U.S. patent application number 13/539119 was filed with the patent office on 2012-10-25 for medium for the specific detection of resistant microorganisms.
This patent application is currently assigned to BIOMERIEUX. Invention is credited to Nathalie BAL, Vanessa CHANTEPERDRIX, Sylvain ORENGA, John PERRY, Celine ROGER-DALBERT, Gilles ZAMBARDI.
Application Number | 20120270252 13/539119 |
Document ID | / |
Family ID | 36572019 |
Filed Date | 2012-10-25 |
United States Patent
Application |
20120270252 |
Kind Code |
A1 |
ORENGA; Sylvain ; et
al. |
October 25, 2012 |
MEDIUM FOR THE SPECIFIC DETECTION OF RESISTANT MICROORGANISMS
Abstract
A method for distinguishing among a first group of
microorganisms belonging to a first taxon of Gram negative
bacteria, the first group of bacteria exhibiting a mechanism of
resistance to a treatment; a second group of microorganisms
belonging to a second taxon of Gram negative bacteria, the second
taxon of bacteria being different than said first taxon, and
exhibiting a mechanism of resistance to a treatment identical to
the mechanism of the first group; and a third group of Gram
negative bacteria that is not resistant to the treatment.
Inventors: |
ORENGA; Sylvain;
(Neuville-sur-Ain, FR) ; ROGER-DALBERT; Celine;
(Vaux-en-Bugey, FR) ; PERRY; John;
(Newcastle-Upon-Tyne, GB) ; CHANTEPERDRIX; Vanessa;
(Grenoble, FR) ; ZAMBARDI; Gilles; (Trept, FR)
; BAL; Nathalie; (Villeurbanne, FR) |
Assignee: |
BIOMERIEUX
Marcy L'Etoile
FR
|
Family ID: |
36572019 |
Appl. No.: |
13/539119 |
Filed: |
June 29, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11794907 |
Jul 9, 2007 |
|
|
|
PCT/FR2006/050109 |
Feb 9, 2006 |
|
|
|
13539119 |
|
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Current U.S.
Class: |
435/18 ; 435/25;
435/34; 435/38 |
Current CPC
Class: |
C12Q 1/04 20130101; C12Q
1/10 20130101; C12Q 1/37 20130101; C12Q 1/14 20130101; C12Q 1/34
20130101; C12Q 1/527 20130101; C12Q 1/045 20130101 |
Class at
Publication: |
435/18 ; 435/34;
435/25; 435/38 |
International
Class: |
C12Q 1/10 20060101
C12Q001/10; C12Q 1/34 20060101 C12Q001/34; C12Q 1/26 20060101
C12Q001/26; C12Q 1/04 20060101 C12Q001/04 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 10, 2005 |
FR |
0550394 |
Oct 7, 2005 |
FR |
0553049 |
Claims
1. A method for distinguishing among at least three groups of
microorganisms that may be present in a biological sample, the at
least three groups comprising: a first group of microorganisms
belonging to a first taxon of Gram negative bacteria, the first
group of bacteria exhibiting a mechanism of resistance to a
treatment; a second group of microorganisms belonging to a second
taxon of Gram negative bacteria, the second taxon of bacteria being
different than said first taxon, and exhibiting a mechanism of
resistance to a treatment identical to the mechanism of the first
group; and a third group of Gram negative bacteria that is not
resistant to said treatment, the method comprising: inoculating a
culture medium with the biological sample, wherein the culture
medium comprises: a first substrate for detecting a first enzymatic
or metabolic activity of said first group of microorganisms; a
marker for differentiating the first group of microorganisms and
the second group of microorganisms, said marker being a substrate
for detecting an enzymatic or metabolic activity of said second
group of microorganisms; and an antimicrobial that is active on the
third group of microorganisms; and distinguishing among any members
of the three groups of microorganisms that are present on the
culture medium to determine of which group of microorganisms they
are members on the basis of their interactions with the first
substrate, the markers, and the antimicrobial.
2. The method according to claim 1, wherein: the first group of
microorganisms is selected from E. coli ESBL and HL Case bacteria;
and the second group of microorganisms is selected from KESC ESBL
or HL Case bacteria.
3. The method of claim 1, wherein: the third group of
microorganisms is bacteria not resistant to beta-lactamines and/or
cephalosporins.
4. The method of claim 1, wherein: the first group of
microorganisms is E. coli ESBL bacteria; the second group of
microorganisms is KESC ESBL bacteria; and the third group of
microorganisms is bacteria not resistant to beta-lactamines.
5. The method of claim 1, wherein: the first group of
microorganisms is E. coli HL Case bacteria; the second group of
microorganisms is KESC HL Case bacteria; and the third group of
microorganisms is bacteria not resistant to cephalosporins.
6. The method of claim 1, wherein: the first group of
microorganisms is E. coli ESBL bacteria; the second group of
microorganisms is KESC ESBL bacteria; the third group of
microorganisms is bacteria not resistant to beta-lactamines and/or
cephalosporins; and the biological sample further comprises a
fourth group of microorganisms that is Proteeae ESBL bacteria.
7. The method of claim 1, wherein: the first substrate is a
substrate for detecting a beta-glucuronidase or beta-galactosidase
enzymatic activity; the marker is a second substrate for detecting
a beta-glucosidase, tryptophanase, or desaminase activity; and the
antimicrobial is ceftazidime.
8. The method of claim 1, wherein: the first substrate is a
substrate for detecting a beta-glucuronidase or beta-galactosidase
enzymatic activity; the marker is a second substrate for detecting
a beta-glucosidase, tryptophanase or desaminase activity; and the
antimicrobial is cefpodoxime and cloxacillin.
9. The method of claim 1, wherein: the substrate is a first
substrate for detecting a beta-glucuronidase or beta-galactosidase
enzymatic activity; the marker is a second substrate for detecting
a beta-glucosidase, desaminase or tryptophanase activity; and the
antimicrobial is ceftriaxone and clavulanic acid.
10. The method of claim 1, wherein: the substrate is a first
substrate for detecting a beta-glucuronidase or beta-galactosidase
enzymatic activity; the marker is a second substrate for detecting
a beta-glucosidase activity; the antimicrobial is a combination
comprising: cefpodoxime, cloxacillin, vancomycin, and amphotericin
B; and the culture medium further comprises a third substrate for
detecting a desaminase activity.
11. The method of claim 10, wherein the antimicrobial further
comprises cefsulodine.
12. The method of claim 1, wherein: the first group of
microorganisms is E. coli ESBL bacteria; the second group of
microorganisms is KESC ESBL bacteria; the third group of
microorganisms is bacteria not resistant to beta-lactamines and/or
to cephalosporins; the biological sample further comprises a fourth
group of microorganisms that is Proteeae ESBL bacteria; the first
substrate is a substrate for detecting a beta-glucuronidase or
beta-galactosidase enzymatic activity; the marker is a second
substrate for detecting a beta-glucosidase activity; the
antimicrobial is a combination comprising: cefpodoxime,
cloxacillin, vancomycin, and amphotericin B; and the culture medium
further comprises a third substrate for detecting a desaminase
activity.
13. The method of claim 1, wherein: the first group of
microorganisms is E. coli ESBL bacteria; the second group of
microorganisms is KESC ESBL bacteria; the third group of
microorganisms is bacteria not resistant to beta-lactamines and/or
to cephalosporins; the biological sample further comprises a fourth
group of microorganisms that is Proteeae ESBL bacteria; the first
substrate is a substrate for detecting a beta-glucuronidase or
beta-galactosidase enzymatic activity; the marker is a second
substrate for detecting a beta-glucosidase activity; the
antimicrobial is a combination comprising: cefpodoxime,
cloxacillin, vancomycin, amphotericin B, and cefsulodine; and the
culture medium further comprises a third substrate for detecting a
desaminase activity.
Description
CROSS-REFERENCE TO PRIOR APPLICATIONS
[0001] This is a divisional of application Ser. No. 11/794,907
filed Jul. 9, 2007, which is a National Stage Application of
PCT/FR2006/050109 filed Feb. 9, 2006, and claims the benefit of
French Application Nos. 0550394 filed Feb. 10, 2005 and 0553049
filed Oct. 7, 2005. The entire disclosures of the prior
applications are hereby incorporated by reference herein in their
entirety.
[0002] The field of the invention is that of microbiological
analysis by means of biochemistry, and in particular the detection
and identification of microorganisms, for instance of bacteria or
yeasts.
[0003] Bacterial resistance to antibiotics is a major public health
problem. The resistance of infectious microorganisms to a treatment
has developed at the same time as anti-infectious molecules and
today represents a major obstacle in therapeutics. This resistance
is responsible for many problems, including difficulties in
detection in the laboratory, limited treatment options and a
deleterious impact on clinical outcome.
[0004] In particular, the rapid and irrepressible increase in the
resistance of pathogenic bacteria, over the last 20 years,
represents one of the major current problems in medicine.
Infections caused by these organisms are responsible for extended
periods of hospitalization and are associated with high morbidity
and mortality rates, following therapeutic failures.
[0005] Several resistance mechanisms can be involved simultaneously
in a bacterial strain. They are generally classified in 3
categories: deficient penetration of the antibiotic into the
bacterium, inactivation or excretion of the antibiotic by bacterial
enzymatic systems, and lack of affinity between the bacterial
target and the antibiotic.
[0006] Enzymatic inactivation is the most common mechanism of
acquired resistance in terms of number of species and of
antibiotics involved. Thus, chromosomal class C cephalosporinases
today constitute one of the predominant resistance mechanisms of
gram-negative bacteria, the bacteria expressing such enzymes being
resistant to cephalosporins. Similarly, .beta.-lactamases are
enzymes expressed by certain bacteria, capable of hydrolyzing the
C--N bond of the .beta.-lactame ring, the basic structure of
antibiotics of the .beta.-lactamine family, so as to give a
microbiologically inactive product. Several .beta.-lactamase
inhibitors (BLIs), such as clavulanic acid (CA), tazobactam and
sulbactam, have been developed in order to increase the
antimicrobial activity and broaden the spectrum of the
.beta.-lactamines which are associated therewith. They act as a
suicide subject for .beta.-lactamases, and prevent enzymatic
degradation of the antibiotics and allow them to become effective
against bacteria that were initially resistant. However, by virtue
of the persistent exposure of strains to antibiotic pressure, the
bacteria express their ability to adapt through the continuous and
dynamic production of .beta.-lactamases, which evolves at the same
time as the development of new molecules.
