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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Definitions

• the present invention relates to a specific reaction medium of Clostridium difficile. It also relates to a method for detecting and / or identifying Clostridium difficile using such a medium.
• Clostridium difficile is a commensal germ of the digestive flora. Clostridium difficile spores are found in the soil in hospitals, with the active form found only at the intestinal level. Under the microscope, after Gram stain, they are elongate bacilli with a slightly swollen tip. Being resistant to most antibiotics, it can then develop significantly in case of disruption of the digestive flora by antibiotic therapy. It then secretes two toxins A and B, an enterotoxin and a cytotoxin, causing pseudomembranous colitis or postantibiotic diarrhea. As a result, Clostridium difficile is currently recognized as a major enteropathogen, mainly implicated in nosocomial diarrhea in adults.
• Diagnosis is based on different techniques such as the detection of toxin activity (cytotoxicity activity, CTA).
• CTA cytotoxicity activity
• the diagnosis can also be implemented by immunology, thanks to an ELISA type test. It is, however, recommended to combine the immunological techniques used for the detection of toxins with bacterial culture to isolate Clostridium difficile strains.
• This culture step can be carried out on a Clostridium difficile agar marketed by the applicant, the Clostridium difficile colonies then being identified by biochemical methods such as the 20 A api gallery or the rapid ID 32 A gallery. identification by gallery requires to have a pure culture, the bacterium to identify, on a suitable growth medium to obtain enough biomass (3 to 4 Mc Farland) which takes, at best, 48h (isolation culture from 24 to 72 hours from the sampling giving only presumptive detection plus culture from 24 to 72 hours to generate enough biomass). The gallery is then read after 4 hours of incubation for the Rapid ID 32 A and 24 to 48 hours for the 2OA gallery.
• reaction medium a medium comprising all the elements necessary for the expression of a metabolism and / or the growth of microorganisms.
• the reaction medium may be solid, semi-solid or liquid.
• 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, agarose or other natural or artificial gelling agents.
• a number of preparations are commercially available, such as Columbia agar, Trypcococcus agar, Mac Conkey agar, Sabouraud agar or more generally those described in the Handbook of Microbiological Media (CRC Press).
• the reaction medium according to the invention must allow the growth of C. difficile.
• the reaction medium may comprise one or more elements in combination, such as amino acids, peptones, carbohydrates, nucleotides, minerals, vitamins, etc.
• the medium may also comprise a dye.
• dye of Evans blue, neutral red, sheep blood, horse blood, an opacifier such as titanium oxide, nitroaniline, malachite green, brilliant green , one or more metabolic indicators, one or more metabolic regulators ...
• the reaction medium may be a revelation medium or a culture and revelation medium.
• the culture of microorganisms is carried out before seeding and, in the second case, the detection and / or identification medium also constitutes the culture medium.
• Those skilled in the art can also use a bi-box, which makes it easy to compare two media, comprising different substrates or different selective mixtures, on which a same biological sample has been deposited.
• the reaction medium may comprise one or more ⁇ -glucosidase inducers.
• beta-glucosidase inducer is meant a compound inducing an increase in the expression of the targeted metabolic activity, any experimental condition being equal, the metabolic activity is greater when the inducer is at an appropriate concentration than when he is absent or at an inappropriate concentration.
• Mention may in particular be made of a carbohydrate consisting of a radical bonded in position ⁇ to glucose or a carbohydrate with a ⁇ -glucoside subunit, in particular cellobiose, cellulose, starch, cellotriose, trehalose Methyl- ⁇ -glucoside.
• a concentration of between 100 ng / l and 10 g / l, preferably between 10 mg / l and 3 g / l is particularly suitable for the present invention. .
• the reaction medium may comprise one or more growth promoters for C. difficile strains.
• growth promoter is meant a compound or a group of compounds that stimulates the growth of microorganisms. These include blood, serum, egg yolk.
• a concentration of between 0.1 and 10% is particularly suitable for the present invention.
• the reaction medium may comprise one or more reducing agents.
• reducing agent a compound or a group of compounds which facilitates the growth of anaerobic germs by neutralizing the dissolved O 2 present in the medium.
• the reaction medium may comprise one or more inducers of C. difficile spore germination.
• inducer of spore germination is meant a compound or a group of compounds that promotes the passage from the spore state to the vegetative state of C. difficile.
• Sodium Taurocholate may be mentioned in particular.
• a concentration of between 0.1 and 10 g / l, preferably between 1 and 5 g / l is particularly suitable for the present invention.
• the reaction medium may comprise one or more selective agents.
