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Applied and Environmental Microbiology, August 2006, p. 5384-5395, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.00764-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Exposure to Salt and Organic Acids Increases the Ability of Listeria monocytogenes To Invade Caco-2 Cells but Decreases Its Ability To Survive Gastric Stress{dagger}

Matthew R. Garner, Karen E. James, Michelle C. Callahan, Martin Wiedmann, and Kathryn J. Boor*

Department of Food Science, Cornell University, Ithaca, New York 14853

Received 2 April 2006/ Accepted 1 June 2006


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ABSTRACT
 
The effects of environmental stress exposure on Listeria monocytogenes growth and virulence-associated characteristics were investigated. Specifically, we measured the effects of temperature (7 or 37°C), pH (5.5 or 7.4), the presence of salt and organic acids (375 mM NaCl, 8.45 mM sodium diacetate [SD], 275 mM sodium lactate [SL], or a combination of NaCl, SD, and SL), and deletion of sigB, which encodes a key stress response regulator, on the ability of L. monocytogenes to grow, invade Caco-2 cells, and survive exposure to synthetic gastric fluid (pH 2.5 or 4.5). Our results indicate that (i) L. monocytogenes log-phase generation times and maximum cell numbers are not dependent on the alternative sigma factor {sigma}B in the presence of NaCl and organic acids at concentrations typically found in foods; (ii) growth inhibition of L. monocytogenes through the addition of organic acids is pH dependent; (iii) the ability of L. monocytogenes to invade Caco-2 cells is affected by growth phase, temperature, and the presence of salt and organic acids, with the highest relative invasion capabilities observed for cells grown with SL or NaCl at 37°C and pH 7.4; (iv) growth of L. monocytogenes in the presence of NaCl, SD, or SL reduces its ability to survive exposure to gastric fluid; and (v) exposure of L. monocytogenes to gastric fluid reduces the enhanced invasiveness caused by growth in the presence of NaCl or SL. These findings suggest that virulence-associated characteristics that determine the L. monocytogenes infectious dose are likely to be affected by food-specific properties (e.g., pH or the presence of salt or organic acid).


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INTRODUCTION
 
Listeria monocytogenes, a food-borne bacterial pathogen that negatively affects both the safety of the food supply and the cost of food manufacturing (34), is estimated to cause ~28% of all deaths from known food-borne pathogens in the United States (44). Foods considered at greatest risk for transmission of human listeriosis are ready-to-eat (RTE) products that permit growth of L. monocytogenes and that are held at refrigeration temperatures for sufficient time to enable growth (24). L. monocytogenes can grow in the presence of organic acids and salt as well as at temperatures as low as –0.4°C (7, 17, 23, 52). Consumption of L. monocytogenes-contaminated RTE meat products is currently thought to be the most common cause of human listeriosis infections in the United States (24).

One strategy for reducing human exposure to L. monocytogenes is to formulate RTE foods to inhibit or reduce growth of this organism (36). For example, lactate and diacetate are added to RTE deli meat formulations (48, 56), with the goal of reducing L. monocytogenes growth at refrigeration temperatures (6, 43, 48). Lactate concentrations typically used in RTE meats and deli products range from 2% to 3% (48), with a maximum allowable concentration of 4.8%; acetate or diacetate concentrations typically range from 0.1% to 0.15% (56), with a maximum allowable concentration of 0.25% (24). The growth of L. monocytogenes in food products is also influenced by temperature (10, 27). In a recent survey, the average temperature of retail deli meats and RTE foods was determined to be 44°F (6.7°C), with a maximum temperature of 65°F (18°C) (2). The average product temperature in consumers' refrigerators was 50.4°F (10°C), with 76% of the surveyed products kept above 45°F (7.2°C). A number of studies have shown that product formulations that inhibit L. monocytogenes growth at storage temperatures at or below 4°C may permit growth at higher temperatures (3, 40, 43, 53).

Food formulations that are designed to be inhibitory but that allow some bacterial growth are likely to impose environmental stresses on the bacteria. Exposure of L. monocytogenes to sublethal environmental stress conditions can enhance the organism's survival upon subsequent exposure to lethal conditions (26) and can induce the expression of virulence genes (37), and therefore we hypothesized that exposure of L. monocytogenes to food-associated stress conditions, including exposure to salt and organic acids, could influence L. monocytogenes virulence characteristics. In support of this hypothesis, Conte et al. (18, 19) demonstrated that L. monocytogenes exposed to a sublethal acidic pH (pH 5.1) showed increased invasion of human intestinal epithelial Caco-2 cells and increased survival and multiplication in macrophage-like cells relative to nonexposed bacteria. Furthermore, exposure of L. monocytogenes to osmotic and acid stress activates the expression of genes regulated by {sigma}B, a stress-responsive alternative sigma factor in L. monocytogenes (4, 54). Our research group and others have shown that {sigma}B contributes to L. monocytogenes survival under a variety of stress conditions (25). Specifically, we have shown that relative to its parent strain, an otherwise isogenic L. monocytogenes {sigma}B null mutant strain is more susceptible to environmental stresses, including carbon starvation, reduced pH, and oxidative stresses, and also has attenuated virulence (25, 29, 47). The {sigma}B regulon includes a number of virulence and virulence-associated genes, such as bsh, encoding bile salt hydrolase, inlA, encoding internalin A, which mediates invasion of L. monocytogenes into human intestinal epithelial cells, and gadA, encoding part of the glutamate decarboxylase system, which is important in acid survival (37). {sigma}B also directly contributes to transcriptional activation of prfA, which encodes PrfA (47), a key regulator of virulence gene expression in L. monocytogenes (45). Taken together, these observations from previous studies suggested that exposure of L. monocytogenes to sublethal stresses such as those encountered in RTE meat products might enhance the organism's virulence, e.g., by activating the expression of {sigma}B-regulated virulence genes such as inlA. Thus, the goal of this study was to measure the effects of exposure to salt (NaCl) and organic acids commonly used in deli meat formulations (i.e., diacetate and sodium lactate) on the ability of L. monocytogenes, with and without functional {sigma}B, to grow at 7°C, to invade Caco-2 cells, and to survive exposure to gastric acid.


