Reaction medium for methicillin-resistant Staphylococcus aureus (MRSA) bacteria
The present invention relates to a reaction medium for detecting methicillin-resistant Staphylococcus aureus (MRSA) bacteria. The invention also relates to the use of this medium, and to a method for identifying MRSA bacteria.
Methicillin-resistant Staphylococcus aureus (MRSA) are Staphylococcus aureus strains characterized by their resistance to an antibiotic, methicillin, and to related antibiotics such as oxacillin. MRSA bacteria represent a high percentage of nosocomial infections, and are often responsible for serious and potentially fatal health problems. Most commonly cross-transmitted between patients via the healthcare staff, MRSA are responsible for endemic infections that are very difficult to control. In addition to an appropriate treatment, the screening for MRSA carriers and the isolation of colonized patients constitutes the most effective method recommended today by official organizations such as the Society for Healthcare Epidemiology of America. Early and systematic screening is therefore essential. MRSA can be detected by various techniques.
It is thus possible to detect MRSA by molecular biology techniques. In this respect, mention may in particular be made of Application EP887424. However, such methods remain expensive as a routine test, and require qualified staff.
It is also possible to use conventional culture media for detecting Staphylococcus aureus, such as the medium described in Application EP1390524. The detection of MRSA is carried out in an additional step, by means of a specific agglutination test (Slidex MRSA, bioMerieux) or by means of an agar diffusion method in the presence of an oxacillin, cefoxitin or latamoxef disc (recommendations of the Comite de rAntibiogramme de la Societe Francaise de Microbiologie [Antibiogram Committee of the French Society for Microbiology] and of the Clinical Laboratory Standard Institute).
It is also possible to culture bacteria that may be MRSA on agar media in the presence of antibiotics. Such media may also be chromogenic, thereby facilitating the reading and detection of the MRSA. Mention may in particular be made of the medium described in Application EP1543147. However, since the detection of a phosphatase activity under the conditions described is not very specific, it is necessary to combine it with the detection of several other enzymatic activities, thereby reducing the fertility of the medium and increasing the cost thereof.
The invention proposes to solve the gaps in the prior art by providing a novel sensitive, specific reaction medium that enables isolation and rapid identification of methicillin-resistant Staphylococcus aureus (MRSA).
Surprisingly, the inventors have demonstrated that the use of a particular combination of antibiotics, comprising a cephalosporin and an aminoglycoside, makes it possible to obtain an excellent reaction medium for isolating and identifying methicillin-resistant Staphylococcus aureus (MRSA).
Before going any further, the following definitions, which are in no way limiting, will make it
possible to understand the invention more clearly.
For the purpose of the present invention, the term reaction medium is intended to mean a
medium comprising all the elements necessary for the survival and/or the growth of
microorganisms, such as Staphylococcus aureus.
This reaction medium may serve only as revealing medium, or as culture and revealing
medium. In the first case, the microorganisms are cultured 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. The term "solid medium" is intended
to mean, for example, a gelled medium. Preferably, the medium according to the invention is
a gelled medium. Agar is the conventional gelling agent in microbiology for culturing
microorganisms, but it is also possible to use other gelling agents, for instance gelrite, gelatin
or agarose. A certain number of preparations are commercial available, for instance Columbia
agar, trypticase soy agar, Mac Conkey agar, Chapman 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 possible other additives such as,
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 or of a gel that is ready to use, i.e. ready for inoculation in a tube or a flask, or
on a Petri dish. When the presentation form is in the form of a gel in a flask, a prior
regeneration (change to 100°C) of the medium is preferably carried out before pouring into a
Petri dish.
Preferably, the medium according to the invention is a selective medium, i.e. a medium
comprising inhibitors which favour the growth of Staphylococcus aureus bacteria. Mention
may in particular be made of lithium chloride (LiCl), sodium azide (NaN3), colistin,
amphotericin, aztreonam, colimycin, sodium chloride (NaCl), deferoxamine, and vibriostatic

compound O/129.
For the purpose of the present invention, the substrate for an enzymatic or metabolic activity
is chosen from any substrate that can be hydrolysed to give a product which allows the direct
or indirect detection of an enzymatic activity or of a metabolism, such as, in particular, an
osidase activity, preferably a glucuronidase, glucosidase or galactosidase activity.
It may be a natural or synthetic substrate. The metabolism of the substrate causes a variation
in the physicochemical properties of the reaction medium or of the cells of organisms. This
variation can be detected by physicochemical methods, in particular optical methods visible to
the eye of the operator, or by means of spectrometric, electrical, magnetic, etc., instruments.
