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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
• • 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
  • C12Q1/045—Culture media therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
  • G01N33/56938—Staphylococcus
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
  • G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)

Definitions

• the present invention relates to a specific reaction medium of Staphylococcus aureus. It also relates to a method for detecting and / or identifying Staphylococcus aureus which uses such a medium.
• Staphylococcus are opportunistic pathogens in humans at high risk for skin injury through trauma or direct implantation of a medical product.
• Staphylococcus aureus is undoubtedly the most virulent species, since it produces a large number of toxins and extracellular enzymes. It can cause many and varied pathologies, ranging from simple paronychia, to the most severe infections such as septicemia, endocarditis, pneumopathies, osteoarticular infections, whose prognosis can be very reserved. It is often found in patients who must receive hospital care involving devices such as syringes, catheters. There is therefore a great interest in detecting the presence of this pathogenic bacterium, which is increasingly involved in nosocomial diseases.
• Staphylococcus aureus In bacteriology, it is conventional to oppose the species Staphylococcus aureus, characterized by the production of a coagulase, to other species called coagulase negative.
• the traditional methods for differentiating Staphylococcus aureus from Staphylococcus non-aureus are based on the search for free coagulase, DNase and on obtaining a latex agglutination aiming to demonstrate the presence of affinity factor for fibrinogen, protein A and capsular antigens.
• other potentially pathogenic staphylococci species are able to express coagulase.
• Staphylococcus aureus there are also culture techniques in selective media to evaluate the presence of Staphylococcus aureus, such as Chapman's hypersaline medium (7.5% NaCl), which is generally selective for Staphylococcus aureus and Staphylococci that hydrolyze. Mannitol, and Baird Parker's medium (containing Potassium Tellurite and Lithium Chloride as selective agents), which is used to isolate and enumerate coagulase-positive staphylococci in products. and to demonstrate lecithinase activity.
• Chapman's hypersaline medium (7.5% NaCl which is generally selective for Staphylococcus aureus and Staphylococci that hydrolyze.
• Mannitol, and Baird Parker's medium containing Potassium Tellurite and Lithium Chloride as selective agents
• culture media comprising enzymatic substrates, in particular substrates for phosphatase and / or beta-glucosidase or alpha-glucosidase.
• enzymatic substrates in particular substrates for phosphatase and / or beta-glucosidase or alpha-glucosidase.
• chromogenic culture medium CHROMagar (registered trademark) Staph. aureus which allows the isolation of staphylococci and the identification of Staphylococcus aureus using chromogenic substrates which provide a purple coloration of the latter species (Gaillot O. et al., 2000, J. Clin Microbiol., 38, 4, 1587-1591).
• the other species of the same genus are then detected by the blue or colorless coloration, in theory.
• the identification is essentially based on the activities phosphatase, beta-glucosidase, beta-glucuronidase and beta-galactosidase, as well as on the presence of an inhibitor, deferoxamine, which allows the differentiation of Staphylococcus aureus and Staphylococcus epidermidis.
• deferoxamine an inhibitor which allows the differentiation of Staphylococcus aureus and Staphylococcus epidermidis.
• the differentiation of Staphylococcus aureus and Staphylococcus epidermidis is imperfect because both species produce colonies of the same color on the CHROMagar (trademark) medium. This is due to the lack of specificity of the phosphatase substrates that are positive for Staphylococcus aureus and Staphylococcus epidermidis and the fact that the inhibition of these by Deferoxamine is only partial.
• alpha-glucosidase substrates As described in the patent application WO02079486. Thus, by detecting only the alpha-glucosidase activity, it is possible to separate Staphylococcus aureus from Staphylococcus epidermidis and Staphylococcus saprophyticus which are three of the main species of staphylococci isolated in clinical practice.
• the alpha-glucosidase substrates with an Indoxyl core chromophore are very suitable for use in a solid medium, their use can be problematic in liquid medium, because the color does not diffuse in the liquid medium.
• enzymatic substrate (s) ribosidase allows easy and rapid identification of Staphylococcus aureus.
• enzymatic substrate (s) ribosidase in combination with a regulator and / or another metabolic indicator makes it possible to distinguish S. aureus from other species of saphylococci.
