Salmonella enterica Serovar Typhimurium and Listeria ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2003, p. 3945–3951 0099-2240/03/$08.00⫹0 DOI: 10.1128/AEM.69.7.3945–3951.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 69, No. 7

Salmonella enterica Serovar Typhimurium and Listeria monocytogenes Acid Tolerance Response Induced by Organic Acids at 20°C: Optimization and Modeling E. J. Greenacre,* T. F. Brocklehurst, C. R. Waspe, D. R. Wilson, and P. D. G. Wilson Institute of Food Research, Norwich NR4 7UA, United Kingdom Received 8 November 2002/Accepted 7 February 2003

An acid tolerance response (ATR) has been demonstrated in Listeria monocytogenes and Salmonella enterica serovar Typhimurium in response to low pH poised (i.e., adapted) with acetic or lactic acids at 20°C and modeled by using dynamic differential equations. The ATR was not immediate or prolonged, and optimization occurred after exposure of L. monocytogenes for 3 h at pH 5.5 poised with acetic acid and for 2 h at pH 5.5 poised with lactic acid and after exposure of S. enterica serovar Typhimurium for 2 h at pH 5.5 poised with acetic acid and for 3 h at pH 5.5 poised with lactic acid. An objective mechanistic analysis of the acid inactivation data yielded estimates of the duration of the shoulder (ts), the log-linear decline (kmax), and the magnitude of a critical component (C). The magnitude of kmax gave the best agreement with estimates of conditions for optimum ATR induction made from the raw data. The study reported here determined optimum conditions for the induction of an ATR in S. enterica serovar Typhimurium and L. monocytogenes at 20°C by using acetic or lactic acids as acidulants. The experimental temperature of 20°C was chosen to reflect what could possibly occur in products that are temperature abused. Lower temperatures can offer some preservative effect; therefore, studying the response at 20°C (i.e., room temperature) allowed the worse case scenario to be studied. Acetic and lactic acids were the chosen acidulants since these acids are commonly encountered by microorganisms within food products. We then applied a dynamic mathematical model to the data to obtain an estimate of parameters associated with the acid inactivation kinetics. This provided an objective approach to the quantification of the ATR, and we discuss here the use of such a model as a means to identify key features associated with the response.

Many food-borne pathogenic bacteria exhibit stress responses, which enhance their survival in adverse environmental conditions. One stress commonly encountered in foods is an acidic environment, where enhanced survival can involve induction of an acid tolerance response (ATR). The ATR is defined as the resistance of cells to low pH when they have been grown at moderately low pH or when they have been exposed to a low pH for some time (5). Listeria monocytogenes and Salmonella enterica serovar Typhimurium exhibit an ATR when exposed to mildly acidic pH (7, 14). The ATR is growth phase specific (4, 15), with distinct responses occurring in both logarithmic and stationary phases, and it requires the de novo synthesis of acid-shock proteins (6, 8, 18). The ATR confers cross-resistance to other stresses such as heat, sodium chloride, and ethanol (16, 17) and there is some evidence that it may increase bacterial virulence (9, 18). The ATR, therefore, impacts on both predictive modeling and risk assessment approaches to microbiological food safety. Most studies of the ATR have been conducted at temperatures of 30 or 37°C (6, 14, 18) and have typically used mineral acids, such as hydrochloric acid, as the acidulant. Food-borne pathogenic bacteria are more commonly exposed to temperatures of 20°C or below and to weak organic acids, such as lactic or acetic acids, either as by-products of bacterial metabolism in fermentation processes or as deliberate additions to foods as preservatives. As a result, the ATR expressed in response to these conditions is of most relevance to food. In addition, previous studies have typically measured survival of cells at an arbitrary time as an indicator of the ATR. This ignores certain features of inactivation kinetics, such as the shoulder duration and rate of inactivation, that may be indicators of the mechanisms involved in the ATR.

