1. A method for distinguishing at least 3 groups of microorganisms in a biological sample, said sample comprising: • a first group of Gram positive bacteria, belonging to a first taxon of microorganisms and comprising at least one mechanism of resistance to an antimicrobial; • a second group of Gram positive bacteria, belonging to a second taxon of microorganisms, different than said first taxon, but comprising at least one mechanism of resistance to an antimicrobial, identical to that of the first group; • a third group of Gram positive bacteria, that are not resistant to said antimicrobial, said method consisting in contacting the biological sample with a culture medium comprising: □ at least a first substrate for detecting at least a first enzymatic or metabolic activity of said first group of microorganisms; □ at least one marker for differentiating the first group of microorganisms and the second group of microorganisms, said marker being a substrate for detecting at least one enzymatic or metabolic activity of said second group of microorganisms; □ at least one antimicrobial that is active on said third group of microorganisms.

2. The method according to claim 1, wherein the Gram positive bacteria of the first and the second groups are enterococci.

3. A culture medium comprising: • a first substrate for detecting an alpha-glucosidase enzymatic activity ; • a second substrate for detecting a beta-glucosidase or a beta-galactosidase enzymatic activity; • at least one antibiotic, being preferentially vancomycin.

4. A culture medium comprising: • a first substrate for detecting a beta-glucosidase enzymatic activity, • a second substrate for detecting methyl-alpha-glucoside metabolism, in the presence of a colored indicator, • an antibiotic which is vancomycin.

5. The method as claimed in claim 1, for distinguishing: • a first group of enterococcal bacteria developing an acquired resistance to vancomycin; • a second group of enterococcal bacteria developing a natural resistance to vancomycin; • a third group of enterococcal bacteria that are not resistant to vancomycin; said method consisting in contacting the biological sample with a culture medium comprising: • a first substrate for detecting a beta-glucosidase enzymatic activity, • a second substrate for detecting methyl-alpha-glucoside metabolism, in the presence of a colored indicator, • an antibiotic which is vancomycin.

6. A culture medium comprising: • at least a first substrate for detecting alpha-glucoside metabolism; • at least a second substrate for detecting a second activity different than alpha-glucoside metabolism; • at least one antibiotic, which is vancomycin.

7. The culture medium as claimed in claim 6, wherein the at least second substrate makes it possible to detect a beta-glucosidase or beta-galactosidase activity.

8. The culture medium as claimed in claim 6, wherein the first substrate is preferably methyl~a-glucoside, the second substrate is preferably 5-bromo-4-chloro-3-indolyl-6-D-glucoside or 6-chloro-3-indolyl-6-D-glucoside, and the antimicrobial is preferably vancomycin.

9. A method as claimed in claims 1, for distinguishing: • a first group of vancomycin-resistant microorganisms, comprising Enterococcus faecalis and Enterococcus faecium; • a second group of vancomycin-resistant microorganisms, comprising Enterococcus casseliflavus and Enterococcus gallinarum; • a third group of microorganisms that are not resistant to vancomycin; said method consisting in contacting the biological sample with a culture medium comprising: • at least a first substrate for detecting alpha-glucoside metabolism which is a methyl-alpha-glucoside; • at least a second substrate for detecting a second activity different than alpha-glucoside metabolism, being 5-bromo-4-chloro-3-indolyl-B-D-glucoside or 6-chloro-3-indolyl-B-D-glucoside; • at least one antibiotic, which is vancomycin.

10. The culture medium as claimed in claim 6, wherein the first substrate is 5-bromo-4-chloro-3-indolyl-N-methyl-a-D-glucoside or 5-bromo-4-chloro-3-indolyl-a-D-glucoside, the second substrate is 6-chloro-3-indolyl-B-D-glucoside or alizarine-B-D-galactoside or 5-bromo-6-chloro-3-indolyl-6-D-glucoside or 5-bromo-6-chloro-3-indolyl-6-D-galactoside or 6-chloro-3-indolyl-B-D-galactoside, and the antimicrobial is VEincomycin.

11. The method as claimed in claim 1, for distinguishing: • a first group of vancomycin-resistant microorganisms, comprising Enterococcus faecium; • a second group of vancomycin-resistant microorganisms, comprising Enterococcus faecalis; • a third group of microorganisms that are not resistant to vancomycin or that express a natural resistance; said method consisting in contacting the biological sample with a culture medium comprising: • at least a first substrate for detecting alpha-glucoside metabolism which is 5-bromo-4-chloro-3-indolyl-N-methyl-a-D-glucoside or 5-bromo-4-chloro-3-indolyl-a-D-glucoside; • at least a second substrate for detecting a second activity different than alpha-glucoside metabolism, taken fi-om the list 6-chloro-3-indolyl-6-D-glucoside or alizarine-6-D-galactoside or 5-bromo-6-chloro-3-indolyl-B-D-glucoside or 5-bromo-6-chloro-3-indolyl-B-D-galactoside or 6-chloro-3-indolyl-B-D-galactoside; • at least one antibiotic, which is vancomycin.

12. The culture medium as claimed in claim 6, wherein the first substrate is 5-bromo- 4-chloro-3-indolyl-N-methyl-a-D-glucoside or 5-bromo-4-chloro-3-indolyl- a-D-glucoside, the second substrate is 6-chloro-3-indolyl-6-D-glucoside or 5-bromo-6-chloro-3-indolyl-fi-D-glucoside, and the antimicrobial is vancomycin.

