Medium for detecting and/or identifying bacteria
The field of the invention is that of biochemical microbiological analysis, and in particular of the detection and identification of bacteria.
Pathogenic bacteria, and in particular Gram-negative bacilli, such as enterobacteria, are
responsible each year for many diseases, epidemics, etc.
The species E. coli {Escherichia coli) is the aerobic species most predominantly
represented in the digestive tract. However, the presence of said bacteria in water
indicates fecal contamination, and certain strains are pathogenic and responsible for
peritoneal, biliary, appendicular or genital suppurations.
Early and specific detection of E. coli makes it possible to propose a suitable solution, in
terms of treatment, of decontamination, etc. This detection can be based in particular on
the use of detection media comprising particular substrates, specific for a metabolic
activity, referred to as target metabolic activity, such as an enzymatic activity, of the
bacterium that it is desired to detect: through the choice of substrates, depending on
whether or not there is a reaction, it is possible to characterize the nature of a
microorganism.
The CPS ID 3 medium (bioMerieux) uses a ß-glucuronidase substrate combined with a
ß-glucosidase substrate and, optionally, with the detection of tryptophanase, for detecting
strains of the Escherichia coli species. However, while this medium has excellent
specificity, the use of a ß-glucuronidase substrate for detecting E. coli exhibits imperfect
sensitivity owing to the existence of a small proportion of E. coli strains (5-10%) which
do not express this activity. Furthermore, certain Citrobacter strains can also produce
15-glucuronidase-positive colonies, that are the same color as those of E. coli.
The invention proposes to solve the prior art problems by providing a new medium that is
particularly suitable for identifying E. coli bacteria rapidly and inexpensively and in a
manner that is easy to implement.
Surprisingly, the inventors have shown that a suitable supplementary test enables rapid
and easy detection of E. coli. More specifically, the inventors have in particular shown
that an indole test carried out on the E. coli that do not express the target metabolic
activity makes it possible to increase the sensitivity of the test.
Before proceeding with the disclosure of the invention, the following definitions are
given in order to facilitate the understanding of the invention.
The term biological sample is intended to mean a clinical sample, derived from a sample
of biological fluid, or a food sample, derived from any type of food, or an environmental
sample such as a surface sample, water sample, air sample, etc. This sample may thus be
liquid or solid and mention may be made, in a nonlimiting manner, of a clinical sample
from blood, plasma, urine or feces, samples taken from the nose, from the throat, from
the skin, from wounds or from cerebrospinal fluid, a food sample from water, or from
drinks such as milk or a fruit juice; from yogurt, meat, eggs, vegetables, mayonnaise or
cheese; from fish, etc., or a food sample derived from an animal feed, such as in
particular a sample derived from animal meals.
The term detection medium is intended to mean a medium comprising all the elements
necessary for the survival and/or the growth of microorganisms. This detection medium
can either serve as detection medium only, or as culture and detection medium. In the
first case, the culturing of the microorganisms is carried out before seeding, and in the
second case, the detection medium also constitutes the culture medium. The culture
medium according to the invention may contain other possible additives, for instance:
peptones or extracts of tissues, 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 the form of a ready-to-use gel, i.e. ready for seeding in a tube or flask or
on a Petri dish.
For the purpose of the present invention, the detection can be carried out in liquid
medium, a strip, or another solid support.
The term substrate is intended to mean any molecule capable of directly or indirectly
generating a detectable signal due to an enzymatic or metabolic activity of the
microorganism.
The substrate may in particular be a metabolic substrate, such as a carbon or nitrogen
source, coupled to an indicator that produces a coloration in the presence of one of the
products of the metabolism.