MATERIALS AND METHODS Bacteria. L. monocytogenes strain EGD-e, as sequenced by Glaser et al. (11), was obtained from J. Vazquez-Boland, Departmento Patolgia Animal I, Universidad Complutense, Madrid. S. enterica serovar Typhimurium strain SL1344 was provided by Catherine Lee, Department of Microbiology and Molecular Genetics, Harvard Medical School. Culture media. Stock cultures of L. monocytogenes were maintained on nutrient agar (Oxoid) slopes stored at 0°C. Slopes were subcultured monthly by culture to fresh nutrient agar slopes which were incubated at 25°C for 1 day prior to storage at 0°C. Stock cultures of S. enterica serovar Typhimurium were stored at ⫺80°C in tryptone soya broth (Oxoid) plus glycerol (30% [wt/vol]). This culture was used to inoculate nutrient agar slopes monthly, which were incubated at 25°C for 1 day and subsequently stored at 0°C. Liquid growth medium consisted of Trypticase soy broth (Baltimore Biological Laboratory) plus 1% (wt/vol) glucose (TSBG) adjusted to pH 7.0 with HCl and sterilized by filtration through a cartridge filter unit containing a 0.2-␮m-pore-size membrane filter (Kleenpak, Pall, United Kingdom). Preparation of inocula. Bacteria were grown successively at 25°C for 24 h and then at 20°C for 24 h. The resultant population of ca. 109 viable cells ml⫺1 was diluted in peptone salt dilution fluid (13) to produce a suspension that contained the required number of viable bacteria. Acid tolerance. Sterile TSBG (pH 7.0) (100 ml) was transferred to sterile, 250-ml Erlenmeyer flasks, which were closed with a cover that allowed gaseous exchange. Approximately 104 viable cells ml⫺1 were added to two parallel flasks,

* Corresponding author. Mailing address: Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom. Phone: 44(0)1603-255216. Fax: 44(0)1603-507723. E-mail: [email protected]. 3945

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and these were incubated at 20°C for 15 h ⫾1 h on an orbital platform shaker (IKA-Labortechnik, Esslab, United Kingdom) rotating at 120 rpm to maximize aeration. This resulted in cultures that were in the mid-logarithmic phase of growth (data not shown). The pH of one culture was then adjusted, by the addition of either 1 M lactic acid or 1 M acetic acid, to a range of adaptation pH values and reincubated as described above for between 1 and 6 h. The final concentration of acids required to adjust cultures of L. monocytogenes to pH 5.0 and 5.5 were 0.03 M acetic or lactic acid and 0.02 M acetic or lactic acid, respectively. The final concentrations of acetic acid and lactic acid required to adjust cultures of S. enterica serovar Typhimurium to pH 5.0, 5.5, and 5.8 were 0.02 M acetic or lactic acid, 0.018 M acetic or lactic acid, and 0.015 M acetic or lactic acid, respectively. These adaptation pHs were chosen since they included pH values used for these organisms in previous studies (pH 5.5 and 5.8) (4, 7, 14, 18), as well as a value (pH 5.0) representative of the mildly acidic environment organisms may encounter within certain foods. After the required incubation time, cells were removed from the medium by filtration through a 0.22-␮m-pore-size membrane filter (Millipore). These cells were resuspended in 100 ml of fresh TSBG (pH 7.0) by vortexing to remove the cells from the membrane filter. This flask was adjusted to pH 3.0 ⫾ 0.1 by using 1 M HCl and then reincubated as described above for up to 12 h. At intervals throughout incubation a sample was removed, and the number of viable bacteria was determined by spreading 50 ␮l of this suspension onto the surface of duplicate plates of plate count agar (Oxoid CM325) by using a spiral plate maker (Spiral Systems, Inc.). Plate count agar plates were incubated at 35°C for 24 h before enumeration. Cells were also removed by filtration from the parallel control culture (pH 7.0) incubated as described above. These cells were resuspended in 100 ml of fresh TSBG (pH 7.0), the pH was adjusted to pH 3.0 ⫾ 0.1, and the numbers of viable cells were determined as described above. All experiments were performed at least in duplicate. Modeling inactivation kinetics. The numbers of viable bacteria in the culture medium adjusted to pH 3.0 ⫾ 0.1 were modeled to provide a quantitative and objective comparison of inactivation kinetics. The model of Geeraerd et al. (10) was originally developed for modeling microbial inactivation during a mild heat treatment but has key attributes important in the case of acid inactivation. These attributes include the ability to model a shoulder on the inactivation curve, a log-linear decline in viable numbers and a tail. It is cast as dynamic differential equations to allow time-varying challenges. The shoulder of the death curve was modeled by introducing the concept of a critical component (C, which may or may not be a real substance within or outside the cell) that decays immediately upon acid challenge. The amount of this component at the start of the challenge is a measure of the “physiological condition” of the cells and (together with the actual death rate) determines the length of the shoulder. Component C decays as follows: dC ⫽ ⫺ kmaxC dt