Preferably, it is a variation in optical properties, such as a modification of absorption, of
fluorescence or of luminescence.
In the present application, the term "colouration" is used to cover a colouration, which is
absorption of light in the visible spectrum, or a fluorescence, which is absorption at one
wavelength (A,ex) and emission at a greater wavelength (A.em, A.em > A,ex).
For the purpose of the present invention, the term chromogenic substrate is intended to mean
any substrate that can be hydrolysed to give a product which allows the direct or indirect
detection of an enzymatic activity, such as, in particular, an osidase activity, preferably an
alpha-glucosidase activity, an esterase activity, preferably a phosphatase activity, or a
peptidase activity, preferably a coagulase activity.
Mention may in particular be made of substrates based on indoxyl, flavone, alizarin, acridine,
phenoxazine, nitrophenol, nitroaniline, naphthol, catechol, hydroxyquinoline, coumarin or
hydroxyphenylquinoxazol-4-one. Preferably, the substrate(s) used in the present invention is
(are) indoxyl-based.
As alpha-glucosidase substrate, mention may more particularly be made of the substrates
5-bromo-6-chloro-3-indoxyl-alpha-glucoside; dihydroxyflavone-alpha-glucoside; 3,4-cyclo-
hexenoesculetin-alpha-glucoside; 8-hydroxyquinoline-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-alpha-glucoside; naphtholbenzein-alpha-glucoside; indoxyl-
N-methyl-alpha-glucoside; naphthyl-alpha-glucoside; aminophenyl-alpha-glucoside;
dichloroaminophenyl-alpha-glucoside. Preferably, the alpha-glucosidase substrate used is
5-bromo-4-chloro-3-indoxyl-N-methyl-alpha-glucoside or 5-bromo-4-chloro-3-indoxyl-alpha-
glucoside.
As phosphatase substrate, mention may more particularly be made of the substrates 5-bromo-
6-chloro-3-indoxyl phosphate; 3,4-cyclohexenoesculetin phosphate; 5-bromo-4-chloro-
3-indoxyl phosphate; 5-bromo-4-chloro-3-indoxyl-N-methyl phosphate; 6-chloro-3-indoxyl
phosphate; 5-bromo-3-indoxyl phosphate; 5-iodo-3-indoxyl phosphate; 6-fluoro-3-indoxyl
phosphate; nitrophenyl phosphate; 4-methylumbelliferyl phosphate; indoxyl-N-methyl
phosphate; naphthyl phosphate, ELF97-phosphate. Preferably, the phosphatase substrate used
is 6-chloro-3-indoxyl phosphate.
The substrate used in the present invention may be in combination with other substrates, such
as a substrate for an osidase, esterase, and in particular phosphatase or phospholipase,
peptidase, and in particular coagulase. In particular, the medium may comprise two alpha-
glucosidase substrates.
The substrates of the invention can be used in a wide pH range, in particular between pH 5.5
and 10, preferably between 6.5 and 9.
For the purpose of the present invention, an antibiotic which belongs to the cephalosporin
family is an antibiotic preferably chosen from:
o A first-generation cephalosporin, such as: cefalexin, cefaloridine, cefalotin,
cefazolin, cefadroxil, cefazedone, cefatrizine, cefapirine, cefradine, cefacetrile,
cefrodaxine, ceftezole. o A second-generation cephalosporin, such as: cefoxitin, cefuroxime, cefamandole,
cefaclor, cefotetan, cefonicide, cefotiam, loracarbef, cefmetazole, cefprozil,
ceforanide, cefminox. o A third-generation cephalosporin, such as: cefotaxime, ceftazidime, cefsulodine,
ceftriaxone, cefmenoxime, latamoxef, ceftizoxime, cefixime, cefodizime, cefetamet,
cefpiramide, cefoperazone, cefpodoxime, ceftibuten, cefdinir, cefditoren,
ceftriaxone, cefoperazone, cefbuperazone, cefdinir. o A fourth-generation cephalosporin, such as cefepime, cefpirome. In the context of the present invention, the antibiotic which belongs to the cephalosporin family is preferably cefminox or cefdinir.
For the purpose of the present invention, an antibiotic which belongs to the aminoglycoside family is an antibiotic preferably chosen from amikacin, gentamicin, isepamicin, kanamycin, netilmicin, streptomycin and tobramycin.