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MATERIALS AND METHODS
 
Bacterial strains and growth conditions.
The L. monocytogenes strains used in this study included 10403S (9) and the isogenic deletion mutants 10403S{Delta}sigB (57) and 10403S{Delta}prfA (15). To prepare inocula for growth and invasion assays, strains were streaked onto brain heart infusion (BHI; Difco, Detroit, MI) agar plates and incubated at 30°C for 36 to 48 h. Subsequently, single colonies were inoculated into 5 ml of BHI broth each and incubated for 12 h at 37°C with shaking at 210 rpm.

Growth of L. monocytogenes in BHI broth containing salt and organic acids.
To generate salt and organic acid concentrations encountered in RTE meats (27, 48, 56), BHI broth was prepared to contain (i) 2.2% (375.0 mM) sodium chloride (NaCl), (ii) 0.12% (8.450 mM) sodium diacetate (SD; Niacet, Niagara Falls, NY), (iii) 2.5% (275.0 mM) sodium lactate (SL; EM Science, Gibbstown, NJ), and (iv) a combination of all three additives (2.2% sodium chloride, 0.12% sodium diacetate, and 2.5% sodium lactate), and BHI broth alone was also prepared. All five BHI broth formulations were prepared at either pH 5.5 or 7.4 by adjusting the pH with 6 N HCl or 6 N NaOH. Fifteen-milliliter aliquots of each BHI broth formulation were measured into 18-mm tubes and autoclaved at 121°C for 30 min.

To prepare L. monocytogenes for inoculation into the different BHI broth formulations, 50-µl aliquots of L. monocytogenes that had been grown in BHI broth for 12 h as described above were inoculated into 5 ml of BHI broth and then grown statically, at the same temperature (7 or 37°C) that would be used for subsequent experiments, until the culture suspension reached an optical density at 600 nm (OD600) of 0.4, representing log-phase cells. The log-phase cells were then inoculated to achieve either approximately 5 x 106 CFU/ml (large inoculum) or approximately 5 x 101 CFU/ml (small inoculum) in 15-ml aliquots of the different BHI broth formulations that had been either prechilled to 7°C or prewarmed to 37°C. Tubes were incubated statically at either 7 or 37°C.

Optical density monitoring, pH measurement, and enumeration of L. monocytogenes cells.
OD600 values for L. monocytogenes grown in the presence of different salt and organic acid concentrations and at different pH values and temperatures were monitored using a model DU640 spectrophotometer (Beckman, Fullerton, CA). Cell numbers were determined periodically via enumeration of serial dilutions plated onto BHI agar. BHI agar plates were incubated at 30°C for 48 h before individual colonies were counted. Log-phase generation times were calculated according to standard methods (42). Culture pH values were monitored periodically using a Futura electrode and a model 250 pH meter (Beckman, Fullerton, CA).

Survival in synthetic gastric fluid.
L. monocytogenes strain 10403S, grown statically at either 7 or 37°C in the presence of different combinations of salt and organic acids, was also evaluated for survival in synthetic gastric fluid. For survival assays, L. monocytogenes cultures grown in parallel in the presence of the various organic acids or salt were all harvested at the same time point, i.e., when the culture grown in BHI broth alone entered stationary phase. At this point, a 100-µl aliquot of each culture was added to 4.9 ml of synthetic gastric fluid and incubated at 37°C. Cell numbers were determined before exposure and after <10 s, 60 min, and 120 min of exposure to synthetic gastric fluid. Synthetic gastric fluid was prepared according to the method of Beumer et al. (8) to contain the following reagents (per liter): 8.3 g of proteose peptone, 3.5 g of D-glucose, 2.05 g of NaCl, 0.6 g of KH2PO4, 0.11 g of CaCl2, 0.37 g of KCl, 0.05 g of bile, 0.1 g of lysozyme, and 13.3 mg of pepsin. The fluid was prepared 60 min prior to use, adjusted to either pH 2.5 or pH 4.5 using 6 N HCl, and filter sterilized.

Growth of L. monocytogenes for Caco-2 invasion assays.
Invasion assays were initially performed using L. monocytogenes strains (10403S, 10403S{Delta}sigB, and 10403S{Delta}prfA) grown to different stages (early logarithmic, mid-logarithmic, and stationary phases) with shaking at 210 rpm. For these experiments, L. monocytogenes was grown as described above to an OD600 of 0.4 and diluted 1:100 in 50 ml of sterile BHI broth in a 300-ml Nephlo flask (Belco, Vineland, NJ). The culture was incubated at 37°C with shaking until it reached an OD600 of 0.3, and then three 5-ml aliquots were measured into prewarmed, sterile 16-mm tubes and incubated at 37°C with shaking. Invasion assays were then performed when the cultures reached OD600 values of 0.4 (representing early log phase), 1.0 (mid-log phase), and 1.0 (plus 3 h) (stationary phase).

Invasion experiments were also performed with L. monocytogenes 10403S grown statically at 7 or 37°C in the presence of different combinations of salt and organic acids (in BHI broth adjusted to pH 5.5 or pH 7.4). These experiments were performed only with L. monocytogenes cultures grown to the time point when the culture in BHI broth alone reached stationary phase, as previously described. In addition, invasion assays were performed with L. monocytogenes exposed to gastric fluid (pH 4.5) for <10 s and 60 min.

Tissue culture invasion assays.
Invasion assays using the tumor-derived Caco-2 human colorectal epithelial cell line (ATCC HTB-37) were performed as previously described (29). Briefly, 7.5 x 104 Caco-2 cells were seeded approximately 48 h before infection into 24-well plates (Costar; Corning, NY), using Dulbecco's minimum essential medium with Earle's salts (Gibco, Gaithersburg, MD) containing 20% fetal bovine serum without antibiotics. L. monocytogenes grown or treated as described above was inoculated onto the Caco-2 monolayer; at 30 min postinfection, the medium in each well was aspirated, and the Caco-2 cells were washed three times with 1 ml sterile phosphate-buffered saline to remove any unattached, extracellular L. monocytogenes cells. Subsequently, 1 ml of prewarmed, fresh Caco-2 medium without antibiotics was added. At 45 min postinfection, the medium was aspirated, and 1 ml of fresh Caco-2 medium containing 150 µg/ml gentamicin (Gibco) was added to kill any remaining extracellular L. monocytogenes. At 90 min postinfection, Caco-2 cells were lysed by the addition of 500 µl of ice-cold sterile distilled water, and the resulting suspension was used for bacterial enumeration to quantify invading L. monocytogenes cells. Invasion efficiency is reported as follows: (the number of bacteria recovered from each well following Caco-2 cell lysis divided by the number of bacteria that had been used for inoculation) x 100.

