1. A reaction medium comprising (i) an esterase substrate that Streptococcus agalactiae are incapable of using at less than 18 h after inoculation, (ii) one enzymatic substrate chosen from (3-cellobiosidase substrates, N-acetylglucosaminidase substrates and P-glucosidase substrates, and (iii) a phosphatase substrate.

2. The reaction medium as claimed in claim 1, characterized in that the substrate (ii) a P-cellobiosidase substrate.

3. The reaction medium as claimed in claim 1, characterized in that the substrate (ii) a N-acetyl-glucosaminidase substrate.

4. The reaction medium as claimed in claim 1, characterized in that the substrate (ii) a P-glucosidase substrate.

5. The reaction medium as claimed in any one of claims 1 to 4, characterized in that the esterase substrate is chosen from octanoate, nonanoate and decanoate derivatives.

6. The reaction medium as claimed in claim 5, characterized in that the esterase substrate is an indoxyl substrate.

7. The reaction medium as claimed in any one of claims 1 to 6, characterized in that the concentration in each enzymatic substrate is comprised between 10 and 2000 mg/1.

8. The reaction medium as claimed in any one of claims 1 to 7, characterized in that is it also comprises phosphate solutions. - 9T The reaction medium as claimed in claim 8, characterized in that the phosphate solutions are chosen from Na2HPC>4 and K2HPO4.

10. The reaction medium as claimed in any one of claims 1 to 9, characterized in that is it also comprises a mixture of inhibitors for inhibiting or limiting the growth of unwanted strains.

11. The reaction medium as claimed in claim 10, characterized in that the inhibitors are antibiotics. i

12. The reaction medium as claimed in claim 11, characterized in that the antibiotics are aztreonam and amphotericine B.

Method for detecting Streptococcus agalactiae using esterase activity
The present invention relates to the field of the detection and identification of
Streptococcus agalactiae. More specifically, the invention relates to the use of esterase
5 substrates, optionally in combination with at least one a-glucosidase substrate,
phosphatase substrate, P-cellobiosidase substrate or N-acetylglucosaminidase substrate,
for detecting and identifying Streptococcus agalactiae.
The Streptococcus genus contains numerous species that are very widespread in
nature, on the skin and the mucous membranes of humans and animals, and are
i 10 responsible for multiple infections. They are ubiquitous bacteria that are found in the
free state in the outside environment (soil, air, water), in the saprophyte state or in the
commensal state in humans and animals. They are located in the rhinopharynx for
group A, C, G and H streptococci and salivarius, the intestine for group D fecal
streptococci and the vaginal cavity for group B streptococci. Their pathogenic role is
15 extremely varied and depends on the species in question and on their location in the
organism.
Streptococci are Gram + cocci, 0.5 to 1 urn in diameter, that exhibit grouping in
the form of a small chain and are immobile. They are catalase-negative, have a
fermentative metabolism, and they are optionally anaerobic and are sensitive to
20 variations in temperature (optimal growth 37°C) and to variations in pH (optimal pH 7).
; Streptococcus agalactiae (or streptococcus B) is recognized as one of the main
*:; infectious agents responsible for mastitis in cattle. In humans, it is essentially a
saprophyte of the female genital tract (vagina), but it is also found in the rhinopharynx
and in the intestine, in particular the rectum. In adults, colonization often remains
25 asymptomatic, but Streptococcus agalactiae can be responsible for septicemia,
pneumonia, meningitis, arthritis, urinary infections and deep suppurations. In women
who are pregnant, or after having given birth, the infection may lead to endometritis
and to sterility.
In newborns, the contamination occurs in utero or, most commonly, during
30 birth, due to inhalation of the amniotic fluid or of vaginal secretions. An early infection
often appears immediately after birth or within the first hours of life. Early infection is
promoted by premature birth, rupture of the membranes and a strong colonization of the
mother's vagina. The mortality rate in this type of infection is very high (> 50%). Late
infections are generally reflected by meningitis (infantile meningitis) and arthritis.
Systematic screening for the carrying of Streptococcus agalactiae is
5 recommended at the end of pregnancy, ideally between 34 and 38 weeks of amenorrhea
(35-37 weeks of pregnancy), due in particular to its prevalence (10% in France, i.e. at
least 75 000 pregnant women/year) and to the consequences thereof during full-term
births, which makes it a public health problem.
Selective media and/or media which make it possible to direct the diagnosis are
10 commercially available. However, these media have the drawback that they are not
sufficient on their own for the diagnosis of Streptococcus agalactiae and that it is
necessary to carry out supplementary tests, such as demonstrating group B Lancefield
antigen (polysaccharide with dominant presence of rhamnose) and hippurate hydrolysis
(hippurate broth).
15 The selective media most commonly used are Todd-Hewitt broth, an enrichment
broth for searching for group B streptococci in pregnant women. This broth contains
various antibiotics that inhibit most Gram-negative microorganisms of the
accompanying flora, such as nalixidic acid and gentamycin, or nalixidic acid,
polymyxin and crystal violet.
20 After the enrichment step, the antibiotic-supplemented Todd-Hewitt broth must
^ be subcultured on media for searching for streptococci (see CDC (Center for Disease
Control) recommendations, MMWR (Morbidity and Mortality Weekly Report), 16
August 2002, Vol. 51, No. RR-11).
Lim medium is a variant of Todd-Hewitt broth and it contains 1% of yeast
25 extract, nalixidic acid and colistin.
A Columbia agar containing 5% of blood is also used and makes it possible in
particular to demonstrate the p-hemolytic characteristic of Streptococcus agalactiae.