In the context of the present invention, the antibiotic which belongs to the aminoglycoside family is preferably amikacin or kanamycin. The term biological sample is intended to mean a clinical sample, derived from a bronchial,
tracheal or pulmonary aspiration specimen, a pleural fluid specimen, a bronchoalveolar lavage specimen, an expectoration specimen, a blood specimen or a lung biopsy specimen, or a joint fluid or pericardial fluid specimen; a biological fluid or a food sample, derived from any type of food, or a surface specimen. This sample may thus be liquid or solid and mention may be made, in a nonlimiting manner, of a clinical sample of blood, plasma, urine or faeces, or of specimens from the nose, perineum, throat, skin, wounds or cerebrospinal fluid, or a food sample.
The term "sample" is therefore intended to mean the specimen in itself (swab, stools, foods, etc.) and also colonies of microorganisms derived from said specimen (for example after isolation on a gelled culture medium, or in an enrichment broth inoculated with said specimen).
In this respect, the invention relates to a reaction medium for detecting and/or identifying
methicillin-resistant Staphylococcus aureus (MRSA) bacteria, comprising a chromogenic
substrate, a first antibiotic which belongs to the cephalosporin family and a second antibiotic
which belongs to the aminoglycoside family.
According to one preferred embodiment of the invention, said first antibiotic is cefminox or
cefdinir. Preferably, the cefminox concentration is between 10 and 35 mg/1, preferably
between 12 and 28 mg/1. Preferably, the cefdinir concentration is between 0.1 and 1 mg/1,
preferably between 0.2 and 0.6 mg/1.
According to one preferred embodiment of the invention, said second antibiotic is kanamycin
or amikacin.
Preferably, the kanamycin concentration is between 0.2 and 1.5 mg/1, preferably between 0.5
and 1.25 mg/1. Preferably, the amikacin concentration is between 0.5 and 5 mg/1, preferably
between 1 and 2 mg/1.
According to one preferred embodiment of the invention, the combination of two antibiotics
comprises cefminox and kanamycin.
According to another preferred embodiment of the invention, the combination of two
antibiotics comprises cefminox and amikacin.
According to another preferred embodiment of the invention, the combination of two
antibiotics comprises cefdinir and kanamycin.
According to another preferred embodiment of the invention, the combination of two
antibiotics comprises cefdinir and amikacin.
According to one preferred embodiment of the invention, said chromogenic substrate allows the detection of an osidase, esterase or peptidase activity.
According to one preferred embodiment of the invention, said osidase activity is an alpha-glucosidase activity.
Said substrate for an alpha-glucosidase activity is preferably an indoxyl-alpha-glucoside. Preferably, the substrate used is 5-bromo-4-chloro-3-indoxyl-alpha-glucoside (X-alpha-glucoside) or 5-bromo-4-chloro-3-indoxyl-N-methyl-alpha-glucoside (Green A-alpha-glucoside). Preferably, this substrate is present in the medium at a concentration of between 0.01 and 0.2 g/1, preferably between 0.03 and 0.15 g/1. In fact, at this substrate concentration, a better colouration contrast is obtained.
According to one preferred embodiment of the invention, said esterase activity is a phosphatase activity. Preferably, the substrate used is 6-chloro-3-indoxyl phosphate (pink-phosphate). Preferably, this substrate is present in the medium at a concentration of between 0.05 and 0.5 g/1, preferably between 0.1 and 0.4 g/1. In fact, at this substrate concentration, a better colouration contrast is obtained.
According to one particular embodiment of the invention, said reaction medium also comprises a second enzyme substrate. Preferably, said second substrate is an alpha-glucosidase substrate. Preferably, said first substrate is an alpha-glucosidase substrate, preferably an X-alpha-glucoside (5-bromo-4-chloro-3-indoxyl-alpha-glucoside) and said second substrate is an alpha-glucosidase substrate, preferably Green A-alpha-glucoside (5-bromo-4-chloro-3-indoxyl-N-methyl-alpha-glucoside).
According to one preferred embodiment of the invention, the medium also comprises at least one inhibitor which favours the growth of Staphylococcus aureus bacteria, such as lithium chloride (LiCl), sodium azide (NaN3), colistin, amphotericin, aztreonam, polymyxins, sodium chloride (NaCl) and deferoxamine.
According to one preferred embodiment of the invention, the medium also comprises a mixture of inhibitors, comprising four inhibitors, which favours the growth of Staphylococcus aureus bacteria, said inhibitors being 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 methicillin-resistant Staphylococcus aureus (MRSA) bacteria. The invention also relates to a method for detecting and/or identifying methicillin-resistant Staphylococcus aureus (MRSA) bacteria in a biological sample, comprising:
a) inoculating the biological sample that may contain methicillin-resistant
Staphylococcus aureus (MRSA) bacteria on a reaction medium as defined above;
b) incubating;
c) identifying the colonies as being MRSA colonies.