• 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 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, 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 the microorganisms is carried out before seeding, and in the second case, the detection and / or identification medium also constitutes the culture medium.
• the detection and / or identification medium also constitutes the culture medium.
• Those skilled in the art can also use a bi-box, to easily compare two media, comprising different substrates or different selective mixtures, on which the same biological sample will have been deposited.
• metabolic regulator any compound (s) that regulates the growth of a microorganism.
• This metabolic regulator can be in particular an osidase regulator, a selective compound, such as in particular Lithium Chloride, Potassium Tellurite, Sodium Chloride, or a mixture of selective compounds, an antibiotic, a mixture of antibiotic (s) and or antifungal (s) comprising or not one or more beta-lactamines, one or more aminoglycosides, one or more glycopeptides, one or more quinolones, one or more polypeptides, Amphotericin, one or more azoles, which allows to promote the detection of Staphylococcus aureus, inhibitors that favor the growth of Staphylococcus aureus bacteria, such as in particular Lithium Chloride (LiCl), Sodium Azide (NaN3), Colistin, Amphotericin, Aztreonam, Colimicine, Sodium Chloride (NaCl) , Deferoxamine
• the medium according to the invention comprises a selective mixture for promoting the growth of Staphylococcus aureus and / or inhibiting the growth of other species of microorganisms.
• This mixture preferably comprises lithium chloride, a static vibrio compound 0/129, aztreonam, and amphotericin.
• metabolic regulator of osidase is meant in particular the compounds which can induce or repress the expression of one or more osidase activities in the reaction medium.
• the metabolic regulator concentration is between 0.5 mg / l and 75 g / l.
• Metabolic indicator means any compound (s) which makes it possible to demonstrate the presence of a microorganism.
• This metabolic indicator can be in particular a substrate, such as a ribosidase substrate, or a substrate other than a beta ribosidase substrate, such as in particular a substrate of osidase, esterase, peptidase and more particularly beta-glucosidase. and / or beta galactosidase and / or beta glucuronidase and / or phosphatase, which promotes the detection of S. aureus.
• the medium may also include one or more metabolic indicators different from a beta ribosidase substrate, such as a combination of substrates. The skilled person will adapt the concentration of substrate (s) according to the microorganism that one wishes to identify.
• the concentration of substrate is between 5 and 1000 mg / l, preferably between 50 and 400 mg / l.
• substrate a molecule that can be hydrolysed by an enzyme, such as beta ribosidase 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 referred to as a marker part.
• This marker part can be chromogenic, fluorogenic, luminescent, etc.
• beta ribosidase substrate is meant in particular 2-hydroxyphenyl- ⁇ -D-riboside
• Methylumbelliferyl- ⁇ -D-riboside Naphtol- ⁇ -D-riboside, Dichloro-aminophenyl- ⁇ -D-riboside.
• the concentration of beta ribosidase substrate in the medium according to the invention is between 25 and 1000 mg / l and more preferably between 50 and 400 mg / l.
• beta glucosidase substrate is meant in particular 2-hydroxyphenyl- ⁇ -D-glucoside
• Methylumbelliferyl- ⁇ -D-glucoside Naphtholbenzein- ⁇ -D-glucoside, Indoxyl-N-methyl- ⁇ -D-glucoside, 5-Bromo-4-chloro-3-indoxyl-N-methyl- ⁇ -D-glucoside,
• 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
• beta-galactosidase substrate is meant in particular 2-hydroxyphenyl- ⁇ -D-galactoside (catechol- ⁇ -D-galactoside); Magenta- ⁇ -D-galactoside (5-Bromo-6-chloro-3-indoxyl- ⁇ -D-galactoside); Dihydroxyflavone- ⁇ -D-galactoside; X- ⁇ -D-galactoside (5-Bromo-4-chloro-3-indoxyl- ⁇ -D-galactoside); Rose- ⁇ -D-galactoside (6-chloro-3-indoxyl- ⁇ -D-galactoside); 6-Bromo-3-indoxyl- ⁇ -D-galactoside; 5-Bromo-3-indoxyl- ⁇ -D-galactoside; 6-fluoro-3-indoxyl- ⁇ -D-galactoside; Alizarin- ⁇ -D-galactoside; (P) - Nitrophenyl- ⁇ -D-galactoside
• the concentration of beta-galactosidase substrate in the medium according to the invention is between 25 and 1000 mg / l and more preferably between 50 and 400 mg / l.
