in home-style chicken salad contrasted with the inactivation seen in all nine experimental treatment combinations with the commercial chicken salad, indicating a potentially significant effect of formulation or food particle size on growth and inactivation of L. monocytogenes in chicken salad.

The observations made during storage at 7.2[degrees]C were unexpected; however, they were very consistent over the three trials. These findings warrant further study over a defined pH range to see if other inactivation trends exist. Survival differences within the narrow temperature range of 5.0[degrees]C-7.2[degrees]C could be important, since many retail foods are stored within this range. The significant interaction of temperature and pH is particularly evident at 7.2[degrees]C, but additional observations of the kinetics at pH levels of 4.0, 4.6, and 5.2 at additional temperatures between 7.2[degrees] and 21.1[degrees]C would be necessary to describe the variation in inactivation as temperature increases above 7.2[degrees]C.

The complexity of food systems potentially gives rise to interactions of several factors, such as pH, temperature, antimicrobial ingredients, the physical and chemical properties of the food, and even the physical positioning of ingredients and microorganisms throughout the food, and the interactions can appreciably influence microbial behavior. The identification and quantification of these interactive effects in food systems will be an extensive process. The adaptive nature of L. monocytogenes must also be considered in the development of future experiments, as the response to environmental stresses can affect the survival of this pathogen in foods.

TABLE 1 Adjustments of pH with Acetic Acid and Sodium Acetate Solutions
(per 300-Gram Sample)

        Solution Formula      Solution Formula        Solution Formula
pH      at 70[degrees]F       at 45[degrees]F         at 41[degrees]F

pH 4.0  23 mL 2N acetic acid  23.5 mL 2N acetic acid  23 mL 2N acetic
        17 mL [H.sub.2]O      16.5 mL [H.sub.2]O      acid 17 mL
                                                      [H.sub.2]O
pH 4.6  5 mL 2M sodium        4 mL 2M sodium          5 mL 2M sodium
        acetate 35 mL         acetate 36 mL           acetate 35 mL
        [H.sub.2]O            [H.sub.2]O              [H.sub.2]O
pH 5.2  40 mL 2M sodium       20 mL 2M sodium         40 mL 2M sodium
        acetate 0 mL          acetate 0 mL            acetate 0 mL
        [H.sub.2]O            [H.sub.2]O              [H.sub.2]O

TABLE 2 Conditions That Support Growth, Survival, and Inactivation of L.
monocytogenes*

Kinetics      Temperature                 pH          [a.sub.w]**

Growth         2[degrees]-45[degrees]C     4.8-9.6     0.95
Survival       0[degrees]-2[degrees]C      4.0-4.8     0.90-0.95
Inactivation  <0[degrees], >45[degrees]C  <4.8, >9.6  <0.90

* Adapted from FDA's Bad Bug Book (2001a).
** Water activity.

TABLE 3 Effect Tests, Two-Way ANOVA of the Experimental Data for
Behavior of Listeria monocytogenes in pH-Modified Chicken Salad

Effect Tests                            Statistical Values
Source                     Nparm  DF  Sum of Squares  F Ratio   Prob > F

Temperature                1      1   386.83575       245.4856  <.0001
pH                         1      1     6.03739         3.8313   .0510
Temp*pH                    1      1     1.90880         1.2113   .2717
Time                       1      1   320.96325       203.6830  <.0001
Temperature*time           1      1    35.61825        22.6033  <.0001
pH*Time                    1      1    10.47994         6.6506   .0103
Temperature*pH*time        1      1     2.58854         1.6427   .2007

*Indicates an interaction between factors tested.

TABLE 4 Predicted Behavior of Listeria monocytogenes from the USDA
Pathogen Modeling Program (Version 6.1) at Three pH Levels and Three
Temperatures*

                       Days for         Days for         Days for
     [Log.sub.10]      Growth (+)       Growth (+)       Growth (+)
     Change (Growth    or Death (-) at  or Death (-) at  or Death (-) at
pH   or Inactivation)  5[degrees]C      7.2[degrees]C    21.1[degrees]C

4.0  1-[log.sub.10]    N/A              N/A              N/A
4.0  3-[log.sub.10]    -41.3            -39.8            -21.8
4.0  5-[log.sub.10]    -64.2            -62.1            -34.2
4.6  1-[log.sub.10]    +28.4            +19.0             +2.5
4.6  3-[log.sub.10]    +46.1            +30.4             +3.7
4.6  5-[log.sub.10]    N/A              N/A              N/A
5.2  1-[log.sub.10]    +10.2             +6.9             +0.9
5.2  3-[log.sub.10]    +17.4            +11.6             +1.5
5.2  5-[log.sub.10]    N/A              N/A              N/A

* Data are presented as days required for 1-[log.sub.10],
3-[log.sub.10], and 5-[log.sub.10] changes in population.
N/A = Data not available.

