WO2004111263A1

WO2004111263A1 - Culture medium for detecting salmonella

Info

Publication number: WO2004111263A1
Application number: PCT/FR2004/001463
Authority: WO
Inventors: Ghislain Sanhaji; Anthony Bresin; Jérôme THEPAUT; Frédéric Simon
Original assignee: Agro Industrie Recherches Et Developpements (Ard); Aes Laboratoire
Priority date: 2003-06-11
Filing date: 2004-06-11
Publication date: 2004-12-23
Also published as: FR2856076B1; FR2856076A1

Abstract

The inventive culture medium consists of one type of polysaccharide, an organic nitrogen nutrient and water, said polysaccharide being provided with a given viscosity, the medium comprising a colour indicator and a selective agent which inhibits the growth of a strain other than salmonella-type bacteria. The inventive medium is suitable for detecting the presence of said species.

Description

Culture medium for salmonella research and method of use and preparation

Nowadays bacteria of the genus Salmonella are major pathogens. They are the cause of the majority of foodborne illnesses worldwide, and more particularly in Western countries. The main vector of infection is the ingestion of contaminated food. Salmonella can be present in all types of food with higher prevalence in egg products, dairy products and raw meat. The clinical symptoms of salmonellosis are acute diarrhea associated with significant dehydration which requires hospitalization. Salmonellosis can be fatal, especially for young children and the elderly.

Faced with this significant health risk, many countries have established regulations which prohibit the marketing of foodstuffs in which salmonella can be detected in a test sample of

25 g. Many diagnostic tools have also been developed to carry out this research.

Standardized methods can be summarized in 6 successive stages:

- a pre-enrichment of the test sample to be analyzed (16 to 20 hours)

- an enrichment phase in selective broths (24 to 48 hours)

- an isolation phase on selective agar (24 to 48 hours) - a purification phase (18 to 24 hours)

-a biochemical confirmation step (24 to 48 hours)

- a serological confirmation stage (24 to 48 hours)

Thus the release of batches of foodstuffs may require up to more than 10 days. This delay is highly penalizing for manufacturers in the food industry: blocking batches is costly because it requires large storage areas (sometimes at controlled temperature), the marketing time for the finished product is also reduced.

In the human health sector, response times are also long. They are very often ill-suited to the rapid implementation of appropriate antibiotic therapy. The efforts made by all stakeholders in the food chain have contributed to a significant reduction in the prevalence of batches of foodstuffs contaminated with salmonella at unacceptable levels (less than 5% in Western countries). Thus, analytical laboratories now need techniques that allow them to quickly release lots free of salmonella. Any reagent or any method which makes it possible to reduce the number of steps necessary to obtain the result or which makes it possible to reduce the time of one or more steps is fully in line with this requirement of saving time. Reagents thus make it possible to combine several stages. This is particularly the case for the semi-solid culture medium MSRV (Modified Semi Solid Rappaport Vassiliadis) or the DIASALM medium (Semi Solid Salmonella Medium Diagnosis). The principle of these two culture media is based on the mobility of more than 99% of salmonella in semi-solid medium. Made up of a selective base, they contain only a low level of the gelling agent universally used in analytical microbiology: agar-agar. Thus, the deposit of a fraction of the pre-enriched culture on the edge or the center of a Petri dish containing one of these media will contribute to the selective enrichment of salmonella (if the sample contains it) and to their migration in soft agar. After an incubation phase of 18 to 24 hours at an appropriate temperature (conventionally 42 ° C), the appearance of a typical migration arc is then synonymous with a suspicion of salmonella in the sample analyzed. The absence of a migration arc makes it possible to conclude that there is no salmonella in the sample and therefore authorizes the release of the batch. These two culture media are interesting for the user because they make it possible to rule on the non-contamination of a sample with salmonella within periods of between 40 and 48 hours.