• selective agent is meant any compound capable of preventing or slowing the growth of a microorganism. Without being limiting, a concentration of between 5 mg / l and 5 g / l is particularly suitable for the present invention.
• an antibiotic such as D-cycloserine, cephalosporins, such as Cefoxitin or Cefotaxime, Colistin, Polymixin, Fosfomycin, Tobramycin, Gentamicin, Aztreonam, Trimethoprim, quinolones such as Nalidixic acid, an antifungal such as in particular amphotericin B, fluconazole or itraconazole.
• antibiotic any compound capable of preventing or slowing the growth of a bacterium. They belong in particular to the groups of cephalosporins, aminoglycosides, polypeptides, sulphonamides, quinolones. As an indication, mention may be made in particular of the antibiotics cefotaxime, ceftazidime, cefoxitin, ceftriaxone, cefpodoxime, aztreonam, trimethoprim, tobramycin, moxalactam, fosfomycin, D-cylcoserine, Polymixin, Colistine.
• the reaction medium may comprise a second enzymatic substrate, such as in particular an osidase, esterase or peptidase substrate. It may be a substrate targeting an enzyme expressed by Clostridium difficile such as Proline aminopeptidase or an enzyme not expressed by C. difficile such as ⁇ -ribosidase.
• substrate is meant a molecule that can be hydrolysed by an enzyme, such as beta-glucosidase into a product for the direct or indirect detection of a microorganism.
• This substrate comprises in particular a first specific part of the enzymatic activity to be revealed and a second part serving as a marker, hereinafter called marker part. This marker part can be chromogenic, fluorogenic, luminescent, etc.
• the beta-glucosidase substrate suitable for the identification of C. difficile is a substrate which, under appropriate growth conditions, induces C. difficile staining. Such substrates may in particular be selected by means of the test described in Example 1.
• 2-hydroxyphenyl- ⁇ -glucoside (catechol- ⁇ -glucoside) is meant;
• the beta-glucosidase substrate capable of identifying C. difficile, is chosen from 2-hydroxyphenyl- ⁇ -glucoside (catechol- ⁇ -glucoside); Magenta- ⁇ -glucoside (5-Bromo-6-chloro-3-indoxyl- ⁇ -glucoside); Dihydroxyflavone- ⁇ -glucoside; Esculin (esculetin- ⁇ -glucoside), 3,4-cyclohexenoesculetin- ⁇ -glucoside and Alizarin- ⁇ -glucoside.
• 2-hydroxyphenyl- ⁇ -glucoside catechol- ⁇ -glucoside
• Magenta- ⁇ -glucoside (5-Bromo-6-chloro-3-indoxyl- ⁇ -glucoside)
• Dihydroxyflavone- ⁇ -glucoside Dihydroxyflavone- ⁇ -glucoside
• Esculin esculetin- ⁇ -glucoside
• 3,4-cyclohexenoesculetin- ⁇ -glucoside Alizarin- ⁇ -gluco
• the concentration of beta-glucosidase substrate in the medium according to the invention is between 25 and 1000 mg / l and more preferably between 50 and 400 mg / l.
• the reaction medium may comprise a second enzymatic substrate or several enzymatic substrates, preferentially chosen from an osidase, esterase or peptidase substrate.
• osidase substrate is meant in particular the substrates of alpha-glucosidase, galactosidase, ribosidase, hexosaminidase, glucuronidase, xylosidase, fucosidase, cellobiosidase, arabinosidase, mannosidase.
• riboside is meant ribofuranoside and / or ribopyranoside
• arabinoside is arabinofuranoside and / or arabinopyranoside
• fucoside is D-fucoside and / or
• L-fucoside When not specified, it may be either ⁇ -oside or ⁇ -oside.
