1. A method for distinguishing at least 3 groups of microorganisms in a biological sample, said sample comprising: • a first group of microorganisms, belonging to a first taxon of microorganisms and comprising at least one mechanism of resistance to an antimicrobial; • 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 an antimicrobial, identical to that of the first group; • a third group of microorganisms, that are not resistant to said antimicrobial, said method consisting in contacting the biological sample with a culture mediimi comprising: □ at least a first substrate for detecting at least a first enzymatic or metabolic activity of said first group of microorganisms; a 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 as claimed in claim 1, for distinguishing at least three groups of microorganisms in a biological sample, wherein the microorganisms of said first, second and third groups are Gram negative bacteria.

3. The method as claimed in claim 2, wherein the antimicrobial belongs to the beta-lactamin family.

4. The method as claimed in claim 2, wherein the antimicrobial belongs to the cephalosporin family.

5. A culture medium comprising: • a first substrate for detecting a beta-glucuronidase or a beta-galactosidase enzymatic activity; • a second substrate for detecting a beta-glucosidase or tryptophanase or deaminase activity; • a combination comprising an antimicrobial and an inhibitor of a resistance mechanism.

6. A culture medium comprising: • a first substrate for detecting a beta-glucuronidase or a beta-galactosidase enzymatic activity; • a second substrate for detecting a beta-glucosidase or tryptophanase or deaminase enzymatic activity; • at least two antibiotics.

7. A culture medium comprising: • a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity; • a second substrate for detecting a beta-glucosidase or tryptophanase or desaminase activity; • an antibiotic which is ceftazidime.

8. The method as claimed in claim 2, for distinguishing: • a first group ofE. coli ESBL or HL Case bacteria; • a second group of KESC ESBL or HL Case bacteria; • a third group of bacteria that are not resistant to beta-Iactamines and/or to cephalosporins; said method consisting in contacting the biological sample with a culture mediimi comprising • a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity; • a second substrate for detecting a beta-glucosidase or tryptophanase or desaminase activity; • an antibiotic which is ceftazidime.

9. A culture medium comprising: • a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity; • a second substrate for detecting a beta-glucosidase or tryptophanase or desaminase activity; • an antibiotic which is cefpodoxime; • an antibiotic which is cloxacillin.

10. The method as claimed in claim 2, for distinguishing: • a first group ofE. coli ESBL bacteria; • a second group of KESC ESBL bacteria; • a third group of bacteria that are not resistant to beta-lactamines; said method consisting in contacting the biological sample with a culture medium comprising: • a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity; • a second substrate for detecting a beta-glucosidase or tryptophanase or desaminase activity; • an antibiotic which is cefpodoxime; • an antibiotic which is cloxacillin.

11. A culture medium comprising: • a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity; • a second substrate for detecting a beta-glucosidase or desaminase or tryptophanase activity; • a combination comprising ceftriaxone and clavulanic acid.

12. The method as claimed in claim 2, for distinguishing: • a first group ofE. coli HL Case bacteria; • a second group of KESC HL Case bacteria; • a third group of bacteria that are not resistant to beta-lactamins and/or cephalosporins; said method consisting in contacting the biological sample with a culture mediiun comprising: • a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity; • a second substrate for detecting a beta-glucosidase or desaminase or tryptophanase activity; • a combination comprising ceftriaxone and clavulanic acid.

13. A cultixre medium comprising: • a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity; • a second substrate for detecting a beta-glucosidase or desaminase or tryptophanase activity; • two antibiotics, which are cefpodoxime and aztreonam.

14. The method as claimed in claim 2, for distinguishing: • a first group ofE. coli ESBL or HL Case bacteria; • a second group of KESC ESBL or HL Case bacteria; • a third group of bacteria that are not resistant to beta-lactamines and/or to cephalosporins; said method consisting in contacting the biological sample with a culture medium comprising: • a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity; • a second substrate for detecting a beta-glucosidase or desaminase or tryptophanase activity; • a combination comprising ceftriaxone and clavulanic acid.

15. A culture medium comprising: • a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity; • a second substrate for detecting a beta-glucosidase; • a combination of antibiotics comprising: o cefJMjdoxime, o cloxacillin, o vancomycin, and o amphoB; • a third substrate for detecting a desaminase activity.