[0007] Gram-negative bacteria which produce high-level chromosome
class C cephalosporinases (reference is made to HL Case bacteria),
and also gram-negative bacteria which produce extended-spectrum
.beta.-lactamase (reference is then made to ESBL bacteria) have, as
a result, become an increasing threat, in particular because the
number of bacterial species concerned is increasing. HL Case and
ESBL bacteria are resistant to treatments based on 1st- and
2nd-generation penicillins and cephalosporines, but also on
3rd-generation cephalosporines (C3G) (cefotaxim CTX, ceftazidime
CAZ, cefpodoxime CPD, ceftriaxone CRO) and monobactams (aztreonam
ATM). On the other hand, 7.alpha.-methoxycephalosporins
(cephamycins: cefoxitin, cefotetan) and carbapenems (imipenem,
meropenem, ertapenem) generally conserve their activity. ESBLs are
inhibited by .beta.-lactamase inhibitors (BLIs), which makes it
possible to differentiate them from other cephalosporinases.
[0008] These bacteria thus most commonly simultaneously express
resistances to several treatments, which poses difficulties in
setting up a relevant treatment and avoiding therapeutic failures.
An Escherichia coli bacterium can thus be HL Case and ESBL. In
addition, since ESBL-positive enterobacteria have a tendency to
disseminate the resistance by clonal transmission of strains or
conjugative plasma transfer, they represent a problem in terms of
controlling infections. In most studies, Escherichia coli and
Klebsiella pneumoniae remain the most common ESBL-producing
species. However, over the last few years, ESBLs have greatly
broadened their panel of host species. In fact, many species of
enterobacteria and of nonfermenting gram-negative bacilli (such as
Pseudomonas aeruginosa) have also been reported to ESBL
producers.
[0009] In addition to these ESBL bacteria, mention may also be made
of Staphylococcus aureus bacteria, which are also pathogenic
bacteria that develop many mechanisms of resistance, such as
resistance to methicillin, penicillin, tetracycline, erythromycin,
or vancomycin. Enterococcus faecium is another multiresistant
bacterium found in the hospital environment, which can be resistant
to penicillin, vancomycin and linezolide. Mycobacterium
tuberculosis is commonly resistant to isoniazid and to rifampicin.
Other pathogens offer certain resistances, such as Salmonella,
Campylobacter and Streptococcus.
[0010] It therefore becomes essential, from a public health point
of view, to be able to identify such microorganisms, and such
resistance mechanisms, as rapidly as possible.
[0011] In general, the search for microorganisms resistant to a
treatment is carried out according to the following steps:
[0012] 1. Taking a biological sample that may contain said
microorganisms;
[0013] 2. Seeding and incubating a culture medium (18 to 48 h) in
order to induce exponential growth of the microorganisms;
[0014] 3. Pinpointing, on the culture media, colonies of
potentially significant microorganisms;
[0015] 4. Characterizing the microorganism species;
[0016] 5. Identifying the mechanisms of resistance of the
microorganisms analyzed, their biological significance and,
optionally, the appropriate therapy.
[0017] This succession of steps involves a considerable amount of
time between taking the sample that may contain microorganisms and
prescribing a treatment that is appropriate for the patient.
Furthermore, the user must generally perform steps for transferring
microorganims from a first medium to a second medium manually,
which can induce problems, in particular, of contamination, but
also risks to the handler's health.
[0018] By way of example, in order to detect the presence of
broad-spectrum beta-lactamases (ESBLs) in strains of Escherichia
coli and Klebsiella pneumoniae, use may be made of a diffusion
technique as described in the publication by Jacoby & Han (J
Clin Microbial. 34(4): 908-11, 1996), which does not however give
any information regarding the identification of the strains tested:
it is possible to determine whether or not the bacterium is a
ESBL-producing bacterium, but it is not possible to distinguish
whether such a bacterium is an Escherichia coli or a Klebsiella
pneumoniae.
[0019] Metabolic substrates are also used for detecting the
presence of ESBLs or HL cases. In this respect, AES laboratories
proposes a medium in a biplate combining a Drigalski medium with
cefotaxim and a MacConkey medium with ceftazidime. The Drigalski
and MacConkey media make it possible to reveal lactose
acidification, a metabolism which is present in a very large number
of enterobacterial species. However, such a medium only makes it
possible to distinguish resistant bacteria from non-resistant
bacteria, and does not make it possible to distinguish bacteria
expressing a ESBL from those expressing an HL Case. Neither does
this medium make it possible to identify specific bacterial
species, nor does it make it possible, for example, to discriminate
between E. coli bacteria and K. pneumoniae bacteria.
[0020] In the case of the detection of resistance mechanisms other
than ESBL, mention may be made of patent application EP0954560,
which relates to the search for Vancomycin-resistant enterococcal,
by combining Vancomycin with a chromogenic media that reveals two
enzymatic activities (.beta.-glucosidase and pyrrolidonyl
arylamidase). However, this chromogenic medium makes it possible to
determine only whether or not the vancomycin-resistant strains
belong to the Enterococcus genus, but does not make it possible to
identify the species or the resistance mechanisms involved, in
particular if it is a question of an acquired or wild-type
resistance.
[0021] Thus, the characterization of a species of microorganism,
and then the identification of its resistance to a treatment, is
long and laborious. If the laboratory gives the clinician a
positive screen, whereas the isolate is in fact free of resistant
microorganisms, this can lead to needless and inappropriate
treatment. Conversely, not communicating a positive screen, which
is subsequently confirmed, delays the setting of the isolation of
the patient (and possibly an appropriate therapy) by one day. This
shows the need for a rapid and reliable confirmation test.
[0022] The present invention therefore proposes to improve the
prior art by providing a novel diagnostic tool which allows a gain
in time, in reliability and in relevance with respect to the
therapy implemented. Our invention makes it possible, in a single
step, to identify the species of microorganisms present in a
sample, and to determine their mechanism of resistance in order to
propose a treatment appropriate to each patient. This invention is
particularly suitable for discriminating various species of
microorganisms, which have various mechanisms of resistance to
various treatments, but all of which may be present in the same
sample.
[0023] Before going any further in the disclosure of the invention,
the following definitions are given in order to facilitate
understanding of the invention:
[0024] The term "culture medium" is intended to mean a medium
comprising all the elements required for the survival and/or the
growth of microorganisms. The culture medium according to the
invention may contain any possible additives, for instance:
peptones, one or more growth factors, carbohydrates, one or more
selective agents, buffers, one or more gelling agents, etc. This
culture medium may be in liquid form or in gel form which is ready
to use, i.e. ready for seeding in a tube or flask or on a Petri
plate.
[0025] For the purpose of the present invention, the term
"microorganism" covers gram-positive or gram-negative bacteria,
yeasts and, more generally, organisms that are generally
unicellular, invisible to the naked eye, which can be multiplied
and handled in the laboratory.
[0026] By way of gram-negative bacteria, mention may be made of
bacteria of the following genres: Pseudomonas, Escherichia,
Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus,
Campylobacter, Haemophilus, Morganella, Vibrio, Yersinia,
Acinetobacter, Branhamella, Neisseria, Burkholderia, Citrobacter,
Hafnia, Edwardsiella, Aeromonas, Moraxella, Pasteurella,
Providencia, and Legionella.
[0027] By way of gram-positive bacteria, mention may be made of
bacteria of the following genre: Enterococcus, Streptococcus,
Staphylococcus, Bacillus, Listeria, Clostridium, Gardnerella,
Kocuria, Lactococcus, Leuconostoc, Micrococcus, Mycobacteria and
Corynebacteria.
[0028] By way of yeasts, mention may be made of yeasts of the
following genre: Candida, Cryptococcus, Saccharomyces and
Trichosporon.
[0029] The term "biological sample" is intended to mean a clinical
sample, derived from a specimen of biological fluid, or a food
sample, derived from any type of food. This sample may thus be
liquid or solid and mention may be made, in the nonlimiting manner,
of a clinical blood, plasma, urine or faeces sample, nose, throat,
skin, wound or cephalospinal fluid specimens, a food sample from
water, from drinks such as milk or a fruit juice; from yoghurt,
from meat, from eggs, from vegetables, from mayonnaise, from
cheese; from fish, etc., a food sample derived from a feed intended
for animals, such as, in particular, a sample derived from animal
meals.
[0030] The term "mechanism of resistance" is intended to mean any
type of device which allows a microorganism to render a treatment
partially or completely ineffective on said microorganism,
guaranteeing its survival. The mechanisms of resistance are
generally divided up into three categories: deficient penetration
of the antibiotic into the bacterium, inactivation or excretion of
the antibiotic by means of bacterial enzymatic systems, and lack of
affinity between the bacterial target and the antibiotic.
[0031] By way of indication, mention may in particular be made of
mechanisms of resistance related to the expression of an enzyme
belonging to the broad-spectrum .beta.-lactamase group; of an
enzyme belonging to the chromosomal high level class C
cephalosporinase group; mechanisms of resistance to glycopeptides,
preferably developed by bacteria belonging to the Enterococcus
genus.
[0032] Mention will also be made of mechanisms of resistance to
methicillin, penicillin, tetracycline, erythromycin, or vancomycin
when the microorganism is a Staphylococcus aureus bacterium.
[0033] Mention will also be made of mechanisms of resistance to
penicillin, vancomycin and linezolide when the microorganism is an
Enterococcus faecium bacterium.
[0034] Mention will also be made of mechanisms of resistance to
amphotericin B or to antifungals of the azole family when the
microorganism is a yeast.
[0035] Finally, mention will be made of mechanisms of resistance to
isoniazid and to rifampicin when the microorganism is a
Mycobacterium tuberculosis bacterium.
[0036] The term "treatment" is intended to mean a treatment capable
of preventing or reducing the growth of microorganisms derived from
a patient. This treatment may comprise in particular antimicrobial
compounds, such as antibiotics, for instance penicillins,
conventional cephalosporins, broad-spectrum cephalosporins,
monobactams, glycopeptides or aminosides, or such as antifungals or
resistance-inhibiting compounds. It should be noted that this
treatment can also comprise the isolation of the patient, thereby
preventing propagation of the microorganism among other
patients.
[0037] The term "substrate" which allows the detection of an
enzymatic or metabolic activity is intended to mean any molecule
capable of directing or indirectly generating a detectable signal
due to an enzymatic or metabolic activity of the microorganism.
[0038] When this activity is an enzymatic activity, reference is
then made to an enzymatic substrate. The term "enzymatic substrate"
is intended to mean any substrate that can be hydrolyzed by an
enzyme into a product that allows the direct or indirect detection
of a microorganism. This substrate comprises in particular a first
part that is specific for the enzymatic activity to be revealed and
a second part that acts as a label, hereinafter referred to as
labeling part. This labeling part may be chromogenic, fluorogenic,
luminescent, etc. As chromogenic substrate suitable for solid
supports (filter, agar, electrophoresis gel), mention may in
particular be made of substrates based on indoxyl and its
derivatives, and substrates based on hydroxyquinoline or escultin
and their derivatives, which allow the detection of osidase and
esterase activities. Mention may also be made of substrates based
on nitrophenol and nitroaniline and derivatives, making it possible
to detect osidase and esterase activities in the case of substrates
based on nitrophenol, and peptidase activities in the case of
substrates based on nitroaniline. Finally, mention may be made of
substrates based on naphtol and naphtylamine and their derivatives,
which make it possible to detect osidase and esterase activities
via naphtol, and peptidase activities via naphtylamine. This
substrate may allow, in particular, but in a nonlimiting manner,
the detection of an enzymatic activity such as the activity of an
osidase, peptidase, esterase, etc. The enzymatic substrate can also
be a natural substrate of which the product of hydrolysis is
detected directly or indirectly. As natural substrate, mention may
in particular be made of tryptophan for detecting tryptophanase or
desaminase activity, a cyclic amino acid (tryptophan,
phenylalanine, histidine, tyrosine) for detecting desaminase
activity, phosphatidyl inositol for detecting phospholipase
activity, etc.
