EP2459732A1 - Media for the specific detection of gram-negative bacteria resistant to beta-lactam antibiotics - Google Patents

Media for the specific detection of gram-negative bacteria resistant to beta-lactam antibiotics

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Publication number
EP2459732A1
EP2459732A1 EP10752006A EP10752006A EP2459732A1 EP 2459732 A1 EP2459732 A1 EP 2459732A1 EP 10752006 A EP10752006 A EP 10752006A EP 10752006 A EP10752006 A EP 10752006A EP 2459732 A1 EP2459732 A1 EP 2459732A1
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EP
European Patent Office
Prior art keywords
beta
gram
negative bacteria
reaction medium
medium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP10752006A
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German (de)
French (fr)
Inventor
Gilles Zambardi
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Biomerieux SA
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Biomerieux SA
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Publication of EP2459732A1 publication Critical patent/EP2459732A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/045—Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • the field of the invention is that of microbiological biochemical analysis, and in particular the detection and identification of microorganisms, such as in particular bacteria or yeasts.
  • the resistance of bacteria to antibiotics is a major public health problem.
  • the resistance of infectious microorganisms to a treatment has developed at the same time as the anti-infectious molecules and represents today a major obstacle in therapeutics.
  • This resistance is a source of multiple problems, including difficulties in laboratory detection, limited treatment options, and a deleterious impact on the clinical outcome.
  • the rapid and irrepressible increase in the resistance of pathogenic bacteria over the last 20 years is one of the major current problems of medicine.
  • the infections caused by these organisms are at the origin of the lengthening of the hospital stay and are associated with high morbidity and mortality rates, following therapeutic failures.
  • Enzymatic inactivation is the most common mechanism of acquired resistance in terms of number of species and antibiotics involved.
  • class C chromosomal cephalosporinases now constitute one of the predominant resistance mechanisms of gram-negative bacteria, as bacteria expressing such enzymes are resistant to cephalosporins.
  • ⁇ -lactamases are enzymes expressed by certain bacteria, capable of hydrolyzing the CN bond of the ⁇ -lactam nucleus, the basic structure of antibiotics of the ⁇ -lactam family, to give a microbiologically inactive product.
  • IBLs ⁇ -lactamase inhibitors
  • AC clavulanic acid
  • tazobactam tazobactam
  • sulbactam sulbactam
  • Case HN bacteria Gram-negative bacteria producing high-level class C chromosomal cephalosporinases
  • ESBL bacteria Gram-negative bacteria producing extended spectrum ⁇ -lactamases
  • C3G 3rd generation cephalosporins
  • 7 ⁇ -methoxycephalosporins (cephamycins: cefoxitin, cefotetan) and carbapenems (imipenem, meropenem, ertapenem) generally retain their activity.
  • ESBLs are inhibited by ⁇ -lactamase inhibitors (IBL), which differentiates them from other cephalosporinases.
  • IBL ⁇ -lactamase inhibitors
  • Escherichia coli bacterium can thus be Case HN and ESBL.
  • ESBL-positive enterobacteriaceae tend to disseminate resistance by clonal transmission of strains or conjugative plasmid transfer, they represent a problem for infection control.
  • Escherichia coli and Klebsiella pneumoniae remain the most common ESBL producing species.
  • ESBLs have greatly expanded their range of host species.
  • many species of enterobacteria and non-fermenting gram-negative bacilli such as Pseudomonas aeruginosa
  • the search for microorganisms resistant to a treatment is done according to the following steps 1. removal of a biological sample likely to contain the said microorganisms
  • This succession of steps involves a significant time between the taking of the sample may contain microorganisms, and the prescription of a suitable treatment for the patient.
  • the user must generally manually carry out microorganism transfer steps from a first medium to a second medium, which can cause problems including contamination but also risks to the health of the manipulator.
  • Metabolic substrates are also used to detect the presence of ESBL or Case HN.
  • the AES laboratories propose a medium in a bi-box associating a Drigalski medium with cefotaxime and a MacConkey medium with ceftazidime.
  • Drigalski and MacConkey media reveal the acidification of Lactose, a metabolism present in a very large number of Enterobacteriaceae species.
  • such a medium only makes it possible to distinguish resistant bacteria from non-resistant bacteria, but does not make it possible to distinguish the bacteria expressing an ESBL from those expressing a Case HN.
  • This medium also does not allow the identification of particular species of bacteria, and does not allow, for example to discriminate E. coli bacteria, K. pneumoniae bacteria.
  • the present invention therefore proposes to improve the state of the art by presenting a new diagnostic tool allowing a saving of time, reliability and relevance in the therapy implemented.
  • Our invention makes it possible in a single step to identify the species of gram negative microorganisms present in a sample and to determine their resistance mechanism in order to propose a treatment adapted to each patient.
  • reaction medium means a medium comprising all the elements necessary for the survival and / or growth of microorganisms, such as Staphylococcus aureus.
  • This reaction medium can either serve only as a revelation medium or a culture medium and revelation.
  • the culture of the microorganisms is carried out before inoculation and, in the second case, the reaction medium also constitutes the culture medium.
  • the reaction medium may be solid, semi-solid or liquid.
  • solid medium is meant for example a gelled medium.
  • the medium according to the invention is a gelled medium.
  • Agar is the traditional gelling agent in microbiology for the cultivation of microorganisms, but it is possible to use gelatin or agarose.
  • a number of preparations are commercially available, such as Columbia agar, Trypcase-soy agar, Mac Conkey agar, Sabouraud agar or more generally those described in the Handbook of Microbiological Media (CRC Press).
  • the reaction medium according to the invention may contain any other additives, for example: peptones, one or more growth factors, carbohydrates, one or more selective agents, buffer solutions, one or more gelling agents, etc.
  • This reaction medium may be in the form of a liquid, a ready-to-use gel, that is to say ready for seeding in a tube, a bottle, or on a petri dish.
  • a regeneration passesage to 100 ° C.
  • It can also be a powder medium or a bottle which before being poured into a petri dish, tube or bottle is supplemented with a supplement.
  • the medium according to the invention is a selective medium, that is to say a medium comprising compounds that favor the growth of Gram-negative bacteria. Mention may in particular be made of sodium citrate, sodium sulphite, antibiotics such as vancomycin, antifungals such as amphotericin B, natamycin, cycloheximide, surfactants such as bile salts, sodium deoxycholate, Tergitol, dyes such as brilliant green, crystal violet, fuchsin, eosin, methylene blue.
  • the medium according to the invention is a selective medium comprising compounds that favor the growth of extended-spectrum beta lactamase bacteria (ESBL). In particular, cephalosporins may be mentioned.
  • ESBL extended-spectrum beta lactamase bacteria
  • cephalosporin such as: cefalexin, cefaloridine, cefalotin, cefazoline, cefadroxil, cefazedone, cefatrizine, cefapirine, cefradine, cefacetrile, cefrodaxine, ceftezole
  • cephalosporin such as cefoxitin, cefuroxime, cefamandole, cefaclor, cefotetan, cefonicide, cefotiam, loracarbef, cefmetazole, cefprozil, ceforanide
  • cephalosporins such as cefotaxime, ceftazidime, cefsulodine, ceftriaxone, cefmenoxime, latamoxef, ceftizoxime, cefixime, cefodizime, cefetamet, cefpiramide, cefoperazone, cefpodoxime, ceftibuten, cefdinir, cefditoren, ceftriaxone, cefoperazone, cefbuperazone
  • cephalosporin such as cefepime, cefpirome
  • gram-negative bacteria mention may be made in particular of the following genera: Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus, Morganella, Vibrio, Yersinia, Acinetobacter, Branhamella, Neisseria, Burkholderia, Citrobacter, Hafnia, Edwardsella, Aeromonas, Moraxella, Pasteurella, Providencia, and Legionella.
  • Beta-lactam resistance mechanism means any type of device that allows a microorganism to render a treatment partially or totally inoperative on said microorganism, ensuring its survival, said device being linked to the expression of an enzyme belonging to the group of extended spectrum ⁇ -lactamases; of an enzyme belonging to the class C cephalosporinases group expressed at a high level
  • Beta-lactam resistance mechanism marker is understood to mean a compound making it possible to demonstrate such a mechanism of resistance, such as cefepime and its salts (Masuyoshi S. et al, 1989). antibacterial activities of cefepime (BMY-28142) with ceftazidime, cefuzonam, cefotaxime and cefmenoxime. ”)
  • resistance mechanism inhibitor other than said beta-lactam resistance mechanism, it is meant a compound which indirectly inhibits the growth of organisms developing a particular resistance, without inhibition of gram-negative bacteria expressing said mechanism of resistance to beta-lactams.