(1)

The death rate was moderated by a factor, ␣, cast as: ␣⫽1⫺

1 Cc C ⫽ ⫽1⫺ Kc ⫹ C 1 ⫹ Cc 1 ⫹ Cc

(2)

where Cc is a dimensionless concentration, normalized by a Michaelis constant. In other words, Cc ⫽ C/Kc (i.e., Cc, behaves exactly as C in the first equation). The decay of the population of viable cells is assumed to follow the form: dN ⫽ ⫺k 䡠 N dt

(3)

where the instantaneous rate constant, k, is given by: k ⫽ kmax 䡠 ␣

冉

1⫺

Nres N

冊

(4)

and where Nres represents a residual subpopulation of cells (i.e., the tail of the population). The static solution for the model was as follows: N共t兲 ⫽ Nres ⫹ 共N0⫺Nres兲 e⫺kmaxt where the shoulder time is given by:

冉

1 ⫹ Cc(0) 1 ⫹ Cc(0)e⫺kmaxt

冊

(5)

ts ⫽

ln[Cc共0兲] kmax

(6)

The start of the tail is given by: tres ⫽ ts ⫹

ln(N0/Nres) kmax

(7)

The tail portion models the residual proportion of the population (Nres) that is naturally resistant due to heterogeneity within a bacterial population (reviewed by Booth [2]). The present study was primarily concerned with acid inactivation of a population of acid-adapted cells in comparison with an unadapted population, and the small residual population was not under investigation. Additionally, because the residual population was small, the error associated with its enumeration by the viable count procedure was great. This resulted in poor fitting of the model and thus, for the purposes of the present study, a reduced model was developed that ignored the residual population (Nres). The static solution of this reduced model is cast as: N共t兲 ⫽ N0e⫺kmaxt

冉

1 ⫹ Cc共0兲 1 ⫹ Cc共0兲e⫺kmaxt

冊

(8)

The model was fitted to the data by using the statistical software package “R,” and a nonlinear least-squares method was used. The goodness of fit was determined both by visual inspection and from the value of the residual sum of squares (not shown). The standard errors for the fitted parameters are shown in Table 1.

RESULTS Acid tolerance. All data presented are means of at least duplicate experiments. An integral process in the measurement of the acid tolerance is the removal of cells from one culture onto a filter membrane and resuspension of these cells in fresh medium adjusted to pH 3.0. Measurements showed that ⬎90% of cells were recovered from the filter and that cells exposed to either no organic acid, acetic acid, or lactic acid were recovered equally efficiently (see similar initial values in Fig. 1 and 2). The mid-logarithmic-phase cells of L. monocytogenes and S. enterica serovar Typhimurium grown at pH 7.0 lost viability rapidly when exposed to pH 3.0 (i.e., large values of kmax in Table 1; representative data are shown in Fig. 1 and 2). However, when these cells were first incubated for between 1 and 6 h at pH 5.0, 5.5, or 5.8, adjusted by using either acetic or lactic acid, they demonstrated an enhanced tolerance to the acidic conditions and survived for much longer (i.e., small values of kmax in Table 1; representative data shown in Fig. 1 and 2). The cells had developed an ATR during incubation at a mildly acidic pH. Modeling inactivation kinetics. Comparison of acid inactivation data (such as those given in Fig. 1 and 2) can include simple comparisons of the numbers of cells surviving at an arbitrary time point. However, fitting the model of Geeraerd et al. (10) to the data enabled objective estimation of the parameters of the inactivation curve (for example, see Fig. 3). We used the reduced form of the model (equation 8) to estimate the parameters of the critical component Cc(0) and log-linear decline (kmax). From these parameters can be calculated an additional parameter, the magnitude of shoulder time (ts). The goodness of fit was determined both by visual inspection and by the value of the residual sum of squares (data not shown). Data shown were from model fits of acceptable quality both visually and statistically. The parameters of kmax and Cc(0) are shown in Table 1, and the duration of the shoulder time (ts) is shown in Fig. 4.

