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 Gram negative bacteria, the first group of bacteria exhibiting a mechanism of resistance to a treatment; a second group of microorganisms belonging to a second taxon of Gram negative bacteria, the second taxon of bacteria being different than said first taxon, and exhibiting a mechanism of resistance to a treatment identical to the mechanism of the first group; and a third group of Gram negative bacteria that is not resistant to the treatment.

Claims

1. A method for distinguishing among at least three groups of microorganisms that may be present in a biological sample, the at least three groups comprising: 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, the method comprising: inoculating a culture medium with the biological sample, the culture medium comprising: a first substrate for detecting a first enzymatic activity of said first group of microorganisms; a marker for differentiating the first group of microorganisms and the second group of microorganisms, said marker being a substrate for detecting an enzymatic activity of said second group of microorganisms; and an antimicrobial that is active on the third group of microorganisms, and distinguishing among any members of the three groups of microorganisms that are present on the culture medium to determine of which group of microorganisms they are members on the basis of their interactions with the first substrate, the marker, and the antimicrobial.

2. The method of claim 1, wherein: the first group of microorganisms is enterococcal bacteria that have an acquired resistance to vancomycin; the second group of microorganisms is enterococcal bacteria that have a natural resistance to vancomycin; and the third group of microorganisms is enterococcal bacteria that is not resistant to vancomycin.

3. The method of claim 1, wherein: the first group of vancomycin-resistant microorganisms comprises Enterococcus faecalis and Enterococcus faecium; the second group of vancomycin-resistant microorganisms comprises Enterococcus casseliflavus and Enterococcus gallinarum; and the third group of microorganisms is not resistant to vancomycin.

4. The method of claim 1, wherein: the first group of vancomycin-resistant microorganisms comprises Enterococcus faecium; the second group of vancomycin-resistant microorganisms comprises Enterococcus faecalis; and the third group of microorganisms is enterococcal bacteria that is not resistant to vancomycin or that have a natural resistance to vancomycin.

5. The method of claim 1, wherein: the first group of vancomycin-resistant microorganisms comprises Enterococcus faecalis and Enterococcus faecium; the second group of vancomycin-resistant microorganisms comprises Staphylococcus aureus that is intermediary resistant or resistant to vancomycin; and the third group of microorganisms is not resistant to vancomycin.

6. The method of claim 1, wherein: the first substrate is an enzymatic substrate for detecting alpha-glucosidase activity; the marker is a second substrate for detecting a second activity different than alpha-glucosidase activity; and the antimicrobial is vancomycin.

7. The method of claim 6, wherein the second substrate detects beta-glucosidase or beta-galactosidase activity.

8. The method of claim 6, wherein: the first substrate is 5-bromo-4-chloro-3-indolyl-N-methyl-α-D-glucoside or 5-bromo-4-chloro-3-indolyl-α-D-glucoside, and the second substrate is 6-chloro-3-indolyl-β-D-glucoside, alizarine-β-D-galactoside, 5-bromo-6-chloro-3-indolyl-β-D-glucoside, 5-bromo-6-chloro-3-indolyl-β-D-galactoside or 6-chloro-3-indolyl-β-D-galactoside.

9. The method of claim 6, wherein: the first substrate is 5-bromo-4-chloro-3-indolyl-N-methyl-α-D-glucoside or 5-bromo-4-chloro-3-indolyl-α-D-glucoside, and the second substrate is 6-chloro-3-indolyl-β-D-glucoside or 5-bromo-6-chloro-3-indolyl-β-D-glucoside.

10. The method of claim 1, wherein: the first group of vancomycin-resistant microorganisms comprises Enterococcus faecalis and Enterococcus faecium; the second group of vancomycin-resistant microorganisms comprises Enterococcus casseliflavus and Enterococcus gallinarum; the third group of microorganisms is not resistant to vancomycin; the first substrate is an enzymatic substrate for detecting alpha-glucosidase activity; the marker is a second substrate for detecting a second activity different than alpha-glucosidase activity; and the antimicrobial is vancomycin.

11. The method of claim 1, wherein: the first group of vancomycin-resistant microorganisms comprises Enterococcus faecium; the second group of vancomycin-resistant microorganisms comprises Enterococcus faecalis; the third group of microorganisms is enterococcal bacteria that is not resistant to vancomycin or that have a natural resistance to vancomycin; the first substrate is an enzymatic substrate for detecting alpha-glucosidase activity; the marker is a second substrate for detecting a second activity different than alpha-glucosidase activity; and the antimicrobial is vancomycin.