  • lactams such as cloxacillin (Jack and Richmond, 1970 - "A comparative study of eight distinct beta-lactamases synthesized by gram-negative bacteria." for the inhibition of Class C cephalosporinases
  • the substrate of an enzymatic or metabolic activity is chosen from any substrate that can be hydrolysed into a product that allows the direct or indirect detection of an enzymatic activity or a metabolism, such as, in particular, a osidase activity, preferably a glucuronidase, glucosidase or galactosidase activity.
  • the metabolism of the substrate causes a variation in the physicochemical properties of the reaction medium or the cells of organisms.
  • This variation can be detected by physicochemical methods, especially optical methods by the eye of the operator or by means of instruments, spectrometric, electrical, magnetic, ...
  • it is a question of a change in optical properties, such as a change in absorption, fluorescence or luminescence.
  • a chromogenic substrate there may be mentioned in particular substrates based on indoxyl, flavone, alizarin, acridine, esculetin, phenoxazine, nitrophenol, nitroaniline, naphthol, catechol, hydroxyquinoline, coumarin, aminophenol, dichloroaminophenol.
  • the substrate (s) used in the present invention is (are) based on indoxyl.
  • Fluorescent substrates that may especially be mentioned include umbelliferone-based or coumarin-based substrates based on resorufin, phenoxazine, naphthol, naphthylamine, 2'-hydroxyphenyl-heterocycle or 2'-aminophenyl-heterocycle or based on fluorescein.
  • the substrate used in the present invention is 5-bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside, preferably in combination with 5-bromo-6-chloro-3-indoxyl-beta-D-galactopyranoside .
  • beta-glucosidase substrates 5-Bromo-6-chloro-3-indoxyl-beta-glucoside; Dihydroxyflavone-beta- glucoside; 3-hydroxyflavone-beta-glucoside, 3,4-cyclohexenesculetin-beta-glucoside (not 3,4-cyclopentenoesculetin-beta-glucoside?); 8-Hydroxyquinoline-beta-glucoside; 5-Bromo-4-chloro-3-indoxyl-N-methyl-beta-glucoside; 6-Chloro-3-indoxyl-beta-glucoside; 5-Bromo-3-indoxyl-beta-glucoside; 5-Iodo-3-indoxyl-beta-glucoside, 6-Iodo-3-indoxyl-beta-glucoside 6-Fluoro-3-indoxyl-beta-glucoside
  • Alizarin-beta-glucoside Nitrophenyl-beta-glucoside; 4-Methylumbelliferyl-beta-glucoside; Naphtholbenzein-beta-glucoside; Indoxyl-N-methyl-beta-glucoside; Naphtyl-beta-glucoside; Aminophenyl-beta-glucoside; Dichloroaminophenyl beta-glucoside; beta-galactosidase substrates: 5-Bromo-4-chloro-3-indoxyl-beta-galactoside; Dihydroxyflavone-beta-galactoside; 3,4-Cyclohexenoesculin-beta-galactoside; 8-Hydroxyquinoline-beta-galactoside; 5-Bromo-4-chloro-3-indoxyl-N-methyl-beta-galactoside; 6-Chloro-3-indoxyl-
  • Aminophenyl-beta-glucuronide Dichloroaminophenyl-beta-glucuronide; alpha-glucosidase substrates, alpha-galactosidase substrates, esterase substrates, especially lipase, phosphatase, cellobiosidase substrates, ribosidase substrates, hexosaminidase substrates.
  • the substrates of the invention can be used over a wide pH range, in particular between pH 5.5 and 10, preferably between 6.5 and 10.
  • the medium according to the invention comprises a substrate or several substrates of beta enzymatic activity.
  • the concentration of substrate (s) is preferably between 0.01 and 2 g / l, more preferably between 0.02 and 0.2 g / l, and advantageously between 0.05 and 0, 15 g / 1. In fact, at this concentration of substrate, a better color contrast is obtained.
  • said chromogenic substrate is selected from a glucuronidase, beta-glucosidase and beta-galactosidase substrate.
  • biological sample we mean a clinical sample, from a sample of biological fluid, or a food sample, from any type of food.
  • This sample may thus be liquid or solid and may be mentioned in a nonlimiting manner, a clinical sample of blood, plasma, urine, faeces, rectal samples, nose, throats, skin, wounds. , cerebrospinal fluid, a food sample of water, beverages such as milk, fruit juice; yogurt, meat, eggs, vegetables, mayonnaise, cheese; fish ..., a food sample from a feed intended for animals, such as in particular a sample from animal meal.
  • the invention relates to a reaction medium of gram-negative bacteria having a beta-lactam resistance mechanism comprising:
  • beta-lactam resistance mechanism marker that is cefepime, • a resistance mechanism inhibitor, other than said beta-lactam resistance mechanism
  • said resistance mechanism inhibitor is cloxacillin.
  • concentration of cloxacillin is between 0.05 and 1 g / l and more preferably between 0.1 and 0.3 g / l.
  • it is 0.2 g / l.
  • the concentration of cefepime is between 0.05 and 1 mg / l and more preferably between 0.10 and 0.5 mg / l.
  • it is 0.25 mg / l.
  • the reaction medium further comprises a substrate of an enzymatic or metabolic activity, preferably a chromogenic substrate.
  • said chromogenic substrate is selected from a glucuronidase, beta-glucosidase and beta-galactosidase substrate.
  • the chromogenic substrate concentration is between 0.02 and 2 g / l and more preferably between 0.03 and 0.5 g / l.
  • it is 0.1 g / l.
  • said medium comprises a combination of at least two chromogenic substrates.
  • this combination comprises a glucuronidase substrate and a beta-glucosidase substrate.
  • this combination comprises a beta-galactosidase substrate and a beta-glucosidase substrate.
  • the invention also relates to the use of a medium as defined above for the detection of gram-negative bacteria resistant to beta-lactam antibiotics, preferably extended-spectrum beta lactamase bacteria (ESBL).
  • ESBL extended-spectrum beta lactamase bacteria
  • the invention also relates to a method for detecting gram-negative bacteria resistant to beta-lactams, characterized in that it comprises the following steps: 1. having a reaction medium as defined above,
  • the incubation is preferably carried out at a temperature of between 30 ° C. and 42 ° C.
  • Gram-negative bacteria resistant to beta-lactam antibiotics are preferably detected by a specific glucuronidase, beta-glucosidase or beta-galactosidase activity which makes it possible to obtain colored or fluorescent colonies.
  • Other species appear colorless or of a different color or fluorescence than colonies of gram-negative bacteria resistant to beta-lactam antibiotics.
  • the examples below are given for explanatory purposes and have no limiting character. They will help to better understand the invention.
  • strains making it possible to evaluate the activity of antibiotics with respect to Gram-negative species: enterobacteria and non-fermenting bacilli.
  • strains producing ESBL, high level cephalosporinase (CaseHN) and strains with no particular resistance profile, called wild type have been used.
  • the media tested were media composed of the peptone base of Chrom ID CPS medium (bioMérieux ref 43541) to which was added after autoclaving, in supercooled media, 300 mg / l of Cloxacillin and for T medium: 4 mg / l of cefpodoxime, for medium A: 0.25 mg / l of cefepime, for medium B: 3 mg / l of cefamandole and for medium C: 3 mg / l of cefuroxime.
  • Chrom ID CPS medium bioMérieux ref 43541
  • Inoculation of the media The media are inoculated by isolating using the 3-dial method from 0.5McF bacterial suspensions. The media are then incubated for 24 hours at 37 ° C.
  • Reading media The media are visually observed after 18 hours and 24 hours of incubation, the growth density as well as staining and color intensities are evaluated according to the scale below:

Abstract

The invention relates to a reaction medium for gram-negative bacteria having a beta-lactam antibiotic resistance mechanism. Said reaction medium comprises a marker for the beta-lactam antibiotic resistance mechanism, said marker being cefepime, and an inhibitor of a resistance mechanism other than said beta-lactam antibiotic resistance mechanism.