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TABLE 1. Effects of pretreatment of L. monocytogenes and S. enterica serovar Typhimurium on the parameters of kmax and Cc (0) Organism

L. monocytogenes

Pretreatment conditions Acid

pH

Inactivation parameters (SE) Time (h)

Cc(0)

) ⫺2

7.0

–

17.54 (7.8 ⫻ 10 )

1.25 ⫻ 10 (2.51 ⫻ 103)

Acetic acid

5.0

1 2 1 2 3 5 6

6.984 (6.1 ⫻ 100) 16.19 (4.5 ⫻ 101) 10.89 (2.4 ⫻ 101) 2.6 (7.2 ⫻ 10⫺3) 0.63 (1.3 ⫻ 10⫺3) 1.22 (5.2 ⫻ 10⫺3) 2.44 (2.5 ⫻ 10⫺2)

⫺9.4 ⫻ 10⫺1 (1.24 ⫻ 101) 5.22 ⫻ 102 (7.11 ⫻ 105) 4.03 ⫻ 101 (6.67 ⫻ 103) 3.84 ⫻ 101 (2.08 ⫻ 100) 1.61 ⫻ 101 (7.33 ⫻ 100) 1.24 ⫻ 100 (1.27 ⫻ 100) 5.54 ⫻ 100 (1.12 ⫻ 100)

1 2 1 2 5 6

14.2 (3.6 ⫻ 10⫺1) 18.20 (2.1 ⫻ 100) 9.72 (9.3 ⫻ 10⫺2) 1.11 (4.5 ⫻ 10⫺3) 3.43 (1.7 ⫻ 10⫺2) 4.91 (2.8 ⫻ 10⫺2)

7.29 ⫻ 103 (7.98 ⫻ 104) 3.12 ⫻ 104 (1.95 ⫻ 106) 1.98 ⫻ 102 (5.65 ⫻ 102) 6.99 ⫻ 10⫺1 (8.3 ⫻ 10⫺2) 7.49 ⫻ 100 (7.41 ⫻ 100) 3.57 ⫻ 103 (1.03 ⫻ 104) 3.85 ⫻ 101 (1.29 ⫻ 102)

Lactic acid

5.0 5.5

3

None

7.0

–

28.46 (3.2 ⫻ 10⫺1)

Acetic acid

5.5

1 2 4 5 1 2 3 6

2.32 (1.6 ⫻ 10⫺2) 1.00 (3.4 ⫻ 10⫺3) 1.28 (1.7 ⫻ 10⫺3) 1.48 (2.1 ⫻ 10⫺3) 4.54 (3.7 ⫻ 10⫺2) 1.67 (9.3 ⫻ 10⫺3) 1.62 (3.3 ⫻ 10⫺3) 5.41 (1.9 ⫻ 10⫺2)

1.08 ⫻ 101 (1.58 ⫻ 101) 3.23 ⫻ 100 (1.8 ⫻ 100) 3.92 ⫻ 100 (1.81 ⫻ 100) 1.09 ⫻ 100 (5.93 ⫻ 10⫺1) 2.49 ⫻ 100 (4.49 ⫻ 100) 8.82 ⫻ 10⫺1 (1.31 ⫻ 100) 1.95 ⫻ 100 (8.35 ⫻ 10⫺1) 2.21 ⫻ 101 (1.66 ⫻ 101)