[0039] When this activity is a metabolic activity, the substrate is
then a metabolic substrate, such as a source of carbon or of
nitrogen, coupled to an indicator that produces a coloration in the
presence of one of the metabolic products.
[0040] According to a preferred embodiment of the invention, said
first and/or second enzymatic or metabolic activity is an enzymatic
activity preferably chosen from the enzymatic activities:
beta-glucosidase, desaminase, beta-glucuronidase,
beta-galactosidase, alpha-glucosidase, alpha-galactosidase,
hexosaminidase, N-acetyl-hexosaminidase, phosphatase, esterase, and
aminopeptidase.
[0041] For example, for detecting E. coli, use is preferably made
of beta-glucuronidase or .beta.-galactosidase or tryptophanase or
desaminase activity; for detecting Proteus, use is preferably made
of desaminase activity; for detecting enterococci, use is
preferably made of beta-glucosidase activity. For Candida albicans,
hexosaminidase is preferred, for Listeria monocytogenes,
phospholipase is preferred, for salmonellae, esterase is preferred,
for Pseudomonas aeruginosa, esterase or .beta.-alanine
aminopeptidase is preferred, for Staphylococcus aureus phosphatase
or alpha-glucosidase is preferred.
[0042] The expression "marker for differentiating" two groups of
microorganisms is intended to mean a compound which does not have
the same properties on a first and on a second group. This compound
may thus be: [0043] a specific substrate; [0044] an inhibitor of a
mechanism of resistance, which then makes it possible to inhibit
the growth of the organisms developing a specific resistance,
without any discrimination of the microorganism species.
[0045] In the case of the use of a specific substrate, use is
preferably made of beta-glucuronidase, beta-galactosidase,
tryptophanase or desaminase activity for detecting E. coli, use is
preferably made of desaminase activity for detecting Proteus,
[0046] use is preferably made of beta-glucosidase activity for
detecting enterococci. For Candida albicans, hexosaminidase is
preferred, for Listeria monocytogenes, phospholipase is preferred,
for salmonellae,
[0047] esterase is preferred, for Pseudomonas aeruginosa, esterase
or .beta.-alanine aminopeptidase is preferred, for Staphylococcus
aureus, phosphatase or alpha-glucosidase is preferred.
[0048] In the case of the use of an inhibitor of a mechanism of
resistance, use is preferably made of: [0049] clavulanic acid,
tazobactam or sulbactam when the first group and/or the second
group comprises a mechanism of resistance induced by an expression
of .beta.-lactamases. The clavulanic acid concentration in the
medium is then preferably between 0.05 and 32 mg/l, preferably
between 0.1 and 8 mg/l, and even more preferably between 0.25 and 6
mg/l; [0050] cloxacillin or dicloxacillin when the first group
and/or the second group comprises a mechanism of resistance induced
by the expression of cephalosporinases.
[0051] The term "taxon" is intended to mean a group of
microorganisms having a taxonomic unit. A taxon may be a family, a
genus, a set of genre, a species, a set of species or a subspecies.
By way of indication, mention may be made of enterobacteria,
Klebsiella, Escherichia, Enterobacter, Citrobacter, Serratia, KESC
(Klebsiella, Enterobacter, Serratia, Citrobacter), Proteeae,
Proteus, Morganella, Pseudomonas, Staphylococcus, Streptococcus,
Enterococcus, Candida, Escherichia coli, Escherichia coli O157:H7,
Klebsiella pneumoniae, Citrobacter freundii, Enterococcus faecalis,
Enterococcus faecium, Staphylococcus aureus, coagulase-negative
staphylocoque, Candida albicans, Candida glabrata, Candida krusei,
Candida lusitaniae.
[0052] The term "antimicrobial" is intended to mean any compound
capable of preventing or slowing down the growth of a
microorganism. This compound may be an antibiotic or an
antifungal.
[0053] The term "antibiotic" is intended to mean any compound
capable of preventing or slowing down the growth of a bacterium. By
way of indication, mention may in particular be made of the
antibiotics cefotaxime, ceftazidime, ceftriaxone, cefpodoxime,
aztreonam, vancomycin, tobramycin and ciprofloxacin.
[0054] The term "antifungal" is intended to mean any compound
capable of preventing or slowing down the growth of a yeast or of a
mould. By way of indication, mention may in particular be made of
amphotericin B, fluconazole, itraconazole, voriconazole and
cycloheximide.
[0055] According to a preferred embodiment of the invention, when
the antibiotic is [0056] cefotaxime, the cefotaxime concentration
in the medium is preferably between 0.25 and 8 mg/l, preferably
between 1 and 2 mg/l; [0057] ceftazidime, the ceftazidime
concentration in the medium is preferably between 0.25 and 8 mg/l,
preferably between 2 and 2.5 mg/l; [0058] ceftriaxone, the
ceftriaxone concentration in the medium is preferably between 0.25
and 8 mg/l, preferably between 1 and 2.5 mg/l; [0059] cefpodoxime,
the cefpodoxime concentration in the medium is preferably between
0.1 and 32 mg/l, preferably between 0.75 and 10 mg/l, and even more
preferably between 1 and 6 mg/l; [0060] aztreonam, the aztreonam
concentration in the medium is preferably between 0.1 and 8 mg/l,
preferably between 0.75 and 1.5 mg/l.
[0061] According to a specific embodiment of the invention, the
medium comprises a combination of at least two antibiotics.
Preferably, the combination of at least two antibiotics comprises
cefotaxime and ceftazidime.
[0062] Irrespective of the embodiment of the invention, the medium
may also comprise a dye. By way of indication of a dye, mention may
be made of Evans blue, neutral red, sheep blood, horse blood, and
opacifier such as titanium oxide, nitroaniline, malachite green,
brilliant green, etc.
[0063] All the media may also comprise, in order to increase their
sensitivity: [0064] at least one antimicrobial that is active
against gram-positive bacteria, such as in particular linezolide or
vancomycin; [0065] at least one antimicrobial that is active
against yeasts, such as in particular voriconazole or amphotericin
B.
[0066] In this respect, the invention relates to the use of a
combination of two culture media for distinguishing at least three
groups of microorganisms in a biological sample, comprising: [0067]
a first group of microorganisms, belonging to a first taxon of
microorganisms and comprising at least a first mechanism of
resistance to a first treatment; [0068] a second group of
microorganisms, belonging to a second taxon of microorganisms and
comprising at least a second mechanism of resistance to a second
treatment; [0069] a third group of microorganisms, that are not
resistant to said first and second treatments,
[0070] said combination of two culture media comprising: [0071] a.
at least a first substrate for detecting at least a first enzymatic
or metabolic activity of said first group of microorganisms; [0072]
b. at least two markers for differentiating the first group of
microorganisms and the second group of microorganisms; [0073] c. at
least one antimicrobial that is active on said third group of
microorganisms.
[0074] Preferably, the medium comprises at least two markers for
differentiating the first group of microorganisms and the second
group of microorganisms, at least one of which is an inhibitor of
said first or said second mechanism of resistance. The medium may
also comprise at least two markers for differentiating the first
group of microorganisms and the second group of microorganisms,
which are each an inhibitor of said first or said second mechanism
of resistance.
[0075] This first embodiment of the invention makes it possible to
distinguish, in the same sample, a first and a second group
comprising various species or various taxons of microorganisms,
each of the two groups being resistant to a different
treatment.
[0076] This embodiment of the invention thus makes it possible, for
example, to distinguish, in the same sample, a first group
comprising E. coli ESBL bacteria and a second group comprising KESC
HL Case bacteria. In this specific case, the combination of two
media may be the following: [0077] At least a first substrate which
makes it possible to identify E. coli bacteria, for example a
glucuronidase substrate, such as
6-chloro-3-indolyl-.beta.-D-glucuronide, or a galactosidase
substrate, such as 5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside;
[0078] a first identification marker which is a glucosidase
substrate, such as 5-bromo-4-chloro-3-indolyl-.beta.-D-glucoside,
which makes it possible to identify KESC bacteria; [0079] a second
identification marker which is a resistance mechanism inhibitor,
such as cloxacillin; [0080] at least one antimicrobial which is an
antibiotic, such as cefpodoxime.
[0081] Reference is made to a combination of 2 media, i.e. a first
medium may comprise: [0082] a first substrate which makes it
possible to identify E. coli, for example a glucuronidase
substrate, such as 6-chloro-3-indolyl-.beta.-D-glucuronide or a
galactosidase substrate, such as
5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside; [0083] a first
marker which is a resistance mechanism inhibitor, such as
cloxacillin; [0084] an antimicrobial which is an antibiotic, such
as cefpodoxime,
[0085] or the second medium comprises: [0086] a first substrate
which makes it possible to identify E. coli, for example a
glucuronidase substrate, such as
6-chloro-3-indolyl-.beta.-D-glucuronide, or a galactosidase
substrate, such as 5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside;
[0087] a first marker which is a glucosidase substrate, such as
5-bromo-4-chloro-3-indolyl-.beta.-D-glucoside, which makes it
possible to identify KESC bacteria; [0088] an antimicrobial which
is an antibiotic, such as ceftazidime.
[0089] Another alternative would be to use a first medium
comprising: [0090] a first substrate which makes it possible to
identify E. coli, for example a glucuronidase substrate, such as
6-chloro-3-indolyl-.beta.-D-glucuronide, or a galactosidase
substrate, such as 5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside;
[0091] a first marker which is a glucosidase substrate, such as
5-bromo-4-chloro-3-indolyl-.beta.-D-glucoside, which makes it
possible to identify KESC bacteria; [0092] an antimicrobial which
is an antibiotic, such as aztreonam,
[0093] while the second medium comprises: [0094] a first substrate
which makes it possible to identify E. coli, for example a
glucuronidase substrate, such as
6-chloro-3-indolyl-.beta.-D-glucuronide, or a galactosidase
substrate, such as 5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside;
[0095] a first marker which is a resistance mechanism inhibitor,
such as clavulanic acid; [0096] an antimicrobial which is an
antibiotic, such as ceftazidime.