The substrate may also be an enzymatic substrate, i.e. a substrate that can be hydrolyzed by an enzyme so as to give a product that enables direct or indirect detection of a microorganism. This substrate may in particular comprise a first part which is specific for the enzymatic activity to be revealed and a second part which acts as a label, hereinafter known as label part. This label 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 on esculetin and their derivatives, which enable the detection of osidase and esterase activities. Mention may also be made of substrates based on nitrophenol and nitroaniline and derivatives, for detecting osidase and esterase activities in the case of nitrophenol-based substrates, and peptidase activities in the case of nitroaniline-based substrates. Finally, mention may be made of substrates based on naphthol and naphthylamine and their derivatives, which make it possible to detect osidase and esterase activities by means of naphthol, and peptidase activities by means of naphthylamine. This substrate may in particular, but in a nonlimiting manner, enable the detection of an enzymatic activity such as the activity of an osidase, peptidase, esterase, etc. The enzymatic substrate may also be a natural substrate, the product of hydrolysis of which is detected directly or indirectly. As a natural substrate, mention may in particular be made of tryptophan for detecting a tryptophanase or deaminase activity, a cyclic amino acid (tryptophan, phenylalanine, histidine, tyrosine) for detecting a deaminase activity, phosphatidylinositol for detecting a phospholipase activity, etc. According to the present invention, the substrate is preferably selected from substrates based on indoxyl (3-indoxyl, 5-bromo-3-indoxyl, 5-iodo-3-indoxyl, 4-chloro-3-indoxyl, 5-bromo-4-chloro-3-indoxyl, 6-bromo-3-indoxyl, 6-fiuoro-3-indoxyl, 5-bromo-4-chloro-n-methyl-3-indoxyl, n-methy 1-3-indoxyl, etc.); based on umbelliferone (4-methylumbelliferone, cyclohexenoesculetin, etc.); based on alizarine; based on p-naphtholbenzein; based on nitrophenol (ortho-nitrophenol, para-nitrophenol, etc.); based on aminophenol (para-aminophenol, dichloroaminophenol, etc.); on hydroxyquinoline; on catechol (catechol, dihydroxyflavone, hydroxyflavone, etc.); on resorufin; on chlorophenol red; on fluorescein; on naphthol (alpha-naphthol, 2-naphthol, naphthol-ASBI, etc.); on aminocoumarin (7-amino-4-methylcoumarin, etc.); on
naphthylamide; on acridine (aminophenylacridine, etc.); or on aminophenoxazine (aminobenzophenoxazinone, aminopentylresorufin, etc.).
By way of indication, the substrates used for detecting a beta-glucuronidase activity may in particular be 4-methylumbelliferyl-beta-glucuronide, 5-bromo-4-chloro-3-indolyl-beta-glucuronide, 5-bromo-6-chloro-3-indolyl-beta-glucuronide, 6-chloro-3-indolyl-beta-glucuronide, alizarine-beta-glucuronide or cyclohexenoesculetin-beta-glucuronide, or salts thereof.
The substrates used for detecting a beta-galactosidase activity may in particular be
4-methylumbelliferyl-beta-galactoside, 5-bromo-4-chloro-3-indolyl-beta-galactoside,
5-bromo-6-chloro-3-indolyl-beta-galactoside, 6-chloro-3-indolyl-beta-galactoside,
alizarine-beta-galactoside or cyclohexenoesculetin-beta-galactoside, or salts thereof.
The substrates used for detecting a beta-glucosidase activity may in particular be
4-methylumbelliferyl-beta-glucoside, 5-bromo-4-chloro-3-indolyl-beta-glucoside,
5-bromo-6-chloro-3-indolyl-beta-glucoside, 6-chloro-3-indolyl-beta-glucoside, alizarine-beta-glucoside, cyclohexenoesculetin-beta-glucoside, nitrophenyl-beta-glucoside or dichloroaminophenylglucoside, or salts thereof.