1 2 1 2 3 4 1 2 4 5 6

4.91 (2.7 ⫻ 10⫺3) 2.25 (2.9 ⫻ 10⫺3) 3.31 (1.3 ⫻ 10⫺2) 2.39 (4.9 ⫻ 10⫺3) 1.67 (6.0 ⫻ 10⫺3) 3.06 (6.4 ⫻ 10⫺4) 5.20 (7.3 ⫻ 10⫺3) 5.38 (3.8 ⫻ 10⫺2) 6.13 (1.8 ⫻ 10⫺2) 4.57 (3.0 ⫻ 10⫺2) 12.17 (9.3 ⫻ 102)

5.31 ⫻ 100 (5.77 ⫻ 10⫺1) 3.15 ⫻ 10⫺1 (2.0 ⫻ 10⫺1) 5.02 ⫻ 100 (3.76 ⫻ 100) 3.15 ⫻ 100 (1.27 ⫻ 100) 2.20 ⫻ 100 (1.68 ⫻ 100) 5.02 ⫻ 100 (2.06 ⫻ 10⫺1) 1.04 ⫻ 100 (8.6 ⫻ 10⫺1) 2.24 ⫻ 101 (3.41 ⫻ 101) 3.43 ⫻ 101 (2.29 ⫻ 101) 7.69 ⫻ 100 (9.72 ⫻ 100) ⫺3.44 ⫻ 10⫺1 (1.84 ⫻ 104)

5.8

Lactic acid

5.0 5.5

5.8

a

kmax (h

None

5.5

S. enterica serovar Typhimurium

a

⫺1

–, these cells are the unadapted control population.

As expected, the values of kmax were large for unadapted populations of either L. monocytogenes or S. enterica serovar Typhimurium, indicating that the numbers of viable cells were reduced rapidly upon exposure to pH 3.0. In the case of L. monocytogenes, kmax was decreased in populations exposed to either acetic or lactic acids at any pH during adaptation for 1 h. Subsequent exposure to pH 5.0 poised with (i.e., adapted with) either acid resulted in an increase in kmax. At pH 5.5 poised with acetic acid the kmax decreased to a minimum value after exposure for 3 h, and at pH 5.5 poised with lactic acid the kmax decreased to a minimum value after exposure for 2 h. In the case of S. enterica serovar Typhimurium at pH 5.5 poised with acetic acid the kmax decreased to a minimum value after exposure for 2 h, and at pH 5.5 poised with lactic acid the kmax decreased to a minimum value after exposure for 3 h. The kmax did not show such marked minima during exposure to pH 5.8 poised with either acid.

The values of Cc(0) differed with adaptation time during exposure of both organisms to all conditions investigated. Large standard errors were also associated with these parameter estimates. No clear pattern emerged of the value of Cc(0) with respect to adaptation conditions. In the case of L. monocytogenes, only adaptation at pH 5.5 poised with acetic acid for 3 h resulted in a value of ts markedly greater than that of the unadapted population. In contrast, the values of ts shown by S. enterica serovar Typhimurium were greater than unadapted cells when cells were exposed at pH 5.5 poised with acetic acid for 1 h and for 4 h, at pH 5.5 poised with lactic acid for 1 to 3 h, at pH 5.8 poised with lactic acid for 1 h, and at pH 5.8 poised with acetic acid for 3 h. The parameters in Table 1 and in Fig. 4 are indicative of the effects of exposure to pH 3.0 ⫾ 0.1 on the inactivation kinetics of the bacteria. Use of the parameters offered an objective means to quantify the ATR. Scrutiny of the inactivation data presented in Fig. 1 and 2 indicated that the survival of the