[0097] Those skilled in the art will choose each medium so as to
systematically obtain a combination according to the invention. The
antimicrobial that is active on said third group is present in the
two media. Those skilled in the art may in particular use a
biplate, for readily comparing the two media on which the same
biological sample has been deposited.
[0098] In this respect, the invention also relates to a biplate
comprising a combination of two culture media, said combination
comprising: [0099] a. at least a first substrate for detecting at
least a first enzymatic or metabolic activity of said first group
of microorganisms; [0100] b. at least two markers for
differentiating the first group of microorganisms and the second
group of microorganisms; [0101] c. at least one antimicrobial that
is active on said third group of microorganisms, said antimicrobial
being present on each side of the biplate.
[0102] This first embodiment of the invention is not limited to
distinguishing between 3 groups of microorganisms, but may make it
possible to distinguish between 4, 5 or even more groups of
microorganisms. It is then necessary to add additional
identification markers to the medium or media, in order to
discriminate between the various groups.
[0103] In this respect, the invention also relates to a biplate
comprising a combination of two culture media, said combination
comprising:
[0104] for the first medium: [0105] a first substrate which makes
it possible to identify E. coli, for example a glucuronidase
substrate such as 6-chloro-3-indolyl-.beta.-D-glucuronide, or a
galactosidase substrate, such as
5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside, or a substrate
which makes it possible to identify Proteeae, for example
tryptophan; [0106] a first identification marker which is a
glucosidase substrate, such as
5-bromo-4-chloro-3-indolyl-.beta.-D-glucoside; [0107] a second
identification marker which is a resistance inhibitor, such as
cloxacillin; [0108] at least one antimicrobial which is an
antibiotic, such as cefpodoxime;
[0109] for the second medium: [0110] a first substrate which makes
it possible to identify enterococci, for example a glucosidase
substrate, such as 5-bromo-4-chloro-3-indolyl-.beta.-D-glucoside;
[0111] a first identification marker which is another glucosidase
substrate, such as methyl-.alpha.-glucoside; [0112] at least one
antimicrobial which is an antibiotic, such as vancomycin.
[0113] The invention also relates to the use of a culture medium
for distinguishing at least 3 groups of microorganisms in a
biological sample, comprising: [0114] a first group of
microorganisms, belonging to a first taxon of microorganisms and
comprising at least one mechanism of resistance to a treatment;
[0115] a second group of microorganisms, belonging to a second
taxon of microorganisms, different than said first taxon, but
comprising at least one mechanism of resistance to a treatment,
identical to that of the first group; [0116] a third group of
microorganisms, that are not resistant to said treatment, said
culture medium comprising: [0117] a. at least a first substrate for
detecting at least a first enzymatic or metabolic activity of said
first group of microorganisms; [0118] b. at least one marker for
differentiating the first group of microorganisms and the second
group of microorganisms, said marker being a substrate for
detecting at least one enzymatic or metabolic activity of said
second group of microorganisms; [0119] c. at least one
antimicrobial that is active on said third group of
microorganisms.
[0120] This second embodiment of the invention makes it possible to
distinguish, in the same sample, a first and a second group
comprising various species or various taxons of microorganisms, but
each of the two groups being resistant to the same treatment. In
this specific embodiment of the invention, it is not necessary to
use a combination of media, since a single medium comprising the
characteristics as defined above is sufficient.
[0121] This embodiment of the invention thus makes it possible, for
example, to distinguish, in the same sample, a first group
comprising E. coli ESBL bacteria and a second group comprising KESC
ESBL bacteria.
[0122] In this respect, the invention relates to a culture medium
comprising: [0123] a first substrate for detecting a
beta-glucosidase enzymatic activity, preferably
5-bromo-4-chloro-3-indolyl-.beta.-D-glucoside, at a concentration
of between 25 and 500 mg/l, preferably between 40 and 150 mg/l;
[0124] a second substrate for detecting methyl-alpha-glucoside
metabolism, in the presence of a colored indicator, preferably
neutral red, at a concentration of between 2 and 100 mg/l,
preferably between 4 and 50 mg/l; [0125] an antimicrobial which is
an antibiotic, preferably vancomycin, at a concentration of between
0.5 and 128 mg/l, preferably between 2 and 32 mg/l.
[0126] When the antibiotic is vancomycin, this medium is preferably
used for distinguishing: [0127] a first group of enterococcal
bacteria developing an acquired resistance against vancomycin;
[0128] a second group of enterococcal bacteria developing a natural
resistance against vancomycin; [0129] a third group of enterococcal
bacteria that are not resistant to vancomycin.
[0130] The invention also relates to a culture medium comprising:
[0131] a first substrate for detecting a hexosaminidase enzymatic
activity, preferably
5-bromo-4-chloro-3-indolyl-N-acetyl-.beta.-D-glucosaminide, and a
concentration between 25 and 500 mg/l, preferably between 40 and
150 mg/l; [0132] a second substrate for detecting a
beta-glucosidase activity, preferably
6-chloro-3-indolyl-.beta.-D-glucoside, at a concentration of
between 25 and 500 mg/l, preferably between 40 and 200 mg/l; [0133]
an antimicrobial which is an antifungal, which is preferably
amphotericin B (amphoB), at a concentration of between 0.5 and 64
mg/l, preferably between 1 and 16 mg/l, even more preferably
between 1 and 8 mg/l.
[0134] When the antifungal is amphotericin B, this medium is
preferably used to distinguish: [0135] a first group of yeasts
comprising Candida albicans developing a resistance to amphoB;
[0136] a second group of yeasts comprising Candida tropicalis
and/or C. lusitaniae and/or C. kefyr, developing a resistance to
amphoB; [0137] a third group of yeasts that are not resistant to
amphoB.
[0138] The invention also relates to a culture medium comprising:
[0139] a first substrate for detecting a hexosaminidase enzymatic
activity, preferably
5-bromo-4-chloro-3-indolyl-N-acetyl-.beta.-D-glucosaminide, at a
concentration of between 25 and 500 mg/l, preferably between 40 and
150 mg/l; [0140] a second substrate for detecting a phosphatase
activity, preferably 5-bromo-6-chloro-3-indolylphosphate, at a
concentration of between 25 and 750 mg/l, preferably between 40 and
200 mg/l; [0141] an antimicrobial which is an antifungal, which is
preferably amphoB, at a concentration of between 0.5 and 64 mg/l,
preferably between 1 and 16 mg/l, even more preferably between 1
and 8 mg/l.
[0142] When the antifungal is amphotericin B, this medium is
preferably used to distinguish: [0143] a first group of yeasts
comprising Candida albicans developing a resistance to amphoB;
[0144] a second group of yeasts comprising Candida tropicalis
and/or C. glabrata and/or C. krusei, developing a resistance to
amphoB; [0145] a third group of yeasts that are not resistant to
amphoB.
[0146] The invention also relates to a culture medium comprising:
[0147] a first substrate for detecting the hexosaminidase enzymatic
activity of said first group, preferably
5-bromo-4-chloro-3-indolyl-N-acetyl-.beta.-D-glucosaminide, at a
concentration of between 25 and 500 mg/l, preferably of between 40
and 150 mg/l; [0148] a second substrate for detecting the
beta-glucosidase activity of said second group, preferably
6-chloro-3-indolyl-.beta.-D-glucoside, at a concentration of
between 25 and 500 mg/l, preferably between 40 and 200 mg/l; [0149]
an antimicrobial which is an antifungal, preferably fluconazole, at
a concentration of between 1 and 256 mg/l, preferably between 2 and
128 mg/l, even more preferably between 8 and 64 mg/l.
[0150] When the antifungal is fluconazole, this medium is
preferably used to distinguish: [0151] a first group of yeasts
comprising Candida albicans developing a resistance to fluconazole;
[0152] a second group of yeasts, comprising Candida tropicalis
and/or C. lusitaniae and/or C. kefyr, developing a resistance to
fluconazole; [0153] a third group of yeasts that are not resistant
to fluconazole.
[0154] The invention also relates to a culture medium comprising:
[0155] a first substrate for detecting a hexosaminidase enzymatic
activity, preferably
5-bromo-4-chloro-3-indolyl-N-acetyl-.beta.-D-glucosaminide, at a
concentration of between 25 and 500 mg/l, preferably between 40 and
150 mg/l; [0156] a second substrate for detecting a phosphatase
activity, preferably 5-bromo-6-chloro-3-indolylphosphate, at a
concentration of between 25 and 750 mg/l, preferably between 40 and
200 mg/l; [0157] an antimicrobial which is an antifungal, which is
preferably fluconazole, at a concentration of between 1 and 256
mg/l, preferably between 2 and 128 mg/l, even more preferably
between 8 and 64 mg/l.
[0158] When the antifungal is fluconazole, this medium is
preferably used to distinguish: [0159] a first group of yeasts
comprising Candida albicans developing a resistance to fluconazole;
[0160] a second group of yeasts, comprising Candida tropicalis
and/or C. glabrata and/or C. krusei, developing a resistance to
fluconazole; [0161] a third group of yeasts that are not resistant
to fluconazole.
[0162] The invention also relates to a culture medium comprising:
[0163] a first substrate for detecting a beta-glucuronidase
enzymatic activity, preferably
6-chloro-3-indolyl-.beta.-D-glucuronide, at a concentration of
between 25 and 750 mg/l, preferably between 40 and 300 mg/l, or a
beta-galactosidase enzymatic activity, preferably
5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside, at a concentration
of between 25 and 500 mg/l, preferably between 40 and 150 mg/l;
[0164] a second substrate for detecting a beta-glucosidase
activity, preferably 5-bromo-4-chloro-3-indolyl-.beta.-D-glucoside,
at a concentration of between 25 and 500 mg/l, preferably between
40 and 250 mg/l, or a tryptophanase or desaminase activity,
preferably tryptophan, at a concentration of between 50 and 5000
mg/l, preferably between 250 and 2000 mg/l; [0165] an antimicrobial
which is an antibiotic, preferably ceftazidime. The ceftazidime
concentration in the medium is then preferably between 0.25 and 8
mg/l, preferably between 2 and 2.5 mg/l.
[0166] When the antibiotic is ceftazidime, this medium is
preferably used to distinguish: [0167] a first group of E. coli
ESBL or HL Case bacteria; [0168] a second group of KESC ESBL or HL
Case bacteria; [0169] a third group of bacteria that are not
resistant to beta-lactamines and to cephalosporins.