As an alpha-galactosidase substrate, mention may be made of 4-methylumbelliferyl-
alpha-galactoside, 5-bromo-4-chloro-3-indolyl-alpha-galactoside, 5-bromo-6-chloro-
3-indolyl-alpha-galactoside, 6-chloro-3-indolyl-alpha-galactoside, alizarine-
alpha-galactoside or nitrophenyl-alpha-galactoside or salts thereof. The expression enzyme A or C, expressed by the majority of E. coli is intended to mean an enzyme which is expressed by more than 80% of the E. coli under given conditions. Those skilled in the art are aware of a large number of activities expressed by the majority of E. coli strains. Some are in particular described in the Manual of Clinical Microbiology, 7th edition (P. R. Murray and al. 1999), and also in the bioMerieux identification products databases (api 20E, ID 32E, rapid ID 32, etc.) By way of an enzyme expressed by the majority of E. coli, mention may in particular be made of beta-glucuronidase, alpha-galactosidase, beta-ribosidase, phosphatase, L-alanine aminopeptidase, L-leucine aminopeptidase, beta-galactosidase, lactose acidification enzyme, or tryptophanase.
enzyme which is expressed by less than 20% of the E. coli under given conditions. These activities are also known to those skilled in the art and some of them are described in the documents mentioned above. Mention may in particular be made of beta-glucosidase, N-acetylhexosaminidase, esterase, sulfatase, beta-cellobiosidase, alpha-glucosidase, deaminase, oxydase, pigment synthesis enzyme, beta-alanine aminopeptidase, or elastase.
The term indole test is intended to mean a test which makes it possible to detect the production of indole by microorganisms. In general, the indole produced in a reaction medium is detected by means of a reagent, such as Kovac's reagent, dimethylamino-cinnamaldehyde (DMAC A) or James' reagent. In the presence of indole, a red coloration is obtained with Kovac's reagent and James' reagent and a blue coloration is obtained with DMACA.
The term inducer is intended to mean a compound which induces an increase in the expression of the targeted metabolic activity; all experimental conditions being otherwise equal, the metabolic activity is greater when the inducer is at an appropriate concentration than when it is absent or at an unsuitable concentration. Mention may in particular be made:
• for beta-glucuronidase, of a glucuronide preferably selected from glucuronate and methyl-beta-glucuronide;
• for beta-galactosidase, of a beta-galactoside preferably selected from lactose and isopropyl-beta-thiogalactoside;
• for beta-glucosidase, of a carbohydrate constituted of a carbohydrate linked in the ß-position to the glucose, or a carbohydrate with a ß-glucoside subunit, in particular cellobiose, cellulose, starch, cellotriose or trehalose. Mention may also be made of methyl-ß-glucoside, isopropyl-ß-thioglucoside, indoxyl-ß-glucoside or methyl-ß-thioglucoside;
• for alpha-galactosidase, of an alpha-galactoside preferably selected from melibiose and methyl-alpha-galactoside.
In this respect, the invention relates to a method for detecting and/or identifying Escherichia coli (E. coli) in a biological sample, that comprises:
a) inoculating the biological sample liable to contain E. coli on a detection medium that comprises tryptophan and a substrate for an enzyme A, expressed by the majority of E. coli, in order to obtain bacterial colonies;
b) detecting the colonies expressing the activity of the enzyme A and identifying them as being E. coli; and
c) detecting the colonies that do not express the activity of the enzyme A, carrying out an indole test, and identifying the colonies having a positive indole test as being E. coli.
The inoculation of the microorganisms can be carried out by any of the inoculation
techniques known to those skilled in the art. An incubation step may be carried out at a
temperature for which the enzymatic activity that it is desired to detect is optimal, it being
possible for those skilled in the art to readily select this temperature according to the
enzymatic activity to be detected. The detection/identification can be carried out by
means of a visual examination, by colorimetry or by fluorimetry.
According to one preferred embodiment of the invention, the enzyme A is selected from
beta-glucuronidase, alpha-galactosidase, beta-ribosidase, phosphatase, L-alanine
aminopeptidase, L-leucine aminopeptidase and beta-galactosidase.
According to one preferred embodiment of the invention, the enzyme A is
beta-glucuronidase. Preferably, the substrate for beta-glucuronidase activity is selected
from 4-methylumbelliferyl-beta-glucuronide, 5-bromo-4-chloro-3-indolyl-
beta-glucuronide, 5-bromo-6-chloro-3-indolyl-beta-glucuronide, 6-chloro-3-indolyl-
beta-glucuronide, alizarine-beta-glucuronide or cyclohexenoesculetin-beta-glucuronide,
or salts thereof, at concentrations of preferably between 20 and 1000 mg/1.
