A substrate which can be used for the direct identification of pathogenic bacteria belonging to the genus Listeria by detecting an esterase activity other than Phosphatidyl Inositol-specific Phospholipase C (PI-PLC), an esterase which is specific to the species Listeria monocytogenes. The use of two substrates, one as described above and the other specific for all or some members of the genus Listeria, and a reaction medium containing such a substrate or such a combination of substrates are disclosed. An identification method which exploits such culture media is also disclosed. The invention is particularly applicable in the field of diagnosis.

This application is a U.S. National Stage of International Application PCT/FR01/03627, filed Nov. 19, 2001 and published on May 23, 2002 in the French Language.

This invention concerns a chromogenic substrate which can be used for the direct identification of pathogenic bacteria belonging to the genus Listeri, more precisely those of the species Listeria monocytogenes.

The invention also concerns using a combination of two substrates, one substantially specific for the species Listeria monocytogenes, and the other either specific or not for the genus Listeria. It also concerns a reaction medium containing such a substrate or such a combination of substrates. Finally, it concerns an identification method exploiting such culture media.

For many years, special substrates have been used to determine whether certain enzyme activities typical of microorganisms are present or not. Through the use of specific substrates, it is possible--on the basis of whether a reaction takes place or not--to characterize the nature of a group of microorganisms, or distinguish between different strains and/or species belonging to a given genus.

Synthetic enzyme substrates such as those exploited in this invention are made up of two different parts: the first part is specific to the enzyme activity being tested for and will hereafter be referred to as the target part; the second part acts as a marker and will hereafter be referred to as the marker part.

Such special substrates may be either fluorescent or chromogenic. In fact, the second marker part or the product of its reaction with one or more other compounds becomes fluorescent or chromogenic when it is no longer bound to the first target part (in this context, refer to Patent Application PCT/FR99/00781 filed on behalf of the applicant).

Isolating and identifying the bacterium Listeria monocytogenes is a major problem in safety monitoring in both the food industry and medical bacteriology. Among all the bacterial species belonging to the genus Listeria, only Listeria monocytogenes is known to be pathogenic in humans. It can cause listeriosis, a condition which is sometimes mortal (in 25 to 35% of cases) in the immunodeficient, very young children and pregnant women. Other Listeria species are non-pathogenic, or are only pathogenic in animals. Such is the case in particular for Listeria ivanovii.

Although the danger of listeriosis has diminished in recent years in most developed countries, modern society demands ever more stringent safety and, even if sporadic cases are accepted, more widespread outbreaks are not.

However, in France, for example, risk management policy tends to focus on the products further down the line (e.g. contamination levels in finished products) rather than components higher up in the production process (the infection of livestock). As a result, between 15 and 60% of all poultry carcasses, between 3 and 36% of all pig carcasses, and between 7 and 28% of all cow carcasses are contaminated by Listeria.

Therefore, when it comes to human bacterial infection, it is important to be able to make a definitive distinction between Listeria monocytogenes and the other, non-pathogenic members of the genus Listeria.

Distinction has traditionally been made between different Listeria spp. by using selective culture media. Two particular selective media are in the most widespread use: Palcam (Van Netten et al., J. Food Microbiol. (1988), 6, pp. 187-188) and Oxford (Curtis et al., Lett. Appl. Microbiol. (1989), 8, pp. 85-98). All species belonging to the genus Listeria can be detected using these media. Typical colonies are obtained which must subsequently be identified by means of further tests--microscopic and/or biochemical and/or immunologic and/or genetic--to check whether or not the colony corresponds to the species Listeria monocytogenes.

These extra experiments both slow down and increase the cost of testing. Furthermore, they require many different reagents and necessitate trained technicians. Finally, since picking the colonies to be identified is a random process, the extra experiments often introduce mistakes, or at least compromise the accuracy and reliability of the result. This is a particularly acute problem when the number of colonies of Listeria monocytogenes on the isolation medium is much lower than the number of colonies of other Listeria species.