13. The method as claimed in claim 1, for distinguishing: • a first group of vancomycin-resistant microorganisms, comprising Enterococcus faecalis and Enterococcus faecium; • a second group of microorganisms, comprising Staphylococcus aureus, that are intermediary resistant or resistant to vancomycin; • a third group of microorganisms that are not resistant to vancomycin; said method consisting in contacting the biological sample with a culture medium comprising: • at least a first substrate for detecting alpha-glucoside metabolism which is 5-bromo-4-chloro-3-indolyl-N-methyl-a-D-glucoside or 5-bromo-4-chloro-3-indolyl-a-D-glucoside; • at least a second substrate for detecting a second activity different than alpha-glucoside metabolism, which is 6-chloro-3-indolyl-B-D-glucoside or 5-bromo-6-chloro-3-indolyl-6-D-glucoside; • at least one antibiotic, which is vancomycin. Dated this 17* day of May, 2012 r^^/^ OF RElNdFRY & SAGAR ATTORNEY FOR THE\^PF^ICANT(S)

1
Medium for the specific detection of resistant microorganisms
The field of the invention is that of microbiological analysis by means of biochemistry, and in particular the detection and identification of bacteria.
Bacterial resistance to antibiotics is a major public health problem. The resistance of infectious microorganisms to a treatment has developed at the same time as anti-infectious molecules and today represents a major obstacle in therapeutics. This resistance is responsible for many problems, including difficulties in detection in the laboratory, limited treatment options and a deleterious impact on clinical outcome.
In particular, the rapid and irrepressible increase in the resistance of pathogenic bacteria, over the last 20 years, represents one of the major current problems in medicine. Infections caused by these organisms are responsible for extended periods of hospitalization and are associated with high morbidity and mortality rates, following therapeutic failures. Several resistance mechanisms can be involved simultaneously in a bacterial strain. They are generally classified in 3 categories: deficient penetration of the antibiotic into the bacterium, inactivation or excretion of the antibiotic by bacterial enzymatic systems, and lack of affinity between the bacterial target and the antibiotic.
Enzymatic inactivation is the most common mechanism of acquired resistance in terms of number of species and of antibiotics involved. Thus, chromosomal class C cephalosporinases today constitute one of the predominant resistance mechanisms of gram-negative bacteria, the bacteria expressing such enzymes being resistant to cephalosporins. Similarly, P-lactamases are enzymes expressed by certain bacteria, capable of hydrolyzing the C-N bond of the P-lactame ring, the basic structure of antibiotics of the P-lactamine family, so as to give a microbiologically inactive product. Several P-lactamase inhibitors (BLIs), such as clavulanic acid (CA), tazobactam and sulbactam, have been developed in order to increase the antimicrobial activity and broaden the spectrum of the p-lactamines which are associated therewith. They act as a suicide subject for P-lactamases, and prevent enzymatic degradation of the antibiotics and allow them to become effective against bacteria that were initially resistant. However, by virtue of the persistent exposure of strains to antibiotic pressure, the bacteria express their ability to adapt through the continuous and dynamic production of P-lactamases, which evolves at the same time as the development of new molecules. Gram-negative bacteria which produce high-level chromosome class C cephalosporinases (reference is made to HL Case bacteria), and also gram-negative bacteria which produce extended-spectrum P-lactamase (reference is then made to ESBL bacteria) have, as a result,

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become an increasing threat, in particular because the number of bacterial species concerned is increasing. HL Case and ESBL bacteria are resistant to treatments based on 1st- and 2nd-generation penicillins and cephalosporines, but also on 3rd-generation cephalosporines (C3G) (cefotaxim CTX, ceftazidime CAZ, cefipodoxime CPD, ceftriaxone CRO) and monobactams (aztreonam ATM). On the other hand, 7a-methoxycephalosporins (cephamycins: cefoxitin, cefotetan) and carbapenems (imipenem, meropenem, ertapenem) generally conserve their activity. ESBLs are inhibited by P-lactamase inhibitors (BLIs), which makes it possible to differentiate them from other cephalosporinases.
These bacteria thus most commonly simultaneously express resistances to several treatments, which poses difficulties in setting up a relevant treatment and avoiding therapeutic failures. An Escherichia coli bacterium can thus be HL Case and ESBL. In addition, since ESBL-positive enterobacteria have a tendency to disseminate the resistance by clonal transmission of strains or conjugative plasma transfer, they represent a problem in terms of controlling infections. In most studies, Escherichia coli and Klebsiella pneumoniae remain the most corrmion ESBL-producing species. However, over the last few years, ESBLs have greatly broadened their panel of host species. In fact, many species of enterobacteria and of nonfermenting gram-negative bacilli (such as Pseudomonas aeruginosa) have also been reported to ESBL producers.
In addition to these ESBL bacteria, mention may also be made of Staphylococcus aureus bacteria, which are also pathogenic bacteria that develop many mechanisms of resistance, such as resistance to methicillin, penicillin, tetracycline, erythromycin, or vancomycin. Enterococcus faecium is another multiresistant bacterium found in the hospital environment, which can be resistant to penicillin, vancomycin and linezolide. Mycobacterium tuberculosis is commonly resistant to isoniazid and to rifampicin. Other pathogens offer certain resistances, such as Salmonella, Campylobacter and Streptococcus.
It therefore becomes essential, from a public health point of view, to be able to identify such microorganisms, and such resistance mechanisms, as rapidly as possible.
In general, the search for microorganisms resistant to a treatment is carried out according to the following steps:
1. Taking a biological sample that may contain said microorganisms;
2. Seeding and incubating a culture medium (18 to 48 h) in order to induce exponential growth of the microorganisms;