16. The mediimi as claimed in claim 15, wherein it also comprises a fifth antibiotic which is cefsulodine.

17. The method as claimed in claim 2, for distinguishing: • a first group ofE. coli ESBL bacteria; • a second group ofKESC ESBL bacteria; • a third group of bacteria that are not resistant to beta-lactamines and/or to cephalosporins; • a fourth group of Proteeae ESBL bacteria; said method consisting in contacting the biological sample with a culture medium comprising : • a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity; • a second substrate for detecting a beta-glucosidase; • a combination of antibiotics comprising: o cefpodoxime, o cloxacillin, o vancomycin, and o amphoB; • a third substrate for detecting a desaminase activity. Dated this 17*^ day of May, 2012. , /(JIVESH KUMkfi)^"^'^ OF REMERY & SAGAR ATTORNEY FOR IKE APPLICANT(S)

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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 microorganisms, for instance of bacteria or yeasts.
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

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extended-spectrum P-lactamase (reference is then made to ESBL bacteria) have, as a result, 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, cefpodoxime 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 common 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;

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2. Seeding and incubating a culture medium (18 to 48 h) in order to induce exponential growth of the microorganisms;
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 amoimt 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 fi-om a first mediimi 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 bacterium 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 (B-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

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the species or the resistance mechanisms involved, in particular if it is a question of an acquired or wild-type resistance.
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 imicellular, 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, Hajhia, Edwardsiella, Aeromonas, Moraxella, Pasteurella, Providencia, and Legionella.

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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.
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 fioiit 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 freatment capable of preventing or reducing the
growth of microorganisms derived from a patient. This freatment may comprise in particular

6
antimicrobial compounds, such as antibiotics, for instance penicillins, conventional cephalosporins, broad-spectrum cephalosporins, monobactams, glycopeptides or aminosides, or such as antifungals or resistance-inhibiting compoimds. It should be noted that this 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. When 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, tyrosine) 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:

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beta-glucosidase, desaminase, beta-glucuronidase, beta-galactosidase, alpha-glucosidase, alpha-galactosidase, hexosaminidase, N-acetyl-hexosaminidase, phosphatase, esterase, and aminopeptidase.
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 6-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, phospholipase is preferred, for salmonellae, esterase is preferred, for Pseudomonas aeruginosa, esterase or fi-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-lactamases. 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.

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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), 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 compoimd 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, cefjpodoxime, 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 mediimi 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;
• cefjpodoxime, 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 medium 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.

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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 titanixmi oxide, nitroaniline, malachite green, brilliant green, etc. 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 mediimi 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.
This embodiment of the invention thus makes it possible, for example, to distinguish, in the same sample, a first group comprising E coli ESBL bacteria and a second group comprising KESC ESBL bacteria.

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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;
• 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:
• a first substrate for detecting a hexosaminidase enzymatic activity, preferably 5-bromo-4-chloro-3-indolyl-N-acetyl-B-D-glucosaminide, and a concentration between 25 and 500 mg/1, preferably between 40 and 150 mg/1;
• a second substrate for detecting a beta-glucosidase activity, preferably 6-chloro-3-indolyl-6-D-glucoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 200 mg/1;
• an antimicrobial which is an antifimgal, which is preferably amphotericin B (amphoB), at a concentration of between 0.5 and 64 mg/1, preferably between 1 and 16 mg/1, even more preferably between 1 and 8 mg/1.
When the antifungal is amphotericin B, this medium is preferably used to distinguish:
• a first group of yeasts comprising Candida albicans developing a resistance to amphoB;
• a second group of yeasts comprising Candida tropicalis and/or C. lusitaniae and/or C. kefyr, developing a resistance to amphoB;
• a third group of yeasts that are not resistant to amphoB.

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The invention also relates to a culture medium comprising:
• a first substrate for detecting a hexosaminidase enzymatic activity, preferably 5-bromo-4-chloro-3-indolyl-N-acetyl-fi-D-glucosaminide, at a concentration of between 25 and 500 mg/1, preferably between 40 and 150 mg/1;
• a second substrate for detecting a phosphatase activity, preferably 5-bromo-6-chloro-3-indolylphosphate, at a concentration of between 25 and 750 mg/1, preferably between 40 and 200 mg/1;
• an antimicrobial which is an antifimgal, which is preferably amphoB, at a concentration of between 0.5 and 64 mg/1, preferably between 1 and 16 mg/1, even more preferably between 1 and 8 mg/1.
When the antifimgal is amphotericin B, this medium is preferably used to distinguish:
• a first group of yeasts comprising Candida albicans developing a resistance to amphoB;
• a second group of yeasts comprising Candida tropicalis and/or C. glabrata and/or C krusei, developing a resistance to amphoB;
• a third group of yeasts that are not resistant to amphoB.
The invention also relates to a culture medium comprising:
• a first substrate for detecting the hexosaminidase enzymatic activity of said first group, preferably 5-bromo-4-chloro-3-indolyl-N-acetyl-6-D-glucosaminide, at a concentration of between 25 and 500 mg/1, preferably of between 40 and 150 mg/1;
• a second substrate for detecting the beta-glucosidase activity of said second group, preferably 6-chloro-3-indolyl-B-D-glucoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 200 mg/1;
• an antimicrobial which is an antifimgal, preferably fluconazole, at a concentration of between 1 and 256 mg/1, preferably between 2 and 128 mg/1, even more preferably between 8 and 64 mg/1.
When the antifimgal is fluconazole, this mediimi is preferably used to distinguish:
• a first group of yeasts comprising Candida albicans developing a resistance to
fluconazole;