However, this characteristic is not always apparent: the hemolytic halo around the
colonies may be narrow, giving rather the a-hemolytic, or even y-hemolytic,
30 appearance. On the other hand, this characteristic becomes clear if, in the area of the
! *
Streptococcus agalactiae colonies, there are Staphylococcus aureus colonies (Campfactor).
The drawbacks of these selective media are that they must be supplemented with
biochemical tests and/or immunoassays.
5 Currently, the only commercially available, ready-to-use selective medium that
makes it possible to directly isolate and identify Streptococcus agalactiae from
rectovaginal samples is Granada medium (Biolys SA). This medium has the
characteristic of promoting the production of a carotenoid pigment by Streptococcus
agalactiae strains due tc the presence in the medium of soluble starch, proteose peptone
( 10 No. 3, glucose, sodium pyruvate, magnesium sulfate, methotrexate, colistin, crystal
violet, agar, horse serum, anhydrous Na2HP04, metronidazole, MOPS
(morpholinopropanesulfonic acid) hemi-sodium salt and distilled water, and incubation
under anaerobic conditions. This medium therefore has the drawback that the direct
detection of Streptococcus agalactiae is carried out under anaerobic conditions, which
15 is not easy to implement. Moreover, no detection medium containing one or more
enzymatic substrates is available.
| The applicant has now demonstrated, against all expectations, that it is possible
I to use enzymatic substrates, in particular esterase enzymatic substrates, for specifically
detecting and identifying Streptococcus agalactiae.
20 Specifically, surprisingly, the applicant has demonstrated that only
— Streptococcus agalactiae, among the closest bacterial species most commonly
1 encountered in an associated manner, are incapable of using esterase enzymatic
substrates early on (at less than 18 h after inoculation), such that they are the only ones
not to be revealed early on by esterase substrates, for example with no modification of
25 the colonies being obtained in the medium early on, for example with no modification
of the coloration of the colonies being obtained in the medium when a chromogenic
esterase substrate is used, without there being any diffusion of the coloration in the
reaction medium, and therefore with the coloration being concentrated at the colonies,
without these molecules having, however, a harmful effect on the growth of the
30 bacteria.
i
% 4
Consequently, this enzymatic substrate has the additional advantage that the
results can be read early, in particular at approximately 18-20 h of incubation, with a
very good contrast.
Thus, a subject of the present invention is a method for specifically detecting
5 and identifying Streptococcus agalactiae, characterized in that a reaction medium
comprising at least one esterase enzymatic substrate is used.
The esterase enzymatic substrates that are suitable for the purposes of the
invention are any substrate known to those skilled in the art that makes it possible to
demonstrate such an enzymatic activity. Such substrates may, for example, be
10 chromogenic or fluorescent and are described, for example, in the BIOSYNTH catalog,
Substrates and Reagents or www.biosynth.com, or in the GLYCOSYNTH catalog,
enzyme substrates catalog or www.glycosynth.co.uk.
By way of example of esterase substrates, mention may be made of
indoxyloctanoate, indoxylnonanoate or indoxyldecanqate derivatives, preferably
15 indoxyloctanoate derivatives, more preferably their halogenated derivatives, more
preferably the chlorinated or brominated derivatives such as 5-bromo-6-chloro-3-
indoxyloctanoate and 5-bromo-4-chloro-3-indoxyloctanoate for which the reading is
particularly early.
Since a slight esterase activity is observed after 24 h of incubation (activity less
20 than 0.6 on a scale of 0 to 4), the detection of Streptococcus agalactiae can be
improved by adding at least one other enzymatic substrate. Due to the specific property
of Streptococcus agalactiae of not using the esterase substrate, or of using it very little,
it makes no difference whether or not the other enzymatic substrate is used by
Streptococcus agalactiae and the other species. In addition, since the use of the esterase
25 substrate by Streptococcus agalactiae is only very slight, such that this only slightly
modifies the appearance of the colonies obtained, we will indicate, in the subsequent
text, only that Streptococcus agalactiae are incapable of using the esterase substrate.
Thus, according to one embodiment, the method of the invention uses a reaction
medium also comprising another enzymatic substrate other than an esterase substrate.
30 The enzymatic substrates other than an esterase substrate (non-esterase
substrate) suitable for the purposes of the invention are any substrate of which the use
by a strain confers on the colony an appearance different than the appearance obtained
when the esterase substrate is used. Such a different appearance is, for example, a
different coloration. Moreover, this non-esterase substrate is such that, when a strain
uses both this non-esterase substrate and the esterase substrate (strain other than
5 Streptococcus agalactiae), the appearance of the colonies obtained (for example, their
coloration) is also different than the appearance of the colonies of Streptococcus
agalactiae. Specifically, when both an esterase substrate and a non-esterase substrate
that can be used by the Streptococcus agalactiae strains are combined in a reaction
medium, the Streptococcus agalactiae strains are then negative for the esterase and
10 positive for the non-esterase substrate (they can be marked -/+, the first part of the
, equation corresponding to the esterase substrate and the second part corresponding to
the non-esterase substrate), while the other strains are capable of using either only the
esterase substrate (they are +/-), or both the esterase substrate and the non-esterase
substrate (they are +/+). Similarly, when both an esterase substrate and a non-esterase
15 substrate that cannot be used by the Streptococcus agalactiae strains are combined in a
reaction medium, the Streptococcus agalactiae strains are then negative for the esterase
I and negative for the non-esterase substrate (they are -/-), while the other strains are
capable of using either only the esterase substrate (they are +/-), or both the esterase
substrate and the non-esterase substrate (they are +/+). In summary, the Streptococcus
20 agalactiae strains are always -/+ or -/-, whereas the other species are always +/- or +/+.