12. The method of claim 1, wherein: the first group of vancomycin-resistant microorganisms comprises Enterococcus faecalis and Enterococcus faecium; the second group of vancomycin-resistant microorganisms comprises Staphylococcus aureus that is intermediary resistant or resistant to vancomycin; the third group of microorganisms are not resistant to vancomycin; the first substrate is an enzymatic substrate for detecting alpha-glucosidase activity; the marker is a second substrate for detecting a second activity different than alpha-glucosidase activity; and the antimicrobial is vancomycin.

13. The method of claim 1, wherein: the first group of vancomycin-resistant microorganisms comprises Enterococcus faecalis and Enterococcus faecium; the second group of vancomycin-resistant microorganisms comprises Enterococcus casseliflavus and Enterococcus gallinarum; the third group of microorganisms is not resistant to vancomycin; the first substrate is an enzymatic substrate for detecting alpha-glucosidase activity; the marker is a second substrate for detecting beta-glucosidase or beta-galactosidase activity; and the antimicrobial is vancomycin.

14. The method of claim 1, wherein: the first group of vancomycin-resistant microorganisms comprises Enterococcus faecium; the second group of vancomycin-resistant microorganisms comprises Enterococcus faecalis; the third group of microorganisms is enterococcal bacteria that is not resistant to vancomycin or that have a natural resistance to vancomycin; the first substrate is 5-bromo-4-chloro-3-indolyl-N-methyl-a-D-glucoside or 5-bromo-4-chloro-3-indolyl-a-D-glucoside; the marker is 6-chloro-3-indolyl-β-D-glucoside, alizarine-β-D-galactoside, 5-bromo-6-chloro-3-indolyl-β-D-glucoside, 5-bromo-6-chloro-3-indolyl-β-D-galactoside or 6-chloro-3-indolyl-β-D-galactoside; and the antimicrobial is vancomycin.

15. The method of claim 1, wherein: the first group of vancomycin-resistant microorganisms comprises Enterococcus faecalis and Enterococcus faecium; the second group of vancomycin-resistant microorganisms comprises Staphylococcus aureus that is intermediary resistant or resistant to vancomycin; the third group of microorganisms is not resistant to vancomycin; the first substrate is 5-bromo-4-chloro-3-indolyl-N-methyl-a-D-glucoside or 5-bromo-4-chloro-3-indolyl-a-D-glucoside; the marker is 6-chloro-3-indolyl-β-D-glucoside or 5-bromo-6-chloro-3-indolyl-β-D-glucoside; and the antimicrobial is vancomycin.

16. A method of detecting and distinguishing between any extended-spectrum β-lactamase-producing E. coli bacteria (ESBL E. coli bacteria) and any extended-spectrum β-lactamase-producing KESC bacteria (ESBL KESC bacteria) that are present on a culture medium, the method comprising: inoculating a culture medium with a biological sample, wherein the culture medium comprises: a first substrate for detecting beta-galactosidase activity; a second substrate for detecting beta-glucuronidase activity; a third substrate for detecting desaminase activity; and a combination of antimicrobials throughout the entire culture medium, the combination comprising a cephalosporinase inhibitor and a third-generation cephalosporin; and simultaneously detecting and distinguishing between any ESBL E. coli bacteria and ESBL KESC bacteria that are present on the culture medium.

17. A method of detecting and distinguishing between any extended-spectrum β-lactamase-producing E. coli bacteria (ESBL E. coli bacteria) and any extended-spectrum β-lactamase-producing KESC bacteria (ESBL KESC bacteria) that are present on a culture medium, the method comprising: inoculating a culture medium with a biological sample, wherein the culture medium comprises: a first substrate for detecting beta-galactosidase activity; a second substrate for detecting beta-glucosidase activity; and a combination of antimicrobials throughout the entire culture medium, the combination comprising a cephalosporinase inhibitor and a third-generation cephalosporin.

Description

EXAMPLE 1

[0295] 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 β-lactamases inhibitors. For this, a biplate of CPS ID 3 base (chromogenic medium for detecting microorganisms in urine, and sold by bioMérieux under the reference 43541) with one half-agar containing an antibiotic and one half-agar containing an antibiotic/β-lactamases inhibitor combination was used.

1. Choice of Strains

[0296] 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 ESBLs, were used. ESBL-positive strains, high level cephalosporinase-producing strains (HL Case) and wild-type strains are compared in the trials.

