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Journal of Applied Microbiology, 1999, Mar, 86(3), 469-476

Effect of osmotic, alkaline, acid  or thermal stresses on the growth  and inhibition of Listeria monocytogenes

ABSTRACT

Five strains of Listeria monocytogenes (a, b, c, d and e) isolated from industrial plants have been subjected to different osmotic, alkaline, acid or thermal stresses. The effects of these treatments on lag-phase (L) and growth rate () of cells in mid-log phase have been followed using an automated optical density monitoring system. Increasing the osmotic pressure by the addition of different amounts of NaCl increased the lag phase and decreased the growth rate. The same phenomena were observed after decreasing the pH of the medium to 5·8, 5·6 or 5·4 by addition of acetic, lactic or hydrochloric acids. The inhibitory effect was: acetic acid > lactic acid > hydrochloric acid. The addition of NaOH to attain pH values of 9·5, 10·0, 10·5 or 11·0 in the medium produced a dramatic increase of the lag phase at pH 10·5 and 11. Growth rates were also decreased while the maximal population increased with high pH values. These effects varied according to strains. Strains d and e were the most resistant to acidic and alkaline stresses, and e was the most affected by the addition of NaCl. A cold shock of 30 min at 0 °C had limited effects on growth parameters. On the other hand, hyperthermal shocks (55 or 63 °C, 30 min) led to similar increased lag phases and to significant increases of the maximal population in all five strains.

INTRODUCTION

Listeria monocytogenes is a Gram-positive, aerobic to facultative anaerobic bacterium widespread in the environment and detected in foodstuffs such as meat, dairy products, seafood and vegetables. This bacterium has become one of the most important food-borne pathogens in recent years and a major concern both for the food industry and public health. Numerous outbreaks of listeriosis have been observed since the 1980s in the USA, Canada and Europe ( Bille 1990; Farber & Peterkin 1991). This bacterium is a psychrotroph able to grow at refrigeration temperatures and its increasing importance is due to the increased use of refrigeration in food processing and preservation.

In industrial plants, the micro-organisms are subjected to various stressing agents during food processing or cleaning and disinfection of surfaces. The effects of these treatments on L. monocytogenes are of great interest as they could influence its response and ability to survive. Quantifying the behaviour of this pathogen to such environmental or technological stresses would enable better control in food products and plants.

In this study, the responses of five strains of L. monocytogenes isolated from industrial plants and subjected to various concentrations of NaCl, alkaline treatments by the addition of NaOH, moderate acidic treatments by the addition of three different acids (acetic, lactic or hydrochloric acid), and different thermal stresses (cold and heat shocks), are reported. The effects of these treatments on growth parameters (L and ) in bacterial cells in the mid-exponential phase were used to assess their inhibitory efficiency and the sensitivity of the strains.

MATERIALS AND METHODS

Bacterial strains and growth conditions

The five strains a, b, c, d and e of L. monocytogenes were isolated from industrial plants. They were maintained on Tryptic Soy Agar (TSA; Difco) plates incubated at 37 °C for 24 h and then stored at 4 °C. For the experiments, the strains were grown on TSA slopes at 37 °C for 8 h in order to inoculate a pre-culture in an Erlenmeyer flask containing 100 ml meat broth. The pre-culture was incubated in an agitated (150 rev min ¹) water-bath at 20 °C for 17 h (end of the exponential phase). Cultures consisted of 100 ml Meat Bacto Tryptone with 5 g l ¹ glucose added (MBT₅), buffered with K₂HPO₄-KH₂PO₄ (0·1 mol l ¹) and adjusted to pH 7·0. Sterilization was achieved by filtration through a 0·2 m Nalgene filter (Nalgene Company, NY, USA). The culture media were inoculated to obtain approximately 10⁷ cfu ml ¹. The initial populations were controlled by plate counts on TSA.