• 5-Bromo-6-chloro-3-indoxyl- ⁇ -glucoside Dihydroxyflavone ⁇ -glucoside; 3,4-Cyclohexenesculetin- ⁇ -glucoside; 8-Hydroxiquinoline ⁇ -glucoside; 5-Bromo-4-chloro-3-indoxyl- ⁇ -glucoside; 6-Chloro-3-indoxyl- ⁇ -glucoside; 5-Bromo-3-indoxyl- ⁇ -glucoside; Alizarin- ⁇ -glucoside; Nitrophenyl- ⁇ -glucoside; 4-Methylumbelliferyl- ⁇ -glucoside; Naphtholbenzein- ⁇ -glucoside;
• Dichloroaminophenylgalactoside 2-hydroxyphenyl riboside; 5-Bromo-6-chloro-3-indoxyl riboside; Dihydroxyflavone riboside; 5-Bromo-4-chloro-3-indoxyl riboside; 6-Chloro-3-indoxyl-riboside; 5-Bromo-3-indoxyl riboside; 6-Fluoro-
• 3-indoxyl-riboside Alizarin-riboside; Nitrophenyl riboside; 4-Methylumbelliferyl-riboside; Naphtyl-riboside; Aminophenyl riboside; Dichloroaminophenyl riboside;
• 6-Fluoro-3-indoxyl-N-acetyl-glucosaminide Alizarin-N-acetyl-glucosaminide; Nitrophenyl-N-acetyl-glucosaminide; 4-Methylumbelliferyl-N-acetyl-glucosaminide;
• 6-Fluoro-3-indoxyl-glucuronide Alizarin-glucuronide; Nitrophenyl glucuronide;
• Naphtyl xyloside Aminophenyl xyloside; Dichloroaminophenyl xyloside; 5-Bromo
• 6-chloro-3-indoxylfucoside Dihydroxyflavone fucoside; 3,4-Cyclohexenoesculin-fucoside; 5-Bromo-4-chloro-3-indoxylfucoside; 6-chloro-3-indoxylfucoside;
• 3-indoxylcellobioside Dihydroxyflavone-cellobioside; 3,4-Cyclohexenoesculin-cellobioside; 8-Hydroxiquinoline-cellobioside; 5-Bromo-4-chloro-3-indoxylcellobioside; 6-Chloro-3-indoxylcellobioside; 6-Bromo-3-indoxylcellobioside;
• Naphtholbenzein-cellobioside Indoxyl-N-methylcellobioside; 5-Bromo-4-chloro-3-indoxyl-N-methylcellobioside; Naphtylcellobioside; Aminophenylcellobioside;
• Nitrophenyl-arabinoside 4-Methylumbelliferyl-arabinoside; 5-Bromo-4-chloro-3-indoxyl-N-methyl-arabinoside; Naphthyl arabinoside; Aminophenyl-arabinoside; Dichloroaminophenyl arabinoside; 5-Bromo-6-chloro-3-indoxylmannoside;
• Naphtyl mannoside Aminophenylmannoside; Dichloroaminophenylmannoside.
• the osidase substrate is chosen from the substrates of alpha-galactosidase, beta-galactosidase, hexosaminidase and cellobiosidase and in particular from: 5-Bromo-4-chloro-3-indoxyl-galactoside; 5-Bromo-6-chloro-3-indoxyl-N-acetyl-glucosaminide; 3,4-Cyclohexenoesculin-cellobioside; 5-Bromo-4-chloro-
• esterase substrate is meant in particular 5-Bromo-6-chloro-3-indoxyl-octanoate
• 3-indoxylphosphate 5-Bromo-3-indoxylphosphate; 6-Fluoro-3-indoxylphosphate; Alizarin-phosphate; Nitrophenyl phosphate; 4-Methylumbelliferyl phosphate; Naphtholbenzeinphosphate; Indoxyl-N-methylphosphate; 5-Bromo-4-chloro-3-indoxyl-N-methylphosphate; Naphthyl phosphate; Aminophenylphosphate; Dichloroaminophenyl phosphate.
• a concentration of between 25 and 1000 mg / l is particularly suitable for the present invention.
• the esterase substrate is chosen from butyrate esterase, lipase and phosphatase substrates and especially from: 5-Bromo-6-chloro-3-indoxyloctanoate; 5-Bromo-4-chloro-3-indoxyl-butyrate; 5-Bromo-3-indoxylnonanoate and 5-Bromo-6-chloro-3-indoxylphosphate.
• peptidase substrate is intended to mean, in particular, substrates of Proline aminopeptidase, Alanine aminopeptdidase, Leucine aminopeptidase, Pyrrolidonyl arylamidase, Phenylalanine aminopeptidase and Tyrosine aminopeptidase Ala-Phe-Pro-peptidase. These substrates are in particular the compounds associating via a peptide bond the amino acid or the corresponding peptide and a p-nitroaniline, 7-aminomethylcoumarin, 7-aminophenoxazinone, naphthylamine, aminophenyl and dichloroaminophenyl radical.
• Prolyl-p-nitroanilide Prolyl-7-amido-4-methyl-coumarin
• Prolyl-dichloroamidophenol Alanyl-dichloroamidophenol
• Leucyl-p-nitroanilide Pyrrolidonyl-7-amido-4 methylcoumarin
• phenylalanyl-p-nitroanilide Ala-Phe-Pro-7-amido-1-pentyl-phenoxazin-3-one
• Tyrosyl-dichloro-amidophenol a concentration of between 25 and 1000 mg / l is particularly suitable for the present invention.