Description

Milieux pour la détection spécifique de bactéries gram négatives résistantes aux beta- lactamines  Media for the specific detection of beta-lactam-resistant gram-negative bacteria
Le domaine de l'invention est celui de l'analyse microbiologique par voie biochimique, et en particulier de la détection et de l'identification de microorganismes, tels que notamment de bactéries ou de levures. The field of the invention is that of microbiological biochemical analysis, and in particular the detection and identification of microorganisms, such as in particular bacteria or yeasts.
La résistance des bactéries aux antibiotiques est un problème majeur de santé publique. La résistance de microorganismes infectieux envers un traitement s'est développée en même temps que les molécules anti-infectieuses et représente aujourd'hui un obstacle majeur en thérapeutique. Cette résistance est source de problèmes multiples, incluant les difficultés de détection au laboratoire, les options de traitement limitées et un impact délétère sur l'issue clinique. En particulier, l'augmentation rapide et irrépressible de la résistance des bactéries pathogènes, au cours des 20 dernières années, représente un des problèmes actuels majeurs de la médecine. Les infections causées par ces organismes sont à l'origine de l'allongement de la durée d'hospitalisation et sont associées à des taux élevés de morbidité et de mortalité, suite à des échecs thérapeutiques. The resistance of bacteria to antibiotics is a major public health problem. The resistance of infectious microorganisms to a treatment has developed at the same time as the anti-infectious molecules and represents today a major obstacle in therapeutics. This resistance is a source of multiple problems, including difficulties in laboratory detection, limited treatment options, and a deleterious impact on the clinical outcome. In particular, the rapid and irrepressible increase in the resistance of pathogenic bacteria over the last 20 years is one of the major current problems of medicine. The infections caused by these organisms are at the origin of the lengthening of the hospital stay and are associated with high morbidity and mortality rates, following therapeutic failures.
Plusieurs mécanismes de résistance peuvent être impliqués simultanément chez une souche bactérienne. Ils se classent en général en 3 catégories : défaut de pénétration de l'antibiotique dans la bactérie, inactivation ou excrétion de l'antibiotique par des systèmes enzymatiques bactériens et défaut d'affinité entre la cible bactérienne et l'antibiotique. Several resistance mechanisms can be involved simultaneously in a bacterial strain. They generally fall into three categories: lack of penetration of the antibiotic in the bacterium, inactivation or excretion of the antibiotic by bacterial enzyme systems and lack of affinity between the bacterial target and the antibiotic.
L 'inactivation enzymatique est le mécanisme le plus fréquent de résistance acquise en termes de nombre d'espèces et d'antibiotiques concernés. Ainsi, les céphalosporinases chromosomiques de classe C constituent aujourd'hui un des mécanismes de résistance prédominants des bactéries gram-négatif, les bactéries exprimant de telles enzymes étant résistantes aux céphalosporines. De même, les β-lactamases sont des enzymes exprimées par certaines bactéries, capables d'hydrolyser la liaison C-N du noyau β-lactame, structure de base des antibiotiques de la famille des β-lactamines, pour donner un produit microbiologiquement inactif. Plusieurs inhibiteurs de β-lactamases (IBL), tels que l'acide clavulanique (AC), le tazobactam et le sulbactam, ont été développés pour augmenter l'activité antimicrobienne et élargir le spectre des β-lactamines qui leur sont associées. Agissant comme un substrat suicide des β-lactamases, ils empêchent la dégradation enzymatique des antibiotiques et leur permettent de devenir efficace contre des bactéries initialement résistantes. Cependant, de par l'exposition persistante des souches à la pression antibiotique, les bactéries expriment leur capacité d'adaptation par la production continue et dynamique de β-lactamases, évoluant en même temps que le développement de nouvelles molécules. Enzymatic inactivation is the most common mechanism of acquired resistance in terms of number of species and antibiotics involved. Thus, class C chromosomal cephalosporinases now constitute one of the predominant resistance mechanisms of gram-negative bacteria, as bacteria expressing such enzymes are resistant to cephalosporins. Similarly, β-lactamases are enzymes expressed by certain bacteria, capable of hydrolyzing the CN bond of the β-lactam nucleus, the basic structure of antibiotics of the β-lactam family, to give a microbiologically inactive product. Several β-lactamase inhibitors (IBLs), such as clavulanic acid (AC), tazobactam and sulbactam, have been developed to increase antimicrobial activity and broaden the spectrum of β-lactams associated with them. Acting as a suicide substrate for β-lactamases, they prevent the enzymatic degradation of antibiotics and allow them to become effective against bacteria initially resistant. However, because of the persistent exposure of strains to antibiotic pressure, bacteria express their adaptability through the continuous and dynamic production of β-lactamases, evolving at the same time as the development of new molecules.
Les bactéries Gram-négatif productrices de céphalosporinases chromosomiques de classe C de haut niveau (on parle de bactéries Case HN), ainsi que les bactéries Gram-négatif productrices de β-lactamases à spectre étendu (on parle alors de bactéries BLSE) sont devenues de ce fait une menace grandissante, en particulier parce que le nombre d'espèces bactériennes concernées augmente. Les bactéries Case HN et BLSE sont résistantes à des traitements à base de pénicillines et céphalosporines de 1ère et 2ème générations, mais également de céphalosporines de 3ème génération (C3G) (cefotaxime CTX, ceftazidime CAZ, cefpodoxime CPD, ceftriaxone CRO) et des monobactams (aztreonam ATM). Par contre, les 7α-méthoxycéphalosporines (céphamycines: cefoxitine, cefotetan) et les carbapénèmes (imipénème, méropénème, ertapénème) conservent généralement leur activité. Les BLSE sont inhibées par les inhibiteurs de β- lactamases (IBL), ce qui permet de les différencier des autres céphalosporinases. Gram-negative bacteria producing high-level class C chromosomal cephalosporinases (referred to as Case HN bacteria), as well as Gram-negative bacteria producing extended spectrum β-lactamases (now known as ESBL bacteria) have become this is a growing threat, especially as the number of bacterial species involved increases. Case HN and ESBL bacteria are resistant to treatment with penicillins and cephalosporins of the 1st and 2nd generations, but also of 3rd generation cephalosporins (C3G) (cefotaxime CTX, CAZ ceftazidime, cefpodoxime CPD, ceftriaxone CRO) and monobactams ( ATM aztreonam). On the other hand, 7α-methoxycephalosporins (cephamycins: cefoxitin, cefotetan) and carbapenems (imipenem, meropenem, ertapenem) generally retain their activity. ESBLs are inhibited by β-lactamase inhibitors (IBL), which differentiates them from other cephalosporinases.
Ces bactéries expriment ainsi le plus souvent simultanément des résistances à plusieurs traitements, ce qui pose des difficultés pour installer un traitement pertinent et éviter les échecs thérapeutiques. Une bactérie Escherichia coli peut ainsi être Case HN et BLSE. En outre, comme les entérobactéries BLSE positives ont tendance à disséminer la résistance par transmission clonale de souches ou transfert plasmidique conjugatif, elles représentent un problème pour le contrôle des infections. Dans la plupart des études, Escherichia coli et Klebsiella pneumoniae demeurent les espèces productrices de BLSE les plus communes. Mais les BLSE ont, depuis quelques années, grandement élargi leur panel d'espèces hôtes. En effet, de nombreuses espèces d' entérobactéries et de bacilles gram-négatif non fermentants (comme Pseudomonas aeruginosa) ont également été rapportées comme producteurs de BLSE. These bacteria thus most often express resistance to several treatments simultaneously, which poses difficulties in setting up a relevant treatment and avoiding therapeutic failures. An Escherichia coli bacterium can thus be Case HN and ESBL. In addition, since ESBL-positive enterobacteriaceae tend to disseminate resistance by clonal transmission of strains or conjugative plasmid transfer, they represent a problem for infection control. In most studies, Escherichia coli and Klebsiella pneumoniae remain the most common ESBL producing species. But in recent years, ESBLs have greatly expanded their range of host species. In fact, many species of enterobacteria and non-fermenting gram-negative bacilli (such as Pseudomonas aeruginosa) have also been reported as ESBL producers.
Il devient donc indispensable, d'un point de vue de santé publique, de pouvoir identifier le plus rapidement possible de tels microorganismes, et de tels mécanismes de résistances. It is therefore essential, from a public health point of view, to be able to identify as quickly as possible such microorganisms, and such mechanisms of resistance.
