EP2205976A2

EP2205976A2 - Biochemical test for confirming the presence of<i>l. monocytogenes</i>

Info

Publication number: EP2205976A2
Application number: EP08844892A
Authority: EP
Inventors: Bernadette Blanc; Daniel Monget; Nadine Perrot
Original assignee: Biomerieux SA
Current assignee: Biomerieux SA
Priority date: 2007-10-30
Filing date: 2008-10-29
Publication date: 2010-07-14
Also published as: US20100240091A1; CN101842706A; FR2922896B1; FR2922896A1; JP2011500097A; WO2009056762A3; WO2009056762A2

Abstract

The invention relates to a biochemical test for confirming the presence of L. monocytogenes, characterised in that it comprises at least one phosphatidylinositol phospholipase C (PIPLC) substrate and at least one alpha-mannosidase substrate.

Description

Biochemical test to confirm the presence of L. monocytogenes

The present invention relates to the identification of pathogenic bacteria of the genus Listeria, and more specifically of the species Listeria monocytogenes.

The isolation and identification of the bacterium Listeria monocytogenes is a major problem in the surveillance of agro-food hygiene and medical bacteriology. Of the Listeria bacteria, only Listeria monocytogenes is known to be pathogenic to humans. It can cause listeriosis, sometimes fatal (25 to 30% of cases) in immunocompromised people, young children or pregnant women. Other Listeria species are not pathogenic or are only for animals. This is particularly the case of Listeria ivanovii.

Even though the risk of human listeriosis has declined in most developed countries in recent decades, modern society demands more and more security, which, if accommodated sporadically, can not tolerate epidemics.

In the diagnosis of bacterial diseases in humans, it is important to clearly distinguish Listeria monocytogenes from other species of the genus Listeria sp. which are not pathogenic. The genus Listeria sp now comprises six species, of which only Listeria monocytogenes is pathogenic for humans.

Germs are the cause of severe epidemics in humans, as a result of food contamination. In foods (mainly milk and milk products), two species of Listeria are mainly isolated: nonpathogenic Listeria innocua and pathogenic Listeria monocytogenes. These two species have many common biochemical characters, so it is difficult to differentiate them. The beta-hemolysis test, for example, is based on the determination of a beta-hemolytic activity related to the production by the bacterium of a substance that causes lysis of red blood cells (listeriolysin). This test is performed on sheep or horse blood agar, but the answer is often discreet. This test is therefore unreliable and if it makes it possible to differentiate the two species mainly encountered, ie L. mono cyto genes (positive response) of L. innocua (negative response), it is not specific for the pathogenic species.

The CAMP-test is another test, which is associated with the test of beta-hemolysis previously described. The test is carried out on tryptic soy agar, containing 5% of washed red blood cells. The beta-hemolytic strain of S. aureus CIP 5710 is inoculated into a streak perpendicular to the streaks formed by the culture of the Listeria strain to be tested. The reinforcement of the hemolysis of staphylococcus in contact with the two zones indicates a positive reaction. In practice, the CAMP-test is a heavy technique to implement, and, like hemolysis, is not always very reliable. It is also possible to use culture media or identification media. In this respect, patent EP496680, issued to the applicant, describes a bacteriological analysis method to differentiate the species Listeria monocytogenes from other bacteria of the genus Listeria sp. According to this method, a reaction medium comprising a chromogenic or fluoro -ogenic substrate capable of being hydrolyzed by an enzyme called Glycine aminopeptidase is used. This very interesting technique has the disadvantage that the species Listeria monocytogenes is the only one that does not have enzymatic Glycine aminopeptidase activity. The detection of Listeria monocytogenes is carried out by a negative activity, which is not easy, the use of a negative test lacking specificity in case of mutant or if the other species are stressed and do not respond with normal activity .

Listeria monocytogenes and Listeria ivanovii species can be differentiated from other Listeria species by demonstrating phospholipase C activity specific for phosphatidylinositol (PI-PLC). Indeed, it has been demonstrated that PI-PLC is secreted into the culture medium by certain species of the genus Listeria such as Listeria monocytogenes and Listeria ivanovii (Leimeister-Wachter et al., Mol Microbiol. (1991) 5 (2)). ), pp. 361-366). As such, the Ottaviani Agosti Agar medium is a chromogenic medium that uses on the one hand a β-Glucosidase substrate that allows the detection of all species of the genus Listeria and on the other hand the highlighting of PI-PLC activity. of Listeria monocytogenes and Listeria ivanovii by the appearance of a halo around the colony. Whatever the technique used, it is necessary to carry out systematically a confirmation test of Listeria monocytogenes, generally implementing a second culture medium, for which a 24h incubation step is necessary. These include the ALOAConfirmation (AES) confirmation tests, or CHROMagar Identification listeria (BBL). In total, a duration of 48 hours is therefore necessary to identify and confirm the presence of Listeria monocytogenes: use of a first culture medium, which requires 24 hours of incubation, then the second confirmation step which also requires a 24h incubation . This duration of 48 hours is long, and it is desirable to shorten this duration to provide a reliable diagnosis as quickly as possible.

