Classifications

• • 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
  • C12Q1/02—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
  • C12Q1/10—Enterobacteria
• • 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
• • 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
  • C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
  • C12Q2334/50—Indoles
• • 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
  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
  • G01N2333/245—Escherichia (G)

Definitions

• the field of the invention is that of microbiological biochemical analysis, and in particular the detection and identification of bacteria.
• Pathogenic bacteria including Gram-negative bacilli, such as enterobacteria, are responsible for many diseases, epidemics, etc.
• the species E. coli ⁇ Escherichia coli) is the most represented aerobic species in the digestive tract. . However, their presence in water is a witness of faecal contamination, and some strains are pathogenic and responsible for peritoneal, biliary, appendicular or genital suppuration.
• E. coli Early and specific detection of E. coli makes it possible to propose a suitable solution, in terms of treatment, of decontamination ...
• This detection can be based notably on the use of detection media comprising particular substrates, specific for a metabolic activity, called target metabolic activity, such as that an enzymatic activity of the bacterium that one wishes to detect: by the choice of substrates, depending on whether there is reaction or not, it is possible to characterize the nature of a microorganism.
• the CPS ID 3 medium bioMerieux
• the invention proposes to solve the problems of the state of the art by presenting a new medium particularly suitable for identifying E. coli bacteria in a fast, inexpensive and easy to implement manner.
• the inventors have shown that a particular combination of enzyme substrates, at appropriate concentrations, allows rapid and easy detection of E. coli. coli, in particular from a urinary sample.
• the following definitions are given to facilitate the understanding of the invention.
• detection medium is meant a medium comprising all the elements necessary for the survival and / or growth of microorganisms.
• This detection medium can either serve only as a detection medium, or a culture and detection medium.
• the culture of the microorganisms is carried out before seeding, and in the second case, the detection medium also constitutes the culture medium.
• the culture medium according to the invention may contain any other additives, for example: peptones or tissue extracts, one or more growth factors, carbohydrates, one or more selective agents, buffers, one or more gelling agents ...
• This culture medium can 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.
• the detection may be carried out in a liquid medium, strip or other solid support
• substrate any molecule capable of generating directly or indirectly a detectable signal due to an enzymatic or metabolic activity of the microorganism.
• the substrate may in particular be a metabolic substrate, such as a source of carbon or nitrogen, coupled to an indicator producing a coloration in the presence of one of the products of the metabolism.
• a metabolic substrate such as a source of carbon or nitrogen
• the substrate can also be an enzymatic substrate, that is to say a substrate that can be hydrolyzed by an enzyme into a product allowing the direct or indirect detection of a microorganism.
• This substrate may in particular comprise a first specific part of the enzymatic activity to be revealed and a second part serving as a marker, hereinafter referred to as a marker part.
• This marker part can be chromogenic, fluorogenic, luminescent, etc.
• chromogenic substrate well suited to solid supports (filter, agar, electrophoresis gel), mention may be made in particular of substrates based on indoxyl and its derivatives, and substrates based on hydroxyquinoline or esculetin and their derivatives, which allow the detection of osidase and esterase activities. Mention may also be made of nitrophenol and nitroaniline substrates 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.
• substrates based on naphthol and naphthylamine and their derivatives which make it possible to detect the osidase and esterase activities via naphthol, and the peptidase activities via naphthylamine.
• This substrate may in particular, but in a nonlimiting manner, allow the detection of an enzymatic activity such as the activity of an osidase, peptidase, esterase, etc.
• the enzyme substrate may also be a natural substrate, the product of which hydrolysis is detected directly or indirectly.
• Tryptophan for detecting tryptophanase or desaminase activity
• a cyclic amino acid for detecting a desaminase activity
• Phosphatidyl Inositol for detecting phospholipase activity
• the substrate is preferably chosen from substrates based on Indoxyl (3-Indoxyl, 5-Bromo-3-indoxyl, 5-Iodo-3-indoxyl, 4-Chloro-3-indoxyl, 5-Bromo- 4-chloro-3-indoxyl, 5-bromo-6-chloro-3-indoxyl, 6-bromo-3-indoxyl, 6-chloro-3-indoxyl, 6-fluoro-3-indoxyl, 5-bromo-4- chloro-N-methyl-3-indoxyl, N-methyl-3-indoxyl, ...); umbelliferone (4-Methylumbelliferone, Cyclohexenoesculetin, ...); Alizarin; p-Naphtolbenzine; Nitrophenol (ortho-Nitrophenol, para-Nitrophenol, ...); hydroxyquinoline; Cathecol (Cathecol, Dihydroxyflavone, Hydroxyflavone,
• the substrates used for the detection of a beta-glucuronidase activity may especially be 4-methylumbelliferyl-beta-glucuronide, 5-Bromo-4-chloro-3-indolyl-beta-glucuronide, 5-Bromo 6-chloro-3-indolyl-beta-glucuronide, 6-chloro-3-indolyl-beta-glucuronide, Alizarin-beta-glucuronide,
• the substrates used for the detection of a beta-galactosidase activity may especially be 4-methylumbelliferyl-beta-galactoside, 5-Bromo-4-chloro-3-indolyl-beta-galactoside or 5-Bromo-6-chloro.
• the substrates