Bacteria detection and/or identification medium
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 (bioMerieux) medium 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
ß-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 particular
combination of enzymatic substrates, at suitable concentrations, enables rapid and easy
detection of E. coli, in particular using a urine sample.
Before proceeding with the disclosure of the invention, the following definitions are
given in order to facilitate the understanding of the invention.
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 inoculation, 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, 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, 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 a 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, 5-bromo-6-chloro-3-indoxyl, 6-bromo-3-indoxyl, 6-chloro-
3-indoxyl, 6-fluoro-3-indoxyl, 5-bromo-4-chloro-N-methyl-3-indoxyl, N-methyl-
3-indoxyl, etc.); on umbelliferone (4-methylumbelliferone, cyclohexenoesculetin, etc.);
on alizarine; on p-naphtholbenzein; on nitrophenol (ortho-nitrophenol, para-nitrophenol,
etc.); on hydroxyquinoline; on cathechol (cathecol, dihydroxyflavone, hydroxyflavone,
etc.); on resorufin; on Chlorophenol Red; on fluorescein; on aminophenol (paraaminophenol,
dichloroaminophnol, etc.); 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-betaglucuronide,
5-bromo-6-chloro-3-indolyl-beta-glucuronide, 6-chloro-3-indolyl-betaglucuronide,
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, alizarinebeta-
glucoside, cyclohexenoesculetin-beta-glucoside, nitrophenyl-beta-glucoside or
dichloroaminophenylglucoside. or salts thereof.
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 appropriate concentration
than when it is absent or at an unsuitable concentration.
Without being limiting, a concentration of between l00ng/1 and l0g/1, preferably
between 10 mg/1 and 3 g/1, is particularly suitable for the present invention.
Mention may in particular be made of:
• for beta-glucuronidase, a glucuronide preferably selected from glucuronate and
methyl-beta-glucuronide;
• for beta-galactosidase, a galactoside preferably selected from lactose and
isopropyl-beta-thiogalactoside;
• for beta-glucosidase, a carbohydrate constituted of a carbohydrate linked in the
ß-position to 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-6-glucoside or methylß-
thioglucoside.
The term inhibitor is intended to mean a compound which induces a decrease in the
expression of the targeted metabolic activity; all experimental conditions being otherwise
equal, the metabolic activity is weaker when the inducer is at an appropriate
concentration than when it is absent or at an unsuitable concentration.
Without being limiting, a concentration of between 100 ng/1 and 30 g/1, preferably
between 1 mg/1 and 3 g/1, is particularly suitable for the present invention.
Mention may in particular be made of
• for beta-glucuronidase: D-glucose, D-glucaric acid 1,4-lactone
• for beta-galactosidase: 2-deoxygalactose, cellobiose, D-galactose, D-glucose.
The term biological sample is intended to mean 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 yoghurt, 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.
In this respect, the invention relates to a method for detecting and/or identifying E. coli in
a biological sample, preferably a urine sample, that comprises;
a) inoculating the sample, preferably urine sample, liable to contain E. coli on a
detection medium that comprises
• a first substrate selected from a beta-glucuronidase substrate, betagalactosidase
substrate and alpha-galactosidase substrate, and a substrate
for a lactose acidification enzyme, beta-ribosidase, phosphatase, L-alanine
aminopeptidase and L-leucine aminopeptidase, and
• a second substrate, different from said first substrate and selected from a
beta-glucuronidase substrate, beta-galactosidase substrate and alphagalactosidase
substrate, and a substrate for a lactose acidification enzyme,
beta-ribosidase, phosphatase, L-alanine aminopeptidase and L-leucine
aminopeptidase,
for obtaining bacterial colonies;
b) identifying the colonies that react with the first substrate and/or the second
substrate as being colonies of E. coli.
Preferably, said first and second substrates are at a suitable concentration. 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 said 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 fluorimetry.
According to one preferred embodiment of the invention, said first substrate is at a
concentration of between 20 and 1000 mg/1 and said second substrate is at a
concentration of between 20 mg/ and 30 g/1.
According to one preferred embodiment of the invention, said first substrate is a
beta-glucuronidase substrate and the second substrate is a beta-galactosidase substrate.
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.
Preferably, the beta-galactosidase substrate is at a low concentration. Preferably, the
substrate for beta-galactosidase activity is selected from 4-methylumbelliferyl-betagalactoside,
5-bromo-4-chloro-3-indolyl-beta-galactoside, 5-bromo-6-chloro-3-indolylbeta-
galactoside, 6-chloro-3-indolyl-beta-galactoside, alizarine-beta-galactoside or
cyclohexenoesculetin-beta-galactoside, or salts thereof, at a concentration preferably 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 third substrate, preferably selected from a substrate for beta-glucosidase,
beta-lactosidase, N-acetylhexosaminidase, esterase, sulfatase, beta-xylosidase,
phospholipase, alpha-mannosidase, beta-mannosidase, beta-cellobiosidase, alphaglucosidase,
tryptophanase, deaminase, oxydase, pigment synthesis, peptidases (betaalanine
aminopeptidase, elastase, etc.).
