Article pp.284-292 du Vol.23 n°2 (2003)

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ARTICLE ORIGINAL ORIGINAL PAPER

Effect of NaCl pretreatment on the growth and survival of Listeria monocytogenes

at high osmolarity

O. Duche, J. Labadie¹*

RÉSUMÉ

Effet d’un prétraitement au NaCl sur la croissance

et la survie de Listeria monocytogenes à osmolarité élevée.

L. monocytogenes est un pathogène d’origine alimentaire largement répandu dans l’environnement et potentiellement présent dans de nombreux aliments frais ou transformés. La capacité de ce microorganisme à tolérer des environnements salés est importante et dans ce contexte la capacité de croissance et de survie en présence de sel a été étudiée pour sept souches de L. monocytogenes. Il a été montré dans ce travail que certaines souches peuvent se multiplier dans des milieux contenant jusqu’à 11% de NaCl et que le délai de détection d’une croissance varie selon la concentration en sel et les souches utilisées. La suite de cette étude a été effectuée sur la souche LO28 de L. monocytogenes, une souche de référence bien caracté- risée par de nombreux auteurs. Afin d’évaluer l’augmentation de résistance de cette souche au sel, elle a d’abord été exposée à de faibles concentrations de cet agent avant un traitement à des concentrations plus élevées.

Aucune des conditions testées n’a permis d’augmenter la résistance de la souche LO28 au sel, qui reste identique quels que soient les prétraitements appliqués.

Mots clés :

Listeria monocytogenes ; sel ; stress.

SUMMARY

L. monocytogenes is a food-borne pathogen widely distributed in the envi- ronment and potentially found in many kinds of fresh and processed foods.

The ability of this microorganism to tolerate salt is important. Seven strains

1. Station de Recherches sur la Viande, Institut National de la Recherche Agronomique, 63122 Saint-Genès-Champanelle, France.

* Correspondance.

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Effect of NaCl pretreatment on the growth and survival of Listeria monocytogenes at high osmolarity 285

of L. monocytogenes were tested in order to determine their survival capaci- ties in presence of NaCl. The study has shown that some strain could be cultivated in media containing up to 11% NaCl and that the growth delay observed varied according to the salt concentration and the strains. The second part of this study was carried out on L monocytogenes LO28, a reference strain well characterised by many authors. In order to increase the NaCl resistance of this strain, shifts from low to higher NaCl concentrations were applied to growing cells. None of the tested conditions was able to increase the salt resistance of the LO28 strain.

Key words:

Listeria monocytogenes; salt; stress.

1 – INTRODUCTION

L. monocytogenes is a pathogen of public health significance causing occa- sional outbreaks and sporadic cases of food-borne illness. This organism is of particular concern because of its ability to survive and grow under a wide range of adverse conditions, such as low temperature, low pH and high osmolarity (BARKER and PARK, 2001). For instance, during sausage fermentation, most of the bacteria are stressed by the addition of salt, and the production of lactic acid by the starters generally used. Surviving to these hurdles demands the adaptation of bacteria to these conditions, which are far from those existing in optimal growth conditions, that is generally a neutral pH and isotonic salt concentrations. For that purpose, bacteria can activate mechanisms allowing them to increase their resistance to a more important stress, which sometimes can be different from the first applied

Traditionally, salt (NaCl) is used to preserve many food products, in order to inhibit the growth of spoilage and pathogenic bacteria. L. monocytogenes is a salt-tolerant organism, and some strains can grow up to 12% NaCl or are surviving 150 days in pure salt at ambient temperature (GALDIERO et al., 1997;

GNANOU BESSE et al., 2000). This important survival ability is one of the rea- sons, which may explain the difficulty to control L. monocytogenes in a number of salted food products. In this study, the growth of seven L. monocytogenes strains (reference strains and strains isolated from industrial plants) was evalu- ated at high salt concentration. Additionally, the influence of a pretreatment at low salt concentration on the further resistance of L. monocytogenes LO 28 to high NaCl concentrations was investigated.

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2 – MATERIALS AND METHODS

2.1 Bacterial strains and growth conditions

Seven strains of L. monocytogenes were used in this study: the LO28 strain (serotype 1/2c) and the EGD strain (1/2a) were obtained from P. Cossart (Institut Pasteur, Paris, France). Strains A (serotype 4b), B (4b), C (1/2a), D (4b) and E (1/2a) were isolated from industrial plants: strains A, B and D from three dairy plants, and strains C and E from one meat plant. The strains were grown in MCDB medium (MCDB 202, Cryobiosystem, L’Aigle, France), a chemically defined medium (HEBRAUD and GUZZO, 2000) supplemented with 1% yeast nitrogen base (YNB, Difco Laboratories, Detroit, Mich., USA) and 3.6 g/l glucose. The pH of this medium was adjusted to 7.3. The original MCDB medium contains 0.75% NaCl.