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3. Pinpointing, on the culture media, colonies of potentially significant microorganisms;
4. Characterizing the microorganism species;
5. Identifying the mechanisms of resistance of the microorganisms analyzed, their biological significance and, optionally, the appropriate therapy.
This succession of steps involves a considerable amount of time between taking the sample that may contain microorganisms and prescribing a treatment that is appropriate for the patient. Furthermore, the user must generally perform steps for transferring microorganims from a first medium to a second medium manually, which can induce problems, in particular, of contamination, but also risks to the handler's health.
By way of example, in order to detect the presence of broad-spectrum beta-lactamases (ESBLs) in strains of Escherichia coli and Klebsiella pneumoniae, use may be made of a diffusion technique as described in the publication by Jacoby & Han (J Clin Microbiol. 34(4): 908-11, 1996), which does not however give any information regarding the identification of the strains tested: it is possible to determine whether or not the bacteriimi is a ESBL-producing bacterium, but it is not possible to distinguish whether such a bacterium is an Escherichia coli or a Klebsiella pneumoniae.
Metabolic substrates are also used for detecting the presence of ESBLs or HL cases. In this respect, AES laboratories proposes a medium in a biplate combining a Drigalski medium with cefotaxim and a MacConkey medium with ceftazidime. The Drigalski and MacConkey media make it possible to reveal lactose acidification, a metabolism which is present in a very large number of enterobacterial species. However, such a medium only makes it possible to distinguish resistant bacteria from non-resistant bacteria, and does not make it possible to distinguish bacteria expressing a ESBL fi-om those expressing an HL Case. Neither does this medium make it possible to identify specific bacterial species, nor does it make it possible, for example, to discriminate between E. coli bacteria and K. pneumoniae bacteria. In the case of the detection of resistance mechanisms other than ESBL, mention may be made of patent application EP0954560, which relates to the search for Vancomycin-resistant enterococcal, by combining Vancomycin with a chromogenic media that reveals two enzymatic activities (6-glucosidase and pyrrolidonyl arylamidase). However, this chromogenic medium makes it possible to determine only whether or not the vancomycin-resistant strains belong to the Enterococcus genus, but does not make it possible to identify the species or the resistance mechanisms involved, in particular if it is a question of an acquired or wild-type resistance.

4
Thus, the characterization of a species of microorganism, and then the identification of its resistance to a treatment, is long and laborious. If the laboratory gives the clinician a positive screen, whereas the isolate is in fact free of resistant microorganisms, this can lead to needless and inappropriate treatment. Conversely, not communicating a positive screen, which is subsequently confirmed, delays the setting of the isolation of the patient (and possibly an appropriate therapy) by one day. This shows the need for a rapid and reliable confirmation test.
The present invention therefore proposes to improve the prior art by providing a novel diagnostic tool which allows a gain in time, in reliability and in relevance with respect to the therapy implemented. Our invention makes it possible, in a single step, to identify the species of microorganisms present in a sample, and to determine their mechanism of resistance in order to propose a treatment appropriate to each patient. This invention is particularly suitable for discriminating various species of microorganisms, which have various mechanisms of resistance to various treatments, but all of which may be present in the same sample.
Before going any fiirther in the disclosure of the invention, the following definitions are given
in order to facilitate understanding of the invention:
The term "culture medium" is intended to mean a medium comprising all the elements
required for the survival and/or the growth of microorganisms. The culture medium according
to the invention may contain any possible additives, for instance: peptones, one or more
growth factors, carbohydrates, one or more selective agents, buffers, one or more gelling
agents, etc. This culture medium may be in liquid form or in gel form which is ready to use,
i.e. ready for seeding in a tube or flask or on a Petri plate.
For the purpose of the present invention, the term "microorganism" covers gram-positive or
gram-negative bacteria, yeasts and, more generally, organisms that are generally unicellular,
invisible to the naked eye, which can be multiplied and handled in the laboratory.
By way of gram-negative bacteria, mention may be made of bacteria of the following genres:
Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus,
Campylobacter, Haemophilus, Morganella, Vibrio, Yersinia, Acinetobacter, Branhamella,
Neisseria, Burkholderia, Citrobacter, Hafnia, Edwardsiella, Aeromonas, Moraxella,
Pasteurella, Providencia, and Legionella.
By way of gram-positive bacteria, mention may be made of bacteria of the following genre:
Enterococcus, Streptococcus, Staphylococcus, Bacillus, Listeria, Clostridium, Gardnerella,
Kocuria, Lactococcus, Leuconostoc, Micrococcus, Mycobacteria and Corynebacteria.

5
By way of yeasts, mention may be made of yeasts of the following genre: Candida,
Cryptococcus, Saccharomyces and Trichosporon.
The term "biological sample" is intended to mean a clinical sample, derived from a specimen
of biological fluid, or a food sample, derived from any type of food. This sample may thus be
liquid or solid and mention may be made, in the nonlimiting manner, of a clinical blood,
plasma, urine or faeces sample, nose, throat, skin, wound or cephalospinal fluid specimens, a
food sample from water, from drinks such as milk or a fruit juice; from yoghurt, from meat,
from eggs, from vegetables, from mayonnaise, from cheese; from fish, etc., a food sample
derived from a feed intended for animals, such as, in particular, a sample derived from animal
meals.
The term "mechanism of resistance" is intended to mean any type of device which allows a
microorganism to render a treatment partially or completely ineffective on said
microorganism, guaranteeing its survival. The mechanisms of resistance are generally divided
up into three categories: deficient penetration of the antibiotic into the bacterium, inactivation
or excretion of the antibiotic by means of bacterial enzymatic systems, and lack of affinity
between the bacterial target and the antibiotic.
By way of indication, mention may in particular be made of mechanisms of resistance related
to the expression of an enzyme belonging to the broad-spectrum P-lactamase group; of an
enzyme belonging to the chromosomal high level class C cephalosporinase group;
mechanisms of resistance to glycopeptides, preferably developed by bacteria belonging to the
Enterococcus genus.
Mention will also be made of mechanisms of resistance to methicillin, penicillin, tetracycline,
erythromycin, or vancomycin when the microorganism is a Staphylococcus aureus bacterium.
Mention will also be made of mechanisms of resistance to penicillin, vancomycin and
linezolide when the microorganism is an Enterococcus faecium bacterium.
Mention will also be made of mechanisms of resistance to amphotericin B or to antifimgals of
the azole family when the microorganism is a yeast.
Finally, mention will be made of mechanisms of resistance to isoniazid and to rifampicin
when the microorganism is a Mycobacterium tuberculosis bacterium.
The term "treatment" is intended to mean a treatment capable of preventing or reducing the
growth of microorganisms derived from a patient. This treatment may comprise in particular
antimicrobial compoimds, such as antibiotics, for instance penicillins, conventional
cephalosporins, broad-spectrum cephalosporins, monobactams, glycopeptides or aminosides,
or such as antifungals or resistance-inhibiting compounds. It should be noted that this