Acknowledgements: The authors would like to thank Dr. Mark Tamplin of the Microbial Food Safety Unit at USDA's Eastern Regional Research Center for his suggestions and assistance in this research project.

REFERENCES

Centers for Disease Control and Prevention. (1999). Incidence of foodborne illnesses: Preliminary data from the foodborne diseases active surveillance network: FoodNet 1998. Morbidity and Mortality Weekly Report, 48(9), 189-194.

Erickson, J.P., & Jenkins, P. (1991). Comparative Salmonella spp. and Listeria monocytogenes inactivation rates in four commercial mayonnaise products. Journal of Food Protection, 54, 913-916.

Farber, J.M., & Peterkin, P.I. (2000). Listeria monocytogenes. In B.M. Lund, T.C. Baird-Parker, & G.W. Gould (Eds.), Microbial safety and quality of foods (pp. 1178-1232). Gaithersburg, MD: Aspen Publishers, Inc.

Food and Drug Administration. (2001a). Bad bug book. http://www.vm.cfsan.fda.gov/`mow/intro.html (9 May 2003).

Food and Drug Administration. (2001b). FDA food code. http://www.cfsan.fda.gov/`dms/fc01-toc.html (26 Apr. 2003).

George, A.E., & Levett, P.N. (1990). Effects of pH and temperature on survival of Listeria monocytogenes in cole slaw. International Journal of Food Microbiology, 11, 345-350.

Hitchins, A.D. (2001). Detection and enumeration of Listeria monocytogenes in foods. Bacteriological analytical manual online. U.S. Food and Drug Administration. http://www.cfsan.fda.gov/`ebam/bam-10.html (6 Jan. 2002).

Maturin, L.J., & Peeler, J.T. (2001). Aerobic plate count. Bacteriological analytical manual online. U.S. Food and Drug Administration. http://www.cfsan.fda.gov/~ebam/bam-3.html (8 May 2003).

Rosenow, E.M., & Marth, E.H. (1987). Growth of Listeria monocytogenes in skim, whole and chocolate milk, and in whipping cream during incubation at 4, 8, 13, 21 and 35[degrees]C. Journal of Food Protection, 50, 452-459.

Ryser, E.T., & Donnelly, C.W. (2001). Listeria. In F.P. Downes, & K. Ito (Eds.), Compendium of methods for the microbiological examination of foods (4th ed., pp. 343-356). Washington, DC: American Public Health Association.

Schlech, W.F., Lavigne, R.M., Bortolussi, R.A., Allen, A.C., Haldane, E.V., Wort, A.J., Hightower, A.W., Johnson, S.E., King, S.H., Nicholls, E.S., & Broome, C.V. (1983). Epidemic listeriosis--Evidence for transmission by food. New England Journal of Medicine, 308, 203-206.

Shank, F.R., Elliott, E.L., Wachsmuth, I.K., & Losikoff, M.E. (1996). U.S. position on Listeria monocytogenes in foods. Food Control, 7, 229-234.

Swanson, K.M.J., Petran, R.L., & Hanlin, J.H. (2001). Culture methods for enumeration of microorganisms. In F.P. Downes, & K. Ito (Eds.), Compendium of methods for the microbiological examination of foods (4th ed., pp. 53-62). Washington, DC: American Public Health Association.

U.S. Department of Agriculture. (2004). The United States Department of Agriculture Pathogen Modeling Program (Version 6.1.) [Computer software]. http://www.arserrc.gov/mfs/PMP6_Start.htm (20 July 2004).

U.S. Department of Agriculture. (2001). Draft assessment of the relative risk to public health from Listeria monocytogenes among selected categories of ready-to-eat foods. http://www.foodsafety.gov/~dms/lmrisk.html (2 May, 2002).

Wang, C., & Muriana, P.M. (1994). Incidence of Listeria monocytogenes in packages of retail franks. Journal of Food Protection, 57, 382-386.

A.M. Guentert, M.S.

R.H. Linton, Ph.D.

J.B. Luchansky, Ph.D.

M.A. Cousin, Ph.D.

Corresponding Author: R.H. Linton, Professor of Food Safety and Director of the Center for Food Safety Engineering, Purdue University, Department of Food Science, 745 Agricultural Mall Drive, West Lafayette, IN 47907-2009. E-mail: [e-mail].