Preferably, said third substrate is a substrate for 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-indolylbeta-
glucoside, cyclohexenoesculetin-beta-glucoside, nitrophenyl-beta-glucoside or
dichloroaminophenylglucoside, or salts thereof, at a concentration preferably 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 an inducer.
Preferably, the inducer is at a concentration of between 100 ng/1 and 10 g/1.
According to one preferred embodiment of the invention, the inducer is preferably:
• for beta-glucuronidase, a glucuronide preferably selected from glucuronate and
methyl-beta-glucuronide;
• for beta-galactosidase, a galactoside preferably selected from lactose and
isopropyl-beta-thiogalactoside;
• for beta-glucosidase, a carbohydrate constituted of a carbohydrate linked in the
ß-position to 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 methylß-
thioglucoside.
Preferably, the inducer is cellobiose, at a concentration preferably of between 100ng/l
and 10 g/1.
According to one preferred embodiment of the invention, the detection medium also
comprises an inhibitor. Preferably, the inhibitor is at a concentration of between 100 ng
and 30 g/1.
According to one preferred embodiment of the invention, the inhibitor is preferably:
• for beta-glucuronidase: D-glucose, D-glucaric acid 1,4-lactone
• for beta-galactosidase: 2-deoxygalactose, cellobiose, D-galactose, D-glucose.
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: Evaluation of the combination of 6-chloro-3-indolyl-ß-glucuronide and
5-bromo-6-chloro-3-indolyl-ß-galactoside
Various concentrations of 6-chloro-3-indolyl-ß-glucuronide (0-0.1-0.15 and 0.20 g/1) and
of 5-bromo-6-chloro-3-indolyl-ß-galactoside (0-0.025-0.05 and 0.1 g/1) are added to and
combined with the CPS ID 3 medium (bioMerieux) from which the synthetic enzymatic
substrate for ß-glucuronidase has been removed. These media also comprise 5-bromo-
4-chloro-3-indolyl-ß-glucoside at 50mg/l. They are distributed in a proportion of 20 ml
per Petri dish. The Coli ID medium (bioMerieux) which combines a ß-glucuronidase
substrate (6-chloro-3-indolyl-ß-glucuronide) and a 13-galactosidase substrate (5-bromo-
3-indolyl-ß-galactoside), intended for the detection and counting of E. coli and coliforms
in food samples, is tested in parallel. Microorganisms commonly isolated from urine
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 20 hours, and then the colonies formed are examined
Inh
Table 1: Impact of the combination of 6-chloro-3-indolyl-ß-glucuronide and 5-bomo-6-chloro-
3-indolyl-ß-galactoside in the CPS ID 3 medium on colony coloration
NA = not applicable, - = colorless, Inh = inhibited, P = pink, Pp = pale pink, GP = grayish-pink,
GG = grayish-green, GB = grayish-blue, BG = bluish-green, Vi = violet, O = orangey-brown,
T = turquoise
In table 1, it emerges that only the media 2c, 3b, 3c, 4b and 4c combining 6-chloro-
3-indolyl-6-glucuronide with 5-bromo-6-chloro-3-indolyl-ß-galactoside make it possible
to detect all the E. coli strains. This is not the case of the Coli ID medium, which
nevertheless combines a 6-glucuronidase substrate and a 6-galactosidase substrate.
However, on the media 2c, 3c and 4c, the E. cloacae strain is less readily distinguished
from the E. coli strains. Similarly, the C. freundii strain produces colonies of the same
color as the E. coli strains on all the media having at least 0.05g/l of 5-bromo-6-chloro-
3 -indolyl-ß-galactoside.
Thus, it is possible to determine the media that are the most advantageous for
improving the sensitivity of detection of E. coli strains without being too damaging to the
specificity.