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• a second group of yeasts, comprising Candida tropicalis and/or C. lusitaniae and/or C. kefyr, developing a resistance to fluconazole;
• a third group of yeasts that are not resistant to fluconazole.
The invention also relates to a culture medium comprising:
• a first substrate for detecting a hexosaminidase enzymatic activity, preferably 5-bromo-4-chloro-3-indolyl-N-acetyl-B-D-glucosaminide, at a concentration of between 25 and 500 mg/1, preferably between 40 and 150 mg/1;
• a second substrate for detecting a phosphatjise activity, preferably 5-bromo-6-chloro-3-indolylphosphate, at a concentration of between 25 and 750 mg/1, preferably between 40 and 200 mg/1;
• an antimicrobial which is an antifungal, which is preferably fluconazole, at a concentration of between 1 and 256 mg/1, preferably between 2 and 128 mg/1, even more preferably between 8 and 64 mg/1.
When the antifimgal is fluconazole, this medium is preferably used to distinguish:
• a first group of yeasts comprising Candida albicans developing a resistance to fluconazole;
• a second group of yeasts, comprising Candida tropicalis and/or C glabrata and/or C. krusei, developing a resistance to fluconazole;
• a third group of yeasts that are not resistant to fluconazole.
The invention also relates to a culture medium comprising:
• a first substrate for detecting a beta-glucuronidase enzymatic activity, preferably 6-chloro-3-indolyl-B-D-glucuronide, at a concentration of between 25 and 750 mg/1, preferably between 40 and 300 mg/1, or a beta-galactosidase enzymatic activity, preferably 5-bromo-6-chloro-3-indolyl-B-D-galactoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 150 mg/1;
• a second substrate for detecting a beta-glucosidase 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 250 mg/1, or a tryptophanase or desaminase activity, preferably tryptophan, at a concentration of between 50 and 5000 mg/1, preferably between 250 and 2000 mg/1;

13
• an antimicrobial which is an antibiotic, preferably ceftazidime. The ceftazidime
concentration in the medium is then preferably between 0.25 and 8 mg/1, preferably
between 2 and 2.5 mg/1.
When the antibiotic is ceftazidime, this mediimi is preferably used to distinguish:
• a first group ofE. coli ESBL or HL Case bacteria;
• a second group of KESC ESBL or HL Case bacteria;
• a third group of bacteria that are not resistant to beta-lactamines and to cephalosporins.
The invention also relates to a culture mediimi comprising:
• a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity, preferably 6-chloro-3-indolyl-B-D-glucuronide, at a concentration of between 25 and 750 mg/1, preferably between 40 and 300 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 150 mg/1;
• a second substrate for detecting a beta-glucosidase 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 250 mg/1, or a tryptophanase or desaminase activity, preferably tryptophan, at a concentration of between 50 and 5000 mg/1, preferably between 250 and 2000 mg/1;
• an antimicrobial which is an antibiotic, preferably cefipodoxime, at a concentration of between 0.5 and 32 mg/1, preferably between 0.75 and 10 mg/1, and even more preferably between 1 and 6 mg/1, and cloxacillin, at a concentration of between 10 and 2000 mg/1, preferably between 50 and 500 mg/1.
When the antibiotic is ceQ)odoxime, this medium is preferably used to distinguish:
• a first group oiE. coli ESBL bacteria;
• a second group of KESC ESBL bacteria;
• a third group of bacteria that are not resistant to beta-lactamines.
The invention also relates to a culture medium comprising:
• a first substrate for detecting a beta-glucuronidase enzymatic activity, preferably
6-chloro-3-indolyl-B-D-glucuronide, at a concentration of between 25 and 750 mg/1,
preferably between 40 and 300 mg/1, or a beta-galactosidase enzymatic activity.