Statistical analyses.
Each experiment was repeated at least three times. One-way analysis of variance (ANOVA) was performed using the general linear model procedure, and Tukey's studentized range test was used to determine whether significant differences existed between different experimental groups (Minitab 14, State College, PA). Significance was set at P levels of <0.05 and reported at three different intervals (P < 0.05, P < 0.01, and P < 0.001).


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RESULTS
 
Evaluation of L. monocytogenes growth in the presence of salt and organic acids by using OD measurements.
L. monocytogenes 10403S and the isogenic {Delta}sigB mutant were grown in the presence of NaCl, SL, SD, or all three additives (NaCl, SL, and SD) in BHI broth adjusted to pH 5.5 or 7.4 at a temperature of 7 or 37°C to initially characterize the effects of these additives on L. monocytogenes growth under different environmental conditions. At pH 7.4, while OD600 values increased over time for both L. monocytogenes 10403S (Fig. 1) and the {Delta}sigB mutant (see Fig. S1 in the supplemental material), even in the presence of NaCl and organic acids, OD600 increases for L. monocytogenes grown in the presence of SL or all three additives (NaCl, SL, and SD) were smaller than those for bacteria grown in the presence of NaCl, SD, or BHI broth alone. At pH 5.5, on the other hand, OD600 values did not increase for L. monocytogenes grown in the presence of SL or all three additives (NaCl, SL, and SD), in contrast to the detectable OD600 increases that occurred over time for bacteria grown in BHI broth alone, BHI broth with SD, or BHI broth with NaCl (Fig. 1; see Fig. S2 in the supplemental material). No differences were observed in OD600 measurements over time between the 10403S and {Delta}sigB strains under any of the conditions tested (see Table ST1 in the supplemental material).


Figure 1
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  		FIG. 1. Evaluation of L. monocytogenes growth in the presence of salt and organic acids by using OD600 measurements. L. monocytogenes 10403S was inoculated at approximately 5 x 106 CFU/ml into BHI broth (prepared at pH 7.4 or 5.5) that contained (i) no added salt or organic acids, (ii) 2.2% NaCl, (iii) 0.12% SD, (iv) 2.5% SL, or (v) a combination of 2.2% NaCl, 0.12% SD, and 2.5% SL. Cultures were incubated at 7 or 37°C. The data presented here represent single replicates (of three replicates studied) for the 10403S strain only, since no differences between 10403S and the {Delta}sigB null mutant were apparent and since ANOVA of log-phase generation times (calculated based on bacterial numbers determined for the same experiments) indicated that the trials were not different (P > 0.5). Data for all three replicates for both L. monocytogenes 10403S and the {Delta}sigB null mutant are available (see Fig. S1 and S2 in the supplemental material). L. monocytogenes growth phases were defined based on OD600 measurements of bacteria grown in BHI broth without salt and organic acids as early log (el), mid-log (ml), early stationary (es), stationary (s), late stationary (ls), and very late stationary (vs). Arrows and letters indicate the growth phase designations.

OD600 measurements for L. monocytogenes grown in BHI broth (without added organic acids or NaCl) for each of the four pH/temperature combinations were used to define reproducible sampling points at distinct growth stages, as shown in Fig. 1. Specifically, six distinct growth stages were defined for L. monocytogenes grown at pH 7.4 (early log, mid-log, early stationary, stationary, late stationary, and very late stationary phases). In comparison, due to the overall lower OD600 readings at pH 5.5, five distinct stages, not including early stationary phase, were identified for L. monocytogenes grown at this pH. To enable comparisons among treatments in subsequent survival and invasion assays, samples were collected at the same time points from all cultures grown under the same pH/temperature conditions, even though some conditions (e.g., SL at pH 5.5) did not produce detectable increases in OD600 values.

Evaluation of L. monocytogenes growth in the presence of salt and organic acids by using plate counts.
Since optical density readings do not always correlate with bacterial numbers determined by plate count procedures, especially when comparing bacteria grown under different conditions (28), L. monocytogenes cells exposed to organic acids and NaCl under different temperature and pH conditions were enumerated by plate counts at each growth stage identified by OD600 values. Overall ANOVA indicated that maximum viable cell numbers (Table 1) and log-phase generation times (see Table ST1 in the supplemental material) were not significantly different between the 10403S strain and the {Delta}sigB null mutant, confirming the OD600 measurement results. On the other hand, overall ANOVA indicated that pH had a significant (P < 0.001) effect on maximum viable cell numbers (Table 1), while both pH and temperature had a significant effect (P < 0.001) on log-phase generation times (see Table ST1 in the supplemental material). Not surprisingly, L. monocytogenes cells grown at 7°C consistently showed longer generation times (<12 h) than those grown at 37°C, which consistently showed generation times of <2 h (except for the longer generation times for L. monocytogenes grown in BHI broth with SL alone or in combination with NaCl and SD at pH 5.5 [see Table ST1 in the supplemental material]).


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  		TABLE 1. Maximum L. monocytogenes viable cell numbers for cells grown under different conditions and with different initial inoculum numbers

While maximum viable cell numbers were not affected by the presence of food additives at pH 7.4, regardless of temperature, log-phase generation times were influenced by the presence of additives in a temperature-dependent manner. Specifically, regardless of the growth temperature (7 or 37°C), L. monocytogenes 10403S cells grown at pH 7.4 had very similar maximum viable cell numbers, whether grown in BHI broth alone, in BHI broth with NaCl, SL, or SD, or in BHI broth with all three additives (Fig. 2a and b; Table 1), as confirmed by ANOVA (P > 0.05). ANOVA of log-phase generation times showed a significant effect (P < 0.05) of the presence of the additives on log-phase generation times at 37°C, with longer log-phase generation times observed in the presence of NaCl, SL, or all three additives (NaCl, SL, and SD) (see Table ST1 in the supplemental material). At 7°C, however, there was no significant effect (P > 0.05) of the presence of NaCl, SL, or SD on log-phase generation times at pH 7.4. Bacterial enumeration data and OD600 measurements did not always correlate well across treatments, consistent with previous observations by Bereksi et al. (7), who reported different relative cell sizes, and therefore different relative OD600 values, for a given number of L. monocytogenes cells grown under different conditions. In the present study, for example, L. monocytogenes cells grown at 37°C and pH 7.4 in the presence of SL had lower OD600 measurements at stationary phase (OD600 = 0.49 ± 0.04 [mean ± SD for three experiments]) than cells grown in BHI broth alone (OD600 = 0.94 ± 0.07), while viable cell numbers were very similar (9.04 and 9.02 log CFU/ml, respectively).