• Staphylococcus aureus is any strain of Staphylococcus aureus, including resistant strains such as MRSA (methicillin-resistant Staphylococcus aureus), MSSA, GISA, VISA or VRSA (Staphylococcus aureus with intermediate resistance to glycopeptides, Vancomycin intermediate or Vancomycin resistant respectively).
• MRSA methicillin-resistant Staphylococcus aureus
• MSSA methicillin-resistant Staphylococcus aureus
• GISA methicillin-resistant Staphylococcus aureus
• VISA vancomycin intermediate resistance to glycopeptides
• biological sample is meant 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 food sample of 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 reaction medium for the characterization of Staphylococcus aureus comprising a metabolic indicator which is a beta ribosidase substrate in combination with at least one other metabolic indicator and / or at least one metabolic regulator.
• the invention also relates to the use of a metabolic indicator which is a beta ribosidase substrate in combination with another metabolic indicator and / or a metabolic regulator for the characterization of Staphylococcus aureus.
• said metabolic regulator is chosen from a regulator of the osidase activity, a selective compound, such as in particular lithium chloride, potassium tellurite, sodium chloride, or a mixture of selective compounds, an antibiotic or a mixture of antibiotic (s) and / or antifungal (s), such as in particular one or more beta-lactams, one or more aminoglycosides, one or more glycopeptides, one or more quinolones, one or more polypeptides, Amphotericin, azoles, alone or in combination with one or more selective compounds.
• a selective compound such as in particular lithium chloride, potassium tellurite, sodium chloride, or a mixture of selective compounds
• an antibiotic or a mixture of antibiotic (s) and / or antifungal (s) such as in particular one or more beta-lactams, one or more aminoglycosides, one or more glycopeptides, one or more quinolones, one or more polypeptides, Amphotericin, azoles, alone or
• said other metabolic indicator is an enzymatic substrate, preferably a substrate of osidase, esterase, peptidase, beta-glucosidase and / or beta-galactosidase.
• said other metabolic indicator is selected from a substrate of beta glucosidase and / or beta galactosidase.
• said beta ribosidase substrate is chosen from 2-hydroxyphenyl- ⁇ -D-riboside (catechol- ⁇ -D-riboside); Magenta- ⁇ -D-riboside (5-Bromo-6-chloro-3-indoxyl- ⁇ -D-riboside); Dihydroxyflavone- ⁇ -D-riboside;
• Nitrophenyl- ⁇ -D-riboside 4-methylumbelliferyl- ⁇ -D-riboside.
• said beta ribosidase substrate is the
• said beta ribosidase substrate is X- ⁇ -D-riboside (5-Bromo-4-chloro-3-indoxyl- ⁇ -D-riboside).
• said reaction medium is a solid medium.
• said reaction medium is a liquid medium.
• said beta ribosidase substrate is preferentially Catechol- ⁇ -D-riboside because it allows the detection / identification of S. aureus in approximately
• the invention also relates to a method for detecting and / or identifying
• Staphylococcus aureus characterized in that it comprises or consists of the following steps: a) having a reaction medium as defined above b) seeding the medium with a biological sample to be tested, c) incubating, and d ) characterize the presence of Staphylococcus aureus
• This method may further comprise a confirmation step.
• This confirmatory step can be in particular a biochemical, immunological or molecular identification.
• 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 expression of the enzymatic activity that it is desired to detect is optimal, which the person skilled in the art can easily choose according to the enzymatic activity to be detected.
• step c) it is allowed to incubate during step c) between 16 and 48 h, preferably between 18 and 24 h.
• Step d) can be carried out by visual examination, colorimetry or fluorimetry.
• Strains of S. aureus with acquired resistance to one or more antibiotics and in particular MRSA strains of S. aureus resistant to Meticillin
• GISA strains of S. aureus with intermediate resistance to glycopeptides
• VISA strains of S. Vancomycin intermediate resistance aureus
• VRSA S. aureus strains resistant to Meticillin
• Vancomycin and other resistant phenotypes can be detected by adding the appropriate antibiotic, chosen in particular from ⁇ -lactams, cephalosporins, glycopeptides, aminoglycosides, quinolones, polypeptides, etc.
