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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
  • C12Q1/14—Streptococcus; Staphylococcus

Definitions

• the present invention relates to a culture medium for the detection of Staphylococcus aureus bacteria.
• the invention also relates to the use of this medium, as well as a method for identifying S. aureus bacteria.
• the genus Staphylococcus included forty-one species and twenty-four subspecies, of which at least seventeen were found in humans. Most of these species are opportunistic pathogens in humans at high risk for skin injury through trauma or direct implantation of a medical product. Moreover, S. aureus is a bacterium that 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. Amongst staphylococci, S. aureus is undoubtedly the most virulent species, since it produces a large number of toxins and extracellular enzymes.
• S. aureus Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus saprophyticus, and Staphylococcus hominis account for at least 1% of pathogenic isolates isolated from clinical centers, and five of them account for more than 98% of the most common staphylococcal strains. more frequently encountered. Other species are rarely encountered and have low pathogenicity. In bacteriology, it is traditional to contrast the species S. aureus, characterized by the production of a coagulase, with other species called coagulase negative.
• the coagulase positive species are: S. aureus, Staphylococcus intermedius, Staphylococcus hyicus, Staphylococcus delphinis, Staphylococcus lutrae and Staphylococcus schleiferi.
• the hypersaline Chapman medium (75% NaCl) is generally selective for S. aureus and staphylococci that hydrolyze Mannitol. These bacteria turn the medium from red to yellow. Certain microorganisms and group D enterococci, in particular, can produce the same reaction on the medium; it is therefore necessary to check the catalase (negative for streptococci).
• Baird-Parker egg medium (containing Potassium Tellurite and Lithium Chloride as selective agents) is used to isolate and enumerate coagulase-positive staphylococci in food products and highlight activity lecithinase. On this medium the colonies of S. aureus and other coagulase positive species appear with a black center surrounded by an opaque halo. Other micro-organisms can grow on this medium. These are mainly the following groups:
• Chapman lack of sensitivity (ability to highlight the desired species when it is present in small quantities in a biological sample to be tested) and especially specificity (ability to detect the desired species in the biological sample to be tested containing other species).
• S. aureus which uses the Baird-Parker egg medium technique, lacks sensitivity and specificity.
• some strains not belonging to the species S. aureus in particular Staphylococcus schleiferi and Staphylococcus saprophyticus, can also give colonies surrounded by a halo of lightening. It may also happen that some strains of S. aureus do not express the desired enzyme activity or do not grow on the medium, as they may be present in too small amounts in the biological samples and / or inhibited by the components too much. selective middle.
• Baird-Parker + RPF requires the combination of a source of thrombin and a source of plasminogen. These are obtained from the blood of animals, which poses problems of reliability of supply (quality, quantity, ). In addition, the reading of a halo is not possible in broth (liquid medium) and the contrast between the halo and the medium can be reduced. Finally, in the case of confluent colonies, it is difficult to determine which ones produce the halo of those that do not produce it.
• the ⁇ -glucosidase, ⁇ -glucuronidase, ⁇ -galactosidase and phosphatase activities are essentially used, as well as an inhibitor, deferoxamine, which allows the differentiation of S. aureus and, S. epidermidis.
• deferoxamine an inhibitor which allows the differentiation of S. aureus and, S. epidermidis.
• S. aureus differentiation of S. epidermidis is difficult, with both species producing colonies of the same color on the CHROMagar (trademark) medium. This is due to the lack of specificity of the phosphatase substrates which are positive for S. aureus and S. epidermidis and the fact that the inhibition of these by Deferoxamine is only partial.
• the invention proposes to solve the shortcomings of the state of the art by presenting a novel sensitive, specific, and rapid culture medium for isolating and identifying S. aureus.
• reaction medium means a medium comprising all the elements necessary for the survival and / or growth of microorganisms, such as ⁇ S. aureus.
• This reaction medium can either serve only as a revelation medium or a culture medium and revelation.