^ Thus, for example, if a chromogenic esterase substrate which results in a blue
coloration of the colonies when the colony under consideration uses the substrate is
combined with another chromogenic enzymatic substrate which results in a pink
coloration of the colonies when the colony under consideration uses the substrate, four
25 types of coloration can be obtained:' either pink, or colorless to slightly blue, or blue, or
violet (pink+blue). The pink coloration and the colorless to slightly blue appearance are
only representative of Streptococcus agalactiae as follows: either the strain is capable
of using the non-esterase substrate and the colony becomes pink (-/+ strain), or it is
incapable of using the non-esterase substrate and the colony remains colorless or
30 becomes slightly blue (-/- strain). The blue and violet colorations are representative of
the other species as follows: either the strain is capable only of using the esterase
substrate and it becomes blue (+/- strain), or the strain is capable of using both the
esterase substrate and the other enzymatic substrate and it becomes pink and blue, i.e.
violet (+/+ strain).
Similarly, if a fluorescence-absorbing esterase substrate, which results in
5 quenching of fluorescence when the .colony under consideration uses the substrate, is
combined with another fluorescent enzymatic substrate which results in fluorescence at
the colonies when the colony under consideration uses the substrate, the latter substrate
being used by Streptococcus agalactiae, two types of colonies can be obtained: either
fluorescent colonies, or weakly to non-fluorescent colonies. The fluorescent colonies
10 are representative only of Streptococcus agalactiae since this species is only capable of
i using the enzymatic substrate other than the esterase substrate. The weakly to nonfluorescent
colonies are representative of the other species as follows: either the strain
is only capable of using the esterase substrate and it is non-fluorescent, or the strain is
capable of using both the esterase substrate and the other enzymatic substrate and it is
15 weakly to non-fluorescent.
Examples of such substrates other than an esterase substrate that are suitable for
the purposes of the invention include a-glucosidase substrates, phosphatase substrates,
(3-cellobiosidase substrates, N-acetylglucosaminidase substrates and (3-glucosidase
substrates.
.:" ;. 20 Thus, according to another embodiment, the method of the invention uses, as
v - > ' * ' • ' " ' • ' . . ' ..
reaction medium, a reaction medium comprising, in addition to an esterase substrate, at
( • • : . +
least one enzymatic substrate chosen from a-glucosidase substrates, phosphatase
substrates, (3-cellobiosidase substrates, N-acetylglucosaminidase substrates and 0-
glucosidase substrates.
25 The reaction media comprising or consisting of an esterase substrate and (of) at
least one enzymatic substrate chosen from an a-glucosidase substrate, a phosphatase
substrate and a P-cellobiosidase substrate are novel and constitute another subject of the
invention.
The a-glucosidase enzymatic substrates suitable for the purposes of the
30 invention are any substrate known to those skilled in the art that makes it possible to
7
demonstrate such an enzymatic activity. Such substrates may, for example, be
| chromogenic or fluorescent and are described, for example, in the BIOSYNTH catalog,
Substrates and Reagents or Vvrww.biosynth.com, or in the GLYCOSYNTH catalog,
enzyme substrates catalog or wvvw.glycosynth.co.uk.
5 By way of example of an oc-glucosidase substrate, mention may be made of
indoxyl-derivative-based substrates, umbelliferone-derivative-based substrates and
naphthol-derivative-based substrates.
Preferably, the oc-glucosidase enzymatic substrate suitable for the purposes of
the invention is an indoxyl-derivative-based substrate.
10 Examples of such indoxyl derivatives include derivatives of 3-indolyl-a-Dglucopyranoside,
preferably halogenated derivatives of these compounds. By way of
examples of halogenated 3-indolyl-a-D-glucopyranoside derivatives, mention may be
made of 6-bromo-3-indolyl-a-D-glucopyranoside, 5-bromo-6-chloro-3-indolyl-a-Dglucopyranoside,
5-bromo-4-chloro-3-indolyl-oc-D-glucopyranoside, 5-bromo-4-chloro-
15 3-indolyl-N-mefhyl-cc-D-glucopyranoside and 6-chloro-3-indolyl-a-D-glucopyranoside,
the latter compound being particularly preferred.
The phosphatase enzymatic substrates suitable for the purposes of the invention
are any substrate known to those skilled in the art that makes it possible to demonstrate
such an enzymatic activity. Such substrates may, for example, be chromogenic or
20 fluorescent and are described, for example, in the BIOSYNTH catalog, Substrates and
* Reagents or www.biosynth.com.
By way of example of a phosphatase substrate, mention may be made of indolylderivative-
based substrates, umbelliferone-derivative-based substrates and nitrophenylbased
substrates.
25 Preferably, the phosphatase enzymatic substrate suitable for the purposes of the
invention is an indoxyl-derivative-based substrate.
Examples of such indoxyl derivatives include 3-indolyl phosphate derivatives
such as 5-bromo-4-chloro-3-indolyl phosphate, 5-bromo-6-chloro-3-indolyl phosphate
and 6-chloro-3-indolyl phosphate, the latter compound being particularly preferred.
•
I * •. 8
The P-cellobiosidase enzymatic substrates suitable for the purposes of the
invention are any substrate known to those skilled in the art that makes it possible to
demonstrate such an enzymatic activity. Such substrates may, for example, be
chromogenic or fluorescent and are described, for example, in the BIOSYNTH catalog,
5 Substrates and Reagents or www.biosynth.com or in the GLYCOSYNTH catalog,
enzyme substrates catalog or www.glycosynth.co.uk.
By way of example of P-cellobiosidase substrate, mention may be made of
indolyl-derivative-based substrates, umbelliferone-derivative-based substrates and
nitrophenyl-based substrates.
10 Preferably, the P-cellobiosidase enzymatic substrate suitable for the purposes of
the invention is an indoxyl-derivative-based substrate.