En général, la recherche des micro-organismes résistants à un traitement se fait selon les étapes suivantes 1. prélèvement d'un échantillon biologique susceptible de contenir lesdits microorganismes In general, the search for microorganisms resistant to a treatment is done according to the following steps 1. removal of a biological sample likely to contain the said microorganisms
2. ensemencement et incubation d'un milieu de culture (18 à 48H) pour induire une croissance exponentielle des micro-organismes  2. Inoculation and incubation of a culture medium (18 to 48H) to induce an exponential growth of microorganisms
3. Repérage sur les milieux de culture des colonies de micro-organismes potentiellement significatifs 3. Spotting colonies of potentially significant microorganisms on culture media
4. Caractérisation de l'espèce de micro-organisme  4. Characterization of the microorganism species
5. Identification des mécanismes de résistance des micro-organismes analysés, leur signification biologique et éventuellement la thérapie adaptée.  5. Identification of the resistance mechanisms of the microorganisms analyzed, their biological significance and possibly adapted therapy.
Cette succession d'étapes implique un temps important entre le prélèvement de l'échantillon susceptible de contenir des micro-organismes, et la prescription d'un traitement adapté pour le patient. De plus, l'utilisateur doit généralement réaliser manuellement des étapes de transfert de micro-organismes d'un premier milieu vers un deuxième milieu, ce qui peut induire des problèmes notamment de contamination mais également des risques pour la santé du manipulateur. This succession of steps involves a significant time between the taking of the sample may contain microorganisms, and the prescription of a suitable treatment for the patient. In addition, the user must generally manually carry out microorganism transfer steps from a first medium to a second medium, which can cause problems including contamination but also risks to the health of the manipulator.
A titre d'exemple, pour détecter la présence de béta-lactamases à spectre étendu (BLSE) dans des souches d'Escherichia coli et de Klebsiella pneumoniae, on peut utiliser une technique de diffusion telle que décrite dans la publication de Jacoby & Han (J Clin Microbiol. 34(4): 908-11, 1996) qui ne donne toutefois aucune information sur l'identification des souches testées : on peut déterminer si la bactéries est une bactérie productrice de BLSE ou non, mais on ne peut distinguer si une telle bactérie est une Escherichia coli ou une Klebsiella pneumoniae.  By way of example, to detect the presence of extended spectrum beta-lactamases (ESBL) in Escherichia coli and Klebsiella pneumoniae strains, a diffusion technique as described in the publication by Jacoby & Han ( J Clin Microbiol 34 (4): 908-11, 1996), which, however, gives no information on the identification of the strains tested: it is possible to determine whether the bacteria is an ESBL-producing bacterium or not, but it can not be determined whether such a bacterium is Escherichia coli or Klebsiella pneumoniae.
Des substrats métaboliques sont également utilisés pour détecter la présence de BLSE ou Case HN. A ce titre, les laboratoires AES proposent un milieu dans une bi boîte associant un milieu Drigalski avec du céfotaxime et un milieu MacConkey avec de la ceftazidime. Les milieux Drigalski et MacConkey permettent de révéler l'acidification du Lactose, métabolisme présent chez un très grand nombre d'espèces d'entérobactéries. Toutefois, un tel milieu permet uniquement de distinguer des bactéries résistantes de bactéries non résistantes, mais ne permet pas de distinguer les bactéries exprimant une BLSE de celles exprimant une Case HN. Ce milieu ne permet pas non plus l'identification d'espèces particulières de bactéries, et ne permet pas, par exemple de discriminer les bactéries E. coli, de bactéries K. pneumoniae.  Metabolic substrates are also used to detect the presence of ESBL or Case HN. As such, the AES laboratories propose a medium in a bi-box associating a Drigalski medium with cefotaxime and a MacConkey medium with ceftazidime. Drigalski and MacConkey media reveal the acidification of Lactose, a metabolism present in a very large number of Enterobacteriaceae species. However, such a medium only makes it possible to distinguish resistant bacteria from non-resistant bacteria, but does not make it possible to distinguish the bacteria expressing an ESBL from those expressing a Case HN. This medium also does not allow the identification of particular species of bacteria, and does not allow, for example to discriminate E. coli bacteria, K. pneumoniae bacteria.
Dans le cas de la détection de mécanismes de résistance autre que BLSE, on peut citer la demande de brevet EP0954560 qui concerne la recherche des entérocoques résistants à la Vancomycine, en combinant la Vancomycine avec un milieu chromogène révélant deux activités enzymatiques (β-glucosidase et pyrrolidonyl-arylamidase). Toutefois, ce milieu chromogène permet de déterminer uniquement si les souches résistantes à la vancomycine appartiennent ou n'appartiennent pas au genre Enterococcus, mais ne permet pas d'identifier l'espèce ou les mécanismes de résistance impliqués, notamment s'il s'agit d'une résistance acquise ou sauvage. In the case of the detection of resistance mechanisms other than ESBL, mention may be made of the patent application EP0954560, which relates to the search for enterococci resistant to Vancomycin, by combining Vancomycin with a chromogenic medium revealing two enzymatic activities (β-glucosidase and pyrrolidonyl-arylamidase). However, this chromogenic medium only makes it possible to determine whether the vancomycin-resistant strains belong to or do not belong to the Enterococcus genus, but does not make it possible to identify the species or the mechanisms of resistance involved, particularly in the case of acquired or wild resistance.
Ainsi, la caractérisation d'une espèce de microorganisme, puis l'identification de sa résistance à un traitement est longue et fastidieuse. Si le laboratoire rend au clinicien un dépistage positif alors que l'isolât est en fait dépourvu de microorganismes résistants, cela peut entraîner un traitement inutile et inadapté. Inversement, ne pas communiquer un dépistage positif qui sera ensuite confirmé retarde la mise en place de l'isolement du patient (et éventuellement d'une thérapeutique adaptée) d'une journée. Cela montre le besoin d'un test de confirmation rapide et fiable. Thus, the characterization of a species of microorganism, then the identification of its resistance to treatment is long and tedious. If the laboratory gives the clinician a positive screen while the isolate is in fact devoid of resistant microorganisms, this can lead to unnecessary and inappropriate treatment. Conversely, failure to communicate a positive screening which will then be confirmed delays the establishment of the isolation of the patient (and possibly a suitable therapy) of one day. This shows the need for a fast and reliable confirmation test.
La présente invention se propose donc d'améliorer l'état de la technique en présentant un nouvel outil de diagnostic permettant un gain de temps, de fiabilité et de pertinence dans la thérapie mise en œuvre. Notre invention permet en une seule étape d'identifier les espèces de micro-organismes gram négatifs présents dans un échantillon et déterminer leur mécanisme de résistance afin de proposer un traitement adapté à chaque patient. The present invention therefore proposes to improve the state of the art by presenting a new diagnostic tool allowing a saving of time, reliability and relevance in the therapy implemented. Our invention makes it possible in a single step to identify the species of gram negative microorganisms present in a sample and to determine their resistance mechanism in order to propose a treatment adapted to each patient.
Avant d'aller plus avant dans l'exposé de l'invention, les définitions suivantes sont données afin de faciliter la compréhension de l'invention. Before going further in the disclosure of the invention, the following definitions are given to facilitate the understanding of the invention.
Par milieu réactionnel, on entend un milieu comprenant tous les éléments nécessaires à la survie et/ou à la croissance de microorganismes, tels que les Staphylococcus aureus. Ce milieu réactionnel peut soit servir uniquement de milieu de révélation, soit de milieu de culture et de révélation. Dans le premier cas, la culture des microorganismes est effectuée avant ensemencement et, dans le deuxième cas, le milieu réactionnel constitue également le milieu de culture.  By reaction medium means a medium comprising all the elements necessary for the survival and / or growth of microorganisms, such as Staphylococcus aureus. This reaction medium can either serve only as a revelation medium or a culture medium and revelation. In the first case, the culture of the microorganisms is carried out before inoculation and, in the second case, the reaction medium also constitutes the culture medium.
