Culture medium for detecting pathogenic bacteria of the genus Listeria and method for identifying said bacteria

The present invention relates to a nutritive agar culture medium allowing the direct identification of pathogenic bacteria of the genus Listeria, said medium comprising a chromogenic synthetic substrate specifically cleaved by the phospholipase C specific of phosphatidylinositol (PIPLC).

The present invention relates to a culture medium allowing the
 investigation, the isolation, the counting and the direct identification
 of pathogenic bacteria of the genus Listeria as well as a method employing
 this medium.
 The isolation and the identification of the bacterium Listeria
 monocytogenes is a major problem in the monitoring of agrifood hygiene and
 of medical bacteriology. Among the bacteria of the genus Listeria spp only
 the species monocytogenes is known to be pathogenic for man. The other
 species of Listeria are not pathogenic or are pathogenic only for animals.
 This is the case, especially, for Listeria ivanovii. It is additionally
 known that is possible to inhibit the growth of Listeria monocytogenes by
 using bacteriocins. For example, the Patent Application EP 326 062
 describes a method for inhibition of Listeria monocytogenes which employs
 a bacteriocin originating from Pediococcus acidilactici.
 The route of contamination by Listeria monocytogenes is most often of food
 origin. Thus, it is necessary to provide for the detection of Listeria
 monocytogenes all along the agrifood pathway, from raw materials passing
 through the environment of production facilities up to the finished
 product intended for consumption.
 Within the context of diagnosis of bacterial conditions in man, it is
 likewise important to distinguish Listeria monocytogenes from among the
 other bacteria of the genus Listeria spp. which are not pathogens.
 The detection and the isolation of Listeria spp. are conventionally carried
 out using selective culture media. The Oxford (Curtis et al., Lett. Appl.
 Microbiol. (1989), 8, pp. 85-98) and Palcam (Van Netten et al., J. Food
 Microbiol. (1988), 6, pp. 187-188) selective media are the most currently
 used. These media allow the detection of all the species of the genus
 Listeria spp. Thus, the typical colonies observed must be subjected to
 supplementary identification tests, such as microscopic and/or biochemical
 and/or immunological and/or genetic tests, so as to establish membership
 of the monocytogenes species. However, the supplementary manipulations
 necessary for the identification of Listeria monocytogenes increase the
 length and the cost of the analyses. They require a vast number of
 reagents and the use of qualified personnel. In addition, the withdrawal
 of the colonies subjected to the identification being uncertain, the
 supplementary manipulations are often a source of error or at least the
 cause of lower precision and reliability. This is the case especially when
 the colonies of Listeria monocytogenes on the isolation medium are very
 minor with respect to the colonies formed by the other species of
 Listeria.
 The Patent Application EP 496 680 describes a bacteriological analysis
 method to differentiate Listeria monocytogenes from other bacteria of the
 genus Listeria spp.
 According to this method, an identification medium is used comprising a
 chromogenic or fluorogenic substrate capable of being hydrolysed by
 glycine aminopeptidase. The medium used can likewise possibly contain a
 fermentation substrate and/or a reducible substrate and/or a substrate
 which is hydrolysable enzymatically (such as the substrate of
 .alpha.-mannosidase) whose chemical transformation allows the species
 Listeria present in the sample to be analysed to be characterized.
 It is additionally known that it is possible to discriminate pathogenic
 species of Listeria, Listeria monocytogenes and Listeria ivanovii from
 other Listeria by demonstrating the specific phosphatidylinositol activity
 of specific phospholipase C of phosphatidylinositol (PIPLC).