• the peptidase substrate is chosen from the substrates of Proline-aminopeptidase (or Prolyl-arylamidase), Leucine-aminopeptidase (or Leucyl-arylamidase) and Pyrrolidonyl-arylamidase and in particular: Prolyl-p-nitroanilide, Prolyl- 7-amido-4-methyl-coumarin, Leucyl-p-nitroanilide and Pyrrolidonyl-7-amido-4-methylcoumarin.
• biological sample we mean a clinical sample, from a sample of biological fluid 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, plasma, urine, faeces, nose samples, throats, skin, wounds, cerebrospinal fluid, a dietary sample water, beverages such as milk, fruit juice, yoghurt, meat, eggs, vegetables, mayonnaise, cheese; fish ..., a food sample from a feed intended for animals, such as in particular a sample from animal meal.
• the invention relates to a method for detecting and / or identifying Clostridium difficile, characterized in that it comprises the following steps: a) providing a reaction medium comprising at least one beta glucosidase substrate, capable of identifying C. difficile, b) inoculating the medium with a biological sample to be tested, c) incubating, and d) detecting the hydrolysis of the beta-glucosidase substrate indicating the presence of Clostridium difficile
• step c) the incubation is carried out anaerobically.
• said beta-glucosidase substrate is chosen from Alizarin- ⁇ -glucoside and Magenta- ⁇ -glucoside (5-Bromo-6-chloro
• beta-glucosidase substrate is at a concentration of between 25 and 1000 mg / l, more preferably between 50 and 400 mg / l.
• said reaction medium further comprises esculin.
• esculin is at a concentration of between
• said reaction medium further comprises at least one ⁇ -glucosidase inducer, preferentially chosen from cellobiose or methyl- ⁇ -glucoside.
• said reaction medium further comprises at least one growth promoter for C. difficile strains, preferentially chosen from blood, serum, a reducing agent such as in particular cysteine,
• said reaction medium further comprises at least one inducer of the germination of C. difficile spores, preferably Sodium Taurocholate.
• said reaction medium further comprises at least one selective agent, preferably selected from D cycloserinc, an antibiotic, such as in particular D-cycloserine, cefoxitin, cefotaxime, antifungal, such as such as amphotericin B, fluconazole, itraconazole or voriconazole.
• an antibiotic such as in particular D-cycloserine, cefoxitin, cefotaxime
• antifungal such as such as amphotericin B, fluconazole, itraconazole or voriconazole.
• said reaction medium further comprises at least one second enzymatic substrate, preferably selected from a substrate of osidase, esterase, peptidase.
• said reaction medium further comprises at least one reducing agent, preferably cysteine.
• the invention also relates to a reaction medium for the detection and / or identification of Clostridium difficile comprising at least one beta-glucosidase substrate, capable of identifying C. difficile.
• said beta-glucosidase substrate is selected from Alizarin- ⁇ -glucoside, Magenta- ⁇ -glucoside (5-Bromo-6-chloro-3-indoxyl- ⁇ -glucoside ) and CHE- ⁇ -glucoside (3,4-cyclohexenoesculetin- ⁇ -glucoside)
• said beta-glucosidase substrate is at a concentration of between 25 and 1000 mg / l and more preferably between 50 and 400 mg / l.
• said reaction medium further comprises esculin.
• esculin is at a concentration of between 5 and 500 mg / l, preferably between 10 and 100 mg / l.
• said reaction medium further comprises at least one ⁇ -glucosidase inducer, preferentially chosen from cellobiose or methyl- ⁇ -glucoside.
• said reaction medium further comprises at least one growth promoter for C. difficile strains, preferentially selected from blood, serum, a reducing agent such as in particular cysteine, pyruvate, ferrous sulphide.
• said reaction medium comprises, in addition, at least one inducer of the germination of C. difficile spores, preferably Sodium Taurocholate.
• said reaction medium further comprises at least one selective agent.
• the selective agent is chosen from an antibiotic, preferentially D-cycloserine, cefoxitin or cefotaxime, an antifungal agent, preferably amphotericin B, fluconazole, itraconazole or voriconazole.
• said reaction medium further comprises at least one second enzymatic substrate.
• said second substrate is an osidase, esterase or peptidase substrate.
• said reaction medium further comprises at least one reducing agent, preferably cysteine.
• the invention also relates to the use of a medium as defined above for the detection and / or identification of Clostridium difficile.
• Example 1 Identification of Beta-Glucosidase Substrates Suitable for the Identification of C. difficile
• the ⁇ -glucosidase substrates suitable for the detection of Clostridium difficile strains can be identified by those skilled in the art thanks to the example below.