Preparation and application of the challenge solutions

The challenge solutions were prepared by adding NaCl (Prolabo), NaOH (Prolabo), acetic acid (Carlo Erba), lactic acid (Acros) or HCl (Prolabo) to MBT₅ to obtain the final values, after dilution (1/4; v/v) with the culture medium, of 40 g l ¹, 62·5 g l ¹ or 80 g l ¹ of NaCl, pH 9·5, 10·0, 10·5 or 11·0 with NaOH, pH 5·4, 5·6 or 5·8 with either of the three acid solutions. The undissociated acid concentrations corresponding to these acid pH values are shown in Table 1. All the challenge solutions were sterilized through a 0·2 m filter (Nalgene).

Growth in the culture medium (20 °C, 150 rev min ¹) was followed by optical density measurements at 600 nm with a Shimadzu UV-160 A (Shimadzu Corporation, Japan). Stresses were performed on cells in early exponential phase (O.D._{600 =} 0·3) by adding 7·5 ml bacterial culture to 2·5 ml of the appropriate concentrated challenge solution. Each stressed culture was dispensed (400 l per well) aseptically into six wells of a Honeycomb microplate (Labsystems, Finland). Controls consisted of non-inoculated MBT₅ (four wells), MBT₅ plus the challenge solution (four wells) and MBT₅ with non-stressed bacterial cells (six wells) and were run in parallel. Two microplates could be placed in a Bioscreen C automated optical density monitoring system (Labsystems) regulated at 20 °C. They were shaken automatically every 90 s and the absorbance in each microwell was measured at 600 nm every 10 min.

In parallel with these stresses, the effect of the temperature was also studied. In this case, the stress was applied for a short period and was consequently described as shock.

Application of thermal shocks

The thermal shocks were applied for a period of 30 min by transferring early exponential phase cells (O.D._{600 =} 0·3) from 20 °C to 0, 55 or 63 °C. The shocked cultures were then dispensed into the microplates (400 l per well) of Bioscreen C and growth was monitored at 20 °C as described previously.

Analysis of the Bioscreen data

Calculations of log optical density were performed from the means of the values measured by the Bioscreen. The growth curves were fitted by the modified Gompertz equation ( Zwietering et al. 1990) and the growth parameters (L, ) were determined using a non-linear regression model ( Bégot et al. 1996).

FIGURES

Fig. 1  Lag times (h) of five Listeria monocytogenes strains (a, b, c, d, e) after the addition of NaC...

Fig. 2  Lag times (h) of five Listeria monocytogenes strains (a, b, c, d, e) in culture media adjusted...

Fig. 3  Lag times (h) of five Listeria monocytogenes strains (a, b, c, d, e) in culture media adjusted...

Fig. 4  Lag times (h) of five Listeria monocytogenes strains (a, b, c, d, e) in culture media adjusted...

Fig. 5  Lag times (h) of five Listeria monocytogenes strains (a, b, c, d, e) in culture media adjusted...

Fig. 6& emsp;Lag times (h) of five Listeria monocytogenes strains (a, b, c, d, e) after a cold shock o...

Table 1 Equivalence of initial pH and undissociated acid concentrations of the challenge solutions a...

Table 2 Specific growth rates for the five strains of Listeria monocytogenes according to the tested...

RESULTS
 

The results of the screening tests at 20 °C indicated that all five strains of L. monocytogenes behaved differently, depending on the conditions tested. Table 2 summarizes the specific growth rates observed for the different stresses and strains.

Effect of the NaCl concentration

For all five strains, the increase of NaCl concentration in the growth medium increased the lag phase ( Fig. 1). Lag phase varied in the range of 1·27 -1·58 h (a and b strains, respectively) at 40 g l ¹, 1·51 h (a) - 4·73 h (e) at 62·5 g l ¹, and 2·12 h (d) - 5·32 h (e) at 80 g l ¹ NaCl. At 80 g NaCl l ¹, the generation time was almost double that at 40 g l ¹ (TG, respectively, 5·3 h and 2·9 h for strain d). Strain e appeared to be the most sensitive to the highest NaCl concentration (80 g l ¹) ( Table 2). The five strains were all able to grow under the experimented conditions.

Effect of the pH

Alkaline stress.