14
preferably 5-bromo-6-chloro-3-indolyl-fi-D-galactoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 150 mg/1;
• a second substrate for detecting a beta-glucosidase activity, preferably 5-bromo-4-chloro-3-indolyl-fi-D-glucoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 250 mg/1, or a tryptophanase or desaminase activity, preferably tryptophan, at a concentration of between 50 and 5000 mg/1, preferably between 250 and 2000 mg/1;
• a combination comprising an antimicrobial and a resistance inhibitor. Preferably, this combination comprises ceftriaxone at a concentration of between 0.25 and 8 mg/1, preferably between 1 and 2.5 mg/1, and clavulanic acid at a concentration of between 0.05 and 32 mg/1, preferably between 0.1 and 8 mg/1, and even more preferably between 0.5 and 6 mg/1.
This medium is preferably used to distinguish:
• a first group ofE. coli HL Case bacteria;
• a second group of KESC HL Case bacteria;
• a third group of bacteria that are not resistant to cephalosporins.
The invention also relates to a culture medium comprising:
• a first substrate for detecting a beta-glucuronidase enzymatic activity, preferably 6-chloro-3-indolyl-fi-D-glucuronide, at a concentration of between 25 and 750 mg/1, preferably between 40 and 300 mg/1, or a beta-galactosidase enzymatic activity, preferably 5-bromo-6-chloro-3-indolyl-B-D-galactoside, at a concentration between 25 and 500 mg/1, preferably between 40 and 150 mg/1;
• a second substrate for detecting a beta-glucosidase 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 250 mg/1, or a tryptophanase or desaminase activity, preferably tryptophan, at a concentration of between 50 and 5000 mg/1, preferably between 250 and 2000 mg/1;
• two antimicrobials, which are preferably two antibiotics, preferably ceQ)odoxime at a concentration of between 0.1 and 32 mg/1, preferably between 0.25 and 10 mg/1, and even more preferably between 0.5 and 4 mg/1, and aztreonam at a concentration of between 0.1 and 8 mg/1, preferably between 0.5 and 1.5 mg/1.
This medium is preferably used to distinguish:

15
• a first group ofE. coli ESBL or HL Case bacteria;
• a second group of KESC ESBL or HL Case bacteria;
• a third group of bacteria that are not resistant to beta-lactamines and/or to cephalosporins.
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-cliloro-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 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-chloro-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-gaIactoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 250 mg/I, or 6-chloro-3-indolyl-B-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;
• a second group of vancomycin-resistant microorganisms, comprising Enterococcus casseliflavus and Enterococcus gallinarum;
• a third group of microorganisms that are not resistant to vancomycin.

16
In this case, 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-fi-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-chIoro-3-indolyl-a-D-glucoside, the second substrate is preferably 6-chloro-3-indolyl-fi-D-glucoside or alizarine-B-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-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-6-D-glucoside or 5-bromo-6-chloro-3-indolyl-fi-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:

17
1st group of 2nd group of 3rd group of 1st substrate 2nd substrate Antimicrobial
microorganisms microorganisms microorganisms
E. faecalis and E. casseliflavus 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-
{E. 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 12 mg/1.
The invention also relates to a culture medium comprising:
• an antimicrobial, preferably an antibiotic such as cloxacillin;

18
• a resistance mechanism inhibitor, such as preferably a 3rd-generation cephalosporin
chosen from cefotaxime, ceftazidime, cefpodoxime and ceftriaxone.
This medium is also preferably used to detect ESBL bacteria.
The embodiment of the invention described above is not limited to distinguishing 3 groups of microorganisms, but can make it possible to distinguish 4, 5 or even more groups of microorganisms. It is then necessary to add, to the medium, markers for identification between the various groups. In this respect, the invention also relates to a culture medium comprising:
• a first substrate for detecting a beta-glucuronidase enzymatic activity, preferably 6-chloro-3-indolyl-B-D-glucuronide, at a concentration of between 25 and 750 mg/1, preferably between 40 and 300 mg/1, or a beta-galactosidase enzymatic activity, preferably 5-bromo-6-chloro-3-indolyl-lJ-D-galactoside, at a concentration of between 25 and 500 mg/1, preferably of between 40 and 150 mg/1;
• a second substrate for detecting a beta-glucosidase, 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;
• a combination of antimicrobials, preferably a combination of antibiotics such as
o cefpodoxime at a concentration of between 0.5 and 32 mg/1, preferably
between 0.75 and 10 mg/1, and even more preferably between 1 and 6 mg/1; o cloxacillin at a concentration of between 10 and 2000 mg/1, preferably between
50 and 500 mg/1; o vancomycin at a concentration of between 0.5 and 128 mg/1, preferably
between 2 and 32 mg/1; and o amphoB at a concentration of between 0.5 and 64 mg/1, preferably between 1
and 16 mg/1, even more preferably between 1 and 8 mg/1;
• a third substrate for detecting a desaminase activity, such as histidine, phenylalanine,
tryptophan or tyrosine, at a concentration of between 50 and 5000 mg/1, preferably
between 250 and 2000 mg/1.
This mediimi may also comprise a fifth antibiotic, which is cefsulodine, at a concentration of between 0.5 and 64 mg/1, preferably between 1 and 16 mg/1. This medium is preferably used to distinguish:
• a first group ofE. coli ESBL bacteria;

19
• a second group of KESC ESBL bacteria;
• a third group of bacteria that are not resistant to beta-lactamines and/or to cephalosporins;
• a fourth group of Proteeae ESBL bacteria.
Similarly, the invention also relates to a culture medium comprising:
• 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-6-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-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-gaIactoside, at a concentration of between 25 and 500 mg/1, preferably between 40 and 250 mg/1, or 6-chloro-3-indolyl-B-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 mediimi is preferably used to distinguish:

20
• 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:
• 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 fiiUy.
EXAMPLE 1
The example below is based on the phenotypic detection of ESBLs using the reduction of susceptibility of these strains to antibiotics and their sensitivity to combinations with P-lactamases inhibitors. For this, a biplate of CPS ID 3 base (chromogenic medium for detecting microorganisms in urine, and sold by bioMerieux under the reference 43541) with one half-agar containing an antibiotic and one half-agar containing an antibiotic/p-lactamases inhibitor combination was used.
1. Choice of strains
In the context of the manipulations carried out, for evaluating the activity of the
antibiotics active on gram-negative bacilli, various species of enterobacteria {Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Serratia marcescens, Proteus mirabilis) and of nonfermenting gram-negative bacilli (Pseudomonas aeruginosa), capable of producing

21
ESBLs, were used. ESBL-positive strains, high level cephalosporinase-producing strains (HL Case) and wild-type strains are compared in the trials.
For the manipulations regarding the antibiotics active on gram-positive bacteria, strains of gram-positive cocci (Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, Streptococcus agalactiae) and of gram-positive bacilli (Lactobacillus spp) were tested.
For the trials intended to evaluate the activity of the antifimgals, various strains of yeasts (Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, Candida dubliniensis, Saccharomyces cerevisiae, Geotrichum capitatum) were used.
2. Preparation of media
The medium used was a CPS IDS medium (reference 43541) also comprising at least one antibiotic and at least one resistance mechanism inhibitor. The composition of the media tested was the following:
Chromogenic substrate Antibiotic Resistance inhibitor
Medium A 6-chloro-3-indolyl-B-D-glucuronide Cefotaxime 1 mg/l
5-bromo-4-chloro-3-indolyl-B-D-glucoside
Tryptophan
FeClj
Total: 1.73 g/1
Medium B 6-chloro-3-indolyl-B-D-glucuronide Ceftazidime 1.5 mg/l 0.25 mg/l clavulanic acid
5-bromo-4-chloro-3-indolyl-B-D-glucoside
Tryptophan
FeClj
Total: 1.73 g/1
Osmosed water is added and the whole is homogenized and melted in a waterbath at 100°C. The base medium 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 fim). The media are then poured into biplates 90 mm in diameter (i.e. approximately 9.5 ml/half-plate), and left on a flat surface so that they can set. The surface of the agars is then dried imder a laminar flow hood for 30 min.

22
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 |al 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 from 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 m diameter; 2: colonies 2 mm in diameter; 3: colonies of diameter > 2 mm.
5. Results:
a) Screening of antibiotics The 5 antibiotics reconmiended by the NCCLS (National Committee for Clinical Laboratory Standards) were tested. For each product tested, a range was prepared in order to determine the concentrations that make it possible to inhibit the wild-type strains of the enterobacteria tested, without affecting the growth of the ESBL-positive strains or the HL Case-positive strains. The concentrations selected are listed in table III hereinafter.
Table HI. Limiting values of the concentrations of antibiotics that allow inhibition of the wild-type strains of the enterobacteria tested, without affecting the growth of the ESBL-positive strains
under the conditions tested

23
Low value High value
Cefotaxime 1 mg/1 2 mg/1
Ceftazidime 2 mg/1 2.5 mg/1
Ceftriaxone 1 mg/1 2.5 mg/1
Celpodoxime 2 mg/1 10 mg/1
Aztreonam 1 mg/1 1.5 mg/1
The low value corresponds to the minimum concentration of the antibiotic required to inhibit
the wild-type strains of the enterobacteria tested. The high value corresponds to the maximum
concentration of the antibiotic that can be used without affecting the growth of the ESBL-
positive strains tested.
At these concentrations, the separation of the wild-type and resistant strains is satisfactory,
and the expression of the enzymatic activities on the CPS ID 3 medium is compliant.
In addition, it should be noted that ceftazidime is the only antibiotic tested and used in the
detection of ESBLs which showed an activity on the wild-type strains of P. aeruginosa. A
range was prepared in order to define the minimum concentration for complete inhibition of
these strains, and the limiting value is 1.5 mg/1.
The addition of antibiotics had no effect on the expression of the bacterial enzymatic activities on the chromogenic medium. The groups of microorganisms were separated and identified both on control CPS ID 3 medium and on the media comprising the antibiotics as described above.
b) Screening of fi-lactamase inhibitors Three P-lactamases inhibitors (BLIs), i.e. clavulanic acid, tazobactam and sulbactam, were used. A range was prepared for each one, in the presence of cefotaxime, in order to determine the optimum concentration for inhibiting the ESBL-positive strains without impairing the growth of the HL Case strains.
Clavulanic acid appeared to be the most effective BLI in the presence of cefotaxime. Tazobactam and sulbactam required concentrations of greater than 2 mg/1 in order to inhibit the ESBL-positive strains, whereas clavulanic acid was more active at much lower concentrations, over a broad operating range (fi-om 0.1 to 8 mg/1 when it is used in combination with cefotaxime).