6
treatment can also comprise the isolation of the patient, thereby preventing propagation of the microorganism among other patients.
The term "substrate" which allows the detection of an enzymatic or metabolic activity is intended to mean any molecule capable of directing or indirectly generating a detectable signal due to an enzymatic or metabolic activity of the microorganism. A^en this activity is an enzymatic activity, reference is then made to an enzymatic substrate. The term "enzymatic substrate" is intended to mean any substrate that can be hydrolyzed by an enzyme into a product that allows the direct or indirect detection of a microorganism. This substrate comprises in particular a first part that is specific for the enzymatic activity to be revealed and a second part that acts as a label, hereinafter referred to as labeling part. This labeling part may be chromogenic, fluorogenic, luminescent, etc. As chromogenic substrate suitable for solid supports (filter, agar, electrophoresis gel), mention may in particular be made of substrates based on indoxyl and its derivatives, and substrates based on hydroxyquinoline or escultin and their derivatives, which allow the detection of osidase and esterase activities. Mention may also be made of substrates based on nitrophenol and nitroaniline and derivatives, making it possible to detect osidase and esterase activities in the case of substrates based on nitrophenol, and peptidase activities in the case of substrates based on nitroaniline. Finally, mention may be made of substrates based on naphtol and naphtylamine and their derivatives, which make it possible to detect osidase and esterase activities via naphtol, and peptidase activities via naphtylamine. This substrate may allow, in particular, but in a nonlimiting manner, the detection of an enzymatic activity such as the activity of an osidase, peptidase, esterase, etc. The enzymatic substrate can also be a natural substrate of which the product of hydrolysis is detected directly or indirectly. As natural substrate, mention may in particular be made of tryptophan for detecting tryptophanase or desaminase activity, a cyclic amino acid (tryptophan, phenylalanine, histidine, tjrosine) for detecting desaminase activity, phosphatidyl inositol for detecting phospholipase activity, etc. When this activity is a metabolic activity, the substrate is then a metabolic substrate, such as a source of carbon or of nitrogen, coupled to an indicator that produces a coloration in the presence of one of the metabolic products.
According to a preferred embodiment of the invention, said first and/or second enzymatic or metabolic activity is an enzymatic activity preferably chosen fi-om the enzymatic activities: beta-glucosidase, desaminase, beta-glucuronidase, beta-galactosidase, alpha-glucosidase, alpha-galactosidase, hexosaminidase, N-acetyl-hexosaminidase, phosphatase, esterase, and aminopeptidase.

7
For example, for detecting E. coli, use is preferably made of beta-glucuronidase or B-galactosidase or tryptophanase or desaminase activity; for detecting Proteus, use is preferably made of desaminase activity; for detecting enterococci, use is preferably made of beta-glucosidase activity. For Candida albicans, hexosaminidase is preferred, for Listeria monocytogenes, phospholipase is preferred, for salmonellae, esterase is preferred, for Pseudomonas aeruginosa, esterase or B-alanine aminopeptidase is preferred, for Staphylococcus aureus phosphatase or alpha-glucosidase is preferred.
The expression "marker for differentiating" two groups of microorganisms is intended to mean a compound which does not have the same properties on a first and on a second group. This compound may thus be:
• a specific substrate;
• an inhibitor of a mechanism of resistance, which then makes it possible to inhibit the growth of the organisms developing a specific resistance, without any discrimination of the microorganism species.
In the case of the use of a specific substrate, use is preferably made of beta-glucuronidase, beta-galactosidase, tryptophanase or desaminase activity for detecting E. coli, use is preferably made of desaminase activity for detecting Proteus, use is preferably made of beta-glucosidase activity for detecting enterococci. For Candida albicans, hexosaminidase is preferred, for Listeria monocytogenes, phospholipeise is preferred, for salmonellae, esterase is preferred, for Pseudomonas aeruginosa, esterase or B-alanine aminopeptidase is preferred, for Staphylococcus aureus, phosphatase or alpha-glucosidase is preferred. In the case of the use of an inhibitor of a mechanism of resistance, use is preferably made of:
• clavulanic acid, tazobactam or sulbactam when the first group and/or the second group comprises a mechanism of resistance induced by an expression of P-Iactamases. The clavulanic acid concentration in the medium is then preferably between 0.05 and 32 mg/1, preferably between 0.1 and 8 mg/1, and even more preferably between 0.25 and 6 mg/1;
• cloxacillin or dicloxacillin when the first group and/or the second group comprises a mechanism of resistance induced by the expression of cephalosporinases.
The term "taxon" is intended to mean a group of microorganisms having a taxonomic unit. A taxon may be a family, a genus, a set of genre, a species, a set of species or a subspecies. By way of indication, mention may be made of enterobacteria, Klebsiella, Escherichia, Enterobacter, Citrobacter, Serratia, KESC {Klebsiella, Enterobacter, Serratia, Citrobacter),