• the potential ⁇ -glucosidase substrate is tested by incorporating it into a medium allowing the growth of Clostridium difficile, such as a selective medium, containing, for example, inhibitors to slow the growth of certain bacteria or certain eukaryotes or a non-selective medium.
• a selective medium such as Columbia Agar in flask (ref 41244) or an egg base according to George et al. (see example 2).
• an additional reagent such as in particular an iron salt, an ⁇ -Naphtol derivative, to reveal the hydrolysis product.
• the substrate to be tested is tested at a concentration of between 25 and 1000 mg / l.
• the medium thus produced is distributed in a consumable, for example a petri dish or a tube.
• a collection of strains comprising at least one strain of C. difficile is seeded, preferably a single strain per medium.
• the medium is then incubated anaerobically at an appropriate temperature, generally between 20 and 50 ° C., preferably between 30 and 40 ° C.
• Suitable substrates are those for which hydrolysis by the C. difficile strain (s) is detected after incubation.
• the detection is carried out by observing a variation of the optical properties (colonies and / or colored or fluorescent medium) either visually under natural, artificial or UV light, or with the aid of a suitable apparatus, in particular spectrophotometer, fluorimeter, luminometer, image analyzer.
• the basis of the media is an Egg YoIk basis according to George et al. whose composition is as follows (in g / 1):
• the media are divided into Petri dishes.
• Seeding is performed from pre-cultures of 48h at 37 ° C under anaerobic conditions.
• a suspension in physiological saline at 0.5 McF is carried out then l ⁇ l of each suspension is deposited on each box.
• Clostridium spp were tested on 4 different media each containing a chromogenic substrate of beta-glucosidase and 300 mg / L of iron citrate. ammoniacal, according to a protocol similar to that described in Example 2.
• the base of the media was a base Colombia, and the substrates tested were as follows:
• Alizarin ⁇ -glucoside (1,2-dihydroxyanthraquinone- ⁇ -glucoside) at a concentration of 50 mg / l
• Seeding is performed from pre-cultures of 48h at 37 ° C under anaerobic conditions. A suspension in physiological saline at 0.5 McF is carried out then the strains are seeded at the site of 10 ⁇ l calibrated.
• the readings are performed after 24 hours of incubation at 37 ° C.
• medium 2 the absence of glucose causes a decrease in colony size and esculin has a positive effect on the intensity of the colouration.
• media 3 4, and 5, the 2 compounds were mixed.
• medium 4 and 5 there is a more or less significant diffusion of the staining in the agar.
• Medium 3 represents the best compromise between the improvement of growth and color intensity.
• Seeding is performed from pre-cultures of 48h at 37 ° C under anaerobic conditions. A suspension in physiological saline at 0.5 McF is carried out then the strains are seeded at the site of 10 ⁇ l calibrated. The readings are carried out after 24 and 48 hours of incubation at 37 ° C. 3. RESULTS
• the CHE- ⁇ -glucoside substrate has a good sensitivity towards Clostridium difficile from 24h.
• the selectivity of the medium is also very satisfactory since at 24h only 8 out of 76 strains, other than Clostridium difficile, grow on medium 1. It makes it possible to increase the intensity of coloration of Clostridium difficile and to homogenize strains that have a heterogeneous aspect on the medium 1.
• the CHE- ⁇ -glucoside substrate has a good specificity and a good sensitivity towards Clostridium difficile since it makes it possible to highlight nearly 90% of the strains with a minimum false positives from 24h.
• Example 6 Stool samples from patients likely to be infected with a strain of Clostridium difficile were tested on 4 different media. 1. ENVIRONMENT AND MICROORGANISMS
• the first medium (A) corresponds to the medium of Example 1 to which D-cycloserine (250 mg / l) and cefoxitin (16 mg / l) were added.
• the second medium (B) corresponds to the medium 1 of Example 5.
• the 3 e (C) and 4 e (D) media are based on the chromlD Salmonella medium of the applicant, devoid of its enzymatic substrates, added Cysteine to 0.5 g / l and the selective system was replaced by that of medium 1 of Example 5.
• the medium C further comprises CHE-glucoside 100mg / l of 200mg / l ammoniacal iron citrate, Taurocholate sodium at 2.5 g / l and glucose at 1.25 g / l.
• Medium D also comprises CHE-glucoside at 300 mg / l of ammoniacal iron citrate at 500 mg / l, sodium taurocholate at 1 g / l and horse serum at 3%.
• the media are divided into petri dishes.
• Seeding is performed from homogenized stool samples.
• Nb Number of microorganisms detected

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