• the culture of the microorganisms is carried out before inoculation and, in the second case, the reaction medium also constitutes the culture medium.
• the reaction medium may be solid, semi-solid or liquid.
• solid medium is meant for example a gelled medium.
• the medium according to the invention is 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, 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 may contain any other additives, for example: peptones, one or more growth factors, carbohydrates, one or more selective agents, buffer solutions, one or more gelling agents, etc.
• This reaction medium may be in the form of a liquid, a ready-to-use gel, that is to say ready for seeding in a tube, a bottle, or on a petri dish.
• a regeneration passes to 100 ° C. is preferably carried out beforehand of the medium before pouring into a petri dish.
• the medium according to the invention is a selective medium, that is to say a medium comprising inhibitors which favor the growth of Staphylococcus aureus bacteria.
• inhibitors include lithium chloride (LiCl), sodium azide (NaN3), colistin, amphotericin, aztreonam, colimicine, sodium chloride (NaCl), deferoxamine, static vibrio compound 0/129.
• This medium may also be suitable for the detection of methicillin-resistant S. aureus (MRSA), and include one or more antibiotics against which the MRSA are resistant, such as a cephalosporin or a carbapenem, preferably chosen from: • a first-generation cephalosporin, such as: cefalexin, cefaloridine,
• cephalosporin such as Cefoxitin, Cefuroxime, Cefamandole, Cefaclor, Cefotetan, Cefonicide, Cefotiam, Loracarbef, Cefmetazole, Cefprozil, Ceforanide
• cephalosporin such as Cefotaxime, Ceftazidime, Cefsulodine, Ceftriaxone, Cefmenoxime, Latamoxef, Ceftizoxime, Cefixime,
• Cefodizime Cefetamet, Cefpiramide, Cefoperazone, Cefpodoxime, Ceftibuten, Cefdinir, Cefditoren, Ceftriaxone, Cefoperazone
• cephalosporin such as Cefepime, Cefpirome
• the substrate of an enzymatic activity is chosen from any substrate that can be hydrolyzed to a product that allows the direct or indirect detection of an alpha glucosidase enzyme activity. It can be a natural or synthetic substrate.
• the metabolism of the substrate causes a variation in the physicochemical properties of the reaction medium or the cells of organisms. This variation can be detected by physicochemical methods, especially optical methods by the eye of the operator or by means of instruments, spectrometric, electrical, magnetic, ... Preferably, it is a question of a change in optical properties, such as a change in absorption, fluorescence or luminescence.
• a chromogenic substrate there may be mentioned in particular substrates based on indoxyl, flavone, alizarin, naphtholbenzein, nitrophenol, naphthol, catechol, hydroxyquinoline, coumarin.
• the substrate (s) used in the present invention is based on indoxyl.
• a fluorescent substrate mention may be made in particular of substrates based on umbelliferone or coumarin, based on resorufin, phenoxazine, naphthol and naphthylamine. , 2'-hydroxyphenyl-heterocycle or based on fluorescein.
• 5-Bromo-6-chloro-3-indoxyl-alpha-glucoside substrates Dihydroxyflavone-alpha-glucoside; 3,4-cyclohexenoesculetin-alpha-glucoside; 8-Hydroxiquinoline-alpha-glucoside; 5-Bromo-4-chloro-3-indoxyl-alpha-glucoside; 5-Bromo-4-chloro-3-indoxyl-N-methyl-alpha-glucoside; 6- Chloro-3-indoxyl-alpha-glucoside; 5-Bromo-3-indoxyl-alpha-glucoside; 5-Iodo-3-indoxyl-alpha-glucoside; 6-Fluoro-3-indoxyl-alpha-glucoside; Alizarin-alpha-glucoside; Nitrophenyl-alpha-glucoside; 4-methylumbelliferyl
• the substrate used in the present invention is 5-Bromo-4-chloro-3-indoxyl-alpha-glucoside in combination with 5-Bromo-4-chloro-3-indoxyl-N-methyl-alpha-glucoside.