Examples of such indoxyl derivatives include 3-indolyl-P-D-cellobioside
derivatives such as 6-chloro-3-indolyl-D-D-cellobioside and 5-bromo-4-chloro-3-
indolyl-P-D-cellobioside, the latter compound being particularly preferred.
15 The N-acetylglucosaminidase enzymatic substrates suitable for the purposes of
the invention are any substrate known to those skilled in the art that makes it possible to
demonstrate such an enzymatic activity. Such substrates may, for example, be
chromogenic or fluorescent and are described, for example, in the BIOSYNTH catalog,
Substrates and Reagents or www.biosynth.com or in the GLYCOSYNTH catalog,
v'.'..., 20 enzyme substrates catalog or www.glycosyrtth.co.uk.
» By way of example of an N-acetylglucosamihidase substrate, mention may be
**'y made of indoxyl-derivative-based substrates, umbelliferone-derivative-based substrates
and nitrophenyl-based substrates.
Preferably, the N-acetylglucosaminidase enzymatic substrate suitable for the
25 purposes of the invention is an indoxyl-derivative-based substrate.
Examples of such indoxyl derivatives include 3-indolyl-P-N-acetylglucosaminide
derivatives such as 5-bromo-6-chloro-3-indolyl-N-acetyl-p-Dglucosaminide,
6-chloro-3-indolyl-N-acetyl-P-D-glucosaminide and 5-bromo-4-chloro-
3-indolyl-p-N-acetylglucosaminide, the latter compound being particularly preferred.
9 •
The P-glucosidase enzymatic substrates suitable for the purposes of the
invention are any substrate known to those skilled in the art that makes it possible to
demonstrate such an enzymatic activity. Such substrates may, for example, be
chromogenic or fluorescent and are described, for example, in the BIOSYNTH catalog,
5 Substrates and Reagents or www.biosynth.com or in the GLYCOSYNTH catalog,
enzyme substrates catalog or www.glycosynth.co.uk.
By way of example of a P-glucosidase substrate, mention may be made of
indolyl-derivative-based substrates, umbelliferone-derivative-based substrates and
nitrophenyl-based substrates.
10 Preferably, the p-glucosidase enzymatic substrate suitable for the purposes of
i the invention is an indoxyl-derivative-based substrate.
Examples of such indoxyl derivatives include 3-indolyl-p-D-glucopyranoside
derivatives such as 5-bromo-4-chloro-3-indolyl-P-D-glucopyranoside, 5-bromo-6-
chloro-3-indolyl-P-D-glucopyranoside, 6-chloro-3-indolylrP-D-glucopyranoside and 5-
15 bromo-4-chloro-3-indolyl-N-methyl-P-D-glucopyranoside.
According to one embodiment, the method of the invention uses a reaction
medium comprising i) an esterase substrate and ii) a phosphatase substrate or an aglucosidase
substrate, the esterase substrate/phosphatase substrate combination being
preferred.
20 According to another embodiment, the reaction medium comprises, in addition
• to the esterase substrate and the phosphatase substrate or a-glucosidase substrate, an
enzymatic substrate chosen from a p-cellobiosidase substrate, an Nacetylglucosaminidase
substrate and a p-glucosidase substrate, preferably a Pcellobiosidase
substrate and an N-acetylglucosaminidase substrate.
25 The reaction medium as used in the method of the invention is therefore a
detection reaction medium due to the presence of at least one enzymatic substrate.
This reaction medium can either be used as a visualization medium only, or as a
culture and visualization medium. In the first case, the culturing of the microorganisms
is carried out before inoculation and, in the second case, the reaction medium also
30 constitujes the culture medium.
• -
The reaction medium may be solid, semi-solid or liquid. The term "solid or
semi-solid medium" is intended to mean, for example, a gelled medium.
Agar is the conventional solid medium in microbiology for culturing
microorganisms, but it is possible to use gelatin or agarose. A certain number of
5 preparations are commercially available, for instance Columbia agar, trypcase-soy agar,
MacConkey agar, Sabouraud agar or, more generally, those described in the Handbook
of Microbiological Media (CRC Press).
The amount of agar in the reaction medium is from 2 to 40 g/1. For the solid
media, the amount of agar is preferably from 9 to 25 g/1, more preferably from 12 to
10 14 g/1. For the semi-solid media, the amount of agar is preferably from 2 to 6 g/1.
The enzymatic substrates of the invention can be used in a wide pH range, in
particular between pH 5.5 and 10.
The concentration of the enzymatic substrate(s) in the reaction medium is
between 10 and 2000 mg/1, preferably between 50 and 500 mg/1, more preferably
15 between 80 and 400 mg/1, which constitutes a preferred embodiment of the invention.
Of course, those skilled in the art will determine the concentration of the
enzymatic substrate(s) in the medium within this range, according to the substrate
chosen. Thus, insofar as the esterase substrate used is 5-bromo-4-chloro-3-
indolyloctanoate, a concentration of between 100 and 400 mg/1 is preferred.
20 The reaction medium that can be used for the purposes of the invention may also
comprise other components that are of use for improving the specificity and/or the
*
i;>.': sensitivity of the method of the invention.
Thus, according to one embodiment of the invention, the reaction medium
comprises phosphate solutions such as Na2HP04 and K2HPO4 solutions.
25 This is because the use of such phosphate solutions makes it possible to
• ' . " • . ' • substantially, improve the readability of the medium, which is reflected either by a
strength in coloration sharpness, or by an increase in the expression and/or in the
detection of the phosphatase activity at 18 h. .
The concentration of such phosphate solutions is between 0.3 and 1.5 g/1 for
30 each solution, a concentration of 0.5 g/1 being preferred.
i
i •"••••
!