8
Proteeae, Proteus, Morganella, Pseudomonas, Staphylococcus, Streptococcus, Enterococcus, Candida, Escherichia coli, Escherichia coli 0157:H7, Klebsiella pneumoniae, Citrobacter freundii, Enterococcus faecalis, Enterococcus faecium. Staphylococcus aureus, coagulase-negative staphylocoque, Candida albicans, Candida glabrata, Candida krusei, Candida lusitaniae.
The term "antimicrobial" is intended to mean any compound capable of preventing or slowing down the growth of a microorganism. This compound may be an antibiotic or an antifungal. The term "antibiotic" is intended to mean any compound capable of preventing or slowing down the growth of a bacterium. By way of indication, mention may in particular be made of the antibiotics cefotaxime, ceftazidime, ceftriaxone, cefpodoxime, aztreonam, vancomycin, tobramycin and ciprofloxacin.
The term "antifimgal" is intended to mean any compound capable of preventing or slowing down the growth of a yeast or of a mould. By way of indication, mention may in particular be made of amphotericin B, fluconazole, itraconazole, voriconazole and cycloheximide. According to a preferred embodiment of the invention, when the antibiotic is
• cefotaxime, the cefotaxime concentration in the medium is preferably between 0.25 and 8 mg/1, preferably between 1 and 2 mg/1;
• ceftazidime, the ceftazidime concentration in the medium is preferably between 0.25 and 8 mg/1, preferably between 2 and 2.5 mg/1;
• ceftriaxone, the ceftriaxone concentration in the medium is preferably between 0.25 and 8 mg/1, preferably between 1 and 2.5 mg/1;
• cefpodoxime, the cefpodoxime concentration in the medium is preferably between 0.1 and 32 mg/1, preferably between 0.75 and 10 mg/1, and even more preferably between 1 and 6 mg/1;
• aztreonam, the aztreonam concentration in the mediimi is preferably between 0.1 and 8 mg/1, preferably between 0.75 and 1.5 mg/1.
According to a specific embodiment of the invention, the medium comprises a combination of at least two antibiotics. Preferably, the combination of at least two antibiotics comprises cefotaxime and ceftazidime.
Irrespective of the embodiment of the invention, the medium may also comprise a dye. By way of indication of a dye, mention may be made of Evans blue, neutral red, sheep blood, horse blood, and opacifier such as titanium oxide, nitroaniline, malachite green, brilliant green, etc.

9
All the media may also comprise, in order to increase their sensitivity:
• at least one antimicrobial that is active against gram-positive bacteria, such as in particular linezolide or vancomycin;
• at least one antimicrobial that is active against yeasts, such as in particular voriconazole or amphotericin B.
The invention relates to the use of a culture medium for distinguishing at least 3 groups of microorganisms in a biological sample, comprising:
• a first group of microorganisms, belonging to a first taxon of microorganisms and comprising at least one mechanism of resistance to a treatment;
• a second group of microorganisms, belonging to a second taxon of microorganisms, different than said first taxon, but comprising at least one mechanism of resistance to a treatment, identical to that of the first group;
• a third group of microorganisms, that are not resistant to said treatment,
said culture medium comprising:
a. at least a first substrate for detecting at least a first enzymatic or metabolic
activity of said first group of microorganisms;
b. at least one marker for differentiating the first group of microorganisms and
the second group of microorganisms, said marker being a substrate for
detecting at least one enzymatic or metabolic activity of said second group of
microorganisms;
c. at least one antimicrobial that is active on said third group of microorganisms.
This embodiment of the invention makes it possible to distinguish, in the same sample, a first
and a second group comprising various species or various taxons of microorganisms, but each
of the two groups being resistant to the same treatment.
In this respect, the invention relates to a culture medium comprising:
• a first substrate for detecting a beta-glucosidase enzymatic activity, preferably 5-bromo-4-chloro-3-indolyl-B-D-glucoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 150 mg/1;
• a second substrate for detecting methyl-alpha-glucoside metabolism, in the presence of a colored indicator, preferably neutral red, at a concentration of between 2 and 100 mg/1, preferably between 4 and 50 mg/1;

10
• an antimicrobial which is an antibiotic, preferably vancomycin, at a concentration of
between 0.5 and 128 mg/1, preferably between 2 and 32 mg/1.
When the antibiotic is vancomycin, this medium is preferably used for distinguishing:
• a first group of enterococcal bacteria developing an acquired resistance against vancomycin;
• a second group of enterococcal bacteria developing a natural resistance against vancomycin;
• a third group of enterococcal bacteria that are not resistant to vancomycin.
The invention also relates to a culture medium comprising:
• at least a first substrate for detecting alpha-glucoside metabolism, preferably methyl-a-glucoside, at a concentration of between 1 and 50 g/1, preferably between 5 and 20 g/1, or 5-bromo-4-chloro-3-indolyI-a-D-glucoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 250 mg/1, or 5-bromo-4-chloro-3-indolyl-N-methyl-a-D-glucoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 250 mg/1;
• at least a second substrate for detecting a second activity different than alpha-glucoside metabolism, preferably 5-bromo-4-chloro-3-indolyl-B-D-glucoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 250 mg/1, or 6-chloro-3-indolyl-B-D-glucoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 250 mg/1, or alizarine-B-D-galactoside, at a concentration of between 10 and 500 mg/1, preferably between 20 and 250 mg/1, or 5-bromo-6-cliloro-3-indolyl-6-D-glucoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 250 mg/1, or 5-bromo-6-chloro-3-indolyl-B-D-galactoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 250 mg/1, or 6-chloro-3-indolyl-fi-D-galactoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 250 mg/1. Preferably, this second substrate makes it possible to detect a beta-glucosidase or beta-galactosidase activity;
• at least one antimicrobial, preferably an antibiotic, such as vancomycin, at a concentration of between 0.5 and 128 mg/1, preferably between 2 and 32 mg/1.
When the antibiotic is vancomycin, this medium is preferably used to distinguish:
• a first group of vancomycin-resistant microorganisms, comprising Enterococcus
faecalis and Enterococcus faecium;

11
• a second group of vancomycin-resistant microorganisms, comprising Enterococcus casseliflavus and Enterococcus gallinarum;
• a third group of microorganisms that are not resistant to vancomycin.
In this case, the first substrate is preferably methyl-a-glucoside, the second substrate is preferably 5-bromo-4-chloro-3-indolyl-B-D-glucoside or 6-chloro-3-indolyl-6-D-glucoside, and the antimicrobial is preferably vancomycin. This medium is also preferably used to distinguish:
• a first group of vancomycin-resistant microorganisms, comprising Enterococcus faecalis;
• a second group of vancomycin-resistant microorganisms, comprising Enterococcus faecium;
• a third group of microorganisms that are not resistant to vancomycin or that express a natural resistance (E. casseliflavus and E. gallinarum).
In this case, the first substrate is preferably 5-bromo-4-chloro-3-indolyl-N-methyl-a-D-glucoside or 5-bromo-4-chloro-3-indolyl-a-D-glucoside, the second substrate is preferably 6-chloro-3-indolyl-fl-D-glucoside or alizarine-B-D-galactoside or 5 bromo-6-chloro-3-indolyl-fi-D-glucoside or 5-bromo-6-chloro-3-indolyl-B-D-galactoside or 6-chloro-3-indolyl-P-D-galactoside, and the antimicrobial is preferably vancomycin. This medium is also preferably used to distinguish:
• a first group of vancomycin-resistant microorganisms, comprising Enterococcus
faecalis and Enterococcus faecium;
• a second group of microorganisms, comprising Staphylococcus aureus, that are intermediately resistant or resistant to vancomycin;
• a third group of microorganisms that are not resistant to vancomycin.
In this case, the first substrate is preferably 5-bromo-4-chloro-3-indolyl-N-methyl-a-D-glucoside or 5-bromo-4-chloro-3-indolyl-a-D-glucoside, the second substrate is preferably 6-chloro-3-indolyl-B-D-glucoside or 5-bromo-6-chloro-3-indolyl-B-D-glucoside, and the antimicrobial is preferably vancomycin.
The table below makes it possible to distinguish the appropriate combinations of substrates and antimicrobial according to the species that it is desired to detect:

12
1st group of 2nd group of 3rd group of 1st substrate 2nd substrate Antimicrobial
microorganisms microorganisms microorganisms
E. faecalis and E. casseliflavns Microorganisms Methyl-a-glucoside 5-bromo- vancomycin
E.faecium, and E. that are not 4-chloro-
resistant to gallinarum, resistant to 3-indolyl-
vancomycin resistant to vancomycin P-D-glucoside or
vancomycin 6-chloro-
3-indolyl-
P-D-glucoside
E. faecalis, E. faecium, Microorganisms 5-bromo-4-chloro- 6-chloro- vancomycin
resistant to resistant to that are not 3-indolyl-N-methyl- 3-indolyl-
vancomycin vancomycin resistant to a-D-glucoside or P-D-glucoside or
vancomycin or 5-bromo-4-chloro- alizarine-
that express a 3-indolyl- P-D-galactoside
natural resistance a-D-glucoside or 5-bromo-
(£. casseliflavus 6-chloro-3-
and E- indolyl-p-
gallinarum) D-glucoside or
5-bromo-6-
chloro-3-indolyl-
P-D-galactoside
or 6-chloro-
3-indolyl-
P-D-galactoside
E. faecalis and S. aureus. Microorganisms 5-bromo-4-chloro- 6-chloro- vancomycin
E. faecium, resistant to that are not 3-indolyl-N-methyl- 3-indolyl-
resistant to vancomycin resistant to a-D-glucoside or P-D-glucoside or
vancomycin vancomycin 5-bromo-4-chloro- 5-bromo-
3-indolyl- 6-chloro-3-
a-D-glucoside indolyl-P-D-
glucoside It may be relevant to also adjust the vancomycin concentration, preferably to between 0.5 and 12mg/l.
Similarly, the invention also relates to a culture medium comprising:

13
• at least a first substrate for detecting alpha-glucoside metabolism, preferably 5-bromo-4-chloro-3-indolyl-a-D-glucoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 250 mg/1, or 5-bromo-4-chloro-3-indolyl-N-methyl-a-D-glucoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 250 mg/1;
• at least a second substrate for detecting a second activity different than alpha-glucoside metabolism, preferably 6-chloro-3-indolyl-B-D-glucoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 250 mg/1, or 6-chloro-3-indolyl-P-D-galactoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 250 mg/1, or alizarine-P-galactoside, at a concentration of between 10 and 500 mg/1, preferably between 20 and 250 mg/1, or 5-bromo-6-chloro-3-indolyl-B-D-glucoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 250 mg/1, or 5-bromo-6-chloro-3-indolyl-B-D-galactoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 250 mg/l, or 6-chloro-3-indolyl-6-D-galactoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 250 mg/1. Preferably, this second substrate makes it possible to detect a beta-glucosidase or beta-galactosidase activity;
• a combination of antimicrobials, preferably a combination of antibiotics such as
o vancomycin at a concentration of between 0.5 and 128 mg/1, preferably
between 2 and 32 mg/1; o aztreonam at a concentration of between 1 and 150 mg/1, preferably between 4
and 60 mg/1; o colistine at a concentration of between 1 and 100 mg/1, preferably between 2
and 20 mg/1; o amphotericin B at a concentration of between 0.5 and 50 mg/1, preferably
between 1 and 15 mg/1. When one of the antibiotics is vancomycin, this mediiim is preferably used to distinguish:
• a first group of vancomycin-resistant microorganisms, comprising Enterococcus faecium,
• a second group of vancomycin-resistant microorganisms, comprising Enterococcus faecalis,
• a third group of microorganisms that are not resistant to vancomycin.
This medium is also preferably used to distinguish:

14
• a first group of vancomycin-resistant microorganisms, comprising Enterococcus faecium,
• a second group of microorganisms, comprising Staphylococcus aureus, that are intermediately resistant or resistant to vancomycin;
• a third group of microorganisms that are not resistant to vancomycin.
The combinations of substrates according to the groups of microorganisms that it is desired to identify are presented, for example, in the table on page 21. By using an appropriate combination of antimicrobials, it is possible to distinguish not only three groups of microorganisms, but also 4, 5 or even more groups of microorganisms.
The examples below are given by way of explanation and are no way limiting in nature. They will make it possible to understand the invention more fully.
EXAMPLE 1
This first example is based on the phenotypic detection of enterococci resistant to glycopeptides, with specific distinction of Enterococcus faecalis and E. faecium, using the reduction of susceptibility to antibiotics and the demonstration of an enzymatic activity: 6-glucosidase, and of a metabolic activity: Methyl-a-glucoside acidification.
1. Choice of strains
In the context of the manipulations carried out, for eval\iating the activity of the
antibiotics active on enterococci, various species of Enterococcus (Enterococccus faecalis, Enterococcus faecium, Enterococcus casseliflavus, Enterococcus gallinarum,) were used. In the assays, strains resistant to glycopeptides (VRE) and wild-type strains are compared.
2. Preparation of the medium
The medium used was a Columbia medium (51026), also comprising:
• 5-bromo-4-chloro-3-indolyl-B-D-glucopyranoside (X-Glu) at 100 mg/1,
• methyl-a-D-glucoside at 9 g/1,
• neutral red at 25 mg/1,
• bilial salts at 5 g/1,
• vancomycin at 4 mg/1.