According to another preferred embodiment of the invention, the enzyme A is
beta-galactosidase. Preferably, the substrate for beta-galactosidase activity is selected
from 4-methylumbelliferyl-beta-galactoside, 5-bromo-4-chloro-3-indolyl-
beta-galactoside, 5-bromo-6-chloro-3-indolyl-beta-galactoside, 6-chloro-3-indolyl-
beta-galactoside, alizarine-beta-galactoside or cyclohexenoesculetin-beta-galactoside, or
salts thereof, at a concentration of preferably between 10 and 1000 mg/1, preferably
between 20 and 500 mg/1.
Preferably, the substrate for said enzyme A is at a concentration of between 10 and
1000 mg/1, preferably between 20 and 500 mg/1.
According to one preferred embodiment of the invention, the tryptophan concentration is
greater than or equal to 0.02 g/1, preferably greater than or equal to 0.4 g/1.
According to one preferred embodiment of the invention, the detection medium also
comprises a substrate for an enzyme B, not expressed by the majority of E. coli.
According to one preferred embodiment of the invention, the enzyme B is selected from
beta-glucosidase, N-acetylhexosaminidase, esterase, sulfatase, beta-cellobiosidase, alpha-
glucosidase, deaminase, oxydase, pigment synthesis enzyme, beta-alanine
aminopeptidase and elastase.
Preferably, the enzyme B is beta-glucosidase.
Preferably, the substrate for beta-glucosidase activity is selected from
4-methylumbelliferyl-beta-glucoside, 5 -bromo-4-chloro-3 -indolyl-beta-glucoside,
5-bromo-6-chloro-3-indolyl-beta-glucoside, 6-chloro-3-indolyl-beta-glucoside, alizarine-
beta-glucoside, cyclohexenoesculetin-beta-glucoside, nitrophenyl-beta-glucoside or
dichloroaminophenylglucoside, or salts thereof, at a concentration of preferably between
10 and 1000mg/1, preferably between 20 and 500 mg/1.
Preferably, the substrate for said enzyme B is at a concentration of between 10 and
1000 mg/1, preferably between 20 and 500 mg/1.
According to one preferred embodiment of the invention, the detection medium also
comprises a substrate for an enzyme C, expressed by the majority of E. coli.
Two embodiments are then possible:
• either the enzyme C is identical to the enzyme A; in this case, the substrate should be different. As enzymes A and C, mention may, for example, be made of beta-galactosidase; in this case, the medium comprises, for example, 4-methylumbelliferyl-beta-glucuronide as substrate for the enzyme A, and 5-bromo-6-chloro-3-indolyl-beta-glucuronide as substrate for the enzyme C;
• or the enzyme C is different than the enzyme A; mention may for example be made of beta-galactosidase as enzyme A, in combination with alpha-galactosidase as enzyme C. Mention may also be made of beta-galactosidase as enzyme A, in combination with beta-glucuronidase as enzyme C.
Preferably, the enzyme C is selected from beta-glucuronidase, alpha-galactosidase, beta-
ribosidase, phosphatase, L-alanine aminopeptidase, L-leucine aminopeptidase, beta-
galactosidase.
Preferably, the enzyme C is beta-glucuronidase. Preferably, the substrate for
beta-glucuronidase activity is selected from 4-methylumbelliferyl-beta-glucuronide,
5-bromo-4-chloro-3-indolyl-beta-glucuronide, 5-bromo-6-chloro-3-indolyl-
beta-glucuronide, 6-chloro-3-indolyl-beta-glucuronide, alizarine-beta-glucuronide or
cyclohexenoesculetin-beta-glucuronide, or salts thereof, at concentrations of preferably
between 10 and 1000 mg/1.
Preferably, the enzyme C is alpha-galactosidase. Preferably, the substrate for alpha-
galactosidase activity is selected from 4-methylumbelliferyl-alpha-galactoside, 5-bromo-
4-chloro-3-indolyl-alpha-galactoside, 5-bromo-6-chloro-3-indolyl-alpha-galactoside,
6-chloro-3-indolyl-alpha-galactoside, alizarine-alpha-galactoside or nitrophenyl-alpha-
galactoside at concentrations of between 10 and 1000 mg/1/, preferably between 20 and
500 mg/1.