11
The reaction medium may also contain a mixture of inhibitors for inhibiting or
limiting the growth of unwanted strains, such as false-positive strains, for example
Candida or Staphylococcus saprophyticus, without modifying the detection sensitivity
of the medium.
5 In this respect, the reaction mixture may contain a mixture of antibiotics. The
addition of antibiotics to the reaction medium allows, inter alia, a time to be saved since
the identification of Streptococcus agalactiae is carried out directly.
Examples of antibiotics that are suitable for the purposes of the invention
include aztreonam and amphotericin B. These antibiotics are commercially available
10 from ICN, Squibb or Sigma.
The amount of each antibiotic in the reaction medium varies according to the (
antibiotic concerned, and will be readily determined by those skilled in the art.
i
The reaction medium may also comprise one or more elements in combination, j
such as amino acids, peptones, carbohydrates, nucleotides, minerals, vitamins, I
15 surfactants, buffers, phosphate salts, ammonium salts, sodium salts or metal salts.
Examples of media are described in the applicant's patent applications EP 656 421 and
WO 99/09207.
The implementation of the method of the invention can be carried out according
to the following steps consisting in: [
20 a) inoculating a reaction medium as defined above, with all or part of the sample,
• -
b) incubating the inoculated medium,
*
c) revealing the presence of at least one esterase activity alone or in combination
. with at least one other en2ymatic activity other than an esterase activity,
' •
which constitutes another subject of the invention. |
25 The inoculation and incubation steps are widely known to those skilled in the
art. I
For example, the incubation temperature may be 37°C. As regards the I
incubation atmosphere, it is preferably aerobic.
The revealing is carried out with the naked eye by visualization of a change in
30 coloration that does not diffuse in the reaction medium and is therefore concentrated at
the colonies. In the case of the revealing of the fluorescence, the fluorescence reading
12
devices known to those skilled in the art are used.
The biological samples to be analyzed are any clinical sample liable to contain
Streptococcus agalactiae, such as a vaginal specimen, a urine specimen or any other
sample of which the analysis may aid a clinician in reaching a diagnosis.
5 The invention will be understood more clearly from the following examples
given by way of nonlimiting illustration. |
Example 1: Detection of Streptococcus agalactiae using esterase enzymatic (
I
substrates !
1.1 Preparation of the reaction media j
10 The reaction media were prepared by mixing heart-brain extract (4.84 g/1;
Solabia), meat infusion (1.96 g/1; Solabia), biothione (1 g/1; Solabia), biotrypcase
(7.2 g/1; Solabia), sodium carbonate (0.3 g/1; VWR), sodium pyruvate (2 g/1; Fluka),
I
HEPES buffer (0.4 g/1; Sigma), lactalbumin peptone (2 g/1; DMV), glucose (1 g/1;
Merck), American agar (2 g/1; Sobigel) and European agar (12 g/1; Roko).
15 After autoclaving for 15min at 121 °C, an esterase enzymatic substrate as
indicated below was added at a rate of 0.3 g/1; followed by cooling in a water bath at
50°C:
• 5-bromo-4-chloro-3-indolyloctanoate (X-C8; Inalco), which gives a turquoise
coloration when it is used, and
20 • 5-bromo-6-chloro-3-indolyloctanoate (Magenta-C8; Inalco), which gives a
pink-red coloration when it is used.
1 v>- The media were then poured into a Petri dish for the subsequent inoculation
• .
with bacterial strains.
•
1.2 Inoculation of the microorganism strains
25 Three Streptococcus agalactiae strains and three strains of other bacteria, all
from the applicant's collection, suspended in physiological saline, were inoculated so as
to give isolated colonies on each of the media. The dishes were incubated at 37°C for
48 hours. The colonies formed were examined visually after 18, 24 and more than 40
hours of incubation. The coloration of these colonies, the growth and also the intensity
30 of this coloration (representative of the esterase activity) were noted.
1.3 Results • I
' i
• • t
13
The results are given in Table 1 hereinafter and are expressed: I
- in terms of growth (G) with the size being indicated in mm, 1
- in terms of color (Co) with T = turquoise, R = pink or red,
- in terms of intensity (I) of coloration, based on an arbitrary scale ranging from 0 to 4,
5 0 corresponding to an absence of activity and 4 corresponding to the presence of a very intense coloration, j
- according to the incubation time in hours (T).
1
t
i
|
i
i
I
!
*
Table 1
Strains X-C8 Magenta-C8
(accession No.) T G Co I G Co I
Streptococcus 18 1.2 1.2 agalactiae 24 2 T 0.3 2 R 0.3
(7611003) >40 2.5 T 2.3 2.5 R 1.7
Streptococcus 18 0.4 0.4
agalactiae 24 0.7 T 0.3 0.7 R 0.3
(0101060) >40 1.3 T 3 1.3 R 2
Streptococcus 18 0.3 agalactiae 24 0.2 0.5 j
(8904053) > 40 0.3 T 0.3 1
Enterococcus 18 0.8 T 1.7 0.8 R 1
faecalis 24 2 T 3 1.8 R 1.7 j
l _ ; (0008192) >40 2 T 3.5 2 R 3.5 [
Enterococcus 18 0.5 T 2 0.7 R 1 f
faecium 24 1 T 3 1.7 R 2.7
(7611005) >40 1 T 3 1.7 R 3
Staphylococcus 18 0.5 T 2 0.5 R 2 epidermidis 24 1.5 T 3 1 R 3 j
(7509009) > 40 1.5 T 1 3 1.3 R 3.5 j
. |
• • The results demonstrate that streptococci B can be detected early using an
esterase enzymatic substrate since they exhibit a zero to very weak activity at 18-24 h. S
5
Example 2: Detection of Streptococcus agalactiae using an esterase substrate and
v •• • •.. • . :
an a-glucosidase substrate or phosphatase substrate
.-- -, +
\ : The protocol described above in Example 1 was repeated, with the exception j
that, at the same time as 0.3 g/1 of the esterase substrate X-C8, 0.3 g/1 of 6-chloro-3-
10 indolyl-a-D-glucopyranoside (Rose-a-Glu), or 0.3 g/1 of 6-chloro-3-indolyl phosphate j
!