Le milieu réactionnel peut être solide, semi-solide ou liquide. Par milieu solide, on entend par exemple un milieu gélifié. Préférentiellement, le milieu selon l'invention est un milieu gélifié. L'agar est l'agent gélifiant traditionnel en microbiologie pour la culture des microorganismes, mais il est possible d'utiliser de la gélatine ou de l'agarose. Un certain nombre de préparation sont disponibles dans le commerce, comme par exemple l'agar Columbia, la gélose Trypcase-soja, la gélose Mac Conkey, la gélose Sabouraud ou plus généralement celles décrites dans le Handbook of Microbiological Media (CRC Press). The reaction medium may be solid, semi-solid or liquid. By solid medium is meant for example a gelled medium. Preferably, the medium according to the invention is a gelled medium. Agar is the traditional gelling agent in microbiology for the cultivation of microorganisms, but it is possible to use gelatin or agarose. A number of preparations are commercially available, such as Columbia agar, Trypcase-soy agar, Mac Conkey agar, Sabouraud agar or more generally those described in the Handbook of Microbiological Media (CRC Press).
Le milieu réactionnel selon l'invention peut contenir d'éventuels autres additifs comme par exemple : des peptones, un ou plusieurs facteurs de croissance, des hydrates de carbone, un ou plusieurs agents sélectifs, des solutions tampons, un ou plusieurs gélifiants... Ce milieu réactionnel peut se présenter sous forme de liquide, de gel prêt à l'emploi, c'est à dire prêt à l'ensemencement en tube, flacon, ou sur boite de Pétri. Lorsque la présentation est sous forme de gel en flacon, on réalise préférentiellement une régénération (passage à 1000C) préalable du milieu avant de couler en boîte de Pétri. Il peut également s'agir d'un milieu en poudre ou en flacon qui avant d'être coulé en coulé en boîte de Pétri, tube ou flacon est additionné d'un supplément. The reaction medium according to the invention may contain any other additives, for example: peptones, one or more growth factors, carbohydrates, one or more selective agents, buffer solutions, one or more gelling agents, etc. This reaction medium may be in the form of a liquid, a ready-to-use gel, that is to say ready for seeding in a tube, a bottle, or on a petri dish. When the presentation is in the form of a gel in a vial, a regeneration (passage to 100 ° C.) is preferably carried out beforehand of the medium before pouring into a petri dish. It can also be a powder medium or a bottle which before being poured into a petri dish, tube or bottle is supplemented with a supplement.
Préférentiellement, le milieu selon l'invention est un milieu sélectif, c'est à dire un milieu comprenant des composés qui privilégient la croissance des bactéries Gram-négatif. On peut citer notamment le citrate de sodium, le sulfite de sodium, des antibiotiques tels que la vancomycine, des antifongiques tels que l'amphotéricine B, la natamycine, le cycloheximide, des tensioactifs tels que les sels biliaires, le désoxycholate de sodium, les Tergitol, des colorants tels que le vert brillant, le cristal violet, la fuchsine, l'éosine, le bleu de méthylène. Préférentiellement, le milieu selon l'invention est un milieu sélectif comprenant des composés qui privilégient la croissance des bactéries beta lactamase à spectre étendu (BLSE). On peut citer notamment les céphalosporines.  Preferably, the medium according to the invention is a selective medium, that is to say a medium comprising compounds that favor the growth of Gram-negative bacteria. Mention may in particular be made of sodium citrate, sodium sulphite, antibiotics such as vancomycin, antifungals such as amphotericin B, natamycin, cycloheximide, surfactants such as bile salts, sodium deoxycholate, Tergitol, dyes such as brilliant green, crystal violet, fuchsin, eosin, methylene blue. Preferably, the medium according to the invention is a selective medium comprising compounds that favor the growth of extended-spectrum beta lactamase bacteria (ESBL). In particular, cephalosporins may be mentioned.
o céphalosporine de première génération, telle que : cefalexine, cefaloridine, cefalotine, cefazoline, cefadroxil, cefazedone, cefatrizine, cefapirine, cefradine, cefacetrile, cefrodaxine, ceftezole o first-generation cephalosporin, such as: cefalexin, cefaloridine, cefalotin, cefazoline, cefadroxil, cefazedone, cefatrizine, cefapirine, cefradine, cefacetrile, cefrodaxine, ceftezole
o céphalosporine de deuxième génération, telle que : cefoxitine, cefuroxime, cefamandole, cefaclor, cefotetan, cefonicide, cefotiam, loracarbef, cefmetazole, cefprozil, ceforanide second-generation cephalosporin, such as cefoxitin, cefuroxime, cefamandole, cefaclor, cefotetan, cefonicide, cefotiam, loracarbef, cefmetazole, cefprozil, ceforanide
o céphalosporines de troisième génération, telle que : cefotaxime, ceftazidime, cefsulodine, ceftriaxone, cefmenoxime, latamoxef, ceftizoxime, cefîxime, cefodizime, cefetamet, cefpiramide, cefoperazone, cefpodoxime, ceftibuten, cefdinir, cefditoren, ceftriaxone, cefoperazone, cefbuperazone third-generation cephalosporins, such as cefotaxime, ceftazidime, cefsulodine, ceftriaxone, cefmenoxime, latamoxef, ceftizoxime, cefixime, cefodizime, cefetamet, cefpiramide, cefoperazone, cefpodoxime, ceftibuten, cefdinir, cefditoren, ceftriaxone, cefoperazone, cefbuperazone
o céphalosporine de quatrième génération, telle que cefepime, cefpirome o fourth-generation cephalosporin, such as cefepime, cefpirome
A titre de bactéries gram négatives, on peut citer notamment les bactéries des genres suivants: Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus, Morganella, Vibrio, Yersinia, Acinetobacter, Branhamella, Neisseria, Burkholderia, Citrobacter, Hafnia, Edwardsiella, Aeromonas, Moraxella, Pasteurella, Providencia, et Legionella. As gram-negative bacteria, mention may be made in particular of the following genera: Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus, Morganella, Vibrio, Yersinia, Acinetobacter, Branhamella, Neisseria, Burkholderia, Citrobacter, Hafnia, Edwardsella, Aeromonas, Moraxella, Pasteurella, Providencia, and Legionella.
Par mécanisme de résistance aux beta-lactamines, on entend tout type de dispositif qui permet à un micro-organisme de rendre un traitement partiellement ou totalement inopérant sur ledit microorganisme, garantissant sa survie, ledit dispositif étant lié à l'expression d'une enzyme appartenant au groupe des β-lactamases à spectre étendu ; d'une enzyme appartenant au groupe des céphalosporinases de classe C exprimée à un haut niveau  Beta-lactam resistance mechanism means any type of device that allows a microorganism to render a treatment partially or totally inoperative on said microorganism, ensuring its survival, said device being linked to the expression of an enzyme belonging to the group of extended spectrum β-lactamases; of an enzyme belonging to the class C cephalosporinases group expressed at a high level
Par marqueur de mécanisme de résistance aux beta-lactamines, on entend un composé permettant de mettre en évidence un tel mécanisme de résistance, tel que la cefepime et ses sels (Masuyoshi S. et al, 1989 - « Comparison of the in vitro and in vivo antibacterial activities of cefepime (BMY-28142) with ceftazidime, cefuzonam, cefotaxime and cefmenoxime. ») Beta-lactam resistance mechanism marker is understood to mean a compound making it possible to demonstrate such a mechanism of resistance, such as cefepime and its salts (Masuyoshi S. et al, 1989). antibacterial activities of cefepime (BMY-28142) with ceftazidime, cefuzonam, cefotaxime and cefmenoxime. ")
Par inhibiteur de mécanisme de résistance autre que ledit mécanisme de résistance aux beta-lactamines, on entend un compose qui permet d'inhiber indirectement la croissance des organismes développant une résistance particulière, sans inhibition de bactéries gram négatives exprimant ledit mécanisme de résistance aux beta-lactamines, tel que la cloxacilline ( Jack et Richmond, 1970 - «A comparative study of eight distinct beta- lactamases synthesized by gram-negative bacteria. » ) pour l'inhibition des céphalosporinases de classe C By resistance mechanism inhibitor other than said beta-lactam resistance mechanism, it is meant a compound which indirectly inhibits the growth of organisms developing a particular resistance, without inhibition of gram-negative bacteria expressing said mechanism of resistance to beta-lactams. lactams, such as cloxacillin (Jack and Richmond, 1970 - "A comparative study of eight distinct beta-lactamases synthesized by gram-negative bacteria.") for the inhibition of Class C cephalosporinases
Au sens de la présente invention, le substrat d'une activité enzymatique ou métabolique est choisi parmi tout substrat pouvant être hydrolyse en un produit qui permet la détection, directe ou indirecte d'une activité enzymatique ou d'un métabolisme, telle que notamment une activité osidase, préférentiellement une activité glucuronidase, glucosidase ou galactosidase.  For the purposes of the present invention, the substrate of an enzymatic or metabolic activity is chosen from any substrate that can be hydrolysed into a product that allows the direct or indirect detection of an enzymatic activity or a metabolism, such as, in particular, a osidase activity, preferably a glucuronidase, glucosidase or galactosidase activity.