Figure 2
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  		FIG. 2. Viability of L. monocytogenes inoculated into BHI broth containing food additives. L. monocytogenes 10403S was inoculated at approximately 5 x 106 CFU/ml into BHI broth (prepared at pH 7.4 or 5.5) that contained (i) no added salt or organic acids, (ii) 2.2% NaCl, (iii) 0.12% SD, (iv) 2.5% SL, or (v) a combination of 2.2% NaCl, 0.12% SD, and 2.5% SL. Cultures were incubated at 7 or 37°C. The optical density was monitored throughout incubation, and viable cell numbers were determined (CFU/ml) at the defined growth stages. The data shown represent the average values of three independent experiments; error bars indicate standard deviations. Viable cell numbers for the L. monocytogenes {Delta}sigB mutant grown under the same conditions are available (see Fig. S3 in the supplemental material).

At pH 5.5, the presence of SL (either alone or in combination with NaCl and SD) significantly affected L. monocytogenes maximum viable cell numbers and log-phase generation times (Fig. 2c and d and Table 1; see Table ST1 in the supplemental material). L. monocytogenes inoculated into BHI broth with SL or into BHI broth with all three additives (NaCl, SL, and SD) at pH 5.5 consistently showed either no growth or reductions in viable cell numbers (Fig. 2). The maximum viable cell numbers reached by L. monocytogenes at 37°C and pH 5.5 in BHI broth alone were significantly (P < 0.001) higher (8.60 log) than those reached in BHI broth with SL (6.51 log) or in BHI broth with all three additives (6.57 log) (Table 1). The maximum viable cell numbers were observed at 4.5 h postinoculation (Table 1); viable cell numbers in BHI broth with SL or in BHI broth with all three additives decreased to 1.57 and 3.39 log, respectively, at 72 h postinoculation (Fig. 2c). At 7°C and pH 5.5, maximum L. monocytogenes cell numbers in BHI broth alone were also significantly (P < 0.001) higher than those in BHI broth with SL or in BHI broth with all three additives (Table 1); however, in contrast to viable cell numbers in BHI broth with SL or in BHI broth with all three additives at 37°C, at 7°C, the numbers decreased only slightly under the same conditions (<0.6 log) over the total incubation time (28 days) (Fig. 2d).

Growth of small initial L. monocytogenes inoculum in the presence of salt and sodium lactate.
Initial experiments evaluated the growth and survival of L. monocytogenes in the presence of NaCl, SL, and SD, using an inoculum of approximately 5 x 106 CFU/ml. Since inoculum size has been reported to be a critical factor affecting microbial growth in the presence of growth inhibitors (40), additional experiments were performed to expose a smaller inoculum (approximately 5 x 101 CFU/ml) of L. monocytogenes 10403S to (i) BHI broth alone, (ii) BHI broth and NaCl, and (iii) BHI broth and SL (Table 1) at both pH 5.5 and pH 7.4 as well as at both 7 and 37°C. While the absolute time required for the small inoculum to reach stationary phase was longer than that required with a larger inoculum size (Table 1; see Fig. S4 in the supplemental material), the maximum viable L. monocytogenes cell numbers were similar, regardless of inoculum size, except in the presence of SL (Table 1). Consistent with the observation of a reduction in viable cell number for L. monocytogenes exposed to SL at pH 5.5 (with a starting inoculum of approximately 5 x 106 CFU/ml) (Fig. 2), no L. monocytogenes cells were recovered under these conditions when a small inoculum (~5 x 101 CFU/ml) was used. Since the detection limit for the enumeration method is 1 x 101 CFU/ml, these data further support reductions in viability of L. monocytogenes upon exposure to SL at pH 5.5.

Growth phase dependence of L. monocytogenes invasion of Caco-2 cells.
Stationary-phase L. monocytogenes 10403S cells and {Delta}prfA and {Delta}sigB null mutants grown with shaking at 37°C had the highest relative invasion efficiencies in Caco-2 cells, followed by mid- and early-log-phase bacteria (Fig. 3). Growth phase effects on invasion efficiency were less pronounced for the {Delta}sigB null mutant than for the 10403S and {Delta}prfA strains. In addition, the {Delta}sigB strain showed significantly lower invasion efficiencies (P < 0.001 by ANOVA) than the 10403S and {Delta}prfA strains, which had similar invasion efficiencies (P ≥ 0.3) (Fig. 3). Based on these initial data, to evaluate the effects of NaCl, SL, or SD on the L. monocytogenes Caco-2 invasion capacity, 10403S was grown in the presence of salt and organic acids to a time point equivalent to stationary phase for L. monocytogenes grown in BHI broth alone.


Figure 3
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  		FIG.3. Box plot of L. monocytogenes invasion efficiencies for Caco-2 tissue culture cells at different stages of bacterial growth. Invasion assays were performed with L. monocytogenes strains grown to the early log, mid-log, and stationary phases, as determined by the optical density. Strain designations (10403S and isogenic {Delta}prfA and {Delta}sigB mutants) are indicated on the x axis, and invasion efficiencies (number of bacteria recovered from lysed Caco-2 cells divided by number of bacteria used for infection x 100) are indicated on the y axis. The data shown represent invasion assay data from three independent experiments. Each box encloses the central 50th percentile of the data, with the median value displayed as a horizontal line. Vertical lines protruding from each box represent the maximum and minimum values. Boxes with different letters represent invasion efficiencies that differed significantly (P < 0.05).