 In fact, it has been shown that PIPLC is secreted into the culture medium
 of pathogenic species of the genus Listeria such as Listeria monocytogenes
 and Listeria invanovii (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 likewise known that it is possible to identify these two
 pathogenic species by means of indirect methods (Notermans et al., App.
 and Env. Microbiology (1991), Vol. 57 No. 9, pp. 2666-2670). According to
 the method proposed by Notermans et al., the strain of Listeria to be
 tested, which has previously been isolated, is inoculated as a spot onto
 the surface of a TY (tryptone yeast extract) agar. After 24 to 48 hours'
 incubation at 37.degree. C., a second agar layer is poured into the Petri
 dishes. This second layer contains agarose, chloramphenicol and
 L-.alpha.-phosphatidylinositol, the natural substrate of PIPLC. The dishes
 are then incubated again at 37.degree. C. and observed for 5 days. This
 method thus requires several steps, such as the isolation of the strain,
 the inoculation as a spot onto agar and the distribution of a second layer
 of agar containing the PIPLC substrate. In addition, this method does not
 allow Listeria monocytogenes bacteria, which are pathogenic for man, or
 Listeria ivanovii bacteria to be distinguished which, as indicated
 previously, are not pathogenic for man.
 The subject of the invention is a medium allowing the identification of
 pathogenic bacteria of the genus Listeria in a single step.
 The present invention relates more especially to a specific culture medium
 allowing the investigation, the isolation, the counting and the direct
 identification of pathogenic species of the genus Listeria, the said
 medium containing a synthetic chromogenic substrate specifically cleaved
 by PIPLC, especially 5-bromo-4-chloro-3-indolylphosphatidyl-myoinositol.
 This chromogenic substrate is preferably used in salt form, for example in
 sodium, potassium or ammonium salt form. Among the salts preferred is
 ammonium 5-bromo-4-chloro-3-indolylmyoinositol-1-yl-phosphate.
 The medium according to the invention allows direct distinction between, on
 the one hand, Listeria monocytogenes and Listeria ivanovii which form
 coloured colonies and, on the other hand, the other species of Listeria
 whose colonies are not coloured.
 According to a preferred method of carrying out the invention, the use of
 5-bromo-4-chloro-3-indolyl-phosphatidylmyoinositol allows the detection of
 the species Listeria monocytogenes and Listeria ivanovii which form blue
 colonies, the other species of Listeria remaining non-coloured.
 Preferentially, the nutritive agar culture medium according to the
 invention contains 5-bromo-4-chloro-3-indolylphosphatidylmyoinositol in
 free form or in salt form, at a concentration of 100 to 500 mg/l,
 preferably at a concentration of 150 to 300 mg/l.
 The nutritive agar culture medium used in the invention must allow the
 growth of Listeria spp. It is, for example, possible to use Columbia agar,
 made up of peptones, starch, sodium chloride and agar and well known to
 the person skilled in the art.
 The present invention also relates to the use of
 5-bromo-4-chloro-3-indolylphosphatidylmyoinositol or one of its salts for
 the preparation of a specific culture medium allowing the investigation,
 the isolation, the counting and the direct identification of pathogenic
 species of the genus Listeria and especially of the bacterium Listeria
 monocytogenes.
 Surprisingly, it has also been found that the addition of blood or of its
 derivatives, such as, for example, plasma or serum, to the culture medium
 allows positive PIPLC colonies having a very clear colour to be obtained.
 The present invention likewise relates to a specific nutritive agar culture
 medium, allowing the investigation, the isolation, the counting and the
 direct identification of pathogenic species of the genus Listeria and
 especially of the bacterium Listeria monocytogenes, said medium containing
 5-bromo-4-chloro-3-indolylphosphatidylmyoinositol or one of its salts and
 blood or one of its derivatives, preferably serum.
 The proportion of blood or of its constituents in the medium according to
 the invention can be between 20 and 80 ml, more especially between 40 and
 60 ml, preferably 50 ml per liter of culture medium. Culture media are
 preferred comprising 5-bromo-4-chloro-3-indolylphosphatidylmyoinositol or
 one of its salts at a concentration of 100 to 200 mg/ml and serum in a
 proportion of 40 to 60 ml per liter of medium.
 The addition to the culture medium of a pulverulent agent such as kaolin or
 silica contributes in intensifying the positive colouration of cultures of
 colonies of PIPLC, thus making the test easier to interpret.
 Preferably, the pulverulent agent is added to the culture medium made up
 with serum.