[0297] 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.

[0298] For the trials intended to evaluate the activity of the antifungals, various strains of yeasts (Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, Candida dubliniensis, Saccharomyces cerevisiae, Geotrichum capitatum) were used.

2. Preparation of Media

[0299] The medium used was a CPS ID3 medium (reference 43541) also comprising at least one antibiotic and at least one resistance mechanism inhibitor.

[0300] The composition of the media tested was the following:

TABLE-US-00002 Chromogenic substrate Antibiotic Resistance inhibitor Medium A 6-chloro-3-indolyl-β-D-glucuronide Cefotaxime 1 mg/l 5-bromo-4-chloro-3-indolyl-β-D-glucoside Tryptophan FeCl.sub.3 Total: 1.73 g/l Medium B 6-chloro-3-indolyl-β-D-glucuronide Ceftazidime 1.5 mg/l 0.25 mg/l clavulanic acid 5-bromo-4-chloro-3-indolyl-β-D-glucoside Tryptophan FeCl.sub.3 Total: 1.73 g/l
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 μm). 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 under a laminar flow hood for 30 min.

3. Inoculation of Media

[0301] 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 μl 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.sup.3 to 10.sup.4 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.

[0302] 4. Reading of media: the readings are carried out at 18 hours (±30 min), 24 h (±1h) and 48 h (±4 h). The density and the size of the colonies, and the appearance, the color and the coloration intensities of the mass and of the isolated colonies were observed, according to the following reading scales 1 to 3: 0: no growth; 0.1: trace of growth; 0.25: colonies of diameter<0.5 mm; 0.5: colonies of 0.5 mm in diameter; 0.75: 0.5 mm<diameter<1 mm; 1: colonies of 1 mm in diameter; 1.25: 1 mm<diameter<1.5 mm; 1.5: colonies 1.5 mm in diameter; 2: colonies 2 mm in diameter; 3: colonies of diameter>2 mm.

5. Results

a) Screening of Antibiotics

[0303] The 5 antibiotics recommended 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.

[0304] The concentrations selected are listed in table III hereinafter.

TABLE-US-00003 TABLE III 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 Low value High value Cefotaxime 1 mg/l   2 mg/l Ceftazidime 2 mg/l 2.5 mg/l Ceftriaxone 1 mg/l 2.5 mg/l Cefpodoxime 2 mg/l  10 mg/l Aztreonam 1 mg/l 1.5 mg/l

[0305] 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.

[0306] 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/l.

[0307] 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 β-lactamase inhibitors

[0308] Three β-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.

[0309] Clavulanic acid appeared to be the most effective BLI in the presence of cefotaxime. Tazobactam and sulbactam required concentrations of greater than 2 mg/l in order to inhibit the ESBL-positive strains, whereas clavulanic acid was more active at much lower concentrations, over a broad operating range (from 0.1 to 8 mg/l when it is used in combination with cefotaxime).

[0310] Each antibiotic (cefotaxime, ceftazidime, ceftriaxone, cefpodoxime, 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-US-00004 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/l + CA  0.1 mg/l Ceftazidime 2.5 mg/l + CA   2 mg/l Ceftriaxone   2 mg/l + CA 0.25 mg/l Cefpodoxime   9 mg/l + CA 0.25 mg/l Aztreonam   1 mg/l + CA  0.5 mg/l

[0311] 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-US-00005 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

[0312] The addition of β-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 β-lactamase inhibitors.

c) Combination of antibiotics

[0313] Given the relative activities of the antibiotics and of clavulanic acid, the following were combined: [0314] cefotaxime (antibiotic active on ESBL-positive strains in combination with the lowest concentration of clavulanic acid), [0315] ceftazidime (antibiotic active on wild-type strains of P. aeruginosa), and [0316] 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.

[0317] 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/l of CAZ (ceftazidime), 1 mg/l of CTX (cefotaxime) and 0.25 mg/l 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.

[0318] 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 Assay

[0319] 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 ID 3 medium base is used on either side, these two sides were differentiated by the presence of a dye.

[0320] 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: [0321] 1.5 or 2 mg/l if the dye is added after autoclaving. [0322] between 2 and 5 mg/l if it is added before autoclaving.

e). Inhibition of Gram-positive Bacteria

[0323] ESBLs are a mechanism of resistance to β-lactamines that is found 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.

[0324] For vancomycin, under the conditions tested, a concentration between 2 and 32 mg/l, and in particular 2 to 5 mg/l, makes it possible to inhibit the sensitive gram-positive bacteria without interfering with the detection of the ESBL bacteria.