Il peut s'agir d'un substrat naturel ou synthétique. Le métabolisme du substrat provoque une variation des propriétés physico-chimiques du milieu réactionnel ou des cellules d'organismes. Cette variation peut être détectée par des méthodes physico-chimiques, notamment des méthodes optiques par l'œil de l'opérateur ou à l'aide d'instruments, spectrométriques, électriques, magnétiques, ... Préférentiellement, il s'agit d'une variation des propriétés optiques, telles qu'une modification d'absorption, de fluorescence ou de luminescence. Comme substrat chromogène, on peut citer notamment les substrats à base d'indoxyl, flavone, alizarine, acridine, esculetine, phénoxazine, nitrophénol, nitroaniline, naphtol, catéchol, hydroxyquinoline, coumarine, aminophénol, dichloroaminophénol. Préférentiellement, le(s) substrat(s) utilisé(s) dans la présente invention est(sont) à base d'indoxyl. It can be a natural or synthetic substrate. The metabolism of the substrate causes a variation in the physicochemical properties of the reaction medium or the cells of organisms. This variation can be detected by physicochemical methods, especially optical methods by the eye of the operator or by means of instruments, spectrometric, electrical, magnetic, ... Preferably, it is a question of a change in optical properties, such as a change in absorption, fluorescence or luminescence. As a chromogenic substrate, there may be mentioned in particular substrates based on indoxyl, flavone, alizarin, acridine, esculetin, phenoxazine, nitrophenol, nitroaniline, naphthol, catechol, hydroxyquinoline, coumarin, aminophenol, dichloroaminophenol. Preferably, the substrate (s) used in the present invention is (are) based on indoxyl.
Comme substrat fluorescent, on peut citer notamment les substrats à base d'umbelliférone ou de coumarine, à base de résorufïne, phénoxazine, naphtol, naphtylamine, 2'-hydroxyphényl-hétérocycle ou 2'-aminophényl-hétérocycle ou encore à base de fluorescéïne.  Fluorescent substrates that may especially be mentioned include umbelliferone-based or coumarin-based substrates based on resorufin, phenoxazine, naphthol, naphthylamine, 2'-hydroxyphenyl-heterocycle or 2'-aminophenyl-heterocycle or based on fluorescein.
Préférentiellement, le substrat utilisé dans la présente invention est 5-bromo-4-chloro- 3-indoxyl-beta-D-glucopyranoside, préférentiellement en combinaison avec le 5-bromo- 6-chloro-3-indoxyl-beta-D-galactopyranoside. Autres substrats possibles ; substrats de beta-glucosidase : 5-Bromo-6-chloro-3-indoxyl-beta-glucoside ; Dihydroxyflavone-beta- glucoside ; 3-hydroxyflavone-beta-glucoside, 3,4-Cyclohexénoesculétine-beta-glucoside (on ne parle pas du 3,4-cyclopentenoesculetin-beta-glucoside ?) ; 8-Hydroxyquinoline- beta-glucoside ; 5-Bromo-4-chloro-3-indoxyl-N-méthyl-beta-glucoside ; 6-Chloro-3- indoxyl-beta-glucoside ; 5-Bromo-3-indoxyl-beta-glucoside ; 5-Iodo-3-indoxyl-beta- glucoside ;6-Iodo-3-indoxyl-beta-glucoside 6-Fluoro-3-indoxyl-beta-glucoside ;Preferably, the substrate used in the present invention is 5-bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside, preferably in combination with 5-bromo-6-chloro-3-indoxyl-beta-D-galactopyranoside . Other substrates possible; beta-glucosidase substrates: 5-Bromo-6-chloro-3-indoxyl-beta-glucoside; Dihydroxyflavone-beta- glucoside; 3-hydroxyflavone-beta-glucoside, 3,4-cyclohexenesculetin-beta-glucoside (not 3,4-cyclopentenoesculetin-beta-glucoside?); 8-Hydroxyquinoline-beta-glucoside; 5-Bromo-4-chloro-3-indoxyl-N-methyl-beta-glucoside; 6-Chloro-3-indoxyl-beta-glucoside; 5-Bromo-3-indoxyl-beta-glucoside; 5-Iodo-3-indoxyl-beta-glucoside, 6-Iodo-3-indoxyl-beta-glucoside 6-Fluoro-3-indoxyl-beta-glucoside;
Alizarine-beta-glucoside ; Nitrophényl-beta-glucoside ; 4-Méthylumbelliferyl-beta- glucoside ; Naphtholbenzein-beta-glucoside ; Indoxyl-N-méthyl-beta-glucoside ; Naphtyl-beta-glucoside ; Aminophényl-beta-glucoside ; Dichloroaminophényl-beta- glucoside; substrats de beta-galactosidase : 5-Bromo-4-chloro-3-indoxyl-beta-galactoside ; Dihydroxyflavone-beta-galactoside ; 3,4-Cyclohexénoesculétine-beta-galactoside ; 8- Hydroxyquinoline-beta-galactoside ; 5-Bromo-4-chloro-3-indoxyl-N-méthyl-beta- galactoside ; 6-Chloro-3-indoxyl-beta-galactoside ; 5-Bromo-3-indoxyl-beta-galactoside ; 5-Iodo-3-indoxyl-beta-galactoside ; 6-Fluoro-3-indoxyl-beta-galactoside ; Alizarine-beta- galactoside ; Nitrophényl-beta-galactoside ; 4-Méthylumbelliferyl-beta-galactoside ; Naphtholbenzein-beta-galactoside ; Indoxyl-N-méthyl-beta-galactoside ; Naphtyl-beta- galactoside ; Aminophényl-beta-galactoside ; Dichloroaminophényl-beta-galactoside ; substrats de beta-glucuronidase : 5-Bromo-6-chloro-3-indoxyl-beta-glucuronide ; Dihydroxyflavone-beta-glucuronide ; 3,4-Cyclohexénoesculétine-beta-glucuronide ; 8- Hydroxyquinoline-beta-glucuronide ; 5-Bromo-4-chloro-3-indoxyl- beta-glucuronide ; 5-Bromo-4-chloro-3-indoxyl-N-méthyl-beta-glucuronide ; 6-Chloro-3-indoxyl-beta- glucuronide ; 5-Bromo-3-indoxyl-beta-glucuronide ; 5-Iodo-3-indoxyl-beta-glucuronide ; 6-Fluoro-3-indoxyl-beta-glucuronide ; Alizarine-beta-glucuronide ; Nitrophényl-beta- glucuronide ; 4-Méthylumbelliferyl-beta-glucuronide ; Naphtholbenzein-beta- glucuronide ; Indoxyl-N-méthyl-beta-glucuronide ; Naphtyl-beta-glucuronide ;Alizarin-beta-glucoside; Nitrophenyl-beta-glucoside; 4-Methylumbelliferyl-beta-glucoside; Naphtholbenzein-beta-glucoside; Indoxyl-N-methyl-beta-glucoside; Naphtyl-beta-glucoside; Aminophenyl-beta-glucoside; Dichloroaminophenyl beta-glucoside; beta-galactosidase substrates: 5-Bromo-4-chloro-3-indoxyl-beta-galactoside; Dihydroxyflavone-beta-galactoside; 3,4-Cyclohexenoesculin-beta-galactoside; 8-Hydroxyquinoline-beta-galactoside; 5-Bromo-4-chloro-3-indoxyl-N-methyl-beta-galactoside; 6-Chloro-3-indoxyl-beta-galactoside; 5-Bromo-3-indoxyl-beta-galactoside; 5-Iodo-3-indoxyl-beta-galactoside; 6-Fluoro-3-indoxyl-beta-galactoside; Alizarin-beta galactoside; Nitrophenyl-beta-galactoside; 4-Methylumbelliferyl-beta-galactoside; Naphtholbenzein-beta-galactoside; Indoxyl-N-methyl-beta-galactoside; Naphtyl beta-galactoside; Aminophenyl-beta-galactoside; Dichloroaminophenyl-beta-galactoside; beta-glucuronidase substrates: 5-Bromo-6-chloro-3-indoxyl-beta-glucuronide; Dihydroxyflavone beta-glucuronide; 3,4-Cyclohexenoesculin-beta-glucuronide; 8- Hydroxyquinoline-beta-glucuronide; 5-Bromo-4-chloro-3-indoxyl-beta-glucuronide; 5-Bromo-4-chloro-3-indoxyl-N-methyl-beta-glucuronide; 6-Chloro-3-indoxyl-beta-glucuronide; 5-Bromo-3-indoxyl-beta-glucuronide; 5-Iodo-3-indoxyl-beta-glucuronide; 6-Fluoro-3-indoxyl-beta-glucuronide; Alizarin-beta-glucuronide; Nitrophenyl-beta- glucuronide; 4-Methylumbelliferyl-beta-glucuronide; Naphtholbenzein-beta-glucuronide; Indoxyl-N-methyl-beta-glucuronide; Naphtyl-beta-glucuronide;
Aminophényl-beta-glucuronide ; Dichloroaminophényl-beta-glucuronide ; substrats d'alpha-glucosidase, substrats d'alpha-galactosidase, substrats d'estérase, notamment de lipase, de phosphatase, substrats de cellobiosidase, substrats de ribosidase, substrats d ' hexosaminidase . Aminophenyl-beta-glucuronide; Dichloroaminophenyl-beta-glucuronide; alpha-glucosidase substrates, alpha-galactosidase substrates, esterase substrates, especially lipase, phosphatase, cellobiosidase substrates, ribosidase substrates, hexosaminidase substrates.