The Applicant's Patent EP-B-0.496.680 describes a bacteriological test method to differentiate the species Listeria monocytogenes from other bacterial species which belong to the genus Listeria. According to this method, use is made of an identification medium containing a chromogenic or fluorogenic substrate which is hydrolyzed by an enzyme called Glycine aminopeptidase. The medium used may also contain a substrate which can be fermented and/or a substrate which can be reduced and/or a substrate which can be enzymatically hydrolyzed, such as the substrate for .alpha.-mannosidase, the chemical conversion of which makes it possible to characterize the Listeria species present in the test sample.

This useful approach has one major drawback in that the species Listeria monocytogenes is the only one not to express Glycine aminopeptidase activity. It is therefore possible to identify all Listeria species except the one member of the genus which is pathogenic, namely Listeria monocytogenes. Trying to detect Listeria monocytogenes by virtue of the absence of a certain activity is not very reliable since such a negative test lacks specificity because of the possibility of mutation and because another species might not be expressing its normal pattern of activity due to stress.

Methods based on using chromogenic media to detect Listeria genus-specific .beta.-glucosidase activity have been developed. Moreover, other techniques based on chromogenic media can be used to distinguish between Listeria monocytogenes and Listeria ivanovii from other Listeria species by assaying phosphatidylinositol phospholipase C (PI-PLC) activity.

It has been shown that certain species of the genus Listeria (such as Listeria monocytogenes and Listeria ivanovii) secrete PI-PLC into the culture medium (Leimeister-Wachter et al., Mol. Microbiol. (1991) 5(2), pp. 361-366; J. Mengaud et al., Mol. Microbiol. (1991) 5(2), pp. 367-372; and Goldfine et al., Infection and Immunity (1992) 60(10), pp. 4059-4067). It is also known that these two species can be identified using indirect methods (Notermans et al., App. and Env. Microbiology (1991), vol. 57 n.degree.9, pp. 2666-2670.

Patent Application WO-A-99/04032 describes a culture medium containing a chromogenic substrate which is specific for Listeria monocytogenes and Listeria ivanovii, in the form of a phosphatidylinositol derivative such as the ammonium salt of 5-Bromo-4-chloro-3-indolyl-myo-inositol-1-phosphate. This affords direct detection of both these species in a single step, and therefore makes it possible to distinguish these two from all other Listeria species.

Bacteriological detection tests based on PI-PLC are also dealt with in the following documents: WO-A-98/38332 which focuses on a method and a detection test for PI-PLC, based on the cleavage of a substrate by the enzyme, one of the residues of said substrate being chromogenic and making it possible to identify pathogenic Listeria, and WO99/48899 which describes a fluorogenic substrate based on 4-methylumbelliferone which is used to detect the PI-PLC activity expressed by a number of different species, namely Clostridium spp., Listeria ivanovii, Staphylococcus aureus, Bacillus cereus, Bacillus thuringiensis as well as Listeria monocytogenes.

In this document, attention is drawn to the fact that phospholipase C activity is not specific for Listeria monocytogenes since it is also found in other species, including another Listeria species, namely Listeria ivanovii.

However, it should be noted that esterase substrates are more easily and more cheaply synthesized than substrates for PI-PLC. Moreover, the advantage of using a chromogenic substrate (rather than a natural one) is that visual detection is easier, i.e. it is easier to distinguish colored colonies than it is to visualize a halo surrounding colonies. This advantage furthermore applies to cultures containing different microorganisms in that each colony has its own specific color, whereas a halo could spread out below two disparate colonies.

The purpose of this invention is to offer a detection method which makes it possible to differentiate the species Listeria monocytogenes from all other Listeria species. This is achieved by detecting at least one metabolic activity which has not hitherto been used either to detect Listeria or to differentiate Listeria monocytogenes from other species belonging to the genus Listeria. This activity is an esterase which is expressed far more strongly by Listeria monocytogenes than by other species in the genus. In addition, at least one other activity is assayed, namely an activity which is expressed by all or some Listeria such as a saccharidase, a phosphatase or an aminopeptidase, which enhances the contrast between the color of colonies of Listeria monocytogenes and that of colonies of other species belonging to the same genus. This makes it possible to distinguish Listeria monocytogenes from Listeria ivanovii et Listeria innocua, which are the species most commonly isolated, and which have enzyme profiles closely resembling that of Listeria monocytogenes.