The present invention aims to provide a detection method that allows the differentiation of Listeria monocytogenes species compared to all other species of Listeria spp. In particular, the invention proposes a new confirmatory test, which is a very rapid biochemical test since it can be implemented in less than 6 hours.

Before going further in the description of the invention, the definitions below are given to facilitate the disclosure of the invention.

By biochemical test, we mean a test allowing the implementation of a biochemical reaction highlighting the presence of bacteria. Such a test may in particular be implemented directly from an inoculum, without implementing a growth step. It is then the preformed enzymes provided by the bacterial suspension that are detected.

Such tests are notably carried out in the API® range or the Vitek 2 system. These tests, packaged in dehydrated form, are brought into contact, under incubation, with the sample to be analyzed. The desired biochemical reaction is detected by a change in the state of the test (appearance disappearance of a coloration, a fluorescence).

The reading of the test is then done visually or with the aid of a reader, directly or after the addition of a developer reagent. The API® range is based on a probabilistic numerical identification method, associating a biochemical test gallery, in cups and a database. It is the judicious combination of tests that ensures the characterization of a group of germs and the distinction of species between them. The VITEK 2 system is a fully automated identification and antibiogram system that implements a 64 micro-well card. By eliminating manual steps, automation saves lab time and ensures consistent analysis procedures that improve the reliability of results. The identification performed on this system uses a probabilistic method similar to that of API galleries. The automation of the reading allows a kinetic reading of the biochemical tests implemented and thus allows an identification in 4 to 8 hours for the current Gram positive germs as well as for Listeria monocytogenes.

The biochemical test according to the invention is a confirmation test for the presence of L monocytogenes, comprising a substrate of PI-PLC and a substrate of α-mannosidase, but may also comprise inorganic salts, peptones, carbohydrates, one or more compounds limiting pH variations, one or more enzyme inducers.

By substrate is meant any molecule capable of generating directly or indirectly a detectable signal due to an enzymatic or metabolic activity of the microorganism.

The substrate may in particular be an enzymatic substrate, that is to say a substrate that can be hydrolyzed by an enzyme into a product allowing the direct or indirect detection of a microorganism.

For the purposes of the present invention, this substrate is in particular a phosphatidyl inositol phospholipase C substrate (PIPLC) or an α-mannosidase substrate.

This substrate may comprise in particular a first specific part of the enzymatic activity to be revealed and a second part serving as a marker, hereinafter referred to as a marker part. This marker part may be chromogenic, fluorogenic, luminescent ... preferably, the substrates used in the present invention are chromogenic. There may be mentioned in particular substrates based on indoxyl and its derivatives, particularly suitable in the case of phosphatidyl inositol phospholipase C (PI-PLC) substrates, substrates based on nitrophenol and derivatives, particularly suitable in the case of substrates of α-mannosidase, but also the substrates based on hydroxyquinoline or esculetin or alizarin or catechol or aminophenol and naphthol and their derivatives For the fluorogenic markers, mention may be made of coumarin derivatives and especially of umbelliferone, naphthol, resorufin, fluorescein According to the present invention, the substrate is preferably chosen from substrates based on: α indoxyl (3-Indoxyl, 5-Bromo-3-indoxyl, 4-Chloro-3- indoxyl, 5-iodo-3-indoxyl, 5-bromo-4-chloro-3-indoxyl, 5-bromo-6-chloro-3-indoxyl, 6-bromo-3-indoxyl, 6-chloro-3-indoxyl, 6-Fluoro-3-indoxyl, 4,6-Dichloro-3-indoxyl, 6,7-Dichloro-3-indoxyl, 4,6,7-Trichloro-3-indoxyl, 5-Bromo-4-chloro Nitrophenol N-methyl-3-indoxyl, N-methyl-3-indoxyl ...) α (ortho-nitrophenol, para-nitrophenol, etc.)