When the pH was adjusted to 9·5, lag times varied from 1 h for d to 3·83 h for a. The increased alkalinity induced an increase in lag phase for all five studied strains ( Fig. 2). However, a strain still appeared to be the most sensitive (slowest) at pH 10 and 10·5. At pH 11, the growth of the strains a and b appeared to be inhibited while strains c, e and particularly d (complete growth in 38 h) ( Table 2) were more tolerant to alkaline conditions.

Acid stress.

Acid stress was produced using either organic (acetic and lactic acids) or inorganic acid (HCl). The sensitivities of the five L. monocytogenes strains to the various acids were different. For the all strains studied, increased growth rates and lag times were correlated with increasing medium acidity ( Figs 3, 4, 5) ( Table 2).

Comparing the growth parameters obtained for the different acids and pH values tested, strains d and e grew more rapidly (generation time of 14·4 h and 19·29 h, respectively, at pH 5·4) and were the most tolerant to acetic acid. Strains a, b and c were more sensitive to acetic acid ( Fig. 3), with generation times from 20 h (a) to 26 h (c) and lag times of 7 h (2 h for d and e).

With regard to the effects of lactic acid on growth parameters, similar responses were observed, although they were less marked than with acetic acid. For strain d, the generation times were similar at pH 5·8 and 5·4 (2·53 h and 3 h, respectively), whereas the lag times were 1·12 h and 1·54 h; a, b and c appeared to be more sensitive to lactic acid than d and e ( Fig. 4).

The acidification of the medium culture to pH 5·4, 5·6 and 5·8 by adding HCl reduced growth of all five strains. The slower growth was due to a lag phase increase and a growth rate decrease. For instance, the lag phase of the strain d changed only from 0·88 h at pH 5·8 to 1·07 h at pH 5·4 ( Fig. 5). The lag times were less than 3 h.

At these pH values, HCl had the least effect of the three acids on the initiation of growth. Strains a and b appeared to be more sensitive to HCl.

The variable tolerance of the five strains of L. monocytogenes to the different acids tested highlighted the influence of the acid nature and of the pH value. The three acids had an inhibitory effect on the growth of all strains but to varying degrees. Acetic acid appeared to be the most effective of the acids, followed by lactic acid and then by hydrochloric acid, particularly against the strains a, b and c.

Effect of thermal shocks

All five strains responded similarly to each of the three thermal shocks. Cold shock did not lead to visible changes in the growth of the strains studied. The lag phase did not exceed 1·35 h (strain c) ( Fig. 6), but after heat shocks, lag time varied from 17·7 h at 55 °C to 30·5 h at 63 °C. However, growth still occurred ( Table 2).

DISCUSSION

Effect of NaCl

Sodium chloride is mainly used in the food industry in the form of brine. In 1991, L. monocytogenes was detected in Mozarella cheese and investigations revealed that the brine solution, used in the manufacture of the cheese, had been contaminated by this pathogen. Our approach was therefore to study the behaviour of L. monocytogenes after addition of NaCl to the culture medium.

Different NaCl concentrations added to the culture medium led to an increased delay before growth, varying slightly depending on the NaCl concentration and on the strain studied. The growth conditions did not prevent growth of the five strains.

Farber et al. (1992) showed that L. monocytogenes was able to grow in the presence of 130-140 g NaCl l ¹ at 15 and 30 °C; 15 °C was the optimum growth temperature at 80 g NaCl l ¹ medium.

Shahamat et al. (1980) observed a total loss of viability in 9 d at 37 °C for L. monocytogenes in the presence of 130 g NaCl l ¹, whereas at 20 °C, it took at least 36 d before death. Consequently, the influence of the osmotic stress on the bacterial population is dependent on the water activity of the medium (and thus, NaCl concentration) and on the temperature. The osmotic stress is a consequence of changes in osmotic exchanges between the bacterial cell and the extracellular environment.

Different authors have investigated the mechanisms of adaptation to osmotic stress in L. monocytogenes. Patchett et al. (1992) reported that cells grown in the presence of 75 g l ¹ NaCl contained higher concentrations of K⁺, betaine, glycine, alanine and proline than cells grown in the absence of NaCl. Ko et al. (1994) also showed that L. monocytogenes accumulated glycine betaine intracellularly when grown under osmotic stress. In addition, these authors noted that salt-stimulated accumulation of glycine betaine, providing osmotolerance in the strain, occurred by a constitutive transport system from the medium rather than by biosynthesis. Similar observations have been made with regard to cryotolerance.