(Rose-P), which give a pink coloration when they are used, was added. I
i i
The results are given in Table 2 below, in which the growth, the coloration and [
the intensity are given, as in Example 1, and where R = Pink/(Rose)/Red, PB =Pink- f
Brown, T - Turquoise, Gr = Green, Vi = Violet, B = Blue, GVi = Grey-Violet and I
15 GB = Grey-Blue.
i
I
•
^ 15
Table 2
! Strains X-C8 + Rose-alpha-Glu X-C8 + Rose-P
(accession No.) T G Co I G Co I
Streptococcus 18 1.3 R 3 1 R 3 j
agalactiae 24 1.3 R 3 1.7 R 4
(7611003) >40 2 R 4 2 R 4
Streptococcus 18 0.2 R 2 0.5 R 3
agalactiae 24 0.3 R 2 0.5 R 3.5
(8709013) >40 1 R 4 1.7 R 4
Streptococcus 18 1 R 2 0.8 R 3
agalactiae 24 1.7 PB 2.7 1.3 R 4
(7702055) >40 1.7 R 4 1.7 R 4
Enterococcus 18 1.5 T 3. 1.7 GB 3
faecium 24 1.7 T 3 1.7 B 3.5
(7611005) > 40 1.8 T 4 2 GVi 4
Staphylococcus 18 0.7 Gr ' 3 0.6 GVi 3.5
epidermidis 24 1.3 GB 3.5 1.3 GVi 3.5
(7509009) >40 1.3 GB 3.5 1.5 GVi 4
Staphylococcus 18 3 GVi 3 2 Vi 4
aureus 24 3 Vi 4 3 Vi 4
(9202070) >40 | 3 Vi '' 4 | 3 1 Vi [ 4 |
I
i
This table demonstrates that the detection of the Streptococcus agalactiae
. strains is improved when a chromogenic esterase substrate is used in combination with I
i
5 another chromogenic enzymatic substrate, other than an esterase substrate, that can be j
|
used by the Streptococcus agalactiae strains.
Example 3: Detection of Streptococcus agalactiae using an esterase substrate, a
phosphatase substrate and a 3-ceIlobiosidase substrate
10 The protocol described in Example 2 was repeated, using 0.3 g/1 of X-C8 and
0.2 g/1 of Rose-P, with the exception that 0.08 g/1 of 5-bromo-4-chloro-3-indolyl-p-Dcellobioside
(Cellobio) is also added at the same time as the other substrates, along with
0.5 g/1 of Na2HP04 and 0.5 g/1 of K2HP04, before autoclaving. j
As control medium, a medium with only X-C8 and Rose-P was used. 15 The results are given in Table 3 below, in which the growth, the coloration and the intensity are given, as in Example 1, and where R = Pink/Rose)/Red, Ma = Mauve,
Vi = Violet, B = Blue, GB = Grey-Blue and DP = Dark Purple. |
I
I
I
I
I
16 !
i
Table 3 I
Strains Control X-C8 + Rose-P + Cellobio I
(accession No.) T G Co I G Co I j
Streptococcus 18 0.7 R 1.7 0.7 R 1.3 [
agalactiae 24 0.7 R 4 0.7 R 3 (0101060) - >40 1.5 R . 4 1.5 R 4 j
Streptococcus 18 1.3 R 4 1 R 4 j
agalactiae 24 1.5 R 4 1.5 R 4 (7701031) >40 1.5 R 4 1.5 R 4
Streptococcus 18 1 R 2 1 R 2 f
agalactiae 24 1.5 R 4 1.5 R 4 (7702055) >40 1.7 R 4 1.7 R 4 Streptococcus 18 0.3 0.3 B 0.5 f
l
v J anginogus 24 0.5 R 0.1 0.4 B 1.3 I
(8507046) >40 1 R 2.3 1 B 2.7 !
Enterococcus 18 1.5 Ma 1.7 1.3 B 3 ;
faecium 24 1.7 Ma 3 1.5 GB 4 [(0002043) | > 40 I 2 | DP | 4 [ 2 [ DP | 4 | j
The results in Table 3 demonstrate an improvement in the specificity of 5 detection of Streptococcus agalactiae compared with the other strains when three j
j
enzymatic substrates, including an esterase substrate, are used.
• '' i Example 4: Detection of Streptococcus agalactiae using an esterase substrate, a C:: I
- •• • %
phosphatase substrate and an N-acetylglucosaminidase substrate j
* 10 The protocol described in Example 3 was repeated^ with the exception that l
V-:-'--' |
0.4 g/1 of 5-bromo-4-chloro-3-indoIyl-P-N-acetylglucosaminide (X-NAGlu) was used j
in place of the Cellobio.