24
Each antibiotic (cefotaxime, ceftazidime, ceftriaxone, ce^odoxime, aztreonam) was tested in the presence of a range of clavulanic acid in order to define the most effective combinations. The combinations selected are listed in table IV below.
Table IV. Selected combinations of antibiotics and of clavulanic acid (CA) that inhibit the
ESBL-positive enterobacterial strains tested, but allow the HL Case-positive strains to grow
Cefotaxime 2 mg/1 +CA 0.1 mg/1
Ceftazidime 2.5 mg/1 +CA 2 mg/1
Ceftriaxone 2 mg/1 +CA 0.25 mg/1
Celpodoxime 9 mg/1 +CA 0.25 mg/1
Aztreonam | 1 mg/11 + CA [ 0.5 mg/1
The results obtained using a biplate containing the antibiotic alone on one side and the antibiotic + clavulanic acid combination on the other side are listed in table V.
Table V
Antibiotic alone Antibiotic + clavulanic acid
Wild-type E. coli
ESBL-positive E. coli Pink colonies
HL Case-positive E. coli Pink colonies Pink colonies
Wild-type Proteeae
ESBL-positive Proteeae Brown colonies
HL Case-positive Proteeae Brown colonies Brown colonies
Wild-type KESC
ESBL-positive KESC Green colonies
HL Case-positive KESC Green colonies Green colonies
The addition of p-lactamase inhibitors had no effect on the expression of the bacterial enzymatic activities on the chromogenic medium. The groups of microorganisms were separated and identified both on control CPS ID 3 medium and on the media comprising P-lactamase inhibitors.
c) Combination of antibiotics Given the relative activities of the antibiotics and of clavulanic acid, the following were combined:
• cefotaxime (antibiotic active on ESBL-positive strains in combination with the lowest concentration of clavulanic acid),
• ceftazidime (antibiotic active on wild-type strains of P. aeruginosa), and

25
• clavulanic acid, so as to be able to inhibit, firstly, the wild-type strains (including those of pyocyanic bacillus), and the ESBL-positive strains of the enterobacteria tested by the addition of the BIL. Such a combination was tested on strains of P. aeruginosa; this species is naturally resistant to cefotaxime but sensitive to ceftazidime. When 1.5 mg/1 of CAZ (ceftazidime), 1 mg/1 of CTX (cefotaxime) and 0.25 mg/1 of CA (clavulanic acid) were combined, all the wild-type strains and the ESBL-positive strains of the enterobacteria tested were inhibited and only the collection of HL Case strains and the ESBL-positive P aeruginosa strains grew. It involves a biplate with CAZ alone on one side and CTX plus clavulanic acid on the other. The addition of these combinations of antibiotics had no effect on the expression of the bacterial enzymatic activities on the chromogenic medium. The groups of microorganisms were separated and identified both on control CPS ID 3 medium and on the media comprising such combinations of antibiotics.
d). Dye assav A medium according to the invention was also employed for use in a biplate, one of the sides containing an antibiotic, and the second containing another antibiotic or a combination of antibiotics. Given that the same CPS ED 3 medium base is used on either side, these two sides were differentiated by the presence of a dye.
The dye tested, Evans blue, gives the medium a green color. The coloration has made it possible to readily differentiate the 2 sides of the biplate without affecting the fertility of the medium, or impairing the reading of the enzymatic activities of the colonies. After having produced a range of Evans blue, added before or after autoclaving, the values selected were the following:
1.5 or 2 mg/1 if the dye is added after autoclaving.
between 2 and 5 mg/1 if it is added before autoclaving.
e). Inhibition of gram-positive bacteria ESBLs are a mechanism of resistance to p-lactamines that is foimd only in gram-negative bacilli; it is therefore advisable to inhibit the gram-positive bacteria via the medium. Two antibiotics, linezolide and vancomycin, were used in the medium according to the invention for the purpose of inhibiting the sensitive gram-positive bacteria.