 Nutritive agar culture media according to the invention containing
 5-bromo-4-chloro-3-indolyl-phosphatidylmyoinositol or one of its salts as
 synthetic chromogenic substrate specifically cleaved by PIPLC, serum and a
 pulverulent agent, such as kaolin or silica, are also part of the
 invention.
 The concentration of the pulverulent agent in the nutritive agar culture
 medium depends on the nature of the agent used and can vary between 5 and
 30 g/l, preferably between 15 and 25 g/l.
 According to the invention, the culture medium can likewise contain a
 carbohydrate which can be metabolized by Listeria ivanovii but not by
 Listeria monocytogenes, such as xylose, for example. The addition of such
 a carbohydrate to a culture medium containing
 5-bromo-4-chloro-3-indolylphosphatidylmyoinositol or one of its salts thus
 allows the distinction between, on the one hand, Listeria monocytogenes,
 which forms pure blue colonies, and on the other hand, Listeria ivanovii,
 which forms pale blue to green colonies and finally the other species of
 Listeria whose colonies are uncoloured. Some carbohydrates metabolized by
 Listeria monocytogenes are described in the Manual of Clinical
 Microbiology (6th Edition--1995) ASM Press Washington D.C. pp. 343-344.
 The present invention likewise relates to a specific nutritive agar culture
 medium allowing the investigation, the isolation, the counting and the
 direct identification of Listeria monocytogenes, the said medium
 containing 5-bromo-4-chloro-3-indolyl-phosphatidylmyoinositol or one of
 its salts as synthetic chromogenic substrate specifically cleaved by
 PIPLC, blood derivatives, preferably serum, a pulverulent agent such as
 kaolin or silica and a carbohydrate which can be metabolized or fermented
 by Listeria ivanovii, but not by Listeria monocytogenes, preferably
 xylose.
 The concentration of the carbohydrate which can be metabolized by Listeria
 ivanovii, but not by Listeria monocytogenes, in the culture medium can
 vary between 5 and 15 g/l and depends, of course, on the carbohydrate
 used.
 The culture medium according to the invention can likewise contain a pH
 indicator. As pH indicators, it is possible to mention those which are
 currently used in microbiology, for example alizarinsulphonic acid, Methyl
 Red, Chlorophenol Red, litmus, Bromocresol Purple, Bromophenol Red,
 Bromoxylenol Blue, alizarin, Bromothymol Blue, Phenol Red, etc. Of course,
 the pH indicator must be used in the culture medium in an adjusted
 concentration. The latter depends on the nature of the indicator and can
 vary between 50 and 300 mg/l. Among the different pH indicators, it is
 preferred to use Phenol Red. This indicator even improves the distinction
 between Listeria monocytogenes and Listeria ivanovii, the colonies being,
 when the medium contains S-bromo-4-chloro-3-indolylphosphatidylmyoinositol
 or one of its salts, respectively of pure blue colour without colour
 change of the indicator for Listeria monocytogenes and of pale blue colour
 and surrounded by a yellow circle for Listeria ivanovii, this phenomenon
 being connected with the colour change of the indicator.
 According to the invention, a preferred culture medium is a nutritive agar
 culture medium containing
 5-bromo-4-chloro-3-indolylphosphatidylmyoinositol or one of its salts,
 blood derivatives, preferably serum, a pulverulent agent such as kaolin or
 silica, a carbohydrate which can be metabolized by Listeria ivanovii but
 not by Listeria monocytogenes, preferably xylose, and a pH indicator such
 as Phenol Red.