Preferably, the substrate for said enzyme C is at a concentration of between 10 and 1000
mg/1, preferably between 20 and 500 mg/1.
According to one preferred embodiment of the invention, the enzyme A is beta-
galactosidase; the enzyme B is beta-glucosidase and the enzyme C is alpha-galactosidase.
In this particular embodiment of the invention,
© the substrate for the enzyme A is preferably 5-bromo-4-chloro-3-indolyl-beta-galactoside, 5-bromo-6-chloro-3-indolyl-beta-galactoside, 6-chloro-3-indolyl-beta-galactoside or alizarine-beta-galactoside, at a concentration of between 10 and 1000 mg/1,
• the substrate for the enzyme B is preferably 5-bromo-4-chloro-3-indolyl-beta-glucoside, 5-bromo-6-chloro-3-indolyl-beta-glucoside, 6-chloro-3-indolyl-beta-glucoside or alizarine-beta-glucoside, at a concentration of between 10 and 1000 mg/1,
• the substrate for the enzyme C is preferably 5-bromo-4-chloro-3-indolyl-alpha-galactoside, 5-bromo-6-chloro-3-indolyl-alpha-galactoside, 6-chloro-3-indolyl-alpha-galactoside or alizarine-alpha-galactoside, at a concentration of between 10 and 1000 mg/1.
According to another preferred embodiment of the invention, the enzyme A is beta-galactosidase; the enzyme B is beta-glucosidase and the enzyme C is beta-glucuronidase. In this particular embodiment of the invention,
• the substrate for the enzyme A is preferably 5-bromo-4-chloro-3-indolyl-beta-galactoside, 5 -bromo-6-chloro-3 -indolyl-beta-galactoside, 6-chloro-3-indolyl-beta-galactoside or alizarine-beta-galactoside, at a concentration of between 10 and 1000 mg/1,
• the substrate for the enzyme B is preferably 5-bromo-4-chloro-3-indolyl-beta-glucoside, 5-bromo-6-chloro-3-indolyl-beta-glucoside, 6-chloro-3-indolyl-beta-glucoside or alizarine-beta-glucoside, at a concentration of between 10 and 1000 mg/1,
• the substrate for the enzyme C is preferably 5-bromo-4-chloro-3-indolyl-beta-glucuronide, 5-bromo-6-chloro-3-indolyl-beta-glucuronide, 6-chloro-3-indolyl-beta-glucuronide or alizarine-beta-glucuronide, at a concentration of between 10 and 1000 mg/1.
According to one particular embodiment of the invention, the detection medium also
comprises an inducer of the enzyme A, an inducer of the enzyme B and/or an inducer of
the enzyme C.
According to one preferred embodiment of the invention, the inducer of the enzyme A, B
or C is at a concentration of between 100 ng/1 and 10 g/1, preferably between 10 mg/1 and
3 g/1.
When the enzyme A or C is beta-glucuronidase, the inducer of said enzyme A or C is
preferably a glucuronide, preferably selected from glucuronate and methyl-
beta-glucuronide.
When the enzyme A or C is beta-galactosidase, the inducer of said enzyme A or C is
preferably a beta-galactoside, preferably selected from lactose and isopropyl-
beta-thiogalactoside.
When the enzyme B is beta-glucosidase, the inducer of the enzyme B is preferably a beta-
glucoside, preferably selected from methyl-beta-glucose, cellobiose, cellotriose,
trehalose, cellulose and starch. Preferably, the inducer of the enzyme B is cellobiose, at a
concentration of preferably between 10 mg/1 and 10 g/1.
When the enzyme C is alpha-galactosidase, the inducer of the enzyme C is preferably melibiose or methyl-alpha-galactoside.