Smith (1996), studying L. monocytogenes grown in liquid media and on processed meat surfaces, indicated that carnitine and glycine betaine enhanced its osmotic and chill tolerance. The amount of each osmolyte accumulated by the cell appeared to be dependent on the osmolarity of the medium, the temperature and the phase of growth of the culture. Moreover, the results suggested that the accumulation of these solutes influenced the survival of the pathogen in both environments tested.

Effect of pH

Alkaline stress.

Alkaline stress led to an increase in the lag phase at pH 11. Alkaline solutions such as NaOH are generally used in detergents to eliminate carbonized sediment, oil or grease. They facilitate protein denaturation, fats saponification, and have a bactericidal activity. Rowbury & Hussain (1996), in studies on Escherichia coli cells exposed to alkaline pH (pH 8·8 to pH 10), reported damage to the outer membrane, ribosomes, proteins and DNA. In addition, a total dissociation of NaOH into Na⁺ and OH ions disturbed the energetic metabolism of the bacteria. An Na⁺ excess in the cell would modify the transmembrane Na⁺ gradient maintained through an Na⁺/H⁺ antiporter, and when the extracellular pH increased, the antiporter activity would increase too ( Karpel et al. 1991). Two hypotheses are then proposed. (i) At high alkaline pH, the antiporters might be saturated with the excess of Na⁺ entering the cell; consequently, the ion gradients might not be able to be maintained and membrane proteins, implicated in the production of energy, would be inhibited. (ii) Such high pH values could lead to the saponification of membrane lipids and destabilization of those proteins whose activity depended on the integrity of the lipid bilayer. In addition to the loss of energy production, these phenomena would rapidly inhibit growth.

Acid stress.

The experiments performed in this study emphasized that the antilisterial activity of acetic acid was greater than that of lactic or hydrochloric acid, for all five strains. The lag and generation times observed with acetic acid were longer than for the other acids.

These results were consistent with those of other authors on different strains of L. monocytogenes. At the same pH, Sorrells et al. (1989) established that at 10, 25 and 35 °C, acetic and lactic acids were more inhibitory against L. monocytogenes than citric and hydrochloric acids. Conner et al. (1990) also reported that acetic and lactic acids were the most inhibitory. In contrast, Sorrells et al. (1989) observed that with an equimolar concentration of acid, the order of activity was lactic acid, followed by acetic and then hydrochloric acid at 25 and 35 °C.

These results, as well as the findings of El-Shenawy & Marth (1989), highlight the varying influence of pH and temperature, depending on the organic acid used.

A number of investigators have reported the inhibitory effects of low pH and organic acids on L. monocytogenes ( Adams & Hall 1988; Conner et al. 1990; Ita & Hutkins 1991). Two inhibitory mechanisms have been proposed: (i) an intracellular acidification (lost of homeostasis) and (ii) a specific effect of the acid (non-dissociated form) on metabolic activities.

Ita & Hutkins (1991) observed that low intracellular pH was not the major factor in the inhibition of L. monocytogenes at acid pH; indeed, cells treated with organic acids or HCl at pH values as low as 3·5 were able to maintain their cytoplasmic pH at a value near 5. Consequently, the efficiency of the treatments using organic acids would be due to the non-dissociated fraction rather than to proton toxicity.

The inhibitory effect of these acids can be correlated with their dissociation constant (pKa value) and with the greater permeability of the cell membrane to weak acids in their undissociated form. Among the acids we have tested, hydrochloric acid is totally dissociated in aqueous environments whereas acetic acid (pKa ₌ 4·76) has the highest concentration of undissociated acid at pH 5·4 (14·3 mmol l ¹), and lactic acid (pKa ₌ 3·86) has the lowest (2·4 mmol l ¹). Also, acetic acid, a weak acid with the highest pKa value, is more efficient against L. monocytogenes than a stronger hydrochloric acid used at the same pH. These data are consistent with the results obtained by Sorrells et al. (1989).