The control medium is identical to the medium tested, with the exception that it j
does not contain any X-NAGlu. ,
15 The results are given in Table 4 below, in which the growth, the coloration and
•
the intensity are given, as in Example 1, and where R = Pink/Red, B = Blue,
GP = Grey-Pink and Mg = Magenta.
i
1
17 |
Table 4
Strains Control X-C8 + Rose-P + X-NAGlu (accession No.) T G Co I G Co I |
Streptococcus 18 0.5 R 3 0.5 R 2.3 agalactiae 24 1 R 4 1 R 3 i
(7611003) >40 1.2 R 4 1.2 R 4 |
Streptococcus 18 0.5 R 2.7 0.5 R 2.7 . agalactiae 24 0.7 ,R 4 0.7 R 4 (7701031) >40 0.7 R 4 0.7 R 4 j
Enterobacter 18 1.7 R 2.3 1.7 GP 2 j
c/occre 24 2 R 3 2 B 3 j
(0010003) > 40 2.5 B 4 3 B 4 j
Enter ococcus 18 0.5 GP 2 0.5 B 3 [
faecium 24 0.8 GP 2.7 0.8 B 3.5 >•' ~ . [(0002043) I > 40 1 1 | Mg 1 4 | 1 1 B | 4 [ The results in this table demonstrate an improvement in the specificity of j
detection of Streptococcus agalactiae compared with the other strains when three [
5 enzymatic substrates, including an esterase substrate, are used. Example 5; Detection of Streptococcus agalactiae using an esterase substrate, a
phosphatase substrate and a P-glucosidase substrate
The protocol described in Example 4 was repeated, with the exception that I
10 0.08 g/1 of 5-bromo-4-chloro-3-indolyl-p-D-glucopyranoside (X-(3-Glu) and 0.3 g/1 of j
5-bromo-4-chloro-3-indolyl-N-methyl-p-D-glucopyranoside (GreenA-P-Glu) were used * inplaceoftheX-NAGlu. j
The control medium is identical to the medium tested, with the exception that it [
does not contain any X-P-Glu or GreenA-p-Glu. f
15 The results are given in Table 5 below, in which the growth, the coloration and
the intensity are given, as in Example 1, and where R = Pink/Red, Ma = Mauve, j
Vi = Violet, B = Blue and GB = Grey-Blue.
i ' i
! i!
I
i i i
is i
! Tableau 5 !
s t r a i n s X-C8 + Rose-P+ X-C8 + Rose-P+ Control X-P-Glu GreenA-ft-Glu j
(accession No.) T G Co I G Co I G Co I ;
Streptococcus 18 0.4 0.3 0.2 !
agalactiae 24 0.5 R 0.5 0.5 R 0.3 0.4 R 0.3 (9001001) >40 1,7 R 4 1.3 R 4 1.5 R 4 f
Streptococcus 18 1.3 R 4 1,3 R 4 1 R 4 agalactiae 24 1.5 R 4 1.5 R 4 1.5 R 4 (7701031) ' >40 1.5 R 4 1.5 R 4 1.5 R 4 I
Streptococcus 1 8 1 R 2 1 R 2 1 R 2 :'"-, agalactiae 24 1.5 R 4 1.5 R 4 1.3 R 4 (7702055) >40 1.7 R 4 1.7 R 4 1.5 R 4 Streptococcus 18 0.3 0.3 B 3 0.3 B 0.5
anginogus 24 0.5 R 0.1 0.4 B 4 0.4 B 2
(8507046) >40 1 R 2.3 1 B 4 1 B 3.5
Enterococcus 18 1.5 Ma 1.7 1.5 B 4- 1.5 GB 4
Faecium 24 1.7 Ma 3 1.5 B 4 1.7 GB 4
(0002043) | >40 1 2 1 Vi 1 4 J 2- | B 1 4 I 2 |GB1 4 1
The results obtained in Table 5 demonstrate an improvement in the specificity
of detection of Streptococcus agalactiae compared with the other strains when three
5 enzymatic substrates, including an esterase substrate, are used.
Example 6: Improvement in the sensitivity of detection by adding phosphate
solution
'\_j The protocol described in Example 1 was repeated, with the exception that
10 0.3 g/1 of Rose-P and also 0.5 g/1 of Na2HP04 and 0.5 g/l of K2HP04 were added at the
same time as 0.3 g/1 of the esterase substrate X-C8.
The same medium, but with no phosphate solution, was used as control
medium.
The results are given in Table 6 below, in which the growth, the coloration and
15 the intensity are given, as in Example 1, where R = Pink and Mg = Magenta.
19
Table 6 i
„ . . «-. * . Medium Strains Control ph. osph. at,e s wol,iu tht,i. o n ;
(accession No.) T G Co I G Co I j
Streptococcus 18 1.3 R 3 1.3 Mg 3.5 J
agalactiae 24 1.7 Mg 4 1.7 Mg 4 (7611003) >40 1.8 Mg 4 1.8 Mg 4 Streptococcus 18 0.4 R 0.5 0.5 Mg 3 agalactiae 24 0.5 Mg 4 0.7 Mg 3.5
(0101060) >40 1.5 Mg 4 1.5 Mg 4 Streptococcus 18 1 Mg 3 1 R 3.5 '
agalactiae 24 1.5 Mg 4 1.5 Mg 4 j
(7702055) 1 >40 | 1.5 | Mg | 4 | 1.5 | Mg | 4 [ j
The results in this Table 6 demonstrate an improvement in the sharpness of
coloration from 18 h, or an increase in the expression of the S. agalactiae strains. •
5 - Example 7: Comparison of the sensitivity and the specificity of detection of
S. agalactiae using a medium containing an esterase substrate according to the i
invention and the commercially available media
For this sensitivity and specificity study, a medium according to the invention,
10 prepared as described in Example 1, containing 0.3 g/1 of X-C8 and also: 0.2 g/1 of
Rose-P, 0.08 g/1 of Cellobio, 0.5 g/1 of Na2HP04, 0.5 g/1 of K2HP04, 0.012 g/1 of j
aztreonam and 0.004 g/1 of amphotericin B, was used.
As medium for comparison, the Granada medium (ref. 10 077, BIOLYS, j
France) (Granada medium) was used.