• the following media were prepared in 200 ml of a base columbia agar (Oxoid), in combination with the chromogenic substrates of ⁇ -D- ⁇ bosidase.
• the different substrates as well as the concentrations and the additives used in the media are decon the following table 1:
• ⁇ -D-ribosidase substrates are solubilized in DMSO and added to the media to obtain the concentrations indicated in Table 1.
• 191 bacterial strains were seeded on each of the media from a suspension at 0.5 MacFarland diluted to 1000 e .
• the dishes were incubated at 37 ° C. and then read.
• the media were divided into Petri dishes then inoculated with approximately
• the strain of 191 strains included: 79 MRSA, 48 Staphylococcus spp, 61
• reaction media comprising the Catechol- ⁇ -D-riboside or X- ⁇ -riboside substrates are the most specific and the most sensitive since they allowed a good detection of Staphylococcus aureus (MRSA and MSSA), and this, after only 18h of incubation for Catechol- ⁇ -D-riboside and 24h incubation for X- ⁇ -D-riboside with only 3% false-positives in coagulase-negative staphylococci.
• MRSA and MSSA Staphylococcus aureus
• the medium containing the DHF- ⁇ -D-riboside was also very sensitive but less specific since the percentage of coagulase-negative staphylococci ⁇ -ribosidase positive was 68 to 24 and 91% at 48 hours.
• the medium below had the same composition as a S. aureus chromlD base (bioMérieux), without selective agents or agar so as to obtain a liquid medium.
• the substrates and inductors were replaced before autoclaving with 300 mg / L of catechol- ⁇ -D-riboside, solubilized in DMSO, and with 500 mg / l of ammoniacal iron citrate.
• the medium was then distributed in tubes at a rate of 1.5 ml / tube. For this medium, seeding was carried out from pre-cultures of 24 h at 37 ° C. A suspension in 0.5 McF physiological saline was carried out then each tube was inoculated with 10 ⁇ l of bacterial suspension.
• Catechol- ⁇ -D-riboside a GTS medium (bioMérieux) supplemented with 300 mg / L of
• X- ⁇ -D-riboside Modified Chapman medium, without NaCl, autoclaved and supplemented with 60 mg / L of X- ⁇ -D-riboside was used and divided into 55 mm diameter Petri dishes.
• Table 4 Evaluation of Catechol- ⁇ -D-riboside and X- ⁇ -D-riboside substrates on a large panel of Staphylococcus spp.
• Table 5 Abbreviation of different combinations of chromogenic ⁇ -ribosidase substrates and chromogenic substrates specific for other enzymatic activities.
• the different substrates are solubilized in DMSO and added to the media.
• IPTG solubilized in osmosis water is added so as to obtain a concentration of 1 mg / l.
• the media were distributed in a 55 mm petri dish and inoculation was carried out starting from pre-cultures for 24 hours at 37 ° C. A suspension in physiological saline at 0.5 McF was carried out then each strain was seeded, at 1 ° C. 'oese calibrated lO ⁇ l, on a box.
• the readings were carried out after 18, 24 and 48 hours of incubation at 37 ° C.
• a total of 28 strains of staphylococci were tested: 5 MRSA and 3 S. aureus, 4 S. saprophyticus, 4 S. cohnii, 4 S. xylosus, 4 S. sciuri, 2 S. epidermidis and 2 S. haemolyticus.
• C colony staining after incubation
• I the Intensity of this staining
• TI defines the incubation times.
• the intensity of the coloration is an arbitrary scale and can be defined as follows:
• MSSA and most other species of Staphylococci was used and divided into 55 mm diameter Petri dishes.
• the substrate was solubilized in DMSO before being added to the medium.
• Seeding was carried out from pre-cultures of 24 h at 37 ° C. A suspension in 0.5 McF physiological saline was carried out then each strain was sown, at the calibrated level 10 ⁇ l, on a box.
• the readings were carried out after 24 and 48 hours of incubation at 37 ° C.

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• 2007
  • 2007-11-26 FR FR0759309A patent/FR2924127B1/fr not_active Expired - Fee Related
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