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FIG. 1. Survival of L. monocytogenes either unadapted (E) or after prior adaptation at pH 5.5 poised with lactic acid (A) or acetic acid (B) for 1 h (F), 2 h (■), 3 h (Œ), 5 h (), or 6 h (⽧). Datum points are the means of at least duplicate experiments, and the bars show the range.

population varied with adaptation time. Of the objective parameters extracted by the model, the value of kmax best represented the effect of time on adaptation. Although minima were apparent in Table 1, graphical representation of this parameter was inevitably biased by the large values for the unadapted population. We have normalized the value of kmax by reference to unadapted cells. Accordingly, Fig. 5 shows the ratio of the magnitude of the log-linear decline (kmax) of unadapted to adapted cells. This demonstrates that optimization of the ATR occurred after exposure of L. monocytogenes for 3 h at pH 5.5 poised with acetic acid and 2 h at pH 5.5 poised with lactic acid. Optimization of ATR in S. enterica serovar Typhimurium occurred after exposure for 2 h at pH 5.5 poised with acetic acid and after exposure for 3 h at pH 5.5 poised with lactic acid.

DISCUSSION An ATR has previously been determined in S. enterica serovar Typhimurium and L. monocytogenes at 37°C. However, whether this response is also present at lower temperatures is of greater importance to the food industry. The present study demonstrates that exposure of aerobically grown L. monocytogenes and S. enterica serovar Typhimurium to mild acid treatment at 20°C by using either acetic or lactic acid results in an ATR. The ATR observed was not immediate or prolonged but was optimized under a distinct set of conditions, the most important of which appeared to be the adaptation time period and pH. This time phenomenon was also noted in previous studies with L. monocytogenes at 37°C (20). Characterization of the ATR under these conditions provides an opportunity to

FIG. 2. Survival of S. enterica serovar Typhimurium either unadapted (E) or after prior adaptation at pH 5.5 poised with lactic acid (A) and acetic acid (B) for 1 h (F), 2 h (■), 3 h (Œ), 4 h (), or 5 h (⽧). Datum points are means of at least duplicate experiments, and the bars show the range.

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FIG. 3. Survival of unadapted cells of L. monocytogenes EGD-e. Points are experimental data. The line is the model fitted to these data. Parameters estimated by the model are also indicated. These include the initial cell concentration N0 (in CFU per milliliter), the length of the shoulder ts (in minutes), the time to the start of the tail tres (in minutes), and the slope of the log linear region of the curve (the rate constant kmax [per hour]).

compare the ATR with that produced in colonies under immobilized conditions, which are more representative of some food environments (12). The adaptation time may be linked to maintenance of the internal pH (pHi) of the cell. Previous studies have shown that acid death is a direct result of a lowered pHi (7, 19). Acidadapted cells were better able to maintain their pHi and, as a result, survived better in acidic environments. Phan-Thanh et al. (20) also noted that organic acids were more lethal at low pH than inorganic acids. This appeared to be due to their ability to alter the pHi of L. monocytogenes to a lower level than that observed with inorganic acids. Further evidence for the involvement of pHi maintenance in acid tolerance was provided in the studies of Foster and Hall (7) and Cotter et al. (3). It is possible that the adaptation time is directly linked to the ability of the cell to maintain its pHi. The cell may be able to maintain pHi and adapt at mildly acidic conditions but not for a prolonged period. After a critical time, the pHi maintenance system fails and cells become sensitive to the lethal effects of the acid (i.e., the ATR is no longer observed when cells are challenged at pH 3.0). This critical time may be the optimum adaptation time observed in the present study. The present study has also found that adaptation times differed according to the acidulant used to adapt cells. For example, at pH 5.5 S. enterica serovar Typhimurium required an adaptation time of only 2 h for the optimum ATR to be induced by using acetic acid as the acidulant but 3 h when lactic acid was the acidulant. A difference was also observed in the case of L. monocytogenes. However, both organic acids undergo dissociation and, at the relevant pH values used, the protonated (undissociated) concentration was small. For example, in the case of cultures of S. enterica serovar Typhimurium adjusted to pH 5.5, the total concentration of 0.018 M acetic (pK 4.76) or lactic acid (pK 3.86) required to poise the pH was equivalent to a concentration of undissociated acetic or