To this effect, this invention concerns a substrate which can be used to directly identify pathogenic bacteria belonging to the genus Listeria, characterized in that it detects a Listeria monocytogenes-specific esterase activity, an esterase activity which is distinct from PI-PLC activity.

Thus although PI-PLC is an esterase, Listeria ivanovii is esterase negative despite being PI-PLC positive; therefore it could be confused with Listeria monocytogenes. In contrast to the background art and methods based on the detection of PI-PLC activity which do not differentiate Listeria monocytogenes from other Listeria species, this invention makes it possible to detect Listeria monocytogenes in a more specific way.

According to a preferred embodiment, the esterase activity is a specific enzyme activity, i.e. it cleaves the ester linkage between the marker part and the target part of the substrate.

According to a preferred embodiment, the linkage cleaved is an ester bond between an alcohol group on the marker part and an organic acid which constitutes the target.

According to a preferred embodiment, the marker part consists of a chromogenic molecule such as indoxyl which could be constituted by one of the following constituents: 5-Bromo-3-indoxyl, or 5-Bromo-4-chloro-3-indoxyl, or 6-Chloro-3-indoxyl, or 5-Bromo-6-chloro-3-indoxyl, or 6-Bromo-3-indoxyl.

According to a preferred embodiment, the target part consists of a fatty acid with a carbon chain containing between 2 and 20 carbon atoms, preferably between 4 and 10 carbon atoms.

Preferably, the substrate is either 5-Bromo-4-chloro-3-indolyl butyrate, 5-Bromo-4-chloro-3-indolyl octanoate, 5-Bromo-4-chloro-3-indolyl nonanoate or 5-Bromo-4-chloro-3-indolyl decanoate.

According to a modified embodiment, the substrate is paired with at least one other substrate making it possible to detect at least one other enzyme activity expressed by all or some Listeria species.

In the case in which there are two substrates, the substrate to detect esterase activity other than PI-PLC gives a color which is different from that given by the other enzyme activity which is different from the above-mentioned esterase activity.

According to a preferred embodiment, the other enzyme activity expressed by all or some Listeria species is a saccharidase, a phosphatase or an aminopeptidase.

Preferably, the marker part of the other substrate is based on: 5-Bromo-3-indoxyl, or 5-Bromo-4-chloro-3-indoxyl, or 6-Chloro-3-indoxyl, or 5-Bromo-6-chloro-3-indoxyl, or 6-Bromo-3-indoxyl. With respect to -Chloro-3-indoxyl, such a molecule is particularly well described in Patent U.S. Pat. No. 5,364,767 in which it is essentially associated withN-Acetyl-.beta.-D-galactosaminide, with N-Acetyl-.beta.-D-glucosaminide, with Butyrate, with Octanoate, with the salt of p-toluidine phosphate, with Sulfate or with .beta.-D-Glucopyranoside. It can also be based on 5-Bromo-6-chloro-3-indoxyl.

According to a modified embodiment, the other substrate consists of: 5-Bromo-6-chloro-3-indolyl-.beta.-D-glucoside, or 6-Chloro-3-indolyl-.beta.-D-glucoside, or 6-Chloro-3-indolyl-.beta.-D-cellobioside, or 6-Chloro-3-indolyl-N-acetyl-.beta.-D-glucosaminide, or 6-Chloro-3-indolyl-.alpha.-D-mannoside, or 6-Chloro-3-indolylphosphate.

This invention also concerns a reaction medium which allows the direct identification of Listeria monocytogenes, which exploits one or two substrates as defined above.

More precisely, the substrate which detects the esterase activity other than PI-PLC is at a concentration of between 20 mg/l and 3 g/l, or preferably between 50 mgL and 1 g/l, or preferably between 100 and 600 mg/l, or about 250 mg/l.