Caco-2 invasion of L. monocytogenes cells exposed to salt and organic acids.
L. monocytogenes 10403S cells grown in the presence of NaCl, SL, SD, or all three additives at two different pH levels (5.5 and 7.4) and at two different temperatures (7 and 37°C) varied in the ability to invade Caco-2 cells (Fig. 4). Overall ANOVA indicated that the ability of L. monocytogenes to invade Caco-2 cells was affected by growth temperature, pH, and the presence of NaCl and organic acids. In general, L. monocytogenes cells grown at 37°C were more invasive than bacteria grown at 7°C (Fig. 4). For both temperatures, L. monocytogenes cells grown at pH 7.4 were more invasive than bacteria grown at pH 5.5 (Fig. 4).


Figure 4
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  		FIG.4. Box plot of invasion efficiencies for Caco-2 tissue culture cells of L. monocytogenes cells grown to stationary phase in the presence of food additives. L. monocytogenes 10403S was inoculated at approximately 5 x 106 CFU/ml into BHI broth (prepared at pH 7.4 or 5.5) that contained either (i) no added salt or organic acids, (ii) 2.2% NaCl, (iii) 0.12% SD, (iv) 2.5% SL, or (v) a combination of 2.2% NaCl, 0.12% SD, and 2.5% SL. Cultures were incubated at 7 or 37°C until the bacteria grown in BHI broth alone reached stationary phase at a given temperature/pH combination, and then bacteria grown under each condition were directly inoculated onto Caco-2 cell monolayers. Growth conditions are indicated on the x axis, and invasion efficiencies (number of bacteria recovered from lysed Caco-2 cells divided by number of bacteria used for infection x 100) are indicated on the y axis. The data shown represent invasion assay data from three independent experiments. Each box encloses the central 50th percentile of the data, with the median value displayed as a horizontal line. Vertical lines protruding from each box represent the maximum and minimum values. Boxes with different letters represent invasion efficiencies that differed significantly (P < 0.05).

Separate ANOVAs on invasion efficiencies for L. monocytogenes cells grown in BHI broth alone, in the presence of NaCl, SL, or SD, or in the presence of all three additives showed a significant effect of salt and organic acid at each of the four different pH/temperature combinations. Effects of NaCl, SD, and SL on L. monocytogenes invasiveness showed similar trends for bacteria grown at a given pH at both temperatures. At pH 7.4, L. monocytogenes cells grown in the presence of NaCl, SL, or all three additives (NaCl, SL, and SD) showed higher invasion efficiencies than bacteria grown in BHI broth alone or in BHI broth with SD (Fig. 4). These effects were particularly pronounced when bacteria were grown at 37°C and pH 7.4. At 7°C, only bacteria grown in the presence of SL showed significantly higher invasion efficiencies than those of bacteria grown in BHI broth alone at 7°C and pH 7.4 (Fig. 4). At 37°C and pH 7.4, L. monocytogenes cells grown in the presence of NaCl or SL showed the highest invasion efficiencies observed among all conditions tested (33% and 31% invasion efficiencies, respectively); these invasion efficiencies were 10- and 9-fold higher than those for bacteria grown at the same pH (7.4) and temperature (37°C) in BHI broth alone. At pH 5.5, only growth in the presence of NaCl led to an increase in invasion efficiency (compared to bacteria grown in BHI broth alone); this increase was statistically significant only for bacteria grown at 37°C. Furthermore, L. monocytogenes cells grown at pH 5.5 (at either 7 or 37°C) in the presence of SL showed reduced Caco-2 cell invasiveness compared to bacteria grown at the same pH in BHI broth alone. On the other hand, L. monocytogenes cells grown in the presence of all three additives at pH 5.5 were less invasive than bacteria grown in BHI broth alone at 37°C, but not 7°C (Fig. 4).

Since previous studies have shown that centrifugation can activate expression of the {sigma}B regulon (14), which includes inlA (encoding InlA, a protein critical for the invasion of Caco-2 cells), bacterial cells were not collected by centrifugation prior to invasion assays. As a consequence, bacterial cell numbers used for invasion assays differed based on culture growth temperature, pH, and the presence of organic acids. Therefore, to determine if differences in numbers of added bacteria affected the relative invasion efficiencies (i.e., the number of intracellular bacteria recovered divided by the number of bacteria used for inoculation), Caco-2 cell invasion assays were performed with a range of inocula grown to stationary phase in BHI broth (1 x 104 to 1 x 107 CFU) (see Fig. S5 in the supplemental material), which represented the range of inoculum levels used in our experiments. No significant differences (P > 0.05 by ANOVA) in invasion efficiencies were found. We also confirmed that the presence of BHI broth with NaCl or SL (introduced onto Caco-2 cells at 100 µl per 1 ml of tissue culture medium along with the bacterial cells) did not enhance invasion by L. monocytogenes. Specifically, invasion experiments with (i) L. monocytogenes grown in the presence of NaCl or SL and (ii) L. monocytogenes grown in BHI broth alone but added to Caco-2 cells together with a filter-sterilized supernatant from L. monocytogenes grown in the presence of NaCl or SL (thus adding SL or NaCl in BHI broth as well as in the bacterial culture supernatant) showed that the addition of filter-sterilized supernatant with NaCl or SL did not affect Caco-2 invasion of L. monocytogenes grown in BHI broth alone (see Fig. S6 in the supplemental material).