However, given their low level of agar-agar gelling agent, they cause a high number of constraints for the user. They can therefore only be marketed in the form of a dehydrated base which must be reconstituted by the user. The marketing of these media in a petri dish is impossible given their low gel strength. The marketing in ready-to-use bottles is to be avoided because it requires a re-melting of the medium by the user before distribution in a Petri dish. Indeed, these environments are characterized by particularly low pH (between 5 and 5.5), thus repeated heat treatments contribute to hydrolyze the agar-agar, the gel strength finally obtained is no longer suitable (the medium becomes liquid).

Thus the only alternative for the user is to reconstitute the culture medium by himself. This operation, which consumes time, is not always easy to carry out. The final gel strength of the medium is very dependent on the process, the time and the heating temperature during the preparation. However, as the principle of these media is based on the mobility of salmonella, the reproducibility of the results obtained from one production of medium to another will be very strongly linked to the final gel strength and therefore to perfect control of the manufacturing conditions . Those skilled in the art will readily understand that these constraints and that the difficulty of obtaining media of a reproducible specified quality is poorly suited to the needs of the end user. It should also be noted that the same type of difficulty will be encountered with other conventional gelling agents such as agarose. The aim of the present invention is therefore to provide new semi-solid culture media, suitable for the culture and mobility of salmonella, which are characterized in that their rheological properties are compatible with presentations which can be marketed in ready form. for use (Petri dishes and / or flasks). The culture medium makes it possible to detect salmonella rapidly, in particular in less than 48 hours, and in a practical manner, the operator not having to prepare the petri dish, which can be supplied to the 'advance, ready to use, whereas before, he had to do it extemporaneously with the biases that this entails.

The subject of the invention is a culture medium comprising a polysaccharide of repeating unit, having the structure indicated below, a nutrient with organic nitrogen and water, characterized in that the polysaccharide has an apparent viscosity at 20 ° C. with a Brookfield viscometer, model DV II of module 4 at a speed of 6 between 7,000 and 50,000 mPa.s and the medium includes a colored indicator which turns at a pH between 6.4 and 8 and a selective agent inhibiting the growth of strains other than bacteria of the Salmonella species.

Preferably, the polysaccharide is present in an amount of 10 to 30 g / l of the medium, the selective agent in an amount of 5 to 50 mg / l of the medium; the nutrient at a rate of 2 to 20 g / l of the medium; a magnesium growth salt at a rate of 2 to 20 g / l, in particular magnesium chloride; a sodium salt for growth and increase in gel strength at a rate of 2 to 16 g / l of the medium, especially sodium chloride; and the nitrogenous nutrient is a peptone. The colored indicator is phenol red, methyl red, bromothymol blue, bromocresol blue. The medium comprises 0.1 to 5 g / l of the medium of another polysaccharide, which makes the medium more rigid and easier to transport.

The French patent published under No. 02 13 095 describes a polysaccharide allowing optimal bioavailability of the substrates in the culture media. We have demonstrated that, surprisingly, it also makes it possible to obtain culture media the gel strength of which is optimal for the mobility of salmonella and the elasticity of which is suitable for transporting the media ready for use (Petri dishes and / or flasks). This polysaccharide can be prepared, the chemical structure of which is as follows:

n: being an integer between 100 and 300, this polymer containing 7 sugars including 1 glucuronic acid (A), 3 glucose residues (B, C and D), 3 galactose residues (E, F and G), a pyruvate group and 1 to 3 acetate groups not localized by a process in which a heat-treated native polymer can be obtained in water, at a concentration of between 1 and 40 g per liter, which can be obtained by cultivating a bacterial microorganism deposited under the number 1-1809 at the CNCM at the Institut Pasteur on a medium rich in glucose and nitrogenous matter to obtain a fermentation must, by diluting the fermentation must between 1 and 1/20 by volume with water and heating it between 70 and 140 ^° C, then centrifuging or subjecting it to tangential filtration to obtain a supernatant, subjecting the supernatant to filtration at 0.2 μm to obtain a filtrate, concentrating the filtrate and bringing it into contact with ethanol to obtain a precipitate , separating the precipitate and drying it.