The incubation is preferably carried out at a temperature of between 30°C and 42°C. The MRSA are preferably detected by means of one or two a-glucosidase activities or a phosphatase activity which makes it possible to obtained coloured or fluorescent colonies. The other species of Staphylococcus appear colourless or have a colour or fluorescence different from that of the S. aureus colonies. Step a) may be preceded by a step of pre-enrichment in a selective or nonselective medium.
The invention also relates to a reaction medium for detecting and/or identifying methicillin-
resistant Staphylococcus aureus (MRSA) bacteria, comprising a substrate for an enzymatic or
metabolic activity, a first antibiotic chosen from cefdinir and cefminox, and a second
antibiotic which belongs to the aminoglycoside family.
Preferably, the cefminox concentration is between 10 and 35 mg/1, preferably between 12 and
28 mg/1. Preferably, the cefdinir concentration is between 0.1 and 1 mg/1, preferably between
0.2 and 0.6 mg/1.
According to one preferred embodiment of the invention, said second antibiotic is kanamycin
or amikacin.
Preferably, the kanamycin concentration is between 0.2 and 1.5 mg/1, preferably between 0.5
and 1.25 mg/1. Preferably, the amikacin concentration is between 0.5 and 5 mg/1, preferably
between 1 and 2 mg/1.
According to one preferred embodiment of the invention, the combination of two antibiotics
comprises cefminox and kanamycin.
According to another preferred embodiment of the invention, the combination of two
antibiotics comprises cefminox and amikacin.
According to another preferred embodiment of the invention, the combination of two
antibiotics comprises cefdinir and kanamycin.
According to another preferred embodiment of the invention, the combination of two
antibiotics comprises cefdinir and amikacin.
According to one preferred embodiment of the invention, said substrate for an enzymatic or
metabolic activity allows the detection of an osidase, esterase or peptidase activity.
According to one preferred embodiment of the invention, said osidase activity is an alpha-
glucosidase activity.
Said substrate for an alpha-glucosidase activity is preferably an indoxyl-alpha-glucoside. Preferably, the substrate used is 5-bromo-4-chloro-3-indoxyl-alpha-glucoside (X-alpha-glucoside) or 5-bromo-4-chloro-3-indoxyl-N-methyl-alpha-glucoside (Green A-alpha-glucoside). Preferably, this substrate is present in the medium at a concentration of between 0.01 and 0.2 g/1, preferably between 0.03 and 0.15 g/1. In fact, at this substrate concentration, a better colouration contrast is obtained.
According to one preferred embodiment of the invention, said esterase activity is a phosphatase activity. Preferably, the substrate used is 6-chloro-3-indoxyl phosphate (pink-phosphate). Preferably, this substrate is present in the medium at a concentration of between 0.05 and 0.5 g/1, preferably between 0.1 and 0.4 g/1. In fact, at this substrate concentration, a better colouration contrast is obtained.
According to one particular embodiment of the invention, said reaction medium also comprises a second enzyme substrate. Preferably, said second substrate is an alpha-glucosidase substrate. Preferably, said first substrate is an alpha-glucosidase substrate, preferably X-alpha-glucoside (5-bromo-4-chloro-3-indoxyl-alpha-glucoside) and said second substrate is an alpha-glucosidase substrate, preferably Green A-alpha-glucoside (5-bromo-4-chloro-3-indoxyl-N-methyl-alpha-glucoside).
According to one preferred embodiment of the invention, the medium also comprises at least one inhibitor which favours the growth of Staphylococcus aureus bacteria, such as lithium chloride (LiCl), sodium azide (NaN3), colistin, amphotericin, aztreonam, polymyxins, sodium chloride (NaCl) and deferoxamine.
According to one preferred embodiment of the invention, the medium also comprises a mixture of inhibitors, comprising four inhibitors, which favours the growth of Staphylococcus aureus bacteria, said inhibitors being 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 methicillin-resistant Staphylococcus aureus (MRSA) bacteria. The invention also relates to a method for detecting and/or identifying methicillin-resistant Staphylococcus aureus (MRSA) bacteria in a biological sample, comprising:
a) inoculating the biological sample that may contain methicillin-resistant Staphylococcus aureus (MRSA) bacteria on a reaction medium as defined above;
b) incubating;
c) identifying the colonies as being MRSA colonies.