Survival of L. monocytogenes in synthetic gastric fluid.
L. monocytogenes cells grown in the presence of NaCl, SL, or SD at 37°C and at two different pH levels (5.5 or 7.4) were also examined for the ability to survive a 1- or 2-h exposure to synthetic gastric fluid (at pH 2.5 or 4.5) (Fig. 5). Overall ANOVA indicated that (i) the presence of food additives, (ii) the starting pH of the growth medium, (iii) the pH of the gastric fluid, and (iv) the exposure time to gastric fluid (1 or 2 h) each had a significant (P < 0.001 for each of these four factors) effect on L. monocytogenes survival in the gastric fluid. Not surprisingly, L. monocytogenes showed a consistently higher survival rate in gastric fluid at pH 4.5 than at pH 2.5 (Fig. 5). Also, L. monocytogenes grown at pH 5.5 in the presence of NaCl, SD, or BHI broth alone generally showed a greater relative ability to survive exposure to gastric fluid than L. monocytogenes grown under the same conditions at pH 7.4. L. monocytogenes exposed to growth-inhibiting conditions at pH 5.5 (i.e., in the presence of SL or all three additives [NaCl, SD, and SL]) had a reduced ability to survive in pH 2.5 gastric fluid compared to L. monocytogenes grown at pH 7.4 in the presence of SL or all three additives (NaCl, SD, and SL). Importantly, L. monocytogenes grown in the presence of NaCl, SD, SL, or all three additives generally showed reduced survival relative to L. monocytogenes grown in BHI broth alone when exposed to gastric fluid. Among these four conditions, L. monocytogenes grown in the presence of SD generally had the greatest relative survival following exposure to gastric fluid.


Figure 5
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  		FIG. 5. Survival of synthetic gastric fluid exposure by L. monocytogenes grown in the presence of salt and organic acids. L. monocytogenes 10403S was inoculated at approximately 5 x 106 CFU/ml into BHI broth (prepared at pH 7.4 or 5.5) that contained (i) no added salt or organic acids, (ii) 2.2% NaCl, (iii) 0.12% SD, (iv) 2.5% SL, or (v) a combination of 2.2% NaCl, 0.12% SD, and 2.5% SL. Cultures were incubated at 37°C until the bacteria grown in BHI broth alone reached stationary phase at a given temperature/pH combination, and then bacteria grown under each condition were directly exposed to synthetic gastric fluid prepared at pH 4.5 or 2.5. Viable L. monocytogenes numbers (CFU/ml) were determined following exposure to synthetic gastric fluid for 1 or 2 h. Conditions used to grow L. monocytogenes prior to exposure to gastric fluid are indicated on the x axis, and survival rates (number of viable L. monocytogenes cells per ml at 1 or 2 h divided by number of viable cells per ml at 0 h) are indicated on the y axis. The data shown represent the average values of three independent experiments; error bars indicate standard deviations. "XX" indicates that no viable L. monocytogenes cells were recovered.

L. monocytogenes invasion ability following exposure to gastric fluid.
Since L. monocytogenes grown in the presence of NaCl or SL at pH 7.4 and 37°C showed enhanced invasiveness, bacteria grown under these conditions were used to determine the effect of pH 4.5 gastric acid exposure on subsequent invasion of Caco-2 cells. Consistent with the data shown in Fig. 4, L. monocytogenes cells grown at pH 7.4 in the presence of NaCl or SL were more invasive (P < 0.001) than bacteria grown in BHI broth alone at the same pH (Fig. 6). However, following gastric fluid exposure for <10 seconds, the enhanced invasiveness of bacteria grown in the presence of SL or NaCl was greatly diminished; only L. monocytogenes grown in BHI broth with SL maintained a higher level of invasiveness than bacteria grown in BHI broth alone (Fig. 6). Following gastric fluid exposure for 1 h, L. monocytogenes cells grown in BHI broth with NaCl or SL no longer differed from each other or from cells grown in BHI broth alone in the ability to invade Caco-2 cells (P ≥ 0.1). Control experiments in which pH 4.5 gastric fluid was added to Caco-2 cells at the same time as L. monocytogenes grown at pH 7.4 in BHI broth alone or in the presence of NaCl or SL showed that the presence of gastric fluid during the invasion assay did not affect the invasion of Caco-2 cells (see Fig. S7 in the supplemental material).


Figure 6
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  		FIG.6. Invasion into Caco-2 cells of L. monocytogenes subjected to synthetic gastric fluid following growth to stationary phase in the presence of salt or sodium lactate. L. monocytogenes 10403S was inoculated at approximately 5 x 106 CFU/ml into BHI broth (pH 7.4) that contained (i) no added salt or organic acids, (ii) 2.2% NaCl, or (iii) 2.5% SL. Cultures were incubated at 37°C until the bacteria grown in BHI broth alone reached stationary phase, and then bacteria grown under each condition were directly exposed to synthetic gastric fluid prepared at pH 4.5. Bacteria not exposed to gastric fluid as well as bacteria exposed to gastric fluid for <10 s or 1 h were then used for invasion assays; bacteria from these cultures were directly inoculated onto Caco-2 cell monolayers. The data are represented by box plots, with each box enclosing the central 50th percentile of the data, the median value displayed as a horizontal line, and vertical lines protruding from each box representing the maximum and minimum values. The data shown represent invasion assay data from three independent experiments. Boxes with different letters represent invasion efficiencies that differed significantly (P < 0.05).


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DISCUSSION
 
To measure the effects of food-related stress exposure on L. monocytogenes virulence-associated characteristics, we investigated the effects of temperature, pH, and the presence of NaCl and selected organic acids commonly used to inhibit growth in RTE foods on the ability of L. monocytogenes to grow, invade Caco-2 cells, and survive exposure to gastric fluid. An otherwise isogenic L. monocytogenes {Delta}sigB strain was included in key experiments to assess contributions of the stress-responsive alternative sigma factor {sigma}B to growth and survival under these conditions. Our results indicate that (i) L. monocytogenes log-phase generation times and maximum cell numbers are not dependent on the alternative sigma factor {sigma}B in the presence of NaCl and organic acids at concentrations typically found in foods; (ii) growth inhibition of L. monocytogenes through the addition of organic acids is pH dependent; (iii) the ability of L. monocytogenes to invade Caco-2 cells is affected by growth phase, temperature, and the presence of salt and organic acids, with the highest relative invasion capabilities observed for cells grown with SL or NaCl at 37°C and pH 7.4; (iv) growth of L. monocytogenes in the presence of NaCl, SD, or SL reduces its ability to survive exposure to gastric fluid; and (v) exposure of L. monocytogenes to gastric fluid reduces the enhanced invasiveness caused by growth in the presence of NaCl or SL.