Preferably, the PIPLC substrate is a chromogenic substrate. Preferably, the PIPLC substrate is an indolyl-phosphatidyl-myo-inositol, preferentially bromo 4-chloro-3-indoxyl myoinositol phosphate (X-indolyl-phosphatidyl-myo-inositol). Preferably, the α-mannosidase substrate is a chromogenic substrate or fluorogenic. Preferentially, the α-mannosidase substrate is a chromogenic substrate. Preferentially, the α-mannosidase substrate is a mannopyranosidase substrate. Preferably, the mannopyranosidase substrate is nitrophenyl-4-αD mannopyrano side. Preferably, the substrate concentration of PI-PLC is between 5 and 0.1 g / l, preferably between 1.5 and 0.5 g / l.

Preferentially, the concentration of α-mannosidase substrate is between 2 and 0.1 g / l, preferably between 1 and 0.5 g / l. By reaction medium is meant a medium comprising all the elements necessary for the expression of a metabolism and / or the growth of microorganisms. The environment The reaction may be solid, semi-solid or liquid. By solid medium is meant for example a gelled medium. Agar is the traditional gelling agent in microbiology for the cultivation of microorganisms, but it is possible to use gelatin or agarose. A number of preparations are commercially available, for example Columbia agar, Trypcase-soy agar, Mac Conkey agar, Sabouraud agar or more generally those described in the Handbook of Microbiological Media (CRC Press).

The reaction medium may comprise one or more elements in combination, such as amino acids, peptones, carbohydrates, nucleotides, minerals, vitamins, active molecules such as antibiotics, enzymes, surfactants, buffers, phosphate, ammonium, sodium, metal salts, one or more substrates for the detection of enzymatic or metabolic activity ... The medium may also comprise a dye. By way of indication, mention may be made, as dye, of Evans blue, neutral red, sheep blood, horse blood, an opacifier such as titanium oxide, nitroaniline, malachite green, brilliant green ...

For the detection of the genus Listeria, mention may be made in particular of the Palcam and Oxford media, conventional selective media frequently used in industry and cited in the international standards ISO 11290-1 and 11290-2 for the detection and enumeration of Listeria monocytogenes, or chromogenic media. such as the OAA medium which in addition to the detection allows the presumptive identification of L. monocytogenes By biological sample, is meant a clinical sample, from a biological fluid sample or a food sample, from any type of food . This sample may thus be liquid or solid and may be mentioned in a nonlimiting manner, a clinical sample of blood, cerebrospinal fluid, placenta, a food sample of water, drinks such as milk, a fruit juice. ; yoghurt, meat, eggs, vegetables, mayonnaise, cheese, fish, a food sample from a feed intended for animals. In this respect, the invention relates to a biochemical test for confirming the presence of L. monocytogenes, characterized in that it comprises at least one phosphatidyl inositol phospholipase C substrate (PIPLC) and at least one α-mannosidase substrate.

According to a preferred embodiment of the invention, the PIPLC substrate is an indolylphosphatidyl-myo-inositol, preferentially bromo 4 chloro-3 indoxyl myoinositol phosphate.

According to a preferred embodiment of the invention, the alpha mannosidase substrate is a mannopyranosidase substrate, preferentially nitrophenyl-4-αD mannopyrano side. According to a preferred embodiment of the invention, the test further comprises an activator of PIPLC, or several such as in particular alpha-glycerophosphate of

Magnesium, Serum Bovine Albumin.

According to a preferred embodiment of the invention, the concentration of substrate of

PLC is between 5 and 0.1 g / l, preferably between 1.5 and 0.5 g / l. According to a preferred embodiment of the invention, the concentration of α-mannosidase substrate is between 2 and 0.1 g / l, preferably between 1 and 0.5 g / l.

The invention also relates to the use of a biochemical test as described above, ie a biochemical confirmation test comprising a phosphatidyl inositol phospholipase C substrate (PIPLC) and at least one alpha mannosidase substrate, such as previously defined, to confirm the presence of L. monocytogenes.

The test according to the invention is particularly suitable for unitary use or combined with a series of biochemical tests so as to give rise to an identification device API® range or Vitek® system. Currently the identification of L monocytogenes on API or Vitek system uses a reaction profile established by the concatenation of positive or negative responses of several unit biochemical tests. The test according to the invention may advantageously be integrated with such devices so as to reduce the number of tests. The invention also relates to a method of identifying Listeria monocytogenes, characterized in that it comprises the following steps: a) having a reaction medium for detecting the genus Listeria, b) seeding the medium with a biological sample to be tested (c) incubating (d) revealing the presence of the Listeria genus; (e) confirming the presence of L monocytogenes by a biochemical test according to the invention, comprising a phosphatidyl inositol phospholipase C (PIPLC) substrate and at least one alpha mannosidase substrate, as defined above.