The invention also relates to a detection medium comprising tryptophan, a substrate for a beta-galactosidase enzyme, a substrate for a beta-glucosidase enzyme, and cellobiose. According to one preferred embodiment of the invention, the tryptophan concentration is greater than or equal to 0.02 g/1, preferably greater than or equal to 0.4 g/1. Preferably, the substrate for beta-glucosidase activity is selected from 4-methyl-umbelliferyl-beta-glucoside, 5-bromo-4-chloro-3-indolyl-beta-glucoside, 5-bromo-6-chloro-3-indolyl-beta-glucoside, 6-chloro-3-indolyl-beta-glucoside, alizarine-beta-glucoside, cyclohexenoesculetin-beta-glucoside, nitrophenyl-beta-glucoside or dichloroaminophenylglucoside, or salts thereof, at a concentration of preferably between 10 and 1000 mg/1, preferably between 20 and 500 mg/1.
Preferably, the substrate for beta-galactosidase activity is selected from
4-methylumbelliferyl-beta-galactoside, 5-bromo-4-chloro-3-indolyl-beta-galactoside,
5-bromo-6-chloro-3-indolyl-beta-galactoside, 6-chloro-3-indolyl-beta-galactoside,
alizarine-beta-galactoside or cyclohexenoesculetin-beta-galactoside, or salts thereof, at a concentration of preferably between 10 and 1000 mg/1, preferably of between 20 and 500 mg/1. Preferably, the cellobiose is at a concentration of between 10 mg/1 and 10 g/1.
According to one preferred embodiment of the invention, the detection medium also comprises a substrate for an alpha-galactosidase enzyme.
As a substrate for alpha-galactosidase, mention may in particular be made of
4-methylumbelliferyl-alpha-galactoside, 5-bromo-4-chloro-3-indolyl-alpha-galactoside,
5-bromo-6-chloro-3-indolyl-alpha-galactoside, 6-chloro-3-indolyl-alpha-galactoside,
alizarine-alpha-galactoside or nitrophenyl-alpha-galactoside, at concentrations of between 20 and 1000 mg/1/.
The invention also relates to the use of a medium as defined above, for detecting E. coli.
The examples below are given by way of explanation and are in no way limiting in nature. They will make it possible to understand the invention more clearly.
Example 1: Contribution of the indole test on colorless colonies for detecting Escherichia coli
Three thousand (3000) urine samples were inoculated on the commercially available CPS ID 3 medium (bioMerieux). The media were incubated and analyzed in accordance with the supplier's recommendations: for detecting and identifying Escherichia coli strains, the pink-to-red colonies were pre-identified as belonging to the E. coli species. An indole test was carried out in order to confirm this identification. In parallel, the media were used and analyzed in accordance with the present invention: the pink-to-red colonies were identified as belonging to the E. coli species; an indole test was carried out on the colorless colonies; if the reaction was positive, the strain was identified as E. coli. All the identifications were confirmed using suitable biochemical tests commonly used by medical test laboratories.

(Table Removed)
Table 1: Contribution of the indole test on colorless colonies for identifying E. coli
strains on CPS ID 3 medium
It is very clear from Table 1 that, when the indole test is carried out only on the pink-to-red colonies on CPS ID 3 medium, as proposed by the supplier, this results in a decrease in the sensitivity of identification of the E. coli strains. On the other hand, if this test is carried out on the colorless colonies, this makes it possible to obtain a very high identification sensitivity.