[0170] The invention also relates to a culture medium comprising:
[0171] a first substrate for detecting a beta-glucuronidase or
beta-galactosidase enzymatic activity, preferably
6-chloro-3-indolyl-.beta.-D-glucuronide, at a concentration of
between 25 and 750 mg/l, preferably between 40 and 300 mg/l, or
5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside, at a concentration
of between 25 and 500 mg/l, preferably between 40 and 150 mg/l;
[0172] a second substrate for detecting a beta-glucosidase
activity, preferably 5-bromo-4-chloro-3-indolyl-.beta.-D-glucoside,
at a concentration of between 25 and 500 mg/l, preferably between
40 and 250 mg/l, or a tryptophanase or desaminase activity,
preferably tryptophan, at a concentration of between 50 and 5000
mg/l, preferably between 250 and 2000 mg/l; [0173] an antimicrobial
which is an antibiotic, preferably cefpodoxime, at a concentration
of between 0.5 and 32 mg/l, preferably between 0.75 and 10 mg/l,
and even more preferably between 1 and 6 mg/l, and cloxacillin, at
a concentration of between 10 and 2000 mg/l, preferably between 50
and 500 mg/l.
[0174] When the antibiotic is cefpodoxime, this medium is
preferably used to distinguish: [0175] a first group of E. coli
ESBL bacteria; [0176] a second group of KESC ESBL bacteria; [0177]
a third group of bacteria that are not resistant to
beta-lactamines.
[0178] The invention also relates to a culture medium comprising:
[0179] a first substrate for detecting a beta-glucuronidase
enzymatic activity, preferably
6-chloro-3-indolyl-.beta.-D-glucuronide, at a concentration of
between 25 and 750 mg/l, preferably between 40 and 300 mg/l, or a
beta-galactosidase enzymatic activity, preferably
5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside, at a concentration
of between 25 and 500 mg/l, preferably between 40 and 150 mg/l;
[0180] a second substrate for detecting a beta-glucosidase
activity, preferably 5-bromo-4-chloro-3-indolyl-.beta.-D-glucoside,
at a concentration of between 25 and 500 mg/l, preferably between
40 and 250 mg/l, or a tryptophanase or desaminase activity,
preferably tryptophan, at a concentration of between 50 and 5000
mg/l, preferably between 250 and 2000 mg/l; [0181] a combination
comprising an antimicrobial and a resistance inhibitor. Preferably,
this combination comprises ceftriaxone at a concentration of
between 0.25 and 8 mg/l, preferably between 1 and 2.5 mg/l, and
clavulanic acid at a concentration, of between 0.05 and 32 mg/l,
preferably between 0.1 and 8 mg/l, and even more preferably between
0.5 and 6 mg/l.
[0182] This medium is preferably used to distinguish: [0183] a
first group of E. coli HL Case bacteria; [0184] a second group of
KESC HL Case bacteria; [0185] a third group of bacteria that are
not resistant to cephalosporins.
[0186] The invention also relates to a culture medium comprising:
[0187] a first substrate for detecting a beta-glucuronidase
enzymatic activity, preferably
6-chloro-3-indolyl-.beta.-D-glucuronide, at a concentration of
between 25 and 750 mg/l, preferably between 40 and 300 mg/l, or a
beta-galactosidase enzymatic activity, preferably
5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside, at a concentration
between 25 and 500 mg/l, preferably between 40 and 150 mg/l; [0188]
a second substrate for detecting a beta-glucosidase activity,
preferably 5-bromo-4-chloro-3-indolyl-.beta.-D-glucoside, at a
concentration of between 25 and 500 mg/l, preferably between 40 and
250 mg/l, or a tryptophanase or desaminase activity, preferably
tryptophan, at a concentration of between 50 and 5000 mg/l,
preferably between 250 and 2000 mg/l; [0189] two antimicrobials,
which are preferably two antibiotics, preferably cefpodoxime at a
concentration of between 0.1 and 32 mg/l, preferably between 0.25
and 10 mg/l, and even more preferably between 0.5 and 4 mg/l, and
aztreonam at a concentration of between 0.1 and 8 mg/l, preferably
between 0.5 and 1.5 mg/l.
[0190] This medium is preferably used to distinguish: [0191] a
first group of E. coli ESBL or HL Case bacteria; [0192] a second
group of KESC ESBL or HL Case bacteria; [0193] a third group of
bacteria that are not resistant to beta-lactamines and/or to
cephalosporins.
[0194] The invention also relates to a culture medium comprising:
[0195] at least a first substrate for detecting alpha-glucoside
metabolism, preferably methyl-.alpha.-glucoside, at a concentration
of between 1 and 50 g/l, preferably between 5 and 20 g/l, or
5-bromo-4-chloro-3-indolyl-.alpha.-D-glucoside, at a concentration
of between 25 and 500 mg/l, preferably between 40 and 250 mg/l, or
5-bromo-4-chloro-3-indolyl-N-methyl-.alpha.-D-glucoside, at a
concentration of between 25 and 500 mg/l, preferably between 40 and
250 mg/l; [0196] at least a second substrate for detecting a second
activity different than alpha-glucoside metabolism, preferably
5-bromo-4-chloro-3-indolyl-.beta.-D-glucoside, at a concentration
of between 25 and 500 mg/l, preferably between 40 and 250 mg/l, or
6-chloro-3-indolyl-.beta.-D-glucoside, at a concentration of
between 25 and 500 mg/l, preferably between 40 and 250 mg/l, or
alizarine-.beta.-D-galactoside, at a concentration of between 10
and 500 mg/l, preferably between 20 and 250 mg/l, or
5-bromo-6-chloro-3-indolyl-.beta.-D-glucoside, at a concentration
of between 25 and 500 mg/l, preferably between 40 and 250 mg/l, or
5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside, at a concentration
of between 25 and 500 mg/l, preferably between 40 and 250 mg/l, or
6-chloro-3-indolyl-.beta.-D-galactoside, at a concentration of
between 25 and 500 mg/l, preferably between 40 and 250 mg/l.
Preferably, this second substrate makes it possible to detect a
beta-glucosidase or beta-galactosidase activity; [0197] at least
one antimicrobial, preferably an antibiotic, such as vancomycin, at
a concentration of between 0.5 and 128 mg/l, preferably between 2
and 32 mg/l.
[0198] When the antibiotic is vancomycin, this medium is preferably
used to distinguish: [0199] a first group of vancomycin-resistant
microorganisms, comprising Enterococcus faecalis and Enterococcus
faecium; [0200] a second group of vancomycin-resistant
microorganisms, comprising Enterococcus casseliflavus and
Enterococcus gallinarum; [0201] a third group of microorganisms
that are not resistant to vancomycin.
[0202] In this case, the first substrate is preferably
methyl-.alpha.-glucoside, the second substrate is preferably
5-bromo-4-chloro-3-indolyl-.beta.-D-glucoside or
6-chloro-3-indolyl-.beta.-D-glucoside, and the antimicrobial is
preferably vancomycin.
[0203] This medium is also preferably used to distinguish: [0204] a
first group of vancomycin-resistant microorganisms, comprising
Enterococcus faecalis; [0205] a second group of
vancomycin-resistant microorganisms, comprising Enterococcus
faecium; [0206] a third group of microorganisms that are not
resistant to vancomycin or that express a natural resistance (E.
casseliflavus and E. gallinarum).
[0207] In this case, the first substrate is preferably
5-bromo-4-chloro-3-indolyl-N-methyl-.alpha.-D-glucoside or
5-bromo-4-chloro-3-indolyl-.alpha.-D-glucoside, the second
substrate is preferably 6-chloro-3-indolyl-.beta.-D-glucoside or
alizarine-.beta.-D-galactoside or 5
bromo-6-chloro-3-indolyl-.beta.-D-glucoside or
5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside or
6-chloro-3-indolyl-.beta.-D-galactoside, and the antimicrobial is
preferably vancomycin.
[0208] This medium is also preferably used to distinguish: [0209] a
first group of vancomycin-resistant microorganisms, comprising
Enterococcus faecalis and Enterococcus faecium; [0210] a second
group of microorganisms, comprising Staphylococcus aureus, that are
intermediately resistant or resistant to vancomycin; [0211] a third
group of microorganisms that are not resistant to vancomycin.
[0212] In this case, the first substrate is preferably
5-bromo-4-chloro-3-indolyl-N-methyl-.alpha.-D-glucoside or
5-bromo-4-chloro-3-indolyl-.alpha.-D-glucoside, the second
substrate is preferably 6-chloro-3-indolyl-.beta.-D-glucoside or
5-bromo-6-chloro-3-indolyl-.beta.-D-glucoside, and the
antimicrobial is preferably vancomycin.
[0213] The table below makes it possible to distinguish the
appropriate combinations of substrates and antimicrobial according
to the species that it is desired to detect;
TABLE-US-00001 1st group of 2nd group of 3rd group of
microorganisms microorganisms microorganisms 1st substrate 2nd
substrate Antimicrobial E. faecalis and E. casseliflavus
Microorganisms Methyl-.alpha.-glucoside 5-bromo- vancomycin E.
faecium, and E. gallinarum, that are not 4-chloro- resistant to
resistant to resistant to 3-indolyl- vancomycin vancomycin
vancomycin .beta.-D-glucoside or 6-chloro- 3-indolyl-
.beta.-D-glucoside E. faecalis, E. faecium, Microorganisms
5-bromo-4-chloro- 6-chloro- vancomycin resistant to resistant to
that are not 3-indolyl-N-methyl- 3-indolyl- vancomycin vancomycin
resistant to .alpha.-D-glucoside or .beta.-D-glucoside or
vancomycin or 5-bromo-4-chloro- alizarine- that express a
3-indolyl- .beta.-D-galactoside natural resistance
.alpha.-D-glucoside or 5-bromo- (E. casseliflavus 6-chloro-3- and
E. gallinarum) indolyl-.beta.- D-glucoside or 5-bromo-6-
chloro-3-indolyl- .beta.-D-galactoside or 6-chloro- 3-indolyl-
.beta.-D-galactoside E. faecalis and S. aureus, Microorganisms
5-bromo-4-chloro- 6-chloro- vancomycin E. faecium, resistant to
that are not 3-indolyl-N-methyl- 3-indolyl- resistant to vancomycin
resistant to .alpha.-D-glucoside or .beta.-D-glucoside or
vancomycin vancomycin 5-bromo-4-chloro- 5-bromo- 3-indolyl-
6-chloro-3- .alpha.-D-glucoside indolyl-.beta.-D- glucoside
[0214] It may be relevant to also adjust the vancomycin
concentration, preferably to between 0.5 and 12 mg/l.
[0215] The invention also relates to a culture medium comprising:
[0216] an antimicrobial, preferably an antibiotic such as
cloxacillin; [0217] a resistance mechanism inhibitor, such as
preferably a 3rd-generation cephalosporin chosen from cefotaxime,
ceftazidime, cefpodoxime and ceftriaxone.
[0218] This medium is also preferably used to detect ESBL
bacteria.
[0219] The second embodiment of the invention is not limited to
distinguishing 3 groups of microorganisms, but can make it possible
to distinguish 4, 5 or even more groups of microorganisms. It is
then necessary to add, to the medium, markers for identification
between the various groups.