The seeding of the microorganisms can be carried out by all the seeding techniques known to those skilled in the art. An incubation step can be carried out at a temperature for which the enzymatic activity that one wishes to detect is optimal, that the person skilled in the art can easily choose according to the enzymatic activity to be detected. Step d) can be carried out by visual examination, colorimetry or fluorimetry.

To detect the genus Listeria, there may be mentioned OAA medium (bioMerieux, ref 43641) which allows the detection of species of the genus Listeria by colony blue staining (beta-glucosidase activity).

The invention also relates to a method for identifying Listeria monocytogenes, characterized in that it comprises the following steps: a) having a biochemical test or a combination of biochemical tests to detect the genus Listeria, b) revealing the presence of the Listeria genus c) confirming the presence of L monocytogenes by a biochemical test according to the invention, comprising a phosphatidyl inositol phospholipase C substrate (PIPLC) and at least one alpha mannosidase substrate, as defined above. For the detection of the genus Listeria, mention may be made in particular of the morphology and the Gram reaction which is commonly associated with the esculin test, catalase and sugar fermentation tests such as Xylose, Rhamnose, mannitol, ribose, α-methyl-D-mannoside. .

The examples below are given for explanatory purposes and have no limiting character. They will help to better understand the invention.

Example 1

A biochemical test according to the invention A biochemical test according to the invention comprises the following elements in g / 1:

Tris buffer 37.5

Peptone extract 2.5 yeast extract 0.25 alpha-glycerophosphate Mg 1.25 Serum bovine albumin 7.5

X-myo-inositol 1 phosphate 2

4-nitrophenyl-α D-mannopyranoside 2,25

pH = 7 in final. After depositing 20 μl of solution in a plastic support, the test was dehydrated at a temperature of 40 ^° C. for 24 hours.

The test was evaluated to obtain a reaction in 4 to 6 hours at 37 ^° C. +/- 20 ^° C. after rehydration with 50 μl of demineralized water and addition of a colony or rehydration with 50 μl of a bacterial suspension. opacity equivalent to McF 0.5.

B) Reading and interpretation of the test

After 4 and 6 hours of incubation of the test at 37 ^° C. +/- 10 ^° C., the test staining is read and interpreted according to the following grid:

C) Validation of the test according to the invention

The test according to the invention was evaluated, initially using an inoculum of 0.5 McFarland, on 90 pure strains of the genus Listeria previously grown on OAA medium (Ref 43641). The 6 species of the genus Listeria sp were distributed as follows: L. monocytogenes (30 strains), L. ivanovii (20) (including 10 strains of L. ivanovii spp londoniensis and L. ivanovii spp ivanovii subspecies), L. grayii (10), L. seeligeri (10), L. welshimeri (10), L. innocua (10). 4H, 80% of L. monocytogenes strains with a halo on OAA medium (25/25) revealed a positive green color. After 6 hours of incubation all these strains had the expected green color. After 24 hours the reaction is stable and the remainder up to 72 hours incubation at 37 ± 0.2 ° C. None of the 60 strains belonging to species other than L monocytogenes gave a green color. The test therefore has a sensitivity of 100% in 6H and a specificity of 100% in the genus Listeria sp. The performance of the test according to the invention was also evaluated for an inoculum of a colony from pure strains or strains derived from food matrix. In the latter case the matrices were studied after pre-enrichment in Fraser Vi broth as recommended by the AFNOR short protocol (BIO 12-14). This step allows a revitalization of bacterial strains that may have undergone stress related to the treatment of the matrix.

Study on pure strains

Fifty strains of L. monocytogenes cultured 24 h at 35 ⁰ C ± 2 ⁰ C on OAA medium were inoculated in an amount of a colony in the biochemical assay according to the invention rehydrated with 50 .mu.l of sterile water. The test incubated at 35 ⁰ ± 2 was positive in 4H for 94% of strains that had a characteristic colony on OAA medium (blue colony with halo). After 6 hours, all L. monocytogenes strains were detected positive (green) on the biochemical test according to the invention. Matrix study

The search for L. monocytogenes on 22 food matrices was carried out following the AFNOR short protocol (BIO 12-14), ie: 25 g of matrix / stomacher, 1/2 Fraser enrichment (24H-30 ° C), After this phase fortification 0.1 ml of Fraser broth 1/2 are inoculated on OAA medium. The medium is then incubated for 24 hours at 35 ± 20 ^° C. Each of the media is checked and when a characteristic characteristic L. monocytogenes colony (blue with halo) is observed, this colony is taken for inoculation of the test according to the invention. This one is rehydrated beforehand with 50 μl of suspension medium (70,700). Of the 22 matrices tested, 11 showed bacterial growth on OAA medium after 24 hours. Six had characteristic colonies of L. monocytogenes

(blue with halo).