FIG. 4. (A) Effect of adaptation time on the duration of the shoulder (ts) shown by L. monocytogenes EGD-e during exposure to pH 3.0 ⫾ 0.1. Adaptation conditions were acetic acid at pH 5.5 (F), acetic acid at pH 5.0 (Œ), lactic acid at pH 5.5 (■), or lactic acid at pH 5.0. (〫). (B) Effect of adaptation time on the duration of the shoulder (ts) shown by S. enterica serovar Typhimurium during exposure to pH 3.0 ⫾ 0.1. Adaptation conditions were acetic acid at pH 5.5 (F), lactic acid at pH 5.8 (Œ), lactic acid at pH 5.5 (■), acetic acid at pH 5.8 (), or lactic acid at pH 5.0. (⽧).

lactic acid of 3 or 0.4 mM, respectively. Differences in adaptation times between acidulants may be explained by the differing ability of organic acids to alter the pHi of the cell. Certain organic acids may enter into the cell more easily than others and therefore alter the pHi of the cell more readily. Acetic acid is lipid soluble, diffuses rapidly through the plasma membrane and has been shown to have a dramatic effect on the pHi of a cell (21). In contrast, lactic acid demonstrates some lipid solubility and diffuses slowly through the membrane. Disruption of the cell pHi does not appear to be its main mode of inhibition (21). If the pHi of the cell is reduced more readily with acetic acid than with lactic acid, then the optimum adaptation time for acetic acid may also be reduced, and the optimum adaptation

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[Cc(0)] proved in the present study to be inadequate descriptors of the optimized ATR. However, the magnitude of the log-linear decline (kmax) was a good indicator of adaptation. Such a mechanistic modeling approach is worthwhile in that it enables objective study of the effect of changing conditions (adaptation time, acidulant) on the features of the ATR. The present study has identified conditions to produce aerobically grown mid-logarithmic-phase cells of L. monocytogenes and S. enterica serovar Typhimurium that are acid tolerant. The ATR has been shown to be transient and the conditions identified here enable cells to exhibit maximal tolerance to acid shock (which we describe as optimization of the ATR) prior to the decay of the ATR upon continued exposure to organic acids. These cells are the focus of further studies to identify the magnitude and mechanisms of cross-protection of L. monocytogenes and S. enterica serovar Typhimurium to food-relevant inimical conditions. ACKNOWLEDGMENT We are grateful for financial support from the UK Biotechnology and Biological Sciences Research Council. REFERENCES

FIG. 5. (A) Ratio of kmax (unadapted) to kmax (adapted) calculated from the fitted model parameters for L. monocytogenes EGD-e grown at pH 7.0 and adapted with acetic acid at pH 5.5 (F), acetic acid at pH 5.0 (Œ), lactic acid at pH 5.5 (■), or lactic acid at pH 5.0 (⽧). (B) Ratio of kmax (unadapted) to kmax (adapted) calculated from the fitted model parameters for S. enterica serovar Typhimurium grown at pH 7.0 and adapted with acetic acid at pH 5.5 (F), acetic acid at pH 5.8 (⽧), lactic acid at pH 5.8 (Œ), lactic acid at pH 5.5 (■), or with lactic acid at pH 5.0 (Œ).

time may differ according to the organic acid used to poise the system. Differences observed between organisms may result from differences in membrane structure. Previous studies have shown that inorganic acid adaptation provides protection against organic acid stress and vice versa (1, 19). We have demonstrated in the present study that organic acid adaptation provides protection against inorganic acid stress at 20°C (i.e., cross-protection also occurs at this lower temperature). Application of a reduced model derived from the model of Geeraerd et al. (10) enabled an objective mechanistic analysis of the inactivation data. The parameters of the duration of the shoulder (ts), and the magnitude of the critical component

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