At the end of this treatment, the analysis of different industrial batches of this precipitate is carried out to define the molecular mass of the poiysaccharide of the invention. The determination of this molar mass is carried out by gel permeation chromatography on a solution containing the poiysaccharide of the invention (Temperature 30 ^° C.; Eluent: 0.1 M NaNO3; 2 Shodex OH pack columns; SEC equipment with triple detection with : Waters Alliance 2000 V (USA) associated with a Wyatt light scattering device (USA).

The poiysaccharide of the invention obtained according to the process described above has a molar mass of between 3.50.10 ⁵ Da and 2.05.10 ⁵ Da which corresponds to a number of monomers between 150 and 265.

Table 1: Determination of the molar masses of different industrial batches of polymer of the invention.

Advantageously, culture media whose gel strength is optimal for the mobility of salmonella and whose elasticity is suitable for transport include polysaccharide concentrations of between 2 and 30 g / l. They also include the components necessary to obtain the fertility and selectivity properties expected for the cultivation of salmonella.

By way of nonlimiting examples, the additional components may be nutritive agents (carbohydrates, peptones, yeast extracts, casein meat, etc.), selective agents (malachite green, selenite, magnesium chloride, tergitol, etc.). ), pH indicators or chromogenic substrates or colored indicators (phenol red, methyl red, bromothymol blue, bromocresol blue ...). The invention also relates to a process for preparing a medium according to the invention characterized in that the constituents are mixed with the exception of the selective agent in any order when the nutrient does not induce an increase in the pH above 7, but adding the polysaccharide only after having adjusted the pH of the aqueous solution containing a nutrient inducing said increase in pH to a value of 5.5 ± 0.2, then the solution is sterilized containing the mixed components by heating it at least at 100 ° C for at least ten minutes, preferably at least at

110 ° C for 15 minutes, allowed to cool and one of the selective agents is added.

Finally, the invention relates to a method for detecting the presence of the genus Salmonella in a foodstuff or in body tissue constituting a material to be analyzed by doing:

- incubate in an aqueous sterile culture broth containing a nutrient with organic nitrogen buffered to a pH of 7.2 ± 0.2, a sample of at least 5 g and preferably at least 25 g of the material to be analyzed , the amount of broth being between 1 and 10,000 times the volume of the sample for at least 16 hours, between 20 and 45 ° C and, preferably, at 37 ° C to obtain a pre-enriched sample, - deposit in a or several deposition points, preferably at the edge of the dish, at least 50 microliters of pre-enriched sample over less than 2.5% of the surface of a medium according to the invention contained in a Petri dish,

- incubate the Petri dish at a temperature between 20 and 50 ° C, and preferably at 41 ° C, between 12 and 24 hours, and

- one observes the presence or not of a halo of a different coloring from that of the initial medium coupled with an opacification of the medium, of a length, defined by the distance between the point of deposition and the point of the most distant halo from the deposition point greater than 2 cm, the absence of this halo indicating the absence of Salmonella bacteria in the material to be analyzed.

Other characteristics of the media according to the invention will appear in the light of the examples below.

The sample of material to be analyzed can be prepared as follows:

- take 25 grams of the potentially contaminated sample, - add 225 ml of buffered peptone water,

- incubate 16 to 20 hours at 37 ⁰ C +/- 1 ⁰ C - deposit 3 deposits of 0.1 ml of the pre-enriched medium previously in a triangle on the edges of the Petri dish containing the medium of the invention,