The test according to the invention confirmed the belonging of the 6 presumed strains, to the species

L. monocytogenes in 4H. The L. monocytogenes identification of the 6 strains was confirmed by other identification tools (API Listeria gallery, Vitek GP maps and 16S sequencing).

The other 5 matrices were contaminated with Listeria spp. Other than L. monocytogenes or other germs. In all these cases, the test according to the invention implemented on a colony of each of these matrices gave a negative response.

Example 2

The test according to the invention was also validated on 250 pure strains tested with the following distribution:

• L. monocytogenes 150 strains of serotypes and varied origins. • Listeria sp 50 strains: L. grayi 1, L. innocua 11, L. ivanovii 26 (including L. ivanovii spp ivanovii 6, LΛvanovii spp londoniensis 7), L. seeligeri 5, L. welshimeri 7.

• Other genera 50 strains: Bacillus, Lactobacillus, Staphylococci, Enterococci. The strains used are either strains of international collections or isolated strains of food matrices or environment. The identification of these strains was established by the laboratory prior to the study, and is considered the reference identification. The Listeria strains have been identified by API Listeria identification gallery.

Study on Pure Strains

The results obtained on pure strains are presented below.

From OAA agar plates incubated 24H at 37 ⁰ C, 149 strains of Listeria monocytogenes, out of 150 tested, are confirmed by the test according to the invention from 6h. The prolongation of the incubation until 24h does not modify the result.

The 50 strains of Listeria sp developed on OAA medium.

Only 26 strains of L. ivanovii showed characteristic colonies on agar

OAA. All of these isolates tested by the test according to the invention gave a negative result in 6h and 24h.

Of the 50 strains tested, 12 developed on OAA medium without characteristic colonies. The test according to the invention carried out on these isolates is negative.

Reading the test after 4 hours of incubation allowed the detection of 148 isolates. It is therefore possible with the test according to the invention to make a positive result (green) from 4h. A negative result can be returned as early as 6am. The results were unchanged after 48h of culture in OAA medium.

Comparable results were obtained on culture media other than OAA (TSA media (trypcase soy agar and blood agar) .The test according to the invention allowed the confirmation of the presence of L monocytogenes at 6h.

Claims

1) A biochemical test for confirming the presence of L. monocytogenes, characterized in that it comprises at least one phosphatidyl inositol phospholipase C substrate (PIPLC) and at least one alpha mannosidase substrate.

2) A biochemical assay according to claim 1 wherein the PIPLC substrate is an indolyl-phosphatidyl-myo-inositol.

3) biochemical test according to claim 2 wherein the indolyl-phosphatidyl-myosinol is bromo 4 chloro 3 indoxyl myoinositol phosphate.

4) biochemical test according to claim 1 to 3 wherein the concentration of substrate of PI-PLC is between 5 and 0.1 g / 1.

5) The biochemical assay of claim 1 to 4 wherein the alpha mannosidase substrate is a mannopyranosidase substrate.

The biochemical assay of claim 5 wherein the mannopyranosidase substrate is p-nitrophenyl mannopyranoside.

7) Biochemical test according to claim 1 to 6 wherein the concentration of α-mannosidase substrate is between 2 and 0.1 g / 1, preferably between 1 and 0.5 g / 1.

8) Use of a biochemical test according to any one of claims 1 to 7 to confirm the presence of L. monocytogenes ..

9) Process for the identification of Listeria monocytogenes, characterized in that it comprises the following steps: a) having a reaction medium for detecting the genus Listeria, b) seeding the medium with a biological sample to be tested, c) incubate d) reveal the presence of the genus Listeria e) confirm the presence of Listeria monocytogenes by a biochemical test according to any one of claims 1 to 7.

10) A method for identifying Listeria monocytogenes, characterized in that it comprises the following steps: a) having a biochemical test or a combination of biochemical tests to detect the genus Listeria, b) revealing the presence of the genus Listeria, c) confirming the presence of Listeria monocytogenes by a biochemical test according to any one of claims 1 to 7.