The incubation is preferably carried out at a temperature of between 30°C and 42°C.
The MRSA are preferably detected by means of one or two alpha-glucosidase activities or a phosphatase activity which makes it possible to obtain coloured or fluorescent colonies. The other species of Staphylococcus appear colourless or have a colour or fluorescence different from that of the S. aureus colonies. Step a) may be preceded by a step of pre-enrichment in a selective or nonselective medium.
The following examples are given by way of illustration and are in no way limiting in nature. They will make it possible to understand the invention more clearly.
EXAMPLE 1 - MRSA MEDIUM COMPRISING A CEFMINOX-KANAMYCIN ANTIBIOTIC COMBINATION AND VARIOUS CHROMOGENIC SUBSTRATES
1. Preparation of the medium according to the invention
The media tested in the experiments were the following:
Medium T: chromID MRSA control medium (ref. 43 451).
Medium A: medium T, the cefoxitin being substituted with a cefminox (Daewoo Chemical, A8060902; 16mg/l)/kanamycin (Sigma, ref. K4000; 1 mg/1) antibiotic combination, the chromogenic substrate being pink-phosphate (Biosynth, ref. C-5100; 0.25 g/1).
Medium B: medium T, the cefoxitin being substituted with a cefminox (16 mg/1)/ kanamycin (0.75 mg/1) antibiotic combination, the chromogenic substrate being X-alpha-glucoside (Biosynth, B7230; 0.045 g/1) and Green A-alpha-glucoside (Inalco, ref. 1758-0730).
Medium C: medium T, the cefoxitin being substituted with a cefminox (16 mg/1)/ kanamycin (0.75 mg/1) antibiotic combination, the chromogenic substrates being X-alpha-glucoside (Biosynth, B7230; 0.045 g/1) and Green A-alpha-glucoside (Inalco, ref. 1758-0730, 0.08 g/1).
2. Inoculation and reading of media
Various methicillin-resistant (MRSA) or methicillin-sensitive (MSSA) strains of S. aureus bacteria, and coagulase-negative staphylococcal strains (CoNS), all derive from the applicant's collection, were suspended in physiological saline, and then inoculated on the medium, according to the four-quadrant streaking technique. The dishes were incubated at 37°C for 24 hours. The colonies formed were examined visually after 18 h and 24 hours of incubation.
3. Results:
The results obtained are shown in Table 1.
(Table Removed)
These results show an excellent sensitivity of the media according to the invention. In particular, medium A made it possible to detect all MRSA.
EXAMPLE 2 - MRSA MEDIUM COMPRISING CEFMINOX AND KANAMYCIN AT VARIOUS
CONCENTRATIONS
1. Preparation of the medium according to the invention
The media tested in the experiments were the following:
Medium T: chromID MRSA control medium (ref. 43 451).
Medium D: medium T, the cefoxitin being substituted with a cefminox (antibiotic identical to that used in Example l)/kanamycin (antibiotic identical to that used in Example 1) combination of antibiotics, at various concentrations, the chromogenic substrates being X-alpha-glucoside (substrate identical to that used in Example 1; 0.045 g/1) and Green A-alpha-glucoside (substrate identical to that used in Example 1; 0.08 g/1).
(Table Removed)
2. Inoculation and reading of media
This step was carried out as in Example 1.
3. Results:
The results obtained are shown in Table 2 below.
(Table Removed)
These results show that a cefminox concentration of 16 mg/1 and a kanamycin concentration of 0.75 mg/1 made it possible to obtain excellent specificity and sensitivity.
EXAMPLE 3 - MRSA MEDIUM COMPRISING A CEFMINOX-AMIKACIN ANTIBIOTIC
COMBINATION AND VARIOUS CHROMOGENIC SUBSTRATES
1. Preparation of the medium according to the invention
The media tested in the experiments were the following:
Medium T: chromID MRSA control medium (ref. 43 451).
Medium E: medium T, the cefoxitin being substituted with a cefminox (antibiotic identical to that used in Example 1; 16 mg/l)/amikacin (Sigma, ref. 2324; 2 mg/1) antibiotic combination, the chromogenic substrate being pink-phosphate (Biosynth, C-5100; 0.25 g/1).
Medium F: medium T, the cefoxitin being substituted with a cefminox (16 mg/1)/ amikacin (2.5 mg/1) antibiotic combination, the chromogenic substrates being X-alpha-glucoside (substrate identical to that used in Example 1; 0.045 g/1) and Green A-alpha-glucoside (substrate identical to that used in Example 1; 0.08 g/1).