Example 2: Contribution of the indole test on a medium combining cellobiose, 6-chloro-3-indolyl-ß-glucuronide, 5-bromo-6-chloro-3-indolyl-ß-galactoside and 5-bromo-

4-chloro-3-indolyl-ß-glucoside
Tryptophan, 6-chloro-3-indolyl-ß-glucuronide, 5-bromo-6-chloro-3-indolyl-ß-galactoside and 5-bromo-4-chloro-3-indolyl-P-glucoside are added, 0.9 g/1, 0.15 g/1, 0.05 g/1 and 0.1 g/1 respectively, to Trypticase Soy Agar medium (bioMerieux). This medium is supplemented, or not supplemented, with cellobiose at 0.5 g/1. These two media are distributed in a proportion of 20 ml per Petri dish. Microorganisms commonly isolated from urine samples and derived from the applicant's collection are inoculated on these media by semi-quantitative isolation of 10 µl of a suspension at 0.5 McFarland, diluted to 1/20. The dishes are incubated at 37°C for 24 hours, and then the colonies formed are examined visually. The coloration of these colonies is noted. An indole test is carried out on the colorless colonies using James' reagent (bioMerieux). The results are given in Table 2 below:

(Table Removed)
Table 2: Contribution of the indole test on a medium combining 6-chloro-3-indolyl-
ß-glucuronide, 5-bromo-6-chloro-3-indolyl-ß-galactoside and 5-bromo-4-chloro-3-indolyl-ß-glucoside, possibly supplemented with cellobiose, on the identification of
E. coli
It is clear from Table 2 above that, in a medium combining tryptophan, 6-chloro-3-indolyl-ß-glucuronide, 5-bromo-6-chloro-3-indolyl-ß-galactoside and 5-bromo-4-chloro-3-indolyl-ß-glucoside, cellobiose makes it possible to differentiate more clearly between the Citrobacter 009 strain and the E. coli strains. Moreover, in the presence of tryptophan, searching for the production of indole by the colorless colonies makes it
possible to further increase the sensitivity for detecting E. coli without being penalized by damage to the specificity.
Example 3: Impact of the tryptophan concentration on the detection of the colorless colonies of E. coli
Various concentrations of tryptophan (0-0.3-0.6-0.9 g/1) and also cellobiose at 100 mg/1 are added to the CPS ID 3 medium (bioMerieux). These media contain 6-chloro-3-indolyl-beta-glucuronide at 250 mg/1 and 5-bromo-4-chloro-3-indolyl-beta-glucoside at 50 mg/1. These media are distributed in a proportion of 20 ml per Petri dish. Microorganisms derived from the applicant's collection were inoculated on these media by semi-quantitative isolation of 10 µl of a suspension at 0.5 McFarland, diluted to 1/20. The dishes were incubated at 37°C for 24 hours. The colonies formed were examined visually after 24 hours of incubation. The coloration of these colonies is noted. An indole test is carried out on the colorless colonies using James' reagent (bioMerieux). The results are given in Table 3 below:

(Table Removed)
Table 3: Impact of tryptophan concentration on the detection of colorless colonies of
E. coli
It is clear from Table 3 above that, even with a tryptophan concentration of 0.3 g/1, it is possible to detect the production of indole by the E. coli strains; nevertheless, the reaction is more marked at higher concentrations.
Example 4: Contribution of the indole test on a medium combining cellobiose, 5-bromo-6-chIoro-3-indolyl-alpha-galactdside, 5-bromo-6-chloro-3-indolyl-ß-galactoside and 5-bromo-4-chloro-3-indolyl-(3-glucoside
5-Bromo-6-chloro-3-indolyl-alpha-galactoside, 5-bromo-6-chloro-3-indolyl-
ß-galactoside, 5-bromo-4-chloro-3-indolyl-ß-glucoside and isopropyl-thio-ß-galactoside are added, at 75 mg/1, 50 mg/1, 50 mg/1 and 10 mg/1 respectively, to CPS ID 3 medium (bioMerieux) from which the synthetic enzymatic substrates have been removed. This medium is distributed in a proportion of 20 ml per Petri dish. Microorganisms derived from the applicant's collection were inoculated on this medium by semi-quantitative isolation of 10 µl of a suspension at 0.5 McFarland, diluted to 1/20. The dishes were incubated at 37°C for 24 hours. The colonies formed were examined visually after 24 hours of incubation. The coloration of these colonies is noted. An indole test is carried out on the colorless colonies using James' reagent (bioMerieux). The results are given in Table 4 below:
(Table Removed)
Table 4: Impact of tryptophan concentration on the detection of colorless colonies of
E. coli
It is clear from Table 4 above that, in a medium combining 5-bromo-6-chloro-3-indolyl-alpha-galactoside, 5-bromo-6-chloro-3-indolyl-ß-galactoside and 5-bromo-4-chloro-3-indolyl-ß-glucoside, the production of indole by the colorless colonies makes it possible to increase the sensitivity for detecting E. coli.

