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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:

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