26
For vancomycin, under the conditions tested, a concentration between 2 and 32 mg/1, and in
particular 2 to 5 mg/1, makes it possible to inhibit the sensitive gram-positive bacteria without
interfering with the detection of the ESBL bacteria.
For linezolide, under the conditions tested, a concentration between 2 and 64 mg/1, and in
particular 4 to 16 mg/1, makes it possible to inhibit the sensitive gram-positive bacteria
without interfering with the detection of the ESBL bacteria.
The inhibition of the gram-positive bacteria made it possible to improve the detection of the
ESBL bacteria in polymicrobial samples and the specificity of their coloration.
f) Inhibition of yeasts A medium according to the invention can also comprise antifungals in order to inhibit the possible presence of yeasts which could grow on the medium and which could impair microorganism growth.
Two antifungals were therefore tested: voriconazole and amphotericin B. For amphotericin B, under the conditions tested, a concentration between 1 and 32 mg/1, and in particular 2 to 8 mg/1, makes it possible to inhibit the sensitive yeasts without interfering with the detection of the ESBL bacteria.
For voriconazole, under the conditions tested, a concentration of between 1 and 64 mg/1, and in particular 4 to 16 mg/1, makes it possible to inhibit the sensitive gram-positive bacteria without interfering with the detection of the ESBL bacteria.
The inhibition of the yeasts made it possible to improve the detection of ESBL bacteria in polymicrobial samples and the specificity of their coloration.
6. Conclusion
These results demonstrate that the mediimi according to the invention makes it possible to isolate and apparently identify ESBL-producing bacteria, differentiating them from high level cephalosporinase-producing strains. The use of a biplate containing a cephalosporine alone on one side and a cephalosporine/clavulanic acid combination on the other, in a CPS ID 3 base, is particularly advantageous.
EXAMPLE 2
This second example is based on the phenotypic detection of ESBLs using the reduction of susceptibility to antibiotics and the sensitivity of HL Cases to combinations with tobramycin or cloxacillin or dicloxacillin, and presents the use of a cephalosporine mentioned above

27
(CTX, CAZ, CPD, CRO, ATM) in combination with a compound that inhibits cephalosporinases (cloxacillin, dicloxacillin and tobramycin). Such a medium makes it possible to inhibit bacteria which have a "natural" cephalosporinase, most of those which have only a high level cephalosporinase (HL Case), while at the same time allowing growth of ESBL bacteria.
1. Choice of strains
In the context of the manipulations carried out, for evaluating the activity of antibiotics
that are active on gram-negative bacilli, various species of enterobacteria (Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis) and of nonfennenting gram-active bacilli {Pseudomonas aeruginosa) capable of producing ESBLs, were used. In the assays, ESBL-positive strains, strains producing high level cephalosporinase (HL Case) and wild-type strains are compared.
2. Preparation of the medium
The medium used was a CPS IDS medium (43541), also comprising:
• ceftazidime at 2.5 mg/1 and tobramycin at 2 mg/1 (medium A) or
• ceftriaxone at 2 mg/1 and cloxacillin at 150 mg/1 (medium B) or
• ce^odoxime at 2 mg/1 and dicloxacillin at a concentration of between 500 and 1000 mg/1 (medium C).
Osmosed water is added and the whole is homogenized and melted in a waterbath at 100°C. The basic medium is dispensed into flasks, the number of which corresponds to the total niunber 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 ^im). The media are then poured into plates 35 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

28
5. Results:
Medium A comprising ceftazidime and tobramycin made it possible to inhibit all the wild-type strains and all the HL Cases tested. Only the ESBL-positive strains were detected on this medium.
Medium B comprising ceftriaxone and cloxacillin made it possible to inhibit all the HL Cases and all the wild-type strains except HL Case and wild-type P aeruginosa, and grew only the majority of the ESBL-positive strains.
Medium C comprising cefpodoxime and di-cloxacillin made it possible to inhibit all the wild-type strains and the majority of the HL Cases, without affecting the growth of ESBL-positive strains.
EXAMPLE 3
This third 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: B-glucosidase, and of a metabolic activity: Methyl-a-glucoside acidification.
1. Choice of strains
In the context of the manipulations carried out, for evaluating the activity of the
antibiotics active on enterococci, various species of Enterococcus {Enterococccus faecalis, Enterococcus faecium, Enterococcus casseliflavus, Enterococcus gallinarum,) were used, hi 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 pC-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,
• amphotericin B at 2 mg/1.

29
Osmosed water is added and the whole is homogenized and mehed in a waterbath at 100°C. The basic medium 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 ^m). 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 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.
This medium therefore makes it possible to differentiate these two groups of enterococci and
to provide a smtable therapeutic response.
EXAMPLE 4
This fourth 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 P-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, Enterococcus faecium, Enterococcus casseliflavus, Enterococcus gallinarum,) were used. In the assays, strains resistant to glycopeptides (VRE) and wild-type strains are compared.

30
2. Preparation of the medium
The media used were a Columbia 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 |jm).
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.

