Medium for the specific detection of resistant microorganisms   

Abstract: A method for distinguishing among a first group of microorganisms, belonging to a first taxon of bacteria that is vancomycin resistant; a second group of microorganisms, belonging to a second taxon of vancomycin resistant bacteria that is different than said first taxon; and a third group of microorganisms that is not resistant or has a natural resistance to vancomycin.

This is a divisional of application Ser. No. 11/794,907 filed Jul. 9, 2007, which is a National Stage Application of PCT/FR2006/050109 filed Feb. 9, 2006, and claims the benefit of French Application Nos. 0550394 filed Feb. 10, 2005 and 0553049 filed Oct. 7, 2005. The entire disclosures of the prior applications are hereby incorporated by reference herein in their entirety.

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, β-lactamases are enzymes expressed by certain bacteria, capable of hydrolyzing the C—N bond of the β-lactame ring, the basic structure of antibiotics of the β-lactamine family, so as to give a microbiologically inactive product. Several β-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 β-lactamines which are associated therewith. They act as a suicide subject for β-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 β-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 β-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, 7α-methoxycephalosporins (cephamycins: cefoxitin, cefotetan) and carbapenems (imipenem, meropenem, ertapenem) generally conserve their activity. ESBLs are inhibited by β-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;

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 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 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 from 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 andK. 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 (β-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.

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 further 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.

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 β-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 antifungals 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 compounds, such as antibiotics, for instance penicillins, conventional cephalosporins, broad-spectrum cephalosporins, monobactams, glycopeptides or aminosides, or such as anti fungals or resistance-inhibiting compounds. 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 from the enzymatic activities: 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 β-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 β-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 β-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 β-lactamases. The clavulanic acid concentration in the medium is then preferably between 0.05 and 32 mg/l, preferably between 0.1 and 8 mg/l, and even more preferably between 0.25 and 6 mg/l; 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), Proteeae, Proteus, Morganella, Pseudomonas, Staphylococcus, Streptococcus, Enterococcus, Candida, Escherichia coli, Escherichia coli O157: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 “antifungal” 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/l, preferably between 1 and 2 mg/l; ceftazidime, the ceftazidime concentration in the medium is preferably between 0.25 and 8 mg/l, preferably between 2 and 2.5 mg/l; ceftriaxone, the ceftriaxone concentration in the medium is preferably between 0.25 and 8 mg/l, preferably between 1 and 2.5 mg/l; cefpodoxime, the cefpodoxime concentration in the medium is preferably between 0.1 and 32 mg/l, preferably between 0.75 and 10 mg/l, and even more preferably between 1 and 6 mg/l; aztreonam, the aztreonam concentration in the medium is preferably between 0.1 and 8 mg/l, preferably between 0.75 and 1.5 mg/l.

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. 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.

In this respect, the invention relates to the use of a combination of two culture media for distinguishing at least three groups of microorganisms in a biological sample, comprising: a first group of microorganisms, belonging to a first taxon of microorganisms and comprising at least a first mechanism of resistance to a first treatment; a second group of microorganisms, belonging to a second taxon of microorganisms and comprising at least a second mechanism of resistance to a second treatment; a third group of microorganisms, that are not resistant to said first and second treatments, said combination of two culture media 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 two markers for differentiating the first group of microorganisms and the second group of microorganisms; c. at least one antimicrobial that is active on said third group of microorganisms.

Preferably, the medium comprises at least two markers for differentiating the first group of microorganisms and the second group of microorganisms, at least one of which is an inhibitor of said first or said second mechanism of resistance. The medium may also comprise at least two markers for differentiating the first group of microorganisms and the second group of microorganisms, which are each an inhibitor of said first or said second mechanism of resistance.

This first 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, each of the two groups being resistant to a different 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 HL Case bacteria. In this specific case, the combination of two media may be the following: At least a first substrate which makes it possible to identify E. coli bacteria, for example a glucuronidase substrate, such as 6-chloro-3-indolyl-β-D-glucuronide, or a galactosidase substrate, such as 5-bromo-6-chloro-3-indolyl-β-D-galactoside; a first identification marker which is a glucosidase substrate, such as 5-bromo-4-chloro-3-indolyl-β-D-glucoside, which makes it possible to identify KESC bacteria; a second identification marker which is a resistance mechanism inhibitor, such as cloxacillin; at least one antimicrobial which is an antibiotic, such as cefpodoxime.