 On the other hand, the PIPLC activity is not exclusively present in
 Listeria monocytogenes and Listeria ivanovii. This enzymatic activity is
 especially encountered in certain species of Bacillus (Griffith et al.,
 Methods Enzymol. (1991) 197, pp. 493-502) and of Clostridium (Taguchi et
 al., Arch. Biochem. Biophys. (1978), 186, pp. 196-201). In addition, the
 development of a significant saprophytic flora (bacteria or yeasts) on the
 culture medium can be harmful to the detection of Listeria monocytogenes
 by a competition phenomenon. Thus, it is desirable to inhibit the growth
 of interfering microorganisms in order to eliminate the risks of false
 positives (blue colonies formed by bacteria other than Listeria
 monocytogenes) or of false negatives (masking of the blue colouration of
 the colonizes of Listeria monocytogenes by a significant development of
 other contaminants). To avoid these phenomena, it is recommended to make
 up the culture medium with antibacterial and/or antifungal agents with
 respect to which the Listeria spp. are not very sensitive or insensitive.
 These agents can be used alone or in combination. Among these agents, it
 is possible to mention, for example, lithium chloride, acriflavine
 hydrochloride, nalidixic acid, polymixin B, cefotan, colistin sulphate,
 fosfomycin, ceftazidime, moxalactam, cycloheximide and amphotericin B.
 The nutritive agar culture media containing
 5-bromo-4-chloro-3-indolylphosphatidylmyoinositol or one of its salts,
 blood derivatives, preferably serum, a pulverulent agent such as kaolin or
 silica, a carbohydrate which can be metabolized by Listeria ivanovii but
 not by Listeria monocytogenes, preferably xylose, a pH indicator such as,
 especially, Phenol Red, and antibacterial or antifungal agents are
 likewise part of the present invention.
 The present invention also relates to a method for identification of the
 pathogenic bacteria of the genus Listeria, comprising:
 the inoculation of a sample liable to contain the said pathogenic bacteria
 of the genus Listeria onto an agar culture medium containing a specific
 chromogenic substrate of PIPLC,
 the incubation of said inoculated culture medium with said sample,
 and the determination of the presence of said pathogenic bacteria of the
 genus Listeria by the characteristic colour of the substrate. Said sample
 liable to contain pathogenic bacteria of the genus Listeria is inoculated
 in a crude form or is previously diluted or is previously concentrated
 before being inoculated onto a culture medium, such as defined above.
 According to a preferred embodiment of the invention, a crude sample to be
 tested, which is diluted or has been subjected to one or more
 concentration phases on a nutritive agar culture medium which contains a
 synthetic chromogenic substrate specifically cleaved by PIPLC, which is
 5-bromo-4-chloro-3-indolylphosphatidylmyoinositol or one of its salts,
 blood derivatives such as serum, a pulverulent agent such as kaolin or
 silica, a carbohydrate which can be metabolized by Listeria ivanovii but
 not by Listeria monocytogenes, preferably xylose and possibly a pH
 indicator such as, especially, Phenol Red and antibacterial or antifungal
 agents, is cultured. The presence of Listeria monocytogenes is determined
 by the characteristic colour of its colonies.

The following examples are given in a non-limiting manner to illustrate the
 invention.
 EXAMPLE 1
 Differentiation Between Listeria Pathogens, Other Listeria spp. and
 Interfering Substances.

PREATION OF THE CULTURE MEDIA
 Medium 1: base medium
 Columbia agar 39 g/l
 Lithium chloride 10 g/l
 Ceftazidime* 20 mg/l
 Moxalactam* 20 mg/l
 Polymixin B* 20 mg/l
 Amphotericin B* 3 mg/l
 *thermolabile components
 Medium 2: base medium+5-bromo-4-chloro-3-indolyl-phosphatidylmyoinositol
 The composition in grams per liter of this medium is identical to that of
 the medium 1, but the medium 2 additionally contains 300 mg/L of
 5-bromo-4-chloro-3-indolylphosphatidylmyoinositol, in ammonium salt form
 (B-7404 5-bromo-4-chloro-3-indoxylmyoinosi-tol-1-ylphosphate ammonium
 salt, Biosynth Switzerland).
 The preparation of each medium is carried out as follows:
 Dissolution of the non-thermolabile components in osmosed water.
 Heating of the mixtures to boiling.
 Adjustment, if necessary, of the pH to 7.3+/-0.2.
 Distribution into flasks at a rate of 95 ml/flask.