[0220] In this respect, the invention also relates to a culture
medium comprising: [0221] a first substrate for detecting a
beta-glucuronidase enzymatic activity, preferably
6-chloro-3-indolyl-.beta.-D-glucuronide, at a concentration of
between 25 and 750 mg/l, preferably between 40 and 300 mg/l, or a
beta-galactosidase enzymatic activity, preferably
5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside, at a concentration
of between 25 and 500 mg/l, preferably of between 40 and 150 mg/l;
[0222] a second substrate for detecting a beta-glucosidase,
preferably 5-bromo-4-chloro-3-indolyl-.beta.-D-glucoside, at a
concentration of between 25 and 500 mg/l, preferably between 40 and
250 mg/l; [0223] a combination of antimicrobials, preferably a
combination of antibiotics such as [0224] cefpodoxime at a
concentration of between 0.5 and 32 mg/l, preferably between 0.75
and 10 mg/l, and even more preferably between 1 and 6 mg/l; [0225]
cloxacillin at a concentration of between 10 and 2000 mg/l,
preferably between 50 and 500 mg/l; [0226] vancomycin at a
concentration of between 0.5 and 128 mg/l, preferably between 2 and
32 mg/l; and [0227] amphoB at a concentration of between 0.5 and 64
mg/l, preferably between 1 and 16 mg/l, even more preferably
between 1 and 8 mg/l; [0228] a third substrate for detecting a
desaminase activity, such as histidine, phenylalanine, tryptophan
or tyrosine, at a concentration of between 50 and 5000 mg/l,
preferably between 250 and 2000 mg/l.
[0229] This medium may also comprise a fifth antibiotic, which is
cefsulodine, at a concentration of between 0.5 and 64 mg/l,
preferably between 1 and 16 mg/l.
[0230] This medium is preferably used to distinguish: [0231] a
first group of E. coli ESBL bacteria; [0232] a second group of KESC
ESBL bacteria; [0233] a third group of bacteria that are not
resistant to beta-lactamines and/or to cephalosporins; [0234] a
fourth group of Proteeae ESBL bacteria.
[0235] Similarly, the invention also relates to a culture medium
comprising: [0236] at least a first substrate for detecting
alpha-glucoside metabolism, preferably
5-bromo-4-chloro-3-indolyl-.alpha.-D-glucoside, at a concentration
of between 25 and 500 mg/l, preferably between 40 and 250 mg/l, or
5-bromo-4-chloro-3-indolyl-N-methyl-.alpha.-D-glucoside, at a
concentration of between 25 and 500 mg/l, preferably between 40 and
250 mg/l; [0237] at least a second substrate for detecting a second
activity different than alpha-glucoside metabolism, preferably
6-chloro-3-indolyl-.beta.-D-glucoside, at a concentration of
between 25 and 500 mg/l, preferably between 40 and 250 mg/l, or
6-chloro-3-indolyl-.beta.-D-galactoside, at a concentration of
between 25 and 500 mg/l, preferably between 40 and 250 mg/l, or
alizarine-.beta.-galactoside, at a concentration of between 10 and
500 mg/l, preferably between 20 and 250 mg/l, or
5-bromo-6-chloro-3-indolyl-.beta.-D-glucoside, at a concentration
of between 25 and 500 mg/l, preferably between 40 and 250 mg/l, or
5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside, at a concentration
of between 25 and 500 mg/l, preferably between 40 and 250 mg/l, or
6-chloro-3-indolyl-.beta.-D-galactoside, at a concentration of
between 25 and 500 mg/l, preferably between 40 and 250 mg/l.
Preferably, this second substrate makes it possible to detect a
beta-glucosidase or beta-galactosidase activity; [0238] a
combination of antimicrobials, preferably a combination of
antibiotics such as [0239] vancomycin at a concentration of between
0.5 and 128 mg/l, preferably between 2 and 32 mg/l; [0240]
aztreonam at a concentration of between 1 and 150 mg/l, preferably
between 4 and 60 mg/l; [0241] colistine at a concentration of
between 1 and 100 mg/l, preferably between 2 and 20 mg/l; [0242]
amphotericin B at a concentration of between 0.5 and 50 mg/l,
preferably between 1 and 15 mg/l.
[0243] When one of the antibiotics is vancomycin, this medium is
preferably used to distinguish: [0244] a first group of
vancomycin-resistant microorganisms, comprising Enterococcus
faecium, [0245] a second group of vancomycin-resistant
microorganisms, comprising Enterococcus faecalis, [0246] a third
group of microorganisms that are not resistant to vancomycin.
[0247] This medium is also preferably used to distinguish: [0248] a
first group of vancomycin-resistant microorganisms, comprising
Enterococcus faecium, [0249] a second group of microorganisms,
comprising Staphylococcus aureus, that are intermediately resistant
or resistant to vancomycin; [0250] a third group of microorganisms
that are not resistant to vancomycin.
[0251] The combinations of substrates according to the groups of
microorganisms that it is desired to identify are presented, for
example, in the table on page 21. By using an appropriate
combination of antimicrobials, it is possible to distinguish not
only three groups of microorganisms, but also 4, 5 or even more
groups of microorganisms.
[0252] The invention also relates to the use of a combination of
two culture media for distinguishing at least 3 groups of
microorganisms in a biological sample, comprising: [0253] a first
group of microorganisms, belonging to a first species of
microorganisms and comprising a first mechanism of resistance to a
first treatment; [0254] a second group of microorganisms, belonging
to a species of microorganisms that is identical to that of said
first group of microorganisms, but comprising a second mechanism of
resistance to a second treatment, different than that of said first
group; [0255] a third group of microorganisms that are not
resistant to said first and second treatment,
[0256] said combination of two culture media comprising: [0257] a.
at least a first substrate for detecting at least a first enzymatic
or metabolic activity of said first group of microorganisms; [0258]
b. at least one marker for differentiating the first group of
microorganisms and the second group of microorganisms, said marker
being an inhibitor of the mechanism of resistance to the first
treatment and/or to the second treatment; [0259] c. at least one
antimicrobial that is active on said third group of
microorganisms.
[0260] This third embodiment of the invention makes it possible to
distinguish, in the same sample, a first and a second group
comprising the same species of microorganisms or the same taxon,
each of the two groups being resistant to different treatments.
[0261] This embodiment of the embodiment thus makes it possible,
for example, to distinguish, in the same sample, a first group
comprising E. coli ESBL bacteria, and a second group comprising E.
coli HL Case bacteria. In this specific case, the combination of
two media may be the following: [0262] at least a first substrate
which makes it possible to identify E. coli, for example a
beta-glucuronidase substrate, such as
6-chloro-3-indolyl-.beta.-D-glucuronide, or a beta-glucosidase
substrate, such as 5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside;
[0263] a first identification marker which is a resistant mechanism
inhibitor, preferably cloxacillin; [0264] a second identification
marker which is another resistance mechanism inhibitor, preferably
clavulanic acid; [0265] at least one antimicrobial which is
cefpodoxime.
[0266] Reference is made to a combination of 2 media, i.e. a first
medium may comprise: [0267] a first substrate which makes it
possible to identify E. coli, which is
6-chloro-3-indolyl-.beta.-D-glucuronide; [0268] a first marker
which is a resistance mechanism inhibitor, preferably cloxacillin;
[0269] an antimicrobial, preferably an antibiotic, preferably
cefpodoxime,
[0270] while the second medium comprises: [0271] a first substrate
which makes it possible to identify E. coli, such as
6-chloro-3-indolyl-.beta.-D-glucuronide; [0272] a second marker
which is another resistance mechanism inhibitor, preferably
clavulanic acid; [0273] an antimicrobial, preferably an antibiotic,
preferably cefpodoxime.
[0274] Another alternative would be to use a first medium
comprising: [0275] a first substrate which makes it possible to
identify E. coli, which is
5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside; [0276] a first
marker which is a substrate for a desaminase or for a
tryptophanase, preferably tryptophan; [0277] an antimicrobial,
preferably an antibiotic, preferably cefpodoxime,
[0278] or the second medium comprises: [0279] a first substrate
which makes it possible to identify E. coli, which is
6-chloro-3-indolyl-.beta.-D-glucuronide; [0280] a second marker
which is a resistance mechanism inhibitor, preferably clavulanic
acid; [0281] an antimicrobial, preferably an antibiotic, which is
preferably ceftazidime.
[0282] Those skilled in the art will choose each medium in such a
way as to systematically obtain a combination according to the
invention. The antimicrobial that is active on said third group is
present in the two media. Those skilled in the art may in
particular use a biplate, which makes it possible to readily
compare the two media on which the same biological sample would
have been deposited.
[0283] In this respect, the invention also relates to a biplate
comprising a combination of two culture media, said combination
comprising: [0284] a. at least a first substrate for detecting at
least a first enzymatic or metabolic activity of said first group
of microorganisms; [0285] b. at least one marker for
differentiating the first group of microorganisms and the second
group of microorganisms, said marker being an inhibitor of the
mechanism of resistance to the first treatment and/or to the second
treatment; [0286] c. at least one antimicrobial that is active on
said third group of microorganisms.
[0287] This third embodiment of the invention is not limited to
distinguishing 3 groups of microorganisms, but may make it possible
to distinguish 4, 5 or even more groups of microorganisms. It is
then necessary to add additional identification markers to the
medium, in order to discriminate the various groups.
[0288] In this respect, the invention relates to a biplate
comprising a combination of two culture media, said combination
comprising:
[0289] for the first medium, [0290] a first substrate which makes
it possible to identify E. coli, for example a glucuronidase
substrate, such as 6-chloro-3-indolyl-.beta.-D-glucuronide, or a
galactosidase substrate, such as
5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside; [0291] a second
substrate which makes it possible to identify Proteeae, for example
a substrate for a desaminase or for a tryptophanase, such as
tryptophan; [0292] a third substrate which makes it possible to
identify the KESC group, for example a glucosidase substrate, such
as 5-bromo-4-chloro-3-indolyl-.beta.-D-glucoside; [0293] a marker
for differentiating between ESBL strains and HL Case strains,
preferably a resistance inhibitor, preferably clavulanic acid;
[0294] at least one antimicrobial which is preferably an
antibiotic, preferably ceftazidime;
[0295] for the second medium: [0296] a first substrate which makes
it possible to identify E. coli, for example a glucuronidase
substrate, such as in particular
6-chloro-3-indolyl-.beta.-D-glucuronide, or a galactosidase
substrate, such as in particular
5-bromo-6-chloro-3-indolyl-.beta.-D-galactoside; [0297] a second
substrate which makes it possible to identify Proteeae, for example
a substrate for a desaminase or for a tryptophanase, such as
tryptophan; [0298] a third substrate which makes it possible to
identify the KESC group, for example a glucosidase substrate, such
as in particular 5-bromo-4-chloro-3-indolyl-.beta.-D-glucoside;
[0299] at least one antimicrobial, preferably an antibiotic,
preferably ceftriaxone.