CLAIMS
1) A medium for detecting and/or identifying Escherichia coli (E. coli) in a biological
sample, wherein the said medium comprises:
tryptophan and a substrate for an enzyme A, expressed by the majority of E. coli,;
and wherein the said medium is . inoculated with the biological sample liable to
contain E. coli in order to obtain bacterial colonies;
colonies expressing the activity of the enzyme A are detected and are identified as
being E. coli; and
colonies that do not express the activity of the enzyme A are detected by carrying
out an indole test, and the colonies having a positive indole test are identified as
as being E. coli.
2) The medium as claimed in claim 1, wherein the tryptophan concentration is greater than or equal to 0.02 g/1, preferably greater than or equal to 0.4 g/1.
3) The medium as claimed in either one of claims 1 and 2, wherein the said detection medium also comprises a substrate for an enzyme B, not expressed by the majority of E. coli.
4) The medium as claimed in any one of claims 1 to 3, wherein the said detection medium also comprises a substrate for an enzyme C, expressed by the majority of E. coli.
5) The medium as claimed in any one of claims 1 to 4, wherein the enzyme A is selected from beta-glucuronidase, alpha-galactosidase, beta-ribosidase, phosphatase, L-alanine aminopeptidase, L-leucine aminopeptidase and beta-galactosidase.
6) The medium as claimed in any one of claims 1 to 5, wherein the enzyme A is beta-glucuronidase or beta-galactosidase.
7) The medium as claimed in any one of claims 3 to 6, wherein the enzyme B is selected
from beta-glucosidase, N-acetylhexosaminidase, esterase, sulfatase, beta-xylosidase, phospholipase, alpha-mannosidase, beta-mannosidase, beta-cellobiosidase, alpha-glucosidase, deaminase, oxydase, pigment synthesis enzyme, beta-alanine aminopeptidase and elastase.
8) The medium as claimed in claim 7, wherein the enzyme B is beta-glucosidase.
9) The medium as claimed in any one of claims 4 to 8, wherein the enzyme C is selected from beta-glucuronidase, alpha-galactosidase, beta-ribosidase, phosphatase, L-alanine aminopeptidase, L-leucine aminopeptidase and beta-galactosidase.

10) The medium as claimed in claim 4, wherein the enzyme A is beta-galactosidase; the enzyme B is beta-glucosidase and the enzyme C is alpha-galactosidase.
11) The medium as claimed in claim 4, wherein the enzyme A is beta-galactosidase; the enzyme B is beta-glucosidase and the enzyme C is beta-glucuronidase.
12) The medium as claimed in any one of claims 1 to 11, wherein the detection medium also comprises an inducer of the enzyme A, an inducer of the enzyme B and/or an inducer of the enzyme C.
13) The medium as claimed in claim 12, wherein the enzyme is beta-glucuronidase and the inducer of said enzyme A is a glucuronide, preferably selected from glucuronate and methyl-beta-glucuronide.
14) The medium as claimed in claim 12, wherein the enzyme A is beta-galactosidase and the inducer of said enzyme A is a beta-galactoside, preferably selected from lactose and isopropyl-beta-thiogalactoside.
15) The medium as claimed in any one of claims 12 to 14, wherein the enzyme B is beta-glucosidase and the inducer of the enzyme B is a beta-glucoside, preferably selected from
methyl-beta-glucose, cellobiose, cellotriose, trehalose, cellulose and starch.
16) The medium as claimed in claim 15, wherein the inducer of the enzyme B is cellobiose.
17) A detection medium comprising tryptophan, a substrate for a beta-galactosidase enzyme, a substrate for a beta-glucosidase enzyme, and cellobiose.
18) The detection medium as claimed in claim 17, also comprising a substrate for an alpha-galactosidase enzyme.
19) The medium as defined in claim 17 or 18, for use in detecting E. coli.