 Autoclaving of the flasks at 120.degree. C. for 15 minutes.
 Addition of the thermolabile components in sterile solution after cooling
 of the agar to approximately 47.degree. C.
 Distribution of the autoclaved media into sterile Petri dishes (diameter 90
 mm) at a rate of 15 to 20 ml/dish.
 STRAINS TESTED
 The strains tested are the following:
 Listeria spp.:
 Listeria monocytogenes
 Listeria ivanovii
 Listeria innocua
 Listeria seeligeri
 Listeria welshimeri
 Interfering substances:
 Bacillus cereus
 Staphylococcus aureus
 Candida tropicalis
 CULTURING--INCUBATION--READING
 The strains are subcultured in trypto-casein-soya broths and then incubated
 at 37.degree. C.
 After incubation for 18 hours, the broths are diluted in "tryptone salt"
 diluent, made up of tryptone peptone at 1/100 and sodium chloride at
 8.5/1000; so as to obtain suspensions of between 10.sup.6 and 10.sup.7
 cells/ml.
 For each suspension, inoculation is carried out by isolation with a sterile
 loop of 1 .mu.l, a dish of medium 1 and a dish of medium 2. The dishes are
 incubated at 37.degree. C. After incubation for 24 hours and 48 hours, the
 dishes are read. Reading consists in an observation of the colour of the
 colonies formed by each of the strains in each of the media. The results
 obtained are indicated in Table I.
 TABLE I
 COLOUR OF THE COLONIES
 Distinction
 path. Listeria Non-pathogenic Listeria path.
 and non-path.
 MEDIUM L.monoc. L.ivan. L.inn. L.seel. L.welsh Interfering substances
 Listeria
 1 white white white white white inhibition
 impossible
 2 blue blue white white white inhibition
 possible
 Medium 2 allows pathogenic and non-pathogenic Listeria species to be
 differentiated. In addition, the selective supplement allows the bacteria
 Bacillus cereus and Staphylococcus aureus, potentially false positives in
 the culture medium, to be inhibited.
 EXAMPLE 2
 Differentiation Between Pathogenic Listeria Species and Other Listeria
 Species
 PRETION OF THE CULTURE MEDIA
 Medium 2: base medium+5-bromo-4-chloro-3-indolyl-phosphatidylmyoinositol
 Medium 2 is as described in Example 1.
 Medium 3: base medium+5-bromo-4-chloro-3-indolyl-phosphatidylmyoinositol
 The composition of medium 3 is identical to that of medium 2 indicated
 above, but this medium contains 150 mg/l of
 5-bromo-4-chloro-3-indolylphosphatidylmyoinositol in ammonium salt form
 instead of 300 mg/l (medium 2).
 Medium 4: base
 medium+5-bromo-4-chloro-3-indolyl-phosphatidylmyoinositol+blood
 The composition of medium 4 is identical to that of medium 3, but this
 medium additionally contains 50 ml/l of horse blood. For the preparation
 of the medium, the blood is treated as a thermolabile component.
 Medium 5: base
 medium+5-bromo-4-chloro-3-indolyl-phosphatidylmyoinositol+serum
 The composition of medium 5 is identical to that of medium 4, but this
 medium contains 50 ml/l of horse serum instead of blood.
 Medium 6: base
 medium+5-bromo-4-chloro-3-indolyl-phosphatidylmyoinositol+serum+silica
 The composition of medium 6 is identical to that of medium 5, but this
 medium additionally contains 20 g/l of silica, a non-thermolabile
 component.
 The preparation of each medium is carried out as described in Example 1.
 STRAINS TESTED
 The strains tested are the following:
 Listeria spp.:
 Listeeria monocytogenes
 Listeria ivanovii
 Listeria innocua
 Listeria seeligeri
 Listeria welshimeri
 CULTURING--INCUBATION--READING
 For culturing, incubation and reading, the method was as described in
 Example 1. The results obtained are indicated in Table II.