- incubate the dish 12 to 24 hours in the place in an oven at 41 ⁰ C +/- 1 ⁰ C. This eliminates bacteria other than salmonella and the agar is made softer, favoring the mobility of salmonella. read the result: o Presence of a red halo and / or clouding with a diameter greater than 2cm => presumptive presence of salmonella o Presence of a halo less than 2 cm ≈> absence of salmonella As it has As said previously, the present invention also relates to a process for obtaining this culture medium, according to which: a) the nutrients, the colored indicator and the selective elements are mixed; b) The mixture thus obtained is dissolved by adding sufficient water to meet the recommendations for the medium in question; c) The pH is adjusted to 5.5 +/- 0.2; d) The polysaccharide (2 to 30 g / 1) is added to this mixture; e) The whole is sterilized at 110 ^° C.-15 minutes; f) The other selective thermolabile elements are added; g) The sterilized product is poured into ad hoc containers (petri dish, tube, flask, etc.) at 55 ^° C. h) The mixture is left to stand for 24 hours between 2 and 8 ^° C. The embodiments which follow and which are in no way limiting, will enable those skilled in the art to better understand the present invention and its numerous advantages.

EXAMPLE 1 Impact of the Polvsaccharide on the Salmonella Migration

Preparation of culture media: Medium 1:

- Peptones: 10 g / l

- NaCI: 8 g / l

- Malachite green: 0.04 g / l

- Magnesium chloride: 12 g / l - Novobiocin *: 20 mg / l

- Polysaccharide (viscosity: 7000 <n <50,000 mPa.s) * Thermolabile compound

Middle 2:

Medium 2 is again identical to the previous ones, but the polysaccharide used has a longer chain length n> 50,000 mPa.s

Middle 3:

This medium will be the semi-solid agar formulated according to Rappaport and Vassiliadis (MSRV: AES Laboratoire; AEB140672)

100 μl of a bacterial inoculum (prepared from a saturated brain-heart broth) are placed on the edge of the dishes and incubated for 12 to 24 hours at 42 ° C.

The growth of Salmonella will be represented by the presence of a characteristic migration arc.

The results obtained for Salmonella and interfering strains are collated in the table below:

TABLE 1

+: migration arc> 2 cm -: migration arc z 2 cm

On the media comprising the polysaccharide, only the n ° 1 for which 7000 <n <50,000 allows growth of Salmonella, characteristic by their mobility. This highlights the influence of the viscosity of the medium on the mobility of these bacteria.

The migration of salmonella on the MSRV (medium 4) is identical to medium 1. Example 2: Influence of the specific substrate / colored indicator pair on the differential diagnosis of Salmonella

Middle 1:

The composition of this medium is identical to that of medium 1 of Example 1. Medium 2:

The composition of the medium is identical to that of medium 1 of Example 1 with in addition a colored indicator (phenol red) as well as a substrate specifically degraded by Salmonella, such as for example Lysine. The degradation of lysine by Lysine decarboxylase from salmonella will cause alkalization of the medium and a change of indicator from green to red.

Middle 3:

This medium is the MSRV. The same strains as in the previous example were tested and the results are presented in the table below:

TABLE 2

D: Differentiation M: Migration

+: change of color indicator or migration of seeded germs (> 2 cm)

-: no specific differentiation or no migration

The results of Table 2 show that the addition of a substrate degraded specifically by salmonella coupled with a colored indicator allows a visualization as well as a very easy differentiation of the migration of salmonella in the medium:

On the MSRV, the differentiation is not based on the change of a colored indicator, the migration of salmonella is characterized by the presence of a non-specific white halo.

Example 3: Resistance to transport conditions:

Middle 1:

This medium is identical to medium 1 of Example 2

Middle 2:

This medium is identical to medium 1 supplemented with 0.5 g / l of Agar agar. Middle 3:

This medium is identical to medium 1 with substitution of the polysaccharide by PAgar Agar at 2.7 g / l.

Medium 4: This medium is the MSRV.

In this example, the interaction of the agar with the polysaccharide is studied, as well as the effect of the Agar alone in the medium and their influences on the migration of salmonella as well as on the behavior of the medium in Petri dishes. This boxed outfit represents the environment's ability to withstand unfavorable transport conditions.

In order to simulate these conditions, we subjected the media to mechanical stress:

- The “shock” constraint consists in dropping the box from a height of one meter on a solid surface and observing the appearance of the agar.