Reference is made to a combination of 2 media, i.e. a first medium may comprise: a first substrate which makes it possible to identify E. coli, for example a glucuronidase substrate, such as 6-chloro-3-indolyl-β-D-glucuronide or a galactosidase substrate, such as 5-bromo-6-chloro-3-indolyl-β-D-galactoside; a first marker which is a resistance mechanism inhibitor, such as cloxacillin; an antimicrobial which is an antibiotic, such as cefpodoxime, or the second medium comprises: a first substrate which makes it possible to identify E. coli, for example a glucuronidase substrate, such as 6-chloro-3-indolyl-β-D-glucuronide, or a galactosidase substrate, such as 5 -bromo-6-chloro-3-indolyl-β-D-galactoside; a first marker which is a glucosidase substrate, such as 5-bromo-4-chloro-3-indolyl-β-D-glucoside, which makes it possible to identify KESC bacteria; an antimicrobial which is an antibiotic, such as ceftazidime.

Another alternative would be to use a first medium comprising: a first substrate which makes it possible to identify E. coli, for example a glucuronidase substrate, such as G-chloro-3-indolyl-β-D-glucuronide, or a galactosidase substrate, such as 5-bromo-6-chloro-3-indolyl-β-D-galactoside; a first marker which is a glucosidase substrate, such as 5-bromo-4-chloro-3-indolyl-β-D-glucoside, which makes it possible to identify KESC bacteria; an antimicrobial which is an antibiotic, such as aztreonam, while the second medium comprises: a first substrate which makes it possible to identify E. coli, for example a glucuronidase substrate, such as 6-chloro-3-indolyl-β-D-glucuronide, or a galactosidase substrate, such as 5-bromo-6-chloro-3-indolyl-β-D-galactoside; a first marker which is a resistance mechanism inhibitor, such as clavulanic acid; an antimicrobial which is an antibiotic, such as ceftazidime.

Those skilled in the art will choose each medium so as to systematically obtain a combination according to the invention. The antimicrobial that is active on said third group is present in the two media. Those skilled in the art may in particular use a biplate, for readily comparing the two media on which the same biological sample has been deposited. In this respect, the invention also relates to a biplate comprising a combination of two culture media, said combination 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 two markers for differentiating the first group of microorganisms and the second group of microorganisms; c. at least one antimicrobial that is active on said third group of microorganisms, said antimicrobial being present on each side of the biplate.

This first embodiment of the invention is not limited to distinguishing between 3 groups of microorganisms, but may make it possible to distinguish between 4, 5 or even more groups of microorganisms. It is then necessary to add additional identification markers to the medium or media, in order to discriminate between the various groups.

In this respect, the invention also relates to a biplate comprising a combination of two culture media, said combination comprising: for the first medium: a first substrate which makes it possible to identify E. coli, for example a glucuronidase substrate such as 6-chloro-3-indolyl-β-D-glucuronide, or a galactosidase substrate, such as 5-bromo-6-chloro-3-indolyl-β-D-galactoside, or a substrate which makes it possible to identify Proteeae, for example tryptophan; a first identification marker which is a glucosidase substrate, such as 5-bromo-4-chloro-3-indolyl-β-D-glucoside; a second identification marker which is a resistance inhibitor, such as cloxacillin; at least one antimicrobial which is an antibiotic, such as cefpodoxime; for the second medium: a first substrate which makes it possible to identify enterococci, for example a glucosidase substrate, such as 5-bromo-4-chloro-3-indolyl-β-D-glucoside; a first identification marker which is another glucosidase substrate, such as methyl-α-glucoside; at least one antimicrobial which is an antibiotic, such as vancomycin.

The invention also 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 second 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 specific embodiment of the invention, it is not necessary to use a combination of media, since a single medium comprising the characteristics as defined above is sufficient. 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.

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-β-D-glucoside, at a concentration of between 25 and 500 mg/l, preferably between 40 and 150 mg/l; 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/l, preferably between 4 and 50 mg/l; an antimicrobial which is an antibiotic, preferably vancomycin, at a concentration of between 0.5 and 128 mg/l, preferably between 2 and 32 mg/l.

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-β-D-glucosaminide, and a concentration between 25 and 500 mg/l, preferably between 40 and 150 mg/l; a second substrate for detecting a beta-glucosidase activity, preferably 6-chloro-3-indolyl-β-D-glucoside, at a concentration of between 25 and 500 mg/l, preferably between 40 and 200 mg/l; an antimicrobial which is an antifungal, which is preferably amphotericin B (amphoB), at a concentration of between 0.5 and 64 mg/l, preferably between 1 and 16 mg/l, even more preferably between 1 and 8 mg/l.

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;

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