2. Inoculation and reading of media
This step was carried out as in Example 3
3. Results:
The results obtained are shown in Table 3 below.
(Table Removed)
These results show that the antibiotic combination according to the invention made it possible to obtain excellent sensitivity, irrespective of the substrate used.
EXAMPLE 4 - MRSA MEDIUM COMPRISING CEFDINIR AND AMIKACIN 1. Preparation of the medium according to the invention
The media tested in the experiments were the following:
Medium T: chromID MRSA control medium (ref. 43 451).
Medium G: medium T, the cefoxitin being substituted with a cefdinir
(CDR 0806007)/amikacin (antibiotic identical to that used in Example 3) combination of antibiotics at various concentrations, the chromogenic substrate being pink-phosphate (substrate identical to that used in Example 3; 0.25 g/1).
(Table Removed)
Medium H: medium T, the cefoxitin being substituted with a cefdinir/amikacin combination of antibiotics, at various concentrations, the chromogenic substrate being X-alpha-glucoside (substrate identical to that used in Example 1; 0.045 g/1) and Green A-alpha-Glu (substrate identical to that used in Example 1, 0.08 g/1).
(Table Removed)
2. Inoculation and reading of media
This step was carried out as in Example 1.
3. Results:
The results obtained are shown in Table 4 below.
(Table Removed)
These results show that the cefdinir/amikacin antibiotic combination according to the invention made it possible to obtain excellent sensitivity and specificity, irrespective of the concentration used.
EXAMPLE 5 - ANALYSES OF CLINICAL SPECIMENS ON MRSA MEDIA ACCORDING TO THE
INVENTION
1. Preparation of the medium according to the invention
The media tested in the experiments were the following:
Medium T: chromID MRSA control medium (ref. 43 451).
Batch 1: 0.08 g/1 Green A-alpha-Glu + 0.045 g/1 X-alpha-Glu, 16mg/l cefminox +
0.75 mg/1 kanamycin.
Batch 2: 0.08 g/1 Green A-alpha-Glu + 0.045 g/1 X-alpha-Glu, 0.3 mg/1 cefdinir + 2 mg/1 amikacin.
Batch 3: 0.25 g/1 pink-phosphate, 16 mg/1 cefminox + 2 mg/1 amikacin. Batch 4: 0.25 g/1 pink-phosphate, 0.3 mg/1 cefdinir + 2 mg/1 amikacin. The substrates and antibiotics used were the same as those used in the previous examples.
2. Inoculation and reading of media
Two studies were carried out in parallel using various clinical specimens (nose, throat, perineum, wound), which were suspended in physiological saline, and inoculated so as to give isolated colonies on the medium, according to the four-quadrant streaking technique. The dishes were incubated at 37°C for 24 hours. The colonies formed were examined visually after 18 h and 24 hours of incubation.
3. Results:
The results obtained are shown in the tables below. First study:
(Table Removed)
Second study:
(Table Removed)
These results show that the antibiotic combinations according to the invention exhibited excellent detection sensitivity, from 18 h onwards.

CLAIMS
1) Reaction medium for detecting and/or identifying methicillin-resistant Staphylococcus aureus (MRSA) bacteria, comprising a chromogenic substrate, a first antibiotic which belongs to the cephalosporin family and a second antibiotic which belongs to the aminoglycoside family.
2) Reaction medium according to Claim 1, according to which said first antibiotic is cefminox or cefdinir.
3) Reaction medium according to Claim 1 or 2, according to which said second antibiotic is kanamycin or amikacin.

4) Reaction medium according to any one of Claims 1 to 3, according to which said chromogenic substrate allows the detection of an osidase, esterase or peptidase activity.
5) Reaction medium according to Claim 4, according to which said osidase activity is an alpha-glucosidase activity.
6) Reaction medium according to Claim 5, in which said esterase activity is a phosphatase
activity.
7) In vitro use of a reaction medium according to any one of Claims 1 to 6, for isolating and
identifying methicillin-resistant Staphylococcus aureus (MRSA) bacteria.
8) Method for detecting and/or identifying methicillin-resistant Staphylococcus aureus
(MRSA) bacteria in a biological sample, comprising:
a) inoculating the biological sample that may contain methicillin-resistant
Staphylococcus aureus (MRSA) bacteria on a reaction medium according to any one
of Claims 1 to 6;
b) incubating;
c) identifying the colonies as being MRSA colonies.