On the other hand, the boxes were kept vertically overnight, so as to simulate an overturning of the boxes during transport and the appearance of the agar was controlled. The results are summarized in the two tables below:

TABLE 3

+: Migration> 2 cm -: Migration <2cm

TABLE 4

+: Agar intact -: Reversing the agar

The results in Table 3 demonstrate that the supplementation of the medium with Agar agar or the use of PAgar alone in the medium at the concentrations used has no influence on the mobility of salmonella in the medium.

However, as the results in Table 4 show, only the Agar / polysaccharide interaction allows the medium to resist the "Shock" and "Reversal" stress.

In addition, it can be seen that the transport of the MSRV in petri dishes is not possible given its inability to withstand various mechanical stresses. Example 4: Validation of the performance of the medium

The invention was compared, during an AFNOR validation according to the ISO 16140 standard, to the reference method for the search for Salmonella (standard NF EN ISO 6579). The validation, whose attestation number is AES 10 / 04-05 / 04, covers all human and animal food samples, as well as environmental samples.

The main results of this AFNOR validation according to I ¹ ISO 16140 are summarized below: PRACTICABILITY: α Real time handling and flexibility of the technique:

α Time for obtaining results First case: born samples

*: identification of the 1 st day on D6, then 48 additional hours for the other colonies

*: only if the MUCAP test is negative

The SMS medium makes it possible to obtain shorter results times than those obtained by the reference method. 1NCLUSIVITY AND EXCLUSIVITY OF THE ALTERNATIVE METHOD:

The objective of this step is to ensure that all strains of Salmonella are detected by the SMS test (inclusiveness) and that there is no cross-reaction with non-Salmonella strains. Fifty-five strains of Salmonella and thirty strains of non-Salmonella were tested.

No cross-reactions were detected with the non-target strains. The serovars gallinarum and paratyphi A were not detected by the SMS test. The first serovar being immobile and the second having little or no lysine decarboxylase activity. LEVEL OF DETECTION RELATING TO THE ALTERNATIVE AND REFERENCE METHOD:

The objective is to determine the minimum concentration of salmonella sp detectable in a food by the two methods.

METHODS Fifty neck food - strain of Salmonella were tested:

*: CFU / mL or CFU / g

Four contamination levels (1: 0 cell / 25 mL or g, 2: 1 cell / 25 mL or g, 3: 3 cells / 25 mL or g and 4: 9 cells / 25 mL or g) have have been tested for each food - strain pair. Six replications were carried out for each modality after the pre-enrichment. CONCLUSION:

The relative detection levels obtained for the alternative method are identical to those obtained for the reference method regardless of the strain / food matrix couple tested.

This relative detection level is between [0-1] and [0-2] cells per 25 ml or g of product.

RELATIVE ACCURACY. RELATIVE SPECIFICITY AND RELATIVE SENSITIVITY OF THE ALTERNATIVE METHOD AND THE REFERENCE METHOD:

The objective of this study is to assess the performance of the two methods on contaminated or uncontaminated samples. The analyzes are carried out in simple by the two methods and the samples are distributed in the main categories of food products.

RESULTS:

PA: Positive agreement (Positive Reference method / Positive SMS method) 0 NA: Negative agreement (Negative Reference method / Negative SMS method)

ND: Negative deviation (Positive Reference method / Negative SMS method)

PD: Positive deviation (Negative Reference Method / Positive SMS Method)

N: Total number of samples 5 N +: Number of samples found positive with the reference method

N-: Number of samples found negative with the. reference method

ER: Relative accuracy: (PA + NA) / N x 100%, i.e. the agreement between reference method and SMS. SeR: Relative sensitivity: (PA / N +) x 100%, i.e. the ability of the SMS to declare a sample positive when it came out positive by the reference method.

SpR: Relative specificity: (NA / N-) x 100%, ie the ability of the SMS to declare a sample negative when it came out negative by the reference method. CONCLUSION:

During this study, a Salmonella positive meat sample was not detected by the SMS method. On the other hand, the SMS method produced three additional confirmed positives (one seafood and two dairy products) compared to the reference method. COLLABORATIVE STUDY:

The purpose of the collaborative study is to determine the variability of the results obtained in different laboratories analyzing identical samples and to compare these results within the framework of the comparative study of methods.