[0300] The examples below are given by way of explanation and are
no way limiting in nature. They will make it possible to understand
the invention more fully.
EXAMPLE
[0301] The example below is based on the phenotypic detection of
ESBLs using the reduction of susceptibility of these strains to
antibiotics and their sensitivity to combinations with
.beta.-lactamases inhibitors. For this, a biplate of CPS ID 3 base
(chromogenic medium for detecting microorganisms in urine, and sold
by bioMerieux under the reference 43541) with one half-agar
containing an antibiotic and one half-agar containing an
antibiotic/.beta.-lactamases inhibitor combination was used.
1. Choice of Strains
[0302] In the context of the manipulations carried out, for
evaluating the activity of the antibiotics active on gram-negative
bacilli, various species of enterobacteria (Escherichia coli,
Enterobacter aerogenes, Klebsiella pneumoniae, Serratia marcescens,
Proteus mirabilis) and of nonfermenting gram-negative bacilli
(Pseudomonas aeruginosa), capable of producing ESBLs, were used.
ESBL-positive strains, high level cephalosporinase-producing
strains (HL Case) and wild-type strains are compared in the
trials.
[0303] For the manipulations regarding the antibiotics active on
gram-positive bacteria, strains of gram-positive cocci
(Staphylococcus aureus, Staphylococcus saprophyticus,
Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus
faecium, Streptococcus agalactiae) and of gram-positive bacilli
(Lactobacillus spp) were tested.
[0304] For the trials intended to evaluate the activity of the
antifungals, various strains of yeasts (Candida albicans, Candida
glabrata, Candida tropicalis, Candida krusei, Candida dubliniensis,
Saccharomyces cerevisiae, Geotrichum capitatum) were used.
2. Preparation of Media
[0305] The medium used was a CPS ID3 medium (reference 43541) also
comprising at least one antibiotic and at least one resistance
mechanism inhibitor.
[0306] The composition of the media tested was the following:
TABLE-US-00002 Resistance Chromogenic substrate Antibiotic
inhibitor Medium A 6-chloro-3-indolyl-.beta.-D- Cefotaxime
glucuronide 1 mg/l 5-bromo-4-chloro-3- indolyl-.beta.-D-glucoside
Tryptophan FeCl.sub.3 Total: 1.73 g/l Medium B
6-chloro-3-indolyl-.beta.-D- Ceftazidime 0.25 mg/l glucuronide 1.5
mg/l clavulanic acid 5-bromo-4-chloro-3-indolyl- .beta.-D-glucoside
Tryptophan FeCl.sub.3 Total: 1.73 g/l
[0307] Osmosed water is added and the whole is homogenized and
melted in a waterbath at 100.degree. C. The base medium is
dispensed into flasks, the number of which corresponds to the total
number of media to be tested during the process. The flasks are
then autoclaved for 15 min at 121.degree. C. The media are brought
back to and kept molten at 55.+-.3.degree. C. in a waterbath, in
order to sterilely add the thermolabile additives (sterilized
beforehand by filtration through 0.22 .mu.m). The media are then
poured into biplates 90 mm in diameter (i.e. approximately 9.5
ml/half-plate), and left on a flat surface so that they can set.
The surface of the agars is then dried under a laminar flow hood
for 30 min.
3. Inoculation of Media
[0308] An inoculum of 0.5 McF is prepared, in physiological saline,
from 24-hour precultures at 36.degree. C..+-.2.degree. C. in an
aerobic atmosphere on TSA medium, and then 1 .mu.l of this
suspension is transferred into 5 ml of physiological saline. In
order to obtain a sufficient number of isolated colonies, a range
of inocula made it possible to determine that the optimal amount of
bacteria to be inoculated was from 10.sup.3 to 10.sup.4 CFU/ml. The
inoculation is carried out directly on the two half-agars using a
sterile swab. The cultures are then incubated at 37.degree. C. in
an aerobic atmosphere.
[0309] 4. Reading of media: the readings are carried out at 18
hours (.+-.30 min), 24 h (.+-.1 h) and 48 h (.+-.4 h). The density
and the size of the colonies, and the appearance, the color and the
coloration intensities of the mass and of the isolated colonies
were observed, according to the following reading scales 1 to 3: 0:
no growth; 0.1: trace of growth; 0.25: colonies of diameter<0.5
mm; 0.5: colonies of 0.5 mm in diameter; 0.75: 0.5
mm<diameter<1 mm; 1: colonies of 1 mm in diameter; 1.25: 1
mm<diameter<1.5 mm; 1.5: colonies 1.5 mm in diameter; 2:
colonies 2 mm in diameter; 3: colonies of diameter>2 mm.
5. Results:
[0310] a) Screening of Antibiotics
[0311] The 5 antibiotics recommended by the NCCLS (National
Committee for Clinical Laboratory Standards) were tested. For each
product tested, a range was prepared in order to determine the
concentrations that make it possible to inhibit the wild-type
strains of the enterobacteria tested, without affecting the growth
of the ESBL-positive strains or the HL Case-positive strains.
[0312] The concentrations selected are listed in table III
hereinafter.
TABLE-US-00003 TABLE III Limiting values of the concentrations of
antibiotics that allow inhibition of the wild-type strains of the
enterobacteria tested, without affecting the growth of the
ESBL-positive strains under the conditions tested Low value High
value Cefotaxime 1 mg/l 2 mg/l Ceftazidime 2 mg/l 2.5 mg/l
Ceftriaxone 1 mg/l 2.5 mg/l Cefpodoxime 2 mg/l 10 mg/l Aztreonam 1
mg/l 1.5 mg/l
[0313] The low value corresponds to the minimum concentration of
the antibiotic required to inhibit the wild-type strains of the
enterobacteria tested. The high value corresponds to the maximum
concentration of the antibiotic that can be used without affecting
the growth of the ESBL-positive strains tested.
[0314] At these concentrations, the separation of the wild-type and
resistant strains is satisfactory, and the expression of the
enzymatic activities on the CPS ID 3 medium is compliant.
[0315] In addition, it should be noted that ceftazidime is the only
antibiotic tested and used in the detection of ESBLs which showed
an activity on the wild-type strains of P. aeruginosa. A range was
prepared in order to define the minimum concentration for complete
inhibition of these strains, and the limiting value is 1.5
mg/l.
[0316] The addition of antibiotics had no effect on the expression
of the bacterial enzymatic activities on the chromogenic medium.
The groups of microorganisms were separated and identified both on
control CPS ID 3 medium and on the media comprising the antibiotics
as described above.
[0317] b) Screening of .beta.-Lactamase Inhibitors
[0318] Three .beta.-lactamases inhibitors (BLIs), i.e. clavulanic
acid, tazobactam and sulbactam, were used. A range was prepared for
each one, in the presence of cefotaxime, in order to determine the
optimum concentration for inhibiting the ESBL-positive strains
without impairing the growth of the HL Case strains.
[0319] Clavulanic acid appeared to be the most effective BLI in the
presence of cefotaxime. Tazobactam and sulbactam required
concentrations of greater than 2 mg/l in order to inhibit the
ESBL-positive strains, whereas clavulanic acid was more active at
much lower concentrations, over a broad operating range (from 0.1
to 8 mg/l when it is used in combination with cefotaxime).
[0320] Each antibiotic (cefotaxime, ceftazidime, ceftriaxone,
cefpodoxime, aztreonam) was tested in the presence of a range of
clavulanic acid in order to define the most effective combinations.
The combinations selected are listed in table IV below.
TABLE-US-00004 TABLE IV Selected combinations of antibiotics and of
clavulanic acid (CA) that inhibit the ESBL-positive enterobacterial
strains tested, but allow the HL Case-positive strains to grow
Cefotaxime 2 mg/l +CA 0.1 mg/l Ceftazidime 2.5 mg/l +CA 2 mg/l
Ceftriaxone 2 mg/l +CA 0.25 mg/l Cefpodoxime 9 mg/l +CA 0.25 mg/l
Aztreonam 1 mg/l +CA 0.5 mg/l
[0321] The results obtained using a biplate containing the
antibiotic alone on one side and the antibiotic+clavulanic acid
combination on the other side are listed in table V.
TABLE-US-00005 TABLE V Antibiotic Antibiotic + alone clavulanic
acid Wild-type E. coli ESBL-positive E. coli Pink colonies HL
Case-positive E. coli Pink colonies Pink colonies Wild-type
Proteeae ESBL-positive Proteeae Brown colonies HL Case-positive
Proteeae Brown colonies Brown colonies Wild-type KESC ESBL-positive
KESC Green colonies HL Case-positive KESC Green colonies Green
colonies
[0322] The addition of .beta.-lactamase inhibitors had no effect on
the expression of the bacterial enzymatic activities on the
chromogenic medium. The groups of microorganisms were separated and
identified both on control CPS ID 3 medium and on the media
comprising .beta.-lactamase inhibitors.
[0323] c) Combination of Antibiotics
[0324] Given the relative activities of the antibiotics and of
clavulanic acid, the following were combined: [0325] cefotaxime
(antibiotic active on ESBL-positive strains in combination with the
lowest concentration of clavulanic acid), [0326] ceftazidime
(antibiotic active on wild-type strains of P. aeruginosa), and
[0327] clavulanic acid,
[0328] so as to be able to inhibit, firstly, the wild-type strains
(including those of pyocyanic bacillus), and the ESBL-positive
strains of the enterobacteria tested by the addition of the
BIL.
[0329] Such a combination was tested on strains of P. aeruginosa;
this species is naturally resistant to cefotaxime but sensitive to
ceftazidime. When 1.5 mg/l of CAZ (ceftazidime), 1 mg/l of CTX
(cefotaxime) and 0.25 mg/l of CA (clavulanic acid) were combined,
all the wild-type strains and the ESBL-positive strains of the
enterobacteria tested were inhibited and only the collection of HL
Case strains and the ESBL-positive P aeruginosa strains grew. It
involves a biplate with CAZ alone on one side and CTX plus
clavulanic acid on the other.
[0330] The addition of these combinations of antibiotics had no
effect on the expression of the bacterial enzymatic activities on
the chromogenic medium. The groups of microorganisms were separated
and identified both on control CPS ID 3 medium and on the media
comprising such combinations of antibiotics.
[0331] d). Dye Assay
[0332] A medium according to the invention was also employed for
use in a biplate, one of the sides containing an antibiotic, and
the second containing another antibiotic or a combination of
antibiotics. Given that the same CPS ID 3 medium base is used on
either side, these two sides were differentiated by the presence of
a dye.
[0333] The dye tested, Evans blue, gives the medium a green color.
The coloration has made it possible to readily differentiate the 2
sides of the biplate without affecting the fertility of the medium,
or impairing the reading of the enzymatic activities of the
colonies. After having produced a range of Evans blue, added before
or after autoclaving, the values selected were the following:
[0334] 1.5 or 2 mg/l if the dye is added after autoclaving. [0335]
between 2 and 5 mg/l if it is added before autoclaving.