15 69 strains of microorganisms, including 14 of Streptococcus agalactiae, were
inoculated and left to incubate at 37°C for up to 24 h and at ambient temperature
beyond this time. The colonies were visualized as described above. The confirmation of
the colonies suspected of being characteristic of streptococcus B, i.e. appearing to be
pink/red, was carried out by means of an agglutination assay using the Slidex Strepto
20 Kit reagent according to trie supplier's recommendations (bioMerieux, France). The
non-characteristic colonies, i.e. the colonies that were other than pink or that had the
characteristic coloration but gave a negative response in the agglutination assay (false- j
1 ';
• I
I
!
i
i
20 j
positive strains), were identified by means of Galeries ID 32 Strep (bioMerieux, I
France). . !
The results are expressed as % of correct diagnosis relative to all the tests in I
terms of sensitivity and specificity, and are given in Table 7 below, the % sensitivity j
5 corresponding to the number of true positives detected on the medium divided by the j
total number of true positives to be detected (*100), and the % specificity j
corresponding to the number of true negatives detected on the medium divided by the total number of true negatives to be detected (* 100). |
- 10 Table 7 j
% sensitivity and specifity of detection of
5*. agalactiae '
Granada medium Medium of the invention
18h 24h I >40h 18h 24h > 40h
Sensitivity without enrichment 50 50 50 19 79 ' 93
Sensitivity with [
enrichment 50 50 50 .79 86 93 Specificity without
enrichment 100 100 100 87 82 80 !
Specificity with
enrichment 1 100 | 100 | 100 J 89 | 93 | 82 |
The results indicated in this table demonstrate the improvement in the f
sensitivity of detection of streptococci B {Streptococcus agalactiae) using the method [
(:;:; of the invention. Moreover, they also demonstrate that the detection medium of the i
15 invention also has good specificity, which specificity is improved after enrichment due to a passage in Todd-Hewitt broth for 18-24 hours at 35-37°C with or without 5% C02
before inoculation of the agar (see CDC (Center for Disease Control) recommendations,
MMWR (Morbidity and Mortality Weekly Report), 16 August 2002, Vol.51,
No. RR-11). j
20 I
Example 8: Use of the medium based on clinical samples
For this study, the medium according to the invention, as prepared as described
above in Example 7, was used. •• •
21 I
»
A total of 134 samples/swabs originating from vaginal or endocervical I
specimens from pregnant women were used in this study. Each swab was emulsified in 1 ml of sterile physiological saline and 100 ul of
this solution were deposited, firstly, onto a Columbia agar containing 5% of horse
5 blood, and, secondly, onto the medium used in the method of the invention. Moreover,
100 ul of the above solution were used to inoculate a Todd Hewitt broth. After
incubation for 20 hours at 37°C and under aerobic conditions, the blood-agar and the
medium of the invention were inoculated using the Todd Hewitt broth and then
incubated at 37°C for 20 h under aerobic conditions.
- 10 The confirmation of the colonies suspected of being characteristic of
streptococcus B, i.e. that appeared pink/red in color, was carried out by means of an
agglutination assay using the Slidex Strepto Kit reagent according to the supplier's
recommendations (bioMerieux, France).
Among the 134 samples, 112 were inoculated onto the agar media, firstly,
15 directly from the suspension in physiological saline and, secondly, after enrichment in
Todd Hewitt broth. The remaining 22 samples were inoculated onto the agar media
only directly from the suspension in physiological saline.
The results, expressed as average percentage sensitivity and specificity, are
presented in Table 8 below.
0, 20
Table 8
W? Columbia agar Invention agar
Sensitivity 95 100 Specificity 90 99^5
The results in Table 8 above show that the medium of the invention, used with
clinical samples, makes it possible.to improve the sensitivity and the specificity of
25 detection of Streptococcus agalactiae. Specifically, 20/20 specimens containing
Streptococcus agalactiae are detected on the medium of the invention, against 19 on
the Columbia medium, and there is just one false + result on the esterase medium, ;
22
against 24 on the Columbia agar. It can even be noted that the results are better than
when the medium was tested with the laboratory strains.
( :
i

We claim:
1. A reaction medium comprising (i) an esterase substrate that Streptococcus
agalactiae are incapable of using at less than 18 h after inoculation, (ii) one
enzymatic substrate chosen from (3-cellobiosidase substrates, N-acetylglucosaminidase
substrates and P-glucosidase substrates, and (iii) a phosphatase
substrate.
2. The reaction medium as claimed in claim 1, characterized in that the substrate (ii)
a P-cellobiosidase substrate.
3. The reaction medium as claimed in claim 1, characterized in that the substrate (ii)
a N-acetyl-glucosaminidase substrate.
4. The reaction medium as claimed in claim 1, characterized in that the substrate (ii)
a P-glucosidase substrate.
5. The reaction medium as claimed in any one of claims 1 to 4, characterized in that
the esterase substrate is chosen from octanoate, nonanoate and decanoate
derivatives.
6. The reaction medium as claimed in claim 5, characterized in that the esterase
substrate is an indoxyl substrate.
7. The reaction medium as claimed in any one of claims 1 to 6, characterized in that
the concentration in each enzymatic substrate is comprised between 10 and
2000 mg/1.
8. The reaction medium as claimed in any one of claims 1 to 7, characterized in that
is it also comprises phosphate solutions.
-
9T The reaction medium as claimed in claim 8, characterized in that the phosphate
solutions are chosen from Na2HPC>4 and K2HPO4.
10. The reaction medium as claimed in any one of claims 1 to 9, characterized in that
is it also comprises a mixture of inhibitors for inhibiting or limiting the growth of
unwanted strains.
11. The reaction medium as claimed in claim 10, characterized in that the inhibitors
are antibiotics.
i
12. The reaction medium as claimed in claim 11, characterized in that the antibiotics
are aztreonam and amphotericine B.