More precisely, the substrate which detects the other activity such as a saccharidase, a phosphatase or an aminopeptidase is at a concentration of between 10 mg/l and 500 mg/l, preferably between 50 and 300 mg/l, and more preferably still between 100 and 200 mg/l.

The medium is either liquid or semi-solid (with agar).

According to a modified embodiment, the medium includes a means of distinguishing Listeria monocytogenes from Listeria welshimeri and Listeria seeligeri, namely: the addition of at least one acidified carbohydrate (e.g. Xylose and/or D-Tagatose) by Listeria welshimeri and Listeria seeligeri and not by Listeria monocytogenes, or the addition of at least one acidified carbohydrate by Listeria monocytogenes and possibly Listeria innocua and/or Listeria ivanovii and/or Listeria grayii. the addition of at least one substrate to detect saccharidase activity (.beta.-maltosidase) and/or phosphatase and/or aminopeptidase activity (L-glycine aminopeptidase) specific at least to Listeria welshimeri and Listeria seeligeri but not Listeria monocytogenes, or the addition of a natural Phospholipase (PLC) substrate, such as Phosphatidylinositol (PI) and/or Phosphatidylcholine (PC).

According to a particular embodiment, the medium includes a selective means of differentiating Listeria monocytogenes from at least the following species: Staphylococcus aureus, Bacillus thuringiensis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans.

According to a preferred embodiment, the selective means is constituted by one of the following compounds: Lithium chloride Ceftazidime, Amphotericin B, Fosfomycin, and/or Colistin.

This invention furthermore concerns a method for identifying pathogenic bacteria belonging to the species Listeria monocytogenes, comprising the following steps: seeding of a culture medium as described above with a specimen suspected of containing the pathogenic bacteria, incubation of the culture medium seeded with the specimen, and determination of the presence of said pathogenic bacteria by virtue of the color and intensity typical of the substrate(s).

According to a modified embodiment, said specimen suspected of containing pathogenic bacteria is preliminarily concentrated prior to seeding on the culture medium, as defined above.

According to another modified embodiment of the method, whether or not said pathogenic bacteria are present is determined by virtue of the color and intensity typical of the substrate(s) after a period of 18 to 24 hours of incubation.

Finally, the invention proposes using a substrate consisting of a target part as defined above and an inhibitory part which specifically inhibits Listeria monocytogenes when it is released.

This invention therefore essentially concerns the differentiation of Listeria monocytogenes from other species belonging to the genus Listeria.

Esterase enzyme activity is a good marker for differentiating the species Listeria monocytogenes from other species belonging to the genus Listeria, apart from the more specific esterase activity referred to as PI-PLC.

Thus, the background art described above demonstrates the lack of specificity of PI-PLC (which is specific for a certain type of lipid) vis-a-vis differentiating the species Listeria monocytogenes and Listeria ivanovii, for example. PI-PLC hydrolyzes Phosphatidylinositol derivatives at a point between the Glycerol and the inorganic Phosphate. In the case of a chromogenic substrate, the enzyme cleaves between the marker part and the inorganic Phosphate bound to the Inositol, as illustrated below (see Original Patent).

Esterases according to this invention hydrolyze lipids containing one or more fatty acids with a chain preferably containing between 7 and 10 carbon atoms. These esterases hydrolyze ester bonds between an alcohol and an organic fatty acid, as illustrated below (see Original Patent).

Claim 1 of 13 Claims

1. A composition for the direct identification of pathogenic bacteria belonging to the species Listeria monocytogenes, said composition comprising a first substrate comprising a target part comprising a fatty acid having a carbon chain containing between 2 and 20 carbon atoms and a marker part, and wherein said first substrate is cleavable by an esterase activity other than Phosphatidyl Inositol-specific Phospholipase C (PI-PLC), and wherein said esterase activity is specific for Listeria monocytogenes and not specific for other members of the genus Listeria further comprising a second substrate comprising a target part and a marker part, wherein said second substrate detects at least one other enzyme activity expressed by all or some Listeria species, and wherein the marker part of said first substrate is different from the marker part of said second substrate.

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