The highest inhibitory effect of acetic acid can be explained by its ability to diffuse through the cell membrane which is permeable to non-dissociated, non-protonated and lipophilic weak acids. This leads to an accumulation of the acid within the cell cytoplasm, acidification of the cytoplasm, disruption of the proton-motive force and inhibition of substrate transport.

Lactic acid may be less inhibitory as it cannot passively penetrate the cell membrane.

Treatments based on organic acids seem to be the most efficient (against Gram-negative bacteria) and are widely used in decontamination processes such as meat (carcass) decontamination. Lactic acid is suitable for this purpose because it is a natural constituent of meat and is 'generally accepted as safe' ( Van Netten 1996). The intrinsic lactic acid content of meat, together with the buffering capacity, determine the resulting meat surface pH and thus, the bactericidal effectiveness of the agent applied.

Due to its higher pKa, acetic acid is theoretically a better antimicrobial agent than lactic acid. In practice, the lactic acid appeared to be the better meat decontamination agent ( Van Netten 1996). Mixtures of both were also tested on E. coli and Salmonella enteritidis strains ( Adams & Hall 1988) and results confirmed that the undissociated acid was the active antimicrobial species.

Effect of temperature

The cold conditions applied to the five strains of this study did not really affect their growth. Different researchers have worked on the growth of L. monocytogenes under refrigeration. For example, Walker et al. (1990) highlighted the ability of some strains to grow at temperatures as low as - 0·4 °C. These workers also observed that a decrease in the incubation temperature led to an increase in lag and generation times, and to a decrease in the maximal bacterial population.

The ability of L. monocytogenes to proliferate at refrigeration temperatures may be a health concern, particularly with chilled foods. Precaution should therefore be taken to prevent contamination.

Heat shocks of 55 and 63 °C for 30 min induced increased latencies, particularly at 63 °C. These last parameters were chosen to reproduce low pasteurization conditions (62·8 °C, 30 min).

The possible heat resistance of L. monocytogenes is a contentious subject. Bearns & Girard (1958) were the first workers to describe this species as a micro-organism able to survive the pasteurization process of 61·7 °C, 35 min. They concluded that L. monocytogenes was more thermotolerant than most of the non-spore-forming bacterial pathogens. Donnelly et al. (1987) claimed that L. monocytogenes did not survive the lowest legal pasteurization temperature. They stressed the importance of the methodology used to determine the thermal inactivation (whether sealed, immersed, or pre-heated test tubes were used). Consequently, according to these authors, Bearns & Girard 1958 ) could have over-estimated the thermoresistance of the organism.

Many factors such as time, incubation temperature and recovery conditions influence the heat resistance of the strain. Sub-lethal heat shocks appear to be interesting because they induce the rapid synthesis of heat-shock proteins. Knabel et al. (1990) studied the induction of the heat-shock response and reported that stationary cells of L. monocytogenes F5069 preheated to 43 °C had higher heat resistance than cells held at 37 °C.

The stress response has been studied for various bacterial species, including E. coli ( Neidhardt et al. 1987), Bacillus subtilis ( Völker et al. 1994), Pseudomonas fragi ( Michel et al. 1996, 1997) and L. monocytogenes ( Sokolovic et al. 1988; Phan-Thanh & Gormon 1995). The synthesis of heat-shock proteins (Hsp) seems to be strongly stimulated by environmental stress and may aid the cell to overcome temperature-induced damage. Phan-Thanh & Gormon (1995) studied the proteins induced by heat (49 °C) and cold (4 °C) shocks in L. monocytogenes by two-dimensional electrophoresis and showed that induction by heat shock was more intense than that by cold shock. Resistance to different stresses (alkaline or acid) would be increased by induction of the stress proteins.

Perspectives

Following the individual effects of the stresses on the behaviour of L. monocytogenes, work is underway to study the effects of combinations of stresses on cell viability and protein synthesis.

ACKNOWLEDGEMENTS

This work was supported by the Ultrapropre Nutrition Industrie Recherche (U.N.I.R.) French industrial program "Ecologie Microbienne dirigée". Cécile Vasseur was financed by a CIFRE grant and CFPI industries.

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