In all experiments, bacteria from frozen stocks were grown overnight on BHI agar (Difco Laboratories) slopes at 37°C in order to inoculate a flasks containing MCDB medium to an initial optical density of 0.1 at 600 nm (OD₆₀₀). After an overnight growth at 37°C with shaking, the cultures obtained were used to inoculate another culture of the same medium at an OD₆₀₀ of 0.05.

2.2 Growth delay of L. monocytogenes at different concentrations of salt For the 7 strains, NaCl was added at final concentrations raising up to 11%

(w/v) in the culture (OD₆₀₀ of 0.05), and bacteria were incubated at 37°C without shaking. The growth delay was defined as the time necessary to observe a col- our change of the medium, which was also associated with an increase of the opacity of the growth medium.

2.3 Salt shocks of L. monocytogenes LO28 in exponential phase Cultures were grown aerobically at 37°C until they reached exponential phase (OD₆₀₀ of 0.3). Salt treatments were performed on these cultures dis- persed into Erlenmeyer flasks and NaCl was added at final concentrations ranging from 2 to 25% NaCl. The time of the shift was set to zero. The growth curves were followed by optical density measurements (OD₆₀₀) at different times. Then, to test the survival abilities of the strains, final concentrations of salt ranging from 6 to 25% have been tested. In this context, after a serial dilu- tion in tryptone-salt solutions viable cells were streaked on Tryptic Soy Agar (TSA, Difco Laboratories) plates incubated for 24h at 37°C and enumerated.

Each experiment was performed in duplicate.

2.4 Salt pretreatment of L. monocytogenes LO28 in exponential phase The cultures were grown aerobically at 37°C until they reached exponential phase (OD₆₀₀ of 0.3), cells were enumerated in order to calculate the number of initial bacteria corresponding to No. Salt pretreatments were performed by dis- pensing the culture into Erlenmeyer flasks containing different quantities of salt in order to obtain a final concentrations of 3.5%, 4% or 7% NaCl. After two hours, NaCl were added in some cultures containing 3.5% and 7% of salt to get

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higher final concentrations of 25% NaCl. In the same way, NaCl were added in the cultures containing 4% of salt to get a final concentration of 6% and 8% NaCl. Controls without salt were used in each experiments. Viable cells (corresponding to N) were enumerated as described previously. To calculate the ratio of surviving cells or the bacterial growth, the N/No ratio was calcu- lated. Each experiment was performed in duplicate.

3 – RESULTS AND DISCUSSION

3.1 Growth delay of several L. monocytogenes strains at different concentrations of salt

For the seven strains, elevated concentration of NaCl caused a concentration-dependent growth delay (Table 1). All strains exhibited a similar behaviour up to 8% NaCl. The growth delay varied at 9% NaCl in the range of 3 to 8 days for EGD and E strains, respectively. The LO28, the A and D strains were inhib- ited above 10% NaCl, as no growth was observed during the time of the experiment (11 days);other strains were more tolerant to such osmotic conditions. The C and E strains were able to grow in a medium containing 11% NaCl. These results are in good agreement with the literature which indicated that some strains are able to grow up to 12% NaCl (GALDIERO et al., 1997). The C and E strains were isolated from salted meat curing plant. This ecological niche could explain why these strains are able to grow at a higher NaCl concentration.

3.2 Growth and survival of L. monocytogenes LO28 at different salt concentrations

The L. monocytogenes LO28 strain was chosen, because this strain is well characterized (MICHEL and COSSART, 1992; MARRON et al., 1997), and has already shown adaptation mechanisms to various stresses (O'DRIS- COLL et al., 1996).

Table 1

Growth delay of the L. monocytogenes strains after addition of NaCl

Strains Growth delay (days)

0% NaCl 6% NaCl 7% NaCl 8% NaCl 9% NaCl 10% NaCl 11% NaCl

EGD 1 2 2 2 3 8 nd*

LO28 1 2 2 2 7 nd* nd*

A 1 2 2 2 7 nd* nd*

B 1 2 2 3 5 8 nd*

C 1 2 2 3 5 5 5

D 1 2 2 3 6 nd* nd*

E 1 2 2 2 8 8 8

*nd, no growth detected

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In this study, low concentrations of NaCl did not influence the growth of L. monocytogenes LO28, in media containing up to 3% NaCl (Figure 1). A slower growth was observed in concentrations ranging from 4% to 6% NaCl.

This observation is in agreement with previous studies (GALDIERO et al., 1997;

VASSEUR et al., 1999). When the concentrations of salt were above 8% NaCl, growth ceased. Exposure to high salt concentrations (6 to 25%) resulted in a decrease of cultivability (Figure 2), but bacteria were not immediately killed as already shown by Shahamat et al. (1980) who noticed that culturability of L. monocytogenes slowly decreased within 5 days when it was exposed in culture media containing 25.5% NaCl.