31
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 medium 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. casselijlavus 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 microorganisms, belonging to a first taxon of microorganisms and comprising at least one mechanism of resistance to an antimicrobial;
• 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 an antimicrobial, identical to that of the first group;
• a third group of microorganisms, that are not resistant to said antimicrobial,
said method consisting in contacting the biological sample with a culture mediimi comprising:
□ at least a first substrate for detecting at least a first enzymatic or metabolic
activity of said first group of microorganisms;
a 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 as claimed in claim 1, for distinguishing at least three groups of microorganisms in a biological sample, wherein the microorganisms of said first, second and third groups are Gram negative bacteria.
3. The method as claimed in claim 2, wherein the antimicrobial belongs to the beta-lactamin family.
4. The method as claimed in claim 2, wherein the antimicrobial belongs to the cephalosporin family.
5. A culture medium comprising:

• a first substrate for detecting a beta-glucuronidase or a beta-galactosidase enzymatic activity;
• a second substrate for detecting a beta-glucosidase or tryptophanase or deaminase activity;
• a combination comprising an antimicrobial and an inhibitor of a resistance mechanism.

6. A culture medium comprising:
• a first substrate for detecting a beta-glucuronidase or a beta-galactosidase enzymatic activity;
• a second substrate for detecting a beta-glucosidase or tryptophanase or deaminase enzymatic activity;
• at least two antibiotics.
7. A culture medium comprising:
• a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity;
• a second substrate for detecting a beta-glucosidase or tryptophanase or desaminase activity;
• an antibiotic which is ceftazidime.
8. The method as claimed in claim 2, for distinguishing:
• a first group ofE. coli ESBL or HL Case bacteria;
• a second group of KESC ESBL or HL Case bacteria;
• a third group of bacteria that are not resistant to beta-Iactamines and/or to cephalosporins;
said method consisting in contacting the biological sample with a culture mediimi comprising
• a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity;
• a second substrate for detecting a beta-glucosidase or tryptophanase or desaminase activity;
• an antibiotic which is ceftazidime.
9. A culture medium comprising:
• a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity;
• a second substrate for detecting a beta-glucosidase or tryptophanase or desaminase activity;
• an antibiotic which is cefpodoxime;
• an antibiotic which is cloxacillin.
10. The method as claimed in claim 2, for distinguishing:
• a first group ofE. coli ESBL bacteria;

• a second group of KESC ESBL bacteria;
• a third group of bacteria that are not resistant to beta-lactamines;
said method consisting in contacting the biological sample with a culture medium comprising:
• a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity;
• a second substrate for detecting a beta-glucosidase or tryptophanase or desaminase activity;
• an antibiotic which is cefpodoxime;
• an antibiotic which is cloxacillin.
11. A culture medium comprising:
• a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity;
• a second substrate for detecting a beta-glucosidase or desaminase or tryptophanase activity;
• a combination comprising ceftriaxone and clavulanic acid.
12. The method as claimed in claim 2, for distinguishing:
• a first group ofE. coli HL Case bacteria;
• a second group of KESC HL Case bacteria;
• a third group of bacteria that are not resistant to beta-lactamins and/or cephalosporins;
said method consisting in contacting the biological sample with a culture mediiun comprising:
• a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity;
• a second substrate for detecting a beta-glucosidase or desaminase or tryptophanase activity;
• a combination comprising ceftriaxone and clavulanic acid.
13. A cultixre medium comprising:
• a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity;
• a second substrate for detecting a beta-glucosidase or desaminase or tryptophanase activity;
• two antibiotics, which are cefpodoxime and aztreonam.

14. The method as claimed in claim 2, for distinguishing:
• a first group ofE. coli ESBL or HL Case bacteria;
• a second group of KESC ESBL or HL Case bacteria;
• a third group of bacteria that are not resistant to beta-lactamines and/or to cephalosporins;
said method consisting in contacting the biological sample with a culture medium comprising:
• a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity;
• a second substrate for detecting a beta-glucosidase or desaminase or tryptophanase activity;
• a combination comprising ceftriaxone and clavulanic acid.
15. A culture medium comprising:
• a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity;
• a second substrate for detecting a beta-glucosidase;
• a combination of antibiotics comprising: o cefJMjdoxime,
o cloxacillin,
o vancomycin, and
o amphoB;
• a third substrate for detecting a desaminase activity.
16. The mediimi as claimed in claim 15, wherein it also comprises a fifth antibiotic which is cefsulodine.
17. The method as claimed in claim 2, for distinguishing:

• a first group ofE. coli ESBL bacteria;
• a second group ofKESC ESBL bacteria;
• a third group of bacteria that are not resistant to beta-lactamines and/or to cephalosporins;
• a fourth group of Proteeae ESBL bacteria;
said method consisting in contacting the biological sample with a culture medium comprising :
• a first substrate for detecting a beta-glucuronidase or beta-galactosidase enzymatic activity;
• a second substrate for detecting a beta-glucosidase;

• a combination of antibiotics comprising:
o cefpodoxime,
o cloxacillin,
o vancomycin, and
o amphoB;
• a third substrate for detecting a desaminase activity.
Dated this 17*^ day of May, 2012. ,
/(JIVESH KUMkfi)^"^'^ OF REMERY & SAGAR ATTORNEY FOR IKE APPLICANT(S)