[0336] e). Inhibition of Gram-Positive Bacteria
[0337] ESBLs are a mechanism of resistance to .beta.-lactamines
that is found only in gram-negative bacilli; it is therefore
advisable to inhibit the gram-positive bacteria via the medium. Two
antibiotics, linezolide and vancomycin, were used in the medium
according to the invention for the purpose of inhibiting the
sensitive gram-positive bacteria.
[0338] For vancomycin, under the conditions tested, a concentration
between 2 and 32 mg/l, and in particular 2 to 5 mg/l, makes it
possible to inhibit the sensitive grain-positive bacteria without
interfering with the detection of the ESBL bacteria.
[0339] For linezolide, under the conditions tested, a concentration
between 2 and 64 mg/l, and in particular 4 to 16 mg/l, makes it
possible to inhibit the sensitive gram-positive bacteria without
interfering with the detection of the ESBL bacteria.
[0340] The inhibition of the gram-positive bacteria made it
possible to improve the detection of the ESBL bacteria in
polymicrobial samples and the specificity of their coloration.
[0341] f) Inhibition of Yeasts
[0342] A medium according to the invention can also comprise
antifungals in order to inhibit the possible presence of yeasts
which could grow on the medium and which could impair microorganism
growth.
[0343] Two antifungals were therefore tested: voriconazole and
amphotericin B.
[0344] For amphotericin B, under the conditions tested, a
concentration between 1 and 32 mg/l, and in particular 2 to 8 mg/l,
makes it possible to inhibit the sensitive yeasts without
interfering with the detection of the ESBL bacteria.
[0345] For voriconazole, under the conditions tested, a
concentration of between 1 and 64 mg/l, and in particular 4 to 16
mg/l, makes it possible to inhibit the sensitive gram-positive
bacteria without interfering with the detection of the ESBL
bacteria.
[0346] The inhibition of the yeasts made it possible to improve the
detection of ESBL bacteria in polymicrobial samples and the
specificity of their coloration.
[0347] 6. Conclusion
[0348] These results demonstrate that the medium according to the
invention makes it possible to isolate and apparently identify
ESBL-producing bacteria, differentiating them from high level
cephalosporinase-producing strains. The use of a biplate containing
a cephalosporine alone on one side and a cephalosporine/clavulanic
acid combination on the other, in a CPS ID 3 base, is particularly
advantageous.
Example 2
[0349] This second example is based on the phenotypic detection of
ESBLs using the reduction of susceptibility to antibiotics and the
sensitivity of HL Cases to combinations with tobramycin or
cloxacillin or dicloxacillin, and presents the use of a
cephalosporine mentioned above (CTX, CAZ, CPD, CRO, ATM) in
combination with a compound that inhibits cephalosporinases
(cloxacillin, dicloxacillin and tobramycin). Such a medium makes it
possible to inhibit bacteria which have a "natural"
cephalosporinase, most of those which have only a high level
cephalosporinase (HL Case), while at the same time allowing growth
of ESBL bacteria.
1. Choice of Strains
[0350] In the context of the manipulations carried out, for
evaluating the activity of antibiotics that are active on
gram-negative bacilli, various species of enterobacteria
(Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae,
Proteus mirabilis) and of nonfermenting gram-active bacilli
(Pseudomonas aeruginosa) capable of producing ESBLs, were used. In
the assays, ESBL-positive strains, strains producing high level
cephalosporinase (HL Case) and wild-type strains are compared.
2. Preparation of the Medium
[0351] The medium used was a CPS 1D3 medium (43541), also
comprising: [0352] ceftazidime at 2.5 mg/l and tobramycin at 2 mg/l
(medium A) or [0353] ceftriaxone at 2 mg/l and cloxacillin at 150
mg/l (medium B) or [0354] cefpodoxime at 2 mg/l and dicloxacillin
at a concentration of between 500 and 1000 mg/l (medium C).
[0355] Osmosed water is added and the whole is homogenized and
melted in a waterbath at 100.degree. C. The basic medium is
dispensed into flasks, the number of which corresponds to the total
number of media to be tested during the process. The flasks are
then autoclaved for 15 min at 121.degree. C. The media are brought
back to and kept molten at 55.+-.3.degree. C. in a waterbath, in
order to sterilely add the thermolabile additives (sterilized
beforehand by filtration through 0.22 .mu.m). The media are then
poured into plates 35 mm in diameter and left on a flat surface so
that they can set. The surface of the agars is then dried under a
laminar flow hood for 30 min.
3. Inoculation of Media
[0356] This step is carried out as described in example 1.
4. Reading of Media
[0357] This step is carried out as described in example 1
5. Results:
[0358] Medium A comprising ceftazidime and tobramycin made it
possible to inhibit all the wild-type strains and all the HL Cases
tested. Only the ESBL-positive strains were detected on this
medium.
[0359] Medium B comprising ceftriaxone and cloxacillin made it
possible to inhibit all the HL Cases and all the wild-type strains
except HL Case and wild-type P aeruginosa, and grew only the
majority of the ESBL-positive strains.
[0360] Medium C comprising cefpodoxime and di-cloxacillin made it
possible to inhibit all the wild-type strains and the majority of
the HL Cases, without affecting the growth of ESBL-positive
strains.
Example 3
[0361] This third example is based on the phenotypic detection of
enterococci resistant to glycopeptides, with specific distinction
of Enterococcus faecalis and E. faecium, using the reduction of
susceptibility to antibiotics and the demonstration of an enzymatic
activity: .beta.-glucosidase, and of a metabolic activity:
Methyl-.alpha.-glucoside acidification.
1. Choice of Strains
[0362] In the context of the manipulations carried out, for
evaluating the activity of the antibiotics active on enterococci,
various species of Enterococcus (Enterococccus faecalis,
Enterococcus faecium, Enterococcus casseliflavus, Enterococcus
gallinarum,) were used. In the assays, strains resistant to
glycopeptides (VRE) and wild-type strains are compared.
2. Preparation of the Medium
[0363] The medium used was a Columbia medium (51026), also
comprising: [0364]
5-bromo-4-chloro-3-indolyl-.beta.-D-glucopyranoside (X-Glu) at 100
mg/l, [0365] methyl-.alpha.-D-glucoside at 9 g/l, [0366] neutral
red at 25 mg/l, [0367] bilial salts at 5 g/l, [0368] vancomycin at
4 mg/l, [0369] amphotericin B at 2 mg/l.
[0370] Osmosed water is added and the whole is homogenized and
melted in a waterbath at 100.degree. C. The basic medium is
dispensed into flasks, the number of which corresponds to the total
number of media to be tested during the process. The flasks are
then autoclaved for 15 min at 121.degree. C. The media are brought
back to and kept molten at 55.+-.3.degree. C. in a waterbath, in
order to sterilely add the thermolabile additives (sterilized
beforehand by filtration through 0.22 .mu.m).
[0371] The media are then poured into plates 90 mm in diameter and
left on a flat surface so that they can set. The surface of the
agars is then dried under a laminar flow hood for 30 min.
3. Inoculation of Media
[0372] This step is carried out as described in example 1.
4. Reading of Media
[0373] This step is carried out as described in example 1.
5. Results:
[0374] On this medium, only the glycopeptide-resistant enterococcal
strains develop and form colonies.
[0375] The resistant E. faecalis and E. faecium strains form green
colonies, whereas those of E. casseliflavus and of E. gallinarum
form blue-to-violet colonies.
[0376] This medium therefore makes it possible to differentiate
these two groups of enterococci and to provide a suitable
therapeutic response.
Example 4
[0377] This fourth example is based on the phenotypic detection of
glycopeptide-resistant enterococci, with specific distinction of
Enterococcus faecalis and E. faecium, using the reduction of
susceptibility to antibiotics and the demonstration of two
enzymatic activities: .alpha.-glucosidase and .beta.-galactosidase
or .beta.-glucosidase.
1. Choice of Strains
[0378] In the context of the manipulations carried out, for
evaluating the activity of antibiotics active on enterococci,
various species of Enterococcus (Enterococccus faecalis,
Enterococcus faecium, Enterococcus casseliflavus, Enterococcus
gallinarum) were used. In the assays, strains resistant to
glycopeptides (VRE) and wild-type strains are compared.
2. Preparation of the Medium
[0379] The media used were a Columbia medium (51026), also
comprising: [0380]
5-bromo-4-chloro-3-indolyl-N-methyl-.alpha.-D-glucopyranoside
(GreenA-.alpha.-Glu) at 150 mg/l, [0381]
6-chloro-3-indolyl-.beta.-glucopyranoside (Rose-b-Glu [Pink-b-Glu])
at 200 mg/l, or: [0382]
5-bromo-4-chloro-3-indolyl-N-methyl-.alpha.-D-glucopyranoside
(GreenA-.alpha.-Glu) at 150 mg/l, [0383]
alizarine-.beta.-galactopyranoside at 50 mg/l
[0384] and vancomycin at 8 mg/l,
[0385] an amphotericin B at 4 mg/l,
[0386] and colistine at 2 mg/l,
[0387] and aztreonam at 32 mg/l.
[0388] Osmosed water is added and the whole is homogenized and
melted in a waterbath at 100.degree. C. The two basic media are
dispensed into flasks. The flasks are then autoclaved for 15 min at
121.degree. C. The media are brought back to and kept molten at
55.+-.3.degree. C. in a waterbath, in order to sterilely add the
thermolabile additives (sterilized beforehand by filtration through
0.22 .mu.m). The media are then poured into plates 90 mm in
diameter and left on a flat surface so that they can set. The
surface of the agars is then dried under a laminar flow hood for 30
min.
3. Inoculation of Media
[0389] This step is carried out as described in example 1.
4. Reading of Media
[0390] This step is carried out as described in example 1.
5. Results:
[0391] On the medium containing a substrate for a-glucosidase and
for .beta.-glucosidase, the resistant E. faecium strains form
violet colonies, whereas the resistant E. faecalis strains form
pink colonies. The E. casseliflavus and E. gallinarum strains
(natural resistances) are inhibited due to the concentration of
vancomycin.
[0392] This medium therefore makes it possible to differentiate
these two groups of enterococci and to provide a suitable
therapeutic response and also a follow-up of the local
epidemiology.
[0393] On the medium containing a substrate for a-glucosidase and
for .beta.-galactosidase, the resistant E. faecium strains form
violet colonies, whereas the resistant E. faecalis strains form
green colonies. The E. casseliflavus and E. gallinarum strains
(natural resistances) are inhibited due to the concentration of
vancomycin.
[0394] This medium therefore makes it possible to differentiate
these two groups of enterococci and to provide a suitable
therapeutic response and a follow-up of the local epidemiology.
* * * * *