15 • amphotericin B at 2 mg/1.
Osmosed water is added and the whole is homogenized and melted in a waterbath at 100°C. The basic medivim is dispensed into flasks, the number of which corresponds to the total number of media to be tested during the process. The flasks are then autoclaved for 15 min at 121°C. The media are brought back to and kept molten at 55 ± 3°C in a waterbath, in order to sterilely add the thermolabile additives (sterilized beforehand by filtration through 0.22 nm). The media are then poured into plates 90 mm in diameter and left on a flat surface so that they can set. The surface of the agars is then dried under a laminar flow hood for 30 min.
3. Inoculation of media
An inoculum of 0.5 McF is prepared, in physiological saline, from 24-hour precultures at 36°C ± 2°C in an aerobic atmosphere on TSA medium, and then 1 jil of this suspension is transferred into 5 ml of physiological saline. In order to obtain a sufficient number of isolated colonies, a range of inocula made it possible to determine that the optimal amount of bacteria to be inoculated was fi^om 10^ to 10'* CFU/ml. The inoculation is carried out directly on the two half-agars using a sterile swab. The cultures are then incubated at 37°C in an aerobic atmosphere.
4. Reading of media
The readings are carried out at 18 hours (± 30 min), 24h (± Ih) and 48 h (± 4h). The density and the size of the colonies, and the appearance, the color and the coloration intensities of the mass and of the isolated colonies were observed, according to the following reading scales 1 to 3: 0: no growth; 0.1: trace of growth; 0.25: colonies of diameter < 0.5 mm; 0.5: colonies of 0.5 mm in diameter; 0.75: 0.5 mm < diameter < 1 mm; 1: colonies of 1 mm in diameter; 1.25: 1 mm < diameter < 1.5 mm; 1.5: colonies 1.5 mm in diameter; 2: colonies 2 mm in diameter; 3: colonies of diameter > 2 mm.
5. Results:
On this medium, only the glycopeptide-resistant enterococcal strains develop and form
colonies.
The resistant E.faecalis and E.faecium strains form green colonies, whereas those of
E. casseliflavus and ofE. gallinarum form blue-to-violet colonies.

16
This medium therefore makes it possible to dififerentiate these two groups of enterococci and to provide a suitable therapeutic response.
EXAMPLE 2
This second example is based on the phenotypic detection of glycopeptide-resistant enterococci, with specific distinction of Enterococcus faecalis and E. faecium, using the reduction of susceptibility to antibiotics and the demonstration of two enzymatic activities: a-glucosidase and |3-galactosidase or P-glucosidase.
1. Choice of strains
In the context of the manipulations carried out, for evaluating the activity of antibiotics
active on enterococci, various species of Enterococcus (Enterococccus faecalis, Enterococpus faecium, Enterococcus casseliflavus, Enterococcus gallinarum,) were used. In the assays, strains resistant to glycopeptides (VRE) and wild-type strains are compared.
2. Preparation of the medium
The media used were a Colimibia medium (51026), also comprising:
o 5-bromo-4-chloro-3-indolyl-N-methyl-a-D-glucopyranoside (GreenA-a-Glu)
atl50mg/l, o 6-chloro-3-indolyl-P-glucopyranoside (Rose-b-Glu [Pink-b-Glu]) at 200 mg/1, or:
o 5 -bromo-4-chloro-3 -indolyl-N-methyl-a-D-glucopyranoside (GreenA-a-Glu)
at 150 mg/1, o alizarine-P-galactopyranoside at 50 mg/1
and vancomycin at 8 mg/1,
an amphotericin B at 4 mg/1,
and colistine at 2 mg/1,
and aztreonam at 32 mg/1.
Osmosed water is added and the whole is homogenized and melted in a waterbath at 100°C.
The two basic media are dispensed into flasks. The flasks are then autoclaved for 15 min at
121 °C. The media are brought back to and kept molten at 55 ± 3°C in a waterbath, in order to
sterilely add the thermolabile additives (sterilized beforehand by filtration through 0.22 jxm).

17
The media are then poured into plates 90 mm in diameter and left on a flat surface so that they can set. The surface of the agars is then dried under a laminar flow hood for 30 min.
3. Inoculation of media
This step is carried out as described in example 1.
4. Reading of media
This step is carried out as described in example 1.
5. Results:
On the medium containing a substrate for a-glucosidase and for P-glucosidase, the resistant E.faecium strains form violet colonies, whereas the resistant E. faecalis strains form pink colonies. The E. casseliflavus and E. gallinarum strains (natural resistances) are inhibited due to the concentration of vancomycin.
This medium therefore makes it possible to differentiate these two groups of enterococci and to provide a suitable therapeutic response and also a follow-up of the local epidemiology. On the mediimi containing a substrate for a-glucosidase and for P-galactosidase, the resistant E. faecium strains form violet colonies, whereas the resistant E. faecalis strains form green colonies. The E. casseliflavus and E. gallinarum strains (natural resistances) are inhibited due to the concentration of vancomycin.
This medium therefore makes it possible to differentiate these two groups of enterococci and to provide a suitable therapeutic response and a follow-up of the local epidemiology.

We Claim:
1. A method for distinguishing at least 3 groups of microorganisms in a biological sample,
said sample comprising:
• a first group of Gram positive bacteria, belonging to a first taxon of microorganisms and comprising at least one mechanism of resistance to an antimicrobial;
• a second group of Gram positive bacteria, belonging to a second taxon of microorganisms, different than said first taxon, but comprising at least one mechanism of resistance to an antimicrobial, identical to that of the first group;
• a third group of Gram positive bacteria, that are not resistant to said antimicrobial,
said method consisting in contacting the biological sample with a culture medium comprising:
□ at least a first substrate for detecting at least a first enzymatic or metabolic activity of said first group of microorganisms;
□ at least one marker for differentiating the first group of microorganisms and the second group of microorganisms, said marker being a substrate for detecting at least one enzymatic or metabolic activity of said second group of microorganisms;
□ at least one antimicrobial that is active on said third group of microorganisms.