Les substrats de l'invention sont utilisables dans une large gamme de pH, notamment entre pH 5,5 et 10, préférentiellement entre 6,5 et 10. Lorsque le milieu selon l'invention comprend un substrat ou plusieurs substrats d'activité enzymatique beta glucosidase, la concentration en substrat(s) est préférentiellement comprise entre 0,01 et 2 g/1, encore plus préférentiellement entre 0,02 et 0,2 g/1, et, avantageusement, elle est entre 0,05 et 0,15 g/1. En effet, à cette concentration de substrat, on obtient un meilleur contraste de coloration.  The substrates of the invention can be used over a wide pH range, in particular between pH 5.5 and 10, preferably between 6.5 and 10. When the medium according to the invention comprises a substrate or several substrates of beta enzymatic activity. glucosidase, the concentration of substrate (s) is preferably between 0.01 and 2 g / l, more preferably between 0.02 and 0.2 g / l, and advantageously between 0.05 and 0, 15 g / 1. In fact, at this concentration of substrate, a better color contrast is obtained.
Préférentiellement, ledit substrat chromogénique, est choisi parmi un substrat de glucuronidase, de beta glucosidase et de beta galactosidase.  Preferably, said chromogenic substrate is selected from a glucuronidase, beta-glucosidase and beta-galactosidase substrate.
Par échantillon biologique, on entend un échantillon clinique, issu d'un prélèvement de liquide biologique, ou un échantillon alimentaire, issu de tout type d'aliment. Cet échantillon peut être ainsi liquide ou solide et on peut citer d'une manière non limitative, un échantillon clinique de sang, de plasma, d'urines, de fécès, de prélèvements rectal, de nez, de gorges, de peaux, de plaies, de liquide céphalo-rachidien, un échantillon alimentaire d'eau, de boissons tels que le lait, un jus de fruits; de yaourt, de viande, d'œufs, de légumes, de mayonnaise, de fromage ; de poisson..., un échantillon alimentaire issu d'une alimentation destinée aux animaux, tel que notamment un échantillon issu de farines animales.  By biological sample, we mean a clinical sample, from a sample of biological fluid, or a food sample, from any type of food. This sample may thus be liquid or solid and may be mentioned in a nonlimiting manner, a clinical sample of blood, plasma, urine, faeces, rectal samples, nose, throats, skin, wounds. , cerebrospinal fluid, a food sample of water, beverages such as milk, fruit juice; yogurt, meat, eggs, vegetables, mayonnaise, cheese; fish ..., a food sample from a feed intended for animals, such as in particular a sample from animal meal.
A ce titre, l'invention concerne un milieu réactionnel de bactéries gram négatives présentant un mécanisme de résistance aux beta-lactamines comprenant : As such, the invention relates to a reaction medium of gram-negative bacteria having a beta-lactam resistance mechanism comprising:
• Un marqueur de mécanisme de résistance aux beta-lactamines qui est la cefépime, • Un inhibiteur de mécanisme de résistance, autre que ledit mécanisme de résistance aux beta-lactamines • A beta-lactam resistance mechanism marker that is cefepime, • a resistance mechanism inhibitor, other than said beta-lactam resistance mechanism
Selon un mode préféré de réalisation de l'invention, ledit inhibiteur de mécanisme de résistance est la cloxacilline. Préférentiellement, la concentration en cloxacilline est comprise entre 0,05 et lg/1 et plus préférentiellement entre 0,1 et 0,3g/l. Avantageusement, elle est de 0,2g/l. According to a preferred embodiment of the invention, said resistance mechanism inhibitor is cloxacillin. Preferably, the concentration of cloxacillin is between 0.05 and 1 g / l and more preferably between 0.1 and 0.3 g / l. Advantageously, it is 0.2 g / l.
Selon un mode préféré de réalisation de l'invention, la concentration en cefepime est comprise entre 0,05 et lmg/1 et plus préférentiellement entre 0,10 et 0,5mg/l. Avantageusement, elle est de 0,25 mg/1.  According to a preferred embodiment of the invention, the concentration of cefepime is between 0.05 and 1 mg / l and more preferably between 0.10 and 0.5 mg / l. Advantageously, it is 0.25 mg / l.
Selon un mode préféré de réalisation de l'invention, le milieu réactionnel comprend en outre un substrat d'une activité enzymatique ou métabolique, préférentiellement un substrat chromogénique.  According to a preferred embodiment of the invention, the reaction medium further comprises a substrate of an enzymatic or metabolic activity, preferably a chromogenic substrate.
Selon un mode préféré de réalisation de l'invention, ledit substrat chromogénique, est choisi parmi un substrat de glucuronidase, de beta glucosidase et de beta galactosidase.According to a preferred embodiment of the invention, said chromogenic substrate is selected from a glucuronidase, beta-glucosidase and beta-galactosidase substrate.
Selon un mode préféré de réalisation de l'invention, la concentration en substrat chromogénique est comprise entre 0,02 et 2g/l et plus préférentiellement entre 0,03 et 0,5g/l. Avantageusement, elle est de 0,1 g/1. According to a preferred embodiment of the invention, the chromogenic substrate concentration is between 0.02 and 2 g / l and more preferably between 0.03 and 0.5 g / l. Advantageously, it is 0.1 g / l.
Selon un mode préféré de réalisation de l'invention, ledit milieu comprend une combinaison d'au moins deux substrats chromogéniques. Selon un premier mode de réalisation, cette combinaison comprend un substrat de glucuronidase et un substrat de beta glucosidase. Selon un deuxième mode de réalisation, cette combinaison comprend un substrat de beta galactosidase et un substrat de beta glucosidase. L'invention concerne également l'utilisation d'un milieu tel que défini ci avant pour la détection de bactéries gram négatives résistantes aux beta-lactamines, préférentiellement des bactéries beta lactamase à spectre étendu (BLSE) According to a preferred embodiment of the invention, said medium comprises a combination of at least two chromogenic substrates. According to a first embodiment, this combination comprises a glucuronidase substrate and a beta-glucosidase substrate. According to a second embodiment, this combination comprises a beta-galactosidase substrate and a beta-glucosidase substrate. The invention also relates to the use of a medium as defined above for the detection of gram-negative bacteria resistant to beta-lactam antibiotics, preferably extended-spectrum beta lactamase bacteria (ESBL).
L'invention concerne également un procédé de détection de bactéries gram négatives résistantes aux beta-lactamines caractérisé en ce qu'il comprend les étapes suivantes : 1. disposer d'un milieu réactionnel tel que défini ci-avant,  The invention also relates to a method for detecting gram-negative bacteria resistant to beta-lactams, characterized in that it comprises the following steps: 1. having a reaction medium as defined above,
2. ensemencer le milieu avec un échantillon biologique à tester,  2. inoculate the medium with a biological sample to be tested,
3. laisser incuber, et  3. incubate, and
4. détecter la présence de bactéries gram négatives résistantes aux beta-lactamines.  4. detect the presence of gram-negative bacteria resistant to beta-lactams.