Example 2: Impact of cellobiose on a medium combining 6-chloro-3-indolvlß-
glucuronide. 5-bromo-6-chloro-3-indolyl-ß-galactoside and 5-bromo-4-chloro-
3-indolyl-ß-glucoside
6-Chloro-3-indolyl-ß-glucuronide, 5-bromo-6-chloro-3-indolyl-ß-galactoside and
5-bromo-4-chloro-3-indolyl-ß-glucoside are added, at 0.15 g/1, 0.08 g/1 and 0.08 g/1
respectively, to Trypticase Soya Agar Medium (bioMerieux). This medium is
supplemented, or not supplemented, with cellobiose at 0.5g/l. 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 onto 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. The results are given in
table 2 below:
Strains
Escherichia coli 407
Escherichia coli 067
Klebsiella pneumoniae 111
Serratia marcescens 112
Citrobacterfreundii 031
Citrobacterfreundii 009
Streptococcus agalactiae 019
Enterococcus faecalis 117
Concentration of cellobiose in mg/1
0
pink
pink
turquoise
turquoise
gray
pink
mauve
turquoise
500
pink
pink
turquoise
turquoise
gray
violet
mauve
turquoise
Table 2: Impact of cellobiose on a medium combining 6-chloro-3-indolyl-ß-glucuronide,
5-bromo-6-chloro-3-indolyl-6-galactoside and 5-bromo-4-chloro-3-indolyl-8-glucoside with
respect to colony coloration
In table 2 above, it emerges that, in a medium combining 6-chloro-3-indolylß-
glucuronide and 5-bromo-6-chloro-3-indolyl-ß-galactoside, cellobiose makes it
possible to distinguish the Citrobacter 009 strain more clearly from the E. coli strains.
This makes it possible to benefit from the gain in sensitivity for the detection of E. coli
without being penalized by damage to the specificity.
Example 3 - Test for defining the concentration of said first and second substrates
according to the invention
The test below can be carried out in order to define the concentration of said first and
second substrates according to the invention, which is variable depending on the
substrates used and, more generally, on the formulation of the reaction medium. In order
to aid the understanding of this test, it is carried out below in the case of a combination of
ß-glucuronidase and ß-galactosidase, using a kit of microorganism strains, comprising in
particular E. coli strains, including strains which do not express a positive activity or
which express a positive activity weakly or late, and optionally other microorganisms.
This test can be carried out for other types of substrates. Two reaction media comprising
either a suitable concentration of ß-glucuronidase substrate or no ß-glucuronidase
substrate are used to prepare a ß-galactosidase substrate range including a zero
concentration, at least one concentration for obtaining a positive reaction with the E. coli
strains expressing a ß-galactosidase, and also intermediate concentrations. Each of the
media is aliquoted in such a way that each microorganism strain can be inoculated in
culture, pure, on each of the media. After a suitable incubation time, generally between
30 minutes and 72 hours, at an appropriate temperature of preferably between 20 and
50°C, the media are examined so as to select the medium comprising a combination of
substrates for ß-galactosidase and for ß-glucuronidase that makes it possible to reveal the
greatest number of E. coli strains while at the same time distinguishing them from the
greatest number of strains of the other microorganisms. It may be necessary to repeat the
experiment with the concentrations of each of the substrates and also the strain kit being
adjusted. It may be advantageous for the reaction media to also comprise inducers and/or
inhibitors of B-galactosidase and/or ß-glucuronidase.

CLAIMS
1) A medium for detecting and/or identifying E. coli in a urine sample, that comprises:
• a first substrate selected from a beta-glucuronidase substrate, betagalactosidase
substrate and alpha-galactosidase substrate, and a substrate
for a lactose acidification enzyme, beta-ribosidase, phosphatase, L-alanine
aminopeptidase and L-leucine aminopeptidase, and
• a second substrate, different from said first substrate and selected from a
beta-glucuronidase substrate, beta-galactosidase substrate and alphagalactosidase
substrate, and a substrate for a lactose acidification enzyme,
beta-ribosidase, phosphatase, L-alanine aminopeptidase and L-leucine
aminopeptidase,
for obtaining bacterial colonies;
2) The medium as claimed in claim 1, wherein the said first substrate is a beta-glucuronidase
substrate and the second substrate is a beta-galactosidase susbtrate.
3) The medium as claimed in claim 2, wherein the said first substrate is at a concentration of
between 20 and 1000 mg/1 and the second substrate is at a concentration of between
10mg/and30g/l
4) The medium as claimed in any one of claims 1 to 3, wherein the said detection
medium also comprises a third substrate selected from a substrate for beta-glucosidase,
beta-lactosidase, N-acetylhexosaminidase, esterase, sulfatase, beta-xylosidase,
phospholipase, alpha-mannosidase, beta-mannosidase, beta-cellobiosidase, alphaglucosidase,
tryptophanase, deaminase, oxydase, pigment synthesis, peptidases (betaalanine
aminopeptidase, elastase, etc.).
5) The medium as claimed in claim 4, wherein the said third substrate is a beta
glucosidase substrate.
6) The medium as claimed in any one of claims 1 to 5, wherein the said detection medium also
comprises an inducer, preferably cellobiose.
7) The medium as claimed in any one of claims 1 to 6, wherein the said detection
medium also comprises an inhibitor.