 TABLE II
 COLOUR OF THE COLONIES Distinction
 pathogenic Listeria Non-pathogenic Listeria path. and non-path.
 MEDIUM L.monoc. L.ivan. L.inn. L.seel. L.welsh Listeria
 1 blue blue white white White good
 2 bluish bluish white white White average
 3 pure blue pure blue white white White excellent
 4 blue blue white white White good
 5 pure blue pure blue white white White excellent
 The results of Table II demonstrate that in order to carry out a certain
 and precise reading of the test, that is to say to obtain a good
 distinction between the pathogenic Listeria species and the non-pathogenic
 Listeria, it is desirable to make up the culture medium with blood, serum
 or a mixture of serum and silica. The addition to the medium of the blood,
 the serum or a mixture of serum and silica contributes in strongly
 intensifying the blue colouration of the positive PIPLC colonies and thus
 allows the concentration of
 5-bromo-4-chloro-3-indolyl-phosphatidylmyoinositol in the culture medium
 to be reduced very significantly.
 EXAMPLE 3
 Differentiation Between Listeria monocytogenes and Other Listeria Species
 Including Listeria ivanovii
 PREATION OF THE CULTURE MEDIA
 Medium 6: base
 medium+5-bromo-4-chloro-3-indolyl-phosphatidylmyoinositol+serum+silica
 Medium 6 is as described in Example 2.
 Medium 7: base
 medium+5-bromo-4-chloro-3-indolyl-phosphatidylmyoinositol+serum+silica+xyl
 ose
 The composition of medium 7 is identical to that of medium 6, described in
 this example, but this medium additionally contains 10 g/l of xylose, a
 component which for the preparation of the medium is considered as a
 thermolabile component.
 Medium 8: base
 medium+5-bromo-4-chloro-3-indolyl-phosphatidylmyoinositol+serum+silica+xyl
 ose+Phenol Red
 The composition of medium 8 is identical to that of medium 7 indicated
 above, but this medium additionally contains 80 mg/l of Phenol Red
 (non-thermolabile component).
 The preparation of each medium is carried out as described in Example 1.
 STRAINS TESTED
 The strains tested are the following:
 Listeria spp.:
 Listeria monocytogenes
 Listeria ivanovii
 Listeria innocua
 Listeria seeligeri
 CULTURING--INCUBATION--READING
 For culturing, incubation and reading, the method was as described in
 Example 1. The results obtained are indicated in Table III.
 TABLE III
 COLOUR OF THE COLONIES Distinction Distinction
 Non-pathogenic path. and Listeria
 monoc.
 Pathogenic Listeria Listeria non-path. and other
 MEDIUM L.monoc. L.ivan. L.inn. L.seel. Listeria Listeria
 1 pure blue pure blue white White possible impossible
 2 pure blue pale blue to white White possible possible
 green
 3 pure blue pale blue to white White possible possible
 (-) green (-) (+)
 (+)
 (-): no colour change of the coloured indicator, the agar remains red
 around the colonies
 (- xylose strain)
 (+): colour change of coloured indicator, yellow circle around the colonies
 (+ xylose strain)
 The results of Table III demonstrate that the addition of xylose to the
 culture medium, preferably in combination with Phenol Red, allows
 distinction between Listeria monocytogenes and all the other species of
 Listeria, in particular Listeria ivanovii.
 EXAMPLE 4
 Carrying Out the Method According to the Invention
 214 strains of the genus Listeria supplied by the Listeria Reference Centre
 (Institut Pasteur Paris) were tested on medium 8.
 The strains corresponding to the following species were used:
 12 strains of Listeria innocua
 10 strains of Listeria seeligeri
 10 strains of Listeria welshimeri
 10 strains of Listeria ivanovii
 1 strain of Listeria gray
 171 strains of Listeria monocytogenes
 For culturing, incubation and reading, the method was as described in
 Example 1.
 All the strains of Listeria monocytogenes were identified, as well as all
 the strains of Listeria ivanovii, and no false positive result was
 identified.
 This experiment demonstrates the specificity and the reliability of the
 method according to the invention.