[0325] For linezolide, under the conditions tested, a concentration between 2 and 64 mg/l, and in particular 4 to 16 mg/l, makes it possible to inhibit the sensitive gram-positive bacteria without interfering with the detection of the ESBL bacteria.

[0326] 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

[0327] 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.

[0328] Two antifungals were therefore tested: voriconazole and amphotericin B.

[0329] For amphotericin B, under the conditions tested, a concentration between 1 and 32 mg/l, and in particular 2 to 8 mg/l, makes it possible to inhibit the sensitive yeasts without interfering with the detection of the ESBL bacteria.

[0330] For voriconazole, under the conditions tested, a concentration of between 1 and 64 mg/l, and in particular 4 to 16 mg/l, makes it possible to inhibit the sensitive gram-positive bacteria without interfering with the detection of the ESBL bacteria.

[0331] 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

[0332] These results demonstrate that the medium 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

[0333] 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 (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

[0334] 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 nonfermenting 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

[0335] The medium used was a CPS ID3 medium (43541), also comprising: [0336] ceftazidime at 2.5 mg/l and tobramycin at 2 mg/l (medium A) or [0337] ceftriaxone at 2 mg/l and cloxacillin at 150 mg/l (medium B) or [0338] cefpodoxime at 2 mg/l and dicloxacillin at a concentration of between 500 and 1000 mg/l (medium C).

[0339] Osmosed water is added and the whole is homogenized and melted in a waterbath at 100° C.

[0340] 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 μm). 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

[0341] This step is carried out as described in example 1.

4. Reading of Media

[0342] This step is carried out as described in example 1

5. Results

[0343] 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.

[0344] 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.

[0345] 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

[0346] 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: β-glucosidase, and of a metabolic activity: Methyl-α-glucoside acidification.

1. Choice of Strains

[0347] 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. In the assays, strains resistant to glycopeptides (VRE) and wild-type strains are compared.

2. Preparation of the Medium

[0348] The medium used was a Columbia medium (51026), also comprising: [0349] 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside (X-Glu) at 100 mg/l, [0350] methyl-α-D-glucoside at 9 g/l, [0351] neutral red at 25 mg/l, [0352] bilial salts at 5 g/l, [0353] vancomycin at 4 mg/l, [0354] amphotericin B at 2 mg/l.

[0355] 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 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 μm). 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

[0356] This step is carried out as described in example 1.

4. Reading of Media

[0357] This step is carried out as described in example 1.

5. Results:

[0358] On this medium, only the glycopeptide-resistant enterococcal strains develop and form colonies.

[0359] The resistant E. faecalis and E. faecium strains form green colonies, whereas those of E. casseliflavus and of E. gallinarum form blue-to-violet colonies. This medium therefore makes it possible to differentiate these two groups of enterococci and to provide a suitable therapeutic response.

EXAMPLE 4

[0360] 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: α-glucosidase and β-galactosidase or β-glucosidase.

1. Choice of Strains

[0361] 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.

2. Preparation of the Medium

[0362] The media used were a Columbia medium (51026), also comprising: [0363] 5-bromo-4-chloro-3-indolyl-N-methyl-α-D-glucopyranoside (GreenA-α-Glu) at 150 mg/l, [0364] 6-chloro-3-indolyl-β-glucopyranoside (Rose-b-Glu [Pink-b-Glu]) at 200 mg/l,
or: [0365] 5-bromo-4-chloro-3-indolyl-N-methyl-α-D-glucopyranoside (GreenA-α-Glu) at 150 mg/l, [0366] alizarine-β-galactopyranoside at 50 mg/l [0367] and vancomycin at 8 mg/l, [0368] an amphotericin B at 4 mg/l, [0369] and colistine at 2 mg/l, [0370] and aztreonam at 32 mg/l.

[0371] 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 μm). 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

[0372] This step is carried out as described in example 1.

4. Reading of Media

[0373] This step is carried out as described in example 1.

5. Results

[0374] On the medium containing a substrate for α-glucosidase and for β-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.

[0375] 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.

[0376] On the medium containing a substrate for α-glucosidase and for β-galactosidase, the resistant E. faecium strains form violet colonies, whereas the resistant E. faecalis strains form green colonies. The E. casseliflavus and E. gallinarum strains (natural resistances) are inhibited due to the concentration of vancomycin.

[0377] 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.