2. The method according to claim 1, wherein the Gram positive bacteria of the first and the second groups are enterococci.
3. A culture medium comprising:

• a first substrate for detecting an alpha-glucosidase enzymatic activity ;
• a second substrate for detecting a beta-glucosidase or a beta-galactosidase enzymatic activity;
• at least one antibiotic, being preferentially vancomycin.
4. A culture medium comprising:
• a first substrate for detecting a beta-glucosidase enzymatic activity,
• a second substrate for detecting methyl-alpha-glucoside metabolism, in the presence of a colored indicator,
• an antibiotic which is vancomycin.

5. The method as claimed in claim 1, for distinguishing:
• a first group of enterococcal bacteria developing an acquired resistance to vancomycin;
• a second group of enterococcal bacteria developing a natural resistance to vancomycin;
• a third group of enterococcal bacteria that are not resistant to vancomycin;
said method consisting in contacting the biological sample with a culture medium comprising:
• a first substrate for detecting a beta-glucosidase enzymatic activity,
• a second substrate for detecting methyl-alpha-glucoside metabolism, in the presence of a colored indicator,
• an antibiotic which is vancomycin.
6. A culture medium comprising:
• at least a first substrate for detecting alpha-glucoside metabolism;
• at least a second substrate for detecting a second activity different than alpha-glucoside metabolism;
• at least one antibiotic, which is vancomycin.

7. The culture medium as claimed in claim 6, wherein the at least second substrate makes it possible to detect a beta-glucosidase or beta-galactosidase activity.
8. The culture medium as claimed in claim 6, wherein the first substrate is preferably methyl~a-glucoside, the second substrate is preferably 5-bromo-4-chloro-3-indolyl-6-D-glucoside or 6-chloro-3-indolyl-6-D-glucoside, and the antimicrobial is preferably vancomycin.
9. A method as claimed in claims 1, for distinguishing:

• a first group of vancomycin-resistant microorganisms, comprising Enterococcus faecalis and Enterococcus faecium;
• a second group of vancomycin-resistant microorganisms, comprising Enterococcus casseliflavus and Enterococcus gallinarum;
• a third group of microorganisms that are not resistant to vancomycin;
said method consisting in contacting the biological sample with a culture medium comprising:

• at least a first substrate for detecting alpha-glucoside metabolism which is a methyl-alpha-glucoside;
• at least a second substrate for detecting a second activity different than alpha-glucoside metabolism, being 5-bromo-4-chloro-3-indolyl-B-D-glucoside or 6-chloro-3-indolyl-B-D-glucoside;
• at least one antibiotic, which is vancomycin.

10. The culture medium as claimed in claim 6, wherein the first substrate is 5-bromo-4-chloro-3-indolyl-N-methyl-a-D-glucoside or 5-bromo-4-chloro-3-indolyl-a-D-glucoside, the second substrate is 6-chloro-3-indolyl-B-D-glucoside or alizarine-B-D-galactoside or 5-bromo-6-chloro-3-indolyl-6-D-glucoside or 5-bromo-6-chloro-3-indolyl-6-D-galactoside or 6-chloro-3-indolyl-B-D-galactoside, and the antimicrobial is VEincomycin.
11. The method as claimed in claim 1, for distinguishing:

• a first group of vancomycin-resistant microorganisms, comprising Enterococcus faecium;
• a second group of vancomycin-resistant microorganisms, comprising Enterococcus faecalis;
• a third group of microorganisms that are not resistant to vancomycin or that express a natural resistance;
said method consisting in contacting the biological sample with a culture medium comprising:
• at least a first substrate for detecting alpha-glucoside metabolism which is 5-bromo-4-chloro-3-indolyl-N-methyl-a-D-glucoside or 5-bromo-4-chloro-3-indolyl-a-D-glucoside;
• at least a second substrate for detecting a second activity different than alpha-glucoside metabolism, taken fi-om the list 6-chloro-3-indolyl-6-D-glucoside or alizarine-6-D-galactoside or 5-bromo-6-chloro-3-indolyl-B-D-glucoside or 5-bromo-6-chloro-3-indolyl-B-D-galactoside or 6-chloro-3-indolyl-B-D-galactoside;
• at least one antibiotic, which is vancomycin.
12. The culture medium as claimed in claim 6, wherein the first substrate is 5-bromo-
4-chloro-3-indolyl-N-methyl-a-D-glucoside or 5-bromo-4-chloro-3-indolyl-

a-D-glucoside, the second substrate is 6-chloro-3-indolyl-6-D-glucoside or 5-bromo-6-chloro-3-indolyl-fi-D-glucoside, and the antimicrobial is vancomycin.
13. The method as claimed in claim 1, for distinguishing:
• a first group of vancomycin-resistant microorganisms, comprising Enterococcus faecalis and Enterococcus faecium;
• a second group of microorganisms, comprising Staphylococcus aureus, that are intermediary resistant or resistant to vancomycin;
• a third group of microorganisms that are not resistant to vancomycin;
said method consisting in contacting the biological sample with a culture medium comprising:
• at least a first substrate for detecting alpha-glucoside metabolism which is 5-bromo-4-chloro-3-indolyl-N-methyl-a-D-glucoside or 5-bromo-4-chloro-3-indolyl-a-D-glucoside;
• at least a second substrate for detecting a second activity different than alpha-glucoside metabolism, which is 6-chloro-3-indolyl-B-D-glucoside or 5-bromo-6-chloro-3-indolyl-6-D-glucoside;
• at least one antibiotic, which is vancomycin.
Dated this 17* day of May, 2012 r^^/^
OF RElNdFRY & SAGAR ATTORNEY FOR THE\^PF^ICANT(S)