• the substrates of the invention can be used in a wide pH range, in particular between pH 5.5 and 10, preferably between pH 6.5 and 10.
• the substrate concentration is preferably between 0.01 and 2 g / l, more preferably between 0.02 and 0.2 g / l, and advantageously it is 0.1 g / l. In fact, at this concentration of substrate, a better color contrast is obtained.
• Biological sample means a clinical specimen from a bronchial, tracheal or pulmonary aspiration sample, pleural fluid, bronchoalveolar lavage, sputum, blood or lung biopsy, joint fluid or pericardial; 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, perineum, throats, skin, wounds, cerebrospinal fluid, a food sample.
• the invention relates to a reaction medium for the detection and / or identification of S. aureus bacteria comprising a combination of two enzymatic substrates of alpha glucosidase.
• one of the two substrates is an indoxyl-alpha-glucoside substrate, preferentially 5-Bromo-4-chloro-3-indoxyl-N-methyl-alpha-glucoside (XN- methyl-alpha-glucoside) or 5-Bromo-4-chloro-3-indoxyl-alpha-glucoside (X-alpha-glucoside).
• said indoxyl-alpha-glucoside substrate is present in the medium at a concentration of between 0.01 and 2 g / l, preferably between 0.05 and 0.3 g / l.
• said combination of two enzymatic substrates of alpha glucosidase comprises 5-Bromo-4-chloro-3-mdoxyl-N-methyl-alpha-glucoside and 5-Bromo-4-chloro-3-indoxyl-alpha-glucoside .
• said medium further comprises a mixture of inhibitors which favors the growth of Staphylococcus aureus bacteria, preferentially lithium chloride (LiCl), sodium azide (NaN3), colistin, Vibriostatic compound 0/129, Aztreonam, Amphotericin, Colimicine, Sodium Chloride (NaCl), Deferoxamine.
• a mixture of inhibitors which favors the growth of Staphylococcus aureus bacteria, preferentially lithium chloride (LiCl), sodium azide (NaN3), colistin, Vibriostatic compound 0/129, Aztreonam, Amphotericin, Colimicine, Sodium Chloride (NaCl), Deferoxamine.
• the medium further comprises a mixture of inhibitors, comprising four inhibitors, which favors the growth of bacteria of the genus Staphylococcus, which are LiCl, vibriostatic compound 0/129, aztreonam, and. Amphotericin.
• the invention also relates to the in vitro use of a reaction medium as defined above for isolating and identifying S. aureus bacteria.
• the invention finally relates to a method for detecting and / or identifying S. aureus bacteria in a biological sample according to which a) the biological sample capable of containing S. aureus bacteria is seeded on a reaction medium as defined herein. before b) incubates c) the colonies of S. aureus are identified. d) MRSA colonies are identified.
• the incubation is preferably carried out at a temperature of between 30 ° C. and 42 ° C.
• the S. aureus are detected by a specific ⁇ -glucosidase activity which makes it possible to obtain colored or fluorescent colonies.
• Other species of Staphylococcus appear colorless or of a different color or fluorescence than that of S. aureus colonies.
• the invention also relates to a reaction medium for the detection and / or identification of MRSA bacteria comprising a combination of two enzymatic substrates of alpha glucosidase and an antibiotic, against which the MRSA are resistant, preferentially a cephalosporin, such as cefoxitin. .
• This antibiotic may be in combination with another antibiotic.
• Such a combination is preferably chosen from cefoxitin / cefotaxime or cefoxitin / ertapenem.
• Such a medium is particularly suitable for the detection of MRSA.
• one of the two substrates is an indoxyl-alpha-glucoside substrate, preferentially 5-Bromo-4-chloro-3-indoxyl-N-methyl-alpha-glucoside (XN- methyl-alpha-glucoside) or 5-Bromo-4-chloro-3-indoxyl-alpha-glucoside (X-alpha-glucoside).