L. monocytogenes log-phase generation times and maximum cell numbers are not dependent on the alternative sigma factor {sigma}B in the presence of NaCl and organic acids at concentrations typically found in foods.
Our observation that an L. monocytogenes strain bearing an in-frame, nonpolar sigB deletion (57) grows as well as the 10403S strain at pH 5.5 in the presence of organic acids as well as at 7°C may seem somewhat surprising, as {sigma}B has been demonstrated to contribute to lethal acid stress survival in L. monocytogenes (57) and as its activity is induced at low temperatures (4); however, {sigma}B contributions to acid stress survival have been demonstrated only at pH values of <2.5 (25, 26, 57), and no apparent growth defects were reported for a log-phase {Delta}sigB strain grown at 8°C (4). Furthermore, the survival of two different L. monocytogenes {Delta}sigB strains did not differ from that of their respective parent strains following 8 weeks of exposure to pH 5.5 in phosphate-buffered saline (46). We conclude that {sigma}B is of limited importance for L. monocytogenes growth and survival under mild acid and cold stress; however, these findings should not be interpreted as reflecting the absence of induction of {sigma}B activity under these stress conditions. At least some of the stress conditions used here, including exposure to 0.3 M NaCl and cold stress, have previously been shown to lead to induction of {sigma}B activity (4, 5, 37, 54). Furthermore, prior exposure of L. monocytogenes to nonlethal acidic conditions (1 h at pH 4.5) increased subsequent survival of the organism at pH 2.5 through both {sigma}B-dependent and -independent mechanisms (26). Induction of {sigma}B activity upon exposure to organic acid or salt stress, which leads to increased transcription of {sigma}B-dependent virulence genes, e.g., bsh, encoding bile salt hydrolase, and inlA, encoding the InlA molecule, which is critical for invasion of human intestinal epithelial cells (29, 37), thus represents a possible mechanism enabling enhanced invasion of L. monocytogenes cells exposed to different stress conditions, including those that may be encountered in RTE foods.

Growth inhibition of L. monocytogenes through addition of organic acids is pH dependent.
While a number of studies have evaluated the ability of sodium lactate and diacetate, alone as well as in combination, to inhibit or at least retard the growth of L. monocytogenes in broth systems (10) and in foods (31), the importance of medium and food pH on the effectiveness of these food additives has been emphasized less (10, 35). Our data show that sodium lactate and diacetate must be accompanied by a low pH for effective L. monocytogenes growth inhibition. RTE deli meats, such as ham, bologna, sliced chicken, and sliced turkey, are typically formulated with pH values above 6.0 (30). We hypothesize that L. monocytogenes growth-enabling pH values in sliced turkey formulations may have been a contributing factor to the association of these products as vehicles with three human listeriosis outbreaks in the United States since 1998 (11-13).

Our data further indicate that low initial L. monocytogenes concentrations can yield similar maximum bacterial numbers to those reached with large initial inocula, even in the presence of organic acids such as lactate and diacetate, consistent with a study by Glass and Doyle (30), who reported that L. monocytogenes could grow to high concentrations (≥1 x 105 CFU/gram) from low inoculum concentrations (≤1 x 101 CFU/gram), particularly in RTE deli meats with a pH of >6.0. These observations are important since postprocessing contamination of RTE foods is likely to result in a small initial inoculum of L. monocytogenes. Our findings suggest that even low initial L. monocytogenes inoculum levels may multiply sufficiently to represent human health hazards in foods with a growth-permissive pH, given sufficient storage time and conditions. This conclusion is supported by previous studies showing that L. monocytogenes numbers reached 106 to 107 CFU/g after 40 to 45 days of storage at 10°C in frankfurters and bologna formulated with 2.5% lactic acid and inoculated with 102 to 103 CFU/g (or CFU/cm2), despite limited or no growth in these products within the first 8 to 10 days of storage (3, 43). Similarly, turkey ham and bologna formulated with 2 and 2.5% lactic acid, respectively, and inoculated with L. monocytogenes at 102 to 103 CFU/g (or CFU/cm2) allowed no or limited growth during 30 to 35 days of storage at 4 to 5°C, but growth was detectable after 30 to 35 days, with L. monocytogenes cell numbers reaching >104 CFU/cm2 on turkey ham after 42 days of storage at 4°C (58).

The ability of L. monocytogenes to invade Caco-2 cells is affected by growth phase, temperature, and the presence of salt and organic acids.
Our data show that the ability of L. monocytogenes to invade Caco-2 cells is affected by growth phase, consistent with a previous report (39). Specifically, invasion by stationary-phase 10403S cells was >9.5-fold higher than invasion by log-phase 10403S cells, illustrating that exposure of L. monocytogenes cells to different environmental conditions can affect invasiveness and virulence. The observation of increased stationary-phase invasiveness also coincides with stationary-phase induction of {sigma}B activity (54). Since inlA expression also increases in a {sigma}B-dependent manner in stationary-phase L. monocytogenes and since growth phase-dependent effects on invasion appear independent of PrfA (38, 39), which also contributes to inlA transcription (21), future studies on the specific regulatory mechanisms contributing to growth phase- and environmental condition-dependent invasiveness phenotypes in L. monocytogenes should include studies on the role of {sigma}B in these phenomena.

L. monocytogenes growth in the presence of salt and organic acids at different temperatures and different pHs showed that environmental conditions influence the ability of L. monocytogenes to invade Caco-2 cells. Overall, in our experiments, L. monocytogenes invasiveness ranged from a low of 0.014% (for bacteria grown with a combination of NaCl, SL, and SD at pH 5.5 and 37°C) to a high of 33.5% (for bacteria grown with SL at pH 7.4 and 37°C). While it is of particular concern that L. monocytogenes exposed to NaCl and sodium lactate (at pH 7.4 and 37°C) showed the most invasiveness, the low level of invasiveness for bacteria exposed to organic acids at pH 5.5 indicates that the use of these additives in low-pH foods not only inhibits growth (10) but also is unlikely to increase invasiveness. On the other hand, the presence of salt and organic acids in high-pH foods (e.g., ham and sliced turkey) may not inhibit L. monocytogenes growth and also may enhance the pathogen's invasiveness. Exposure to different (food) environments may have additional effects on virulence-related characteristics in L. monocytogenes as well. For example, Conte et al. (18, 19) showed that short-term exposure (1 h) of L. monocytogenes to pH 5.1 not only increased the invasiveness of L. monocytogenes for Caco-2 cells but also increased the ability of L. monocytogenes to survive and multiply in macrophage-like cells, suggesting that exposure of L. monocytogenes cells to a low pH (e.g., in the human stomach) may enhance their overall virulence. The effects of different environmental factors on virulence and virulence-associated characteristics appear complex. A deeper understanding of factors contributing to L. monocytogenes virulence will require the use of multiple experimental systems, including appropriate animal models.