L'incubation est préférentiellement réalisée à une température comprise entre 3O0C et 420C. Les bactéries gram négatives résistantes aux beta-lactamines sont préférentiellement détectées par une activité glucuronidase, beta-glucosidase ou beta-galactosidase spécifique qui permet d'obtenir des colonies colorées ou fluorescentes. Les autres espèces apparaissent incolores ou d'une couleur ou fluorescence différente de celle des colonies de bactéries Gram-négatif résistantes aux beta-lactamines. Les exemples ci dessous sont donnés à titre explicatif et n'ont aucun caractère limitatif. Ils permettront de mieux comprendre l'invention. EXEMPLE 1 The incubation is preferably carried out at a temperature of between 30 ° C. and 42 ° C. Gram-negative bacteria resistant to beta-lactam antibiotics are preferably detected by a specific glucuronidase, beta-glucosidase or beta-galactosidase activity which makes it possible to obtain colored or fluorescent colonies. Other species appear colorless or of a different color or fluorescence than colonies of gram-negative bacteria resistant to beta-lactam antibiotics. The examples below are given for explanatory purposes and have no limiting character. They will help to better understand the invention. EXAMPLE 1
Choix des souches: Les inventeurs ont sélectionné des souches permettant d'évaluer l'activité des antibiotiques vis à vis des espèces à Gram-négatif : entérobactéries et bacilles non- fermentants. En particulier, ont été utilisés des souches productrices de BLSE, de céphalosporinase de haut niveau (CaseHN) et des souches sans profil de résistance particulier, dites sauvages.  Choice of strains: The inventors have selected strains making it possible to evaluate the activity of antibiotics with respect to Gram-negative species: enterobacteria and non-fermenting bacilli. In particular, strains producing ESBL, high level cephalosporinase (CaseHN) and strains with no particular resistance profile, called wild type, have been used.
Préparation des milieux : Les milieux testés étaient des milieux composés de la base peptonée du milieu Chrom ID CPS (bioMérieux réf 43541) à laquelle étaient rajoutés après autoclavage, dans des milieux en surfusion, 300 mg/1 de Cloxacilline et pour le milieu T : 4 mg/1 de Cefpodoxime, pour le milieu A : 0.25 mg/L de Céfépime, pour le milieu B : 3 mg/1 de Céfamandole et pour le milieu C : 3 mg/1 de Céfuroxime.  Preparation of media: The media tested were media composed of the peptone base of Chrom ID CPS medium (bioMérieux ref 43541) to which was added after autoclaving, in supercooled media, 300 mg / l of Cloxacillin and for T medium: 4 mg / l of cefpodoxime, for medium A: 0.25 mg / l of cefepime, for medium B: 3 mg / l of cefamandole and for medium C: 3 mg / l of cefuroxime.
Ensemencement des milieux : Les milieux sont ensemencés en réalisant un isolement selon la méthode des 3 cadrans à partir de suspensions bactériennes à 0.5McF. Les milieux sont ensuite incubés pendant 24 heures à 37 0C Inoculation of the media: The media are inoculated by isolating using the 3-dial method from 0.5McF bacterial suspensions. The media are then incubated for 24 hours at 37 ° C.
Lecture des milieux : Les milieux sont observés visuellement après 18 h et 24 heures d'incubation, la densité de croissance ainsi que les colorations et intensités de coloration sont évaluées suivant l'échelle ci-dessous : Reading media: The media are visually observed after 18 hours and 24 hours of incubation, the growth density as well as staining and color intensities are evaluated according to the scale below:
- ou 0 : absence de croissance ou d'expression d'activité enzymatique (soit aucune coloration)  or 0: absence of growth or expression of enzymatic activity (ie no coloration)
+ : faible croissance, ou activité enzymatique +: low growth, or enzymatic activity
++ : très forte densité de croissance ou forte activité enzymatique (coloration très intense) ++: very high growth density or strong enzymatic activity (very intense coloration)
Résultats : Results:
Conclusion : Les 4 molécules permettent toutes une bonne croissance et expressions des activités enzymatiques pour les souches productrices d'ESBL. Cependant, seule la Céfépime permet une bonne discrimination entre les souches productrices d'ESBL, les souches productrices d'une Case HN ou les souches de taxons sauvages. C'est donc l'antibiotique qui permet de détecter les souches ESBL avec la meilleure sensibilité et spécificité. Conclusion: The 4 molecules all allow good growth and expressions of enzymatic activities for the strains producing ESBL. However, only Cefepime allows good discrimination between ESBL-producing strains, HN Case-producing strains or wild-type taxon strains. It is therefore the antibiotic that can detect ESBL strains with the best sensitivity and specificity.

Claims

REVENDICATIONS
1) Milieu réactionnel de bactéries gram négatives présentant un mécanisme de résistance aux beta-lactamines comprenant : 1) A reaction medium of gram-negative bacteria exhibiting a beta-lactam resistance mechanism comprising:
• un marqueur de mécanisme de résistance aux beta-lactamines qui est le cefepime, A beta-lactam resistance mechanism marker which is cefepime,
• un inhibiteur de mécanisme de résistance, autre que ledit mécanisme de résistance aux beta-lactamines A resistance mechanism inhibitor, other than said beta-lactam resistance mechanism
2) Milieu réactionnel selon la revendication 1 caractérisé en ce que ledit inhibiteur de mécanisme de résistance est la cloxacilline 2) A reaction medium according to claim 1 characterized in that said resistance mechanism inhibitor is cloxacillin
3) Milieu réactionnel selon la revendication 1 ou 2 caractérisé en ce que la concentration en cefepime est comprise entre 0,05 et lmg/1 et plus préférentiellement entre 0,1 et 0,5mg/l. 3) A reaction medium according to claim 1 or 2 characterized in that the cefepime concentration is between 0.05 and 1 mg / l and more preferably between 0.1 and 0.5 mg / l.
4) Milieu réactionnel selon l'une quelconque des revendications 1 a 3 comprenant en outre un substrat d'une activité enzymatique ou métabolique, préférentiellement un substrat chromogénique. 4) A reaction medium according to any one of claims 1 to 3 further comprising a substrate of an enzymatic or metabolic activity, preferably a chromogenic substrate.
5) Milieu réactionnel selon la revendication 4 selon laquelle ledit substrat chromogénique, est choisi parmi un substrat de glucuronidase, de beta glucosidase et de beta galactosidase 5) A reaction medium according to claim 4 wherein said chromogenic substrate is selected from a substrate glucuronidase, beta glucosidase and beta galactosidase
6) Utilisation d'un milieu réactionnel selon l'une quelconque des revendications 1 à 5 pour la détection de bactéries gram négatives résistantes aux beta-lactamines 6) Use of a reaction medium according to any one of claims 1 to 5 for the detection of gram-negative bacteria resistant to beta-lactam antibiotics
7) Utilisation d'un milieu réactionnel selon la revendication 6 selon laquelle les bactéries gram négatives résistantes aux beta-lactamines sont des bactéries beta lactamase à spectre étendu (BLSE) 7) Use of a reaction medium according to claim 6 according to which the gram-negative bacteria resistant to beta-lactam antibiotics are extended spectrum beta lactamase bacteria (ESBL)
8) Procédé de détection de bactéries gram négatives résistantes aux beta-lactamines caractérisé en ce qu'il comprend les étapes suivantes : 8) A method for detecting gram-negative bacteria resistant to beta-lactams, characterized in that it comprises the following steps:
a) disposer d'un milieu réactionnel selon l'une quelconque des revendications 1 à 5, b) ensemencer le milieu avec un échantillon biologique à tester,  a) having a reaction medium according to any one of claims 1 to 5, b) seeding the medium with a biological sample to be tested,
c) laisser incuber, et  c) incubate, and
d) détecter la présence de bactéries gram négatives résistantes aux beta-lactamines.  d) detect the presence of gram-negative bacteria resistant to beta-lactams.
EP10752006A 2009-07-27 2010-07-13 Media for the specific detection of gram-negative bacteria resistant to beta-lactam antibiotics Withdrawn EP2459732A1 (en)

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