• said indoxyl-alpha-glucoside substrate is present in the medium at a concentration of between 0.01 and 2 g / l, preferably between 0.05 and 0.3 g / l.
• said combination of two enzymatic substrates of alpha glucosidase comprises 5-Bromo-4-chloro-3-mdoxyl-N-methyl-alpha-glucoside and 5-Bromo-4-chloro
• said medium further comprises a mixture of inhibitors which favors the growth of Staphylococcus aureus bacteria, preferentially lithium chloride (LiCl), sodium azide (NaN3), colistin, vibriostatic compound 0/129, aztreonam, amphotericin, colimicine, sodium chloride (NaCl), deferoxamine.
• a mixture of inhibitors which favors the growth of Staphylococcus aureus bacteria, preferentially lithium chloride (LiCl), sodium azide (NaN3), colistin, vibriostatic compound 0/129, aztreonam, amphotericin, colimicine, sodium chloride (NaCl), deferoxamine.
• the invention also relates to the in vitro use of a reaction medium as defined above for isolating and identifying MRSA bacteria.
• the invention finally relates to a method for detecting and / or identifying MRSA bacteria in a biological sample according to which a) the biological sample which may contain bacteria is inoculated.
• MRSA on a reaction medium as defined above b) incubates c) the MRSA colonies are identified.
• the incubation is preferably carried out at a temperature between 30 ° C and 42 ° C.
• MRSA are detected by a specific ⁇ -glucosidase activity which makes it possible to obtain colored or fluorescent colonies. Other species appear colorless or of a different color or fluorescence than MRSA colonies.
• the media tested in the following experiments were media comprising MRSA chromlD medium as base medium (bioMérieux 43,451), and comprising the following elements:
• Control medium T control medium chromID MRSA (ref 43 451), comprising in particular an X-N-methylglucoside substrate at a concentration of 0.1 g / l and cefoxitin at 4 mg / l.
• Medium S Medium T, further comprising an X-alpha-glucoside substrate at a concentration of 25, 37, 45 or 50 mg / l.
• Medium F medium S (X-alpha-glucoside concentration: 45 mg / l), Cefoxitin being substituted by a Cefoxitme / Cefbtaxime combination at the concentrations below
• Medium G medium S (X-alpha-glucoside concentration: 45 mg / l), Cefoxitin being substituted with a Cefoxitin / Ceftriaxone combination at the concentrations below
• Medium H Medium S (X-alpha-glucoside concentration: 45 mg / l), Cefoxitin being substituted with a Cefoxitin / Ertapenem combination at the concentrations below
• Medium J medium S (X-alpha-glucoside concentration: 45 mg / l), cefoxitin being substituted with a cefoxitin / cefoperazone combination at the concentrations below
• Medium K medium S (X-alpha-glucoside concentration: 45 mg / l), cefoxitin being substituted with a Cefoxitin / Cefotaxime combination, at the concentrations below, further comprising a mixture of inhibitors of microorganisms not belonging to to the genus Staphylococcus, favoring the growth of S. aureus.
• the strains detected correspond to the strains forming colonies stained on the medium.
• MRSA Medium for detecting MRSA comprising two alpha-glucosidase substrates and a combination of antibiotics selected from cefoxitin / ceftriaxone, cefoxitin / cefotaxime, cefoxitin / ertapenem,
• Combinations of antibiotics above allow performance superior to that of a medium with cefoxitin alone at 4 mg / l.
• 3.3 - Medium for detecting MRSA comprising a combination of Cefoxitine / Cefotaxime antibiotics and a mixture of inhibitors favoring the growth of S. aureus.
• Cefoxitine / Cefotaxime antibiotics combined with a mixture of inhibitors favoring the growth of S. aureus allowed to obtain an excellent specificity and sensitivity, in particular when using Cefoxitin and Cefotaxime concentration of 0 , 75 mg / l.

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• 2008
  • 2008-10-08 FR FR0856815A patent/FR2937052A1/fr active Pending
• 2009
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