Volatile fatty acids (VFAs), which include lactic acid, have been proposed to play a role in regulating virulence and virulence gene expression in other food-borne pathogens. For example, studies of Salmonella enterica serovar Typhimurium have indicated that bacterial sensing of acetate may signal the activation of invasion gene expression (41). We speculate that up-regulation of invasion gene expression in response to VFAs and salt may have evolved as a mechanism that allows bacterial pathogens to sense their presence in the mammalian gastrointestinal tract. Rapid absorption of water from digesta in the first portion of the small intestine typically generates hyperosmolar conditions (>300 mM solute) (16, 20). While acid present in the digesta is rapidly neutralized in the small intestine to yield a neutral pH (1, 20), the VFAs lactate and acetate are rapidly produced (1). Therefore, it is possible that the presence of a high VFA concentration in combination with a mildly acidic pH and high osmolarity could signal the activation of genes involved in attachment to and invasion of the intestinal epithelium, suggesting that the use of VFAs as food additives may potentially enhance the expression of invasion genes in different food-borne bacterial pathogens, not just in L. monocytogenes.

Growth of L. monocytogenes in the presence of NaCl, SD, or SL reduces its ability to survive exposure to gastric fluid.
Our results indicate that exposure of L. monocytogenes to NaCl and organic acids influences the ability of this pathogen to survive exposure to gastric fluid. While our findings are consistent with a previous study (53), which demonstrated that L. monocytogenes cultured on frankfurters with added sodium diacetate and sodium lactate had a decreased ability to survive exposure to gastric fluid compared to control cells with no added organic acids, our data also show that the ability of L. monocytogenes to survive in gastric fluid varies widely and is affected by the gastric fluid pH and prior environmental stress exposure of the pathogen. In particular, L. monocytogenes survival in pH 4.5 gastric fluid can approach 100% even after exposure for 2 h, particularly if L. monocytogenes was exposed to pH 5.5 prior to gastric acid challenge. While a pH of 2.5 may reflect the gastric pH of an empty stomach in a healthy young human (22, 26, 33, 55), a pH of 4.5 is more likely to represent the gastric pH of at-risk, listeriosis-susceptible populations (e.g., the elderly) (49). Specifically, in comparison with younger individuals (average age of 25 years), elderly individuals (average age of 71 years) were found to have stomach pHs that remained as high as pH 4.5 at 120 min postconsumption, in contrast to pH 1.7 at 120 min postconsumption for the younger individuals (22, 49). Listeriosis has also been linked epidemiologically to antacid-consuming populations (32). Furthermore, rats pretreated with cimetidine, which inhibits gastric acid production, developed L. monocytogenes infections with lower infectious doses than untreated animals (50). Our observations of the ability of L. monocytogenes to survive in pH 4.5 gastric fluid are thus biologically relevant for conditions that may lead to food-borne infection with this agent.

Exposure of L. monocytogenes to gastric fluid reduces the enhanced invasiveness caused by growth in the presence of NaCl or SL.
Invasiveness-enhanced L. monocytogenes preexposed to lactate at pH 7.4 survived well in gastric fluid at pH 4.5, suggesting the potential for delivery of "invasion-enhanced" L. monocytogenes into the small intestine, particularly in susceptible individuals, such as the elderly. However, exposure to pH 4.5 gastric fluid, even for as little as <10 seconds, substantially reduced the invasion of L. monocytogenes that had shown enhanced invasion prior to gastric fluid exposure, i.e., due to exposure to lactic acid or NaCl, while not reducing the invasion of L. monocytogenes grown in BHI broth without lactic acid or NaCl. Our findings illustrate the complexity of the effects of different environments on L. monocytogenes virulence-associated phenotypes and further suggest that some effects may be transient. While future experiments using appropriate animal models, e.g., guinea pigs (29), are clearly needed to evaluate the effects of different environmental stress conditions on L. monocytogenes virulence, the in vitro experiments reported here provide a rational starting point for selecting appropriate growth and stress conditions that are most likely to enhance and maximize L. monocytogenes invasiveness and gastric acid survival, which will help to minimize the numbers of experimental animals necessary for future experiments and maximize the likelihood of generating relevant in vivo results that will allow for the design of strategies for formulating foods that will inhibit the growth of L. monocytogenes without enhancing its virulence capacity.

Conclusions.
While previous experiments have shown that exposure to environmental stress alters L. monocytogenes gene expression profiles, which can result in increased virulence gene expression and associated virulence characteristics, as well as enhanced survival in many different model systems (18, 19, 37, 51, 52, 54), our data specifically indicate that food-associated stress conditions can influence L. monocytogenes virulence-associated characteristics, including invasiveness. The fact that invasiveness of the same L. monocytogenes strain can differ by >1,000-fold based on exposure to different environmental conditions clearly indicates that additional quantitative data on virulence modulation by food-related growth and stress conditions are needed and that this information may need to be incorporated into quantitative risk assessments. Our results suggest that environmental stress conditions from specific foods may influence the L. monocytogenes infectious dose and thereby contribute to the association of food-borne infections with specific foods. For example, we speculate that the relatively high pH and presence of NaCl in RTE turkey product formulations may enhance the virulence of L. monocytogenes present in these RTE foods, possibly contributing to their association with a number of recent listeriosis outbreaks.


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ACKNOWLEDGMENTS
 
We thank S. Milillo, P. McGann, H. Marquis, and K. Nightingale for helpful discussions.

This work was supported by National Institutes of Health Award no. RO1-AI052151-01A1 (to K.J.B.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Food Science, Cornell University, 413 Stocking Hall, Ithaca, NY 14853. Phone: (607) 255-3111. Fax: (607) 254-4868. E-mail: kjb4{at}cornell.edu. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back


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Applied and Environmental Microbiology, August 2006, p. 5384-5395, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.00764-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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