3.3 Adaptation treatments to salt of L. monocytogenes LO28

In order to show if a pretreatment at low salt concentration could increase the resistance of the L. monocytogenes LO28 strain to higher salt concentrations, two experiments were carried out (Table 2). In the first, bacteria were pretreated with a salt concentration (3.5% and 7% NaCl) decreasing the growth of the microorganism and then incubated in a more concentrated medium (25% NaCl) (Figure 3). The survival curves in a medium containing 25% NaCl were similar for all the conditions tested independently of the salt concentration during the pretreatment. In the second experiment, a 4% NaCl medium were used in the pretreatment, which were followed by treatments with 6 or 8% NaCl (Figure 4). All the growth curves at 6% and 8% of salt were similar, even if the cells were adapted to a medium containing 4% NaCl. In these two experiments, controls were carried out by a pre-treatment without the addition of salt before the treatment in a more concentrated salt medium. Some adaptation tests were realized in classical growth medium (BHI) but none of the conditions tested allow an

0.1 1 10

0 5 10

Time (h) OD600

Figure 1

Growth curves of L. monocytogenes LO28 in MCDB medium. Without additional salt ( ), with 2% ( ), 3% ( ), 4% ( ), 5% ( ) and 6% NaCl ( ) at 37°C

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increasing of the L. monocytogenes resistance to salt (data not shown). For these specific conditions of pretreatment, adaptation of L. monocytogenes to high salt concentrations was not observed, in contrast to other microorganisms. For instance, Enterococcus faecalis exhibited a higher survival at 28.5% NaCl, if the cells were subjected to a moderate salt shock (6.5% NaCl) (FLAHAUT et al., 1996). In the same way Bacillus subtilis was able to survive toxic concentration of NaCl (6%) by pretreatment with 2% NaCl (VÖLKER et al., 1992).

Table 2

The two experimental conditions applied to L. monocytogenes LO28 in MCDB medium,(Figure 3 and Figure 4).

Conditions Pretreatment (2 h) Final treatments

1 0% NaCl 0% NaCl

3.5% NaCl 7% NaCl 25% NaCl

3.5% NaCl 25% NaCl

7% NaCl 25% NaCl

2 0% NaCl 0% NaCl

4% NaCl 6% NaCl 8% NaCl

4% NaCl 6% NaCl

8% NaCl 7

8 9 10

0 2 4 6

Time (h) Log10 cfu ml-1

Figure 2

Survival curves of L. monocytogenes LO28 in MCDB medium. Without additional salt ( ) with 6% ( ), 8% ( ), 10% ( ), 22% ( ) and 25% NaCl ( ) at 37°C

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Time (h) Log10 (N/No)

-1.0 0.0 1.0 2.0

0 2 4 6

Figure 3

Survival of L. monocytogenes LO28 in MCDB medium. Without additional NaCl ( ), 25% NaCl ( ) and after pretreatment for 2 hours with 3.5% ( ) or 7% NaCl ( )

followed by treatment with 25% NaCl

-0.5 0.0 0.5 1.0 1.5

0 2 4 6

Time (h) Log10 (N/No)

Figure 4

Survival of L. monocytogenes LO28 in MCDB medium. Without additional NaCl ( ), 6% ( ), 8% NaCl ( ) and after pretreatment for 2 hours with 4% followed

by treatment with 6% ( ) and 8% NaCl ( )

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The resistance of L monocytogenes to salt is varying with the strains, some of them being able to grow in media containing 11% NaCl. In the case of the L. monocytogenes LO28 strain, a slower growth was observed above 4% NaCl and none of the pretreatments tested allowed an increase of this strain resistance to higher salt concentrations. However, a previous study has shown that the L. monocytogenes Scott A strain can adapt to salt and ensure a cross pro- tection against other lethal stresses. Thus, an exposure to 7% NaCl for 1 hour increased the resistance of L. monocytogenes to a lethal concentration of H₂O₂ (0.1%), and a pretreatment with 5% ethanol or heat shocking at 45°C for 1 hour increased its resistance to 25% NaCl (LOU and YOUSEF, 1997). Adaptation of this microorganism was only shown for cross protection experiments with the Scott A strain. Complementary studies are necessary to determine if the lack of adaptation after a pretreatment with low concentration of NaCl is strain dependent or due to the conditions tested.

ACKNOWLEDGMENTS

We thank Isabelle Lebert and Sabine Leroy-Setrin for critically reviewing the manuscript. We are grateful to Tania Ngapo for reading the manuscript. This work was supported in part by a grant from the “Région Auvergne”, France.

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