Twenty-four samples contaminated with Salmonella according to three levels of contamination were prepared by the expert laboratory and sent to fourteen collaborating laboratories for analysis by the SMS method and the reference method. CONCLUSION:

During the collaborative study, the SMS method demonstrated a sensitivity and specificity identical to the reference method as well as a relative accuracy of 100% for the three contamination levels. On the other hand, the variability of the SMS method (degree of agreement, agreement and odds ratio) is identical to that of the reference method.

The SMS or alternative method is the method according to the invention.

Claims

1. Culture medium comprising a polysaccharide of repeating unit, the chemical structure of which is as follows:

n: being an integer between 100 and 300, this polymer containing 7 sugars including 1 glucuronic acid (A), 3 glucose residues (B, C and D), 3 galactose residues (E, F and G), a pyruvate group and 1 to 3 non-localized acetate groups, an organic nitrogen and water nutrient, characterized in that the polysaccharide has an apparent viscosity at 20 ° C. with the Brookfield viscometer of model DV II of module 4 at a speed of 6 inclusive , between 7,000 and 50,000 mPa.s and the medium comprises a colored indicator which turns at a pH of between 6.4 and 8 and a selective agent inhibiting the growth of strains other than bacteria of the Salmonella species.

2. Medium according to claim 1, characterized in that the polysaccharide is present in an amount of 10 to 30 g / l of the medium.

3. Medium according to claim 1 or 2, characterized in that the selective agents are present in an amount of 5 to 50 mg / l of the medium.

4. Medium according to one of claims l to 3, characterized in that the nitrogenous nutrient is present in an amount of 2 to 20 g / l of the medium.

5. Medium according to one of claims 1 to 4, characterized in that the medium comprises a magnesium growth salt in an amount of 2 to 20 g / l, in particular magnesium chloride.

6. Medium according to one of claims 1 to 5, characterized in that the medium comprises a sodium salt for growth and for increasing the gel strength at a rate of 2 to 16 g / l of the medium, in particular chloride sodium. 7. Medium according to one of claims 1 to 6, characterized in that the nitrogenous nutrient is a peptone

8. Medium according to one of the preceding claims, characterized in that the colored indicator is phenol red, methyl red, bromothymol blue or bromocresol blue. 9. Medium according to one of claims 1 to 8, characterized in that it comprises from 0.1 to 5 g / l of the medium of another polysaccharide. 10. Method for detecting the presence of bacteria of the genus Salmonella in a foodstuff or in a body tissue constituting a material to be analyzed, characterized in that: - incubation is carried out in an aqueous broth of sterile culture containing a nutrient containing organic nitrogen buffered to a pH of 7.2 ± 0.2, a sample of at least 5 g and preferably at least 25 g of the material to be analyzed, the amount of broth being between 1 and 10,000 per volume of the sample for at least 16 hours, between 20 and 45 ° C and, preferably, at 37 ° C to obtain a pre-enriched sample,

at least 50 microliters of pre-enriched sample are deposited at one or three deposition points, preferably at the edge of the box, on less than 2.5% of the surface of a medium according to claims 1 to 8 contained in a petri dish, - the petri dish is incubated at a temperature between 20 and

50 ° C, and preferably 41 ° C, between 12 and 24 hours, and

11. Method for preparing a medium according to claims 1 to 9, characterized in that the constituents are mixed with the exception of the selective agent in any order when the nutrient does not induce an increase in pH above 7, but adding the polysaccharide only after adjusting the pH of the aqueous solution containing a nutrient inducing said increase in pH to a value of 5.5 ± 0.2, then the solution containing the mixed constituents is sterilized by heating it at least to 100 ° C for at least ten minutes, preferably at least to 110 ° C for 15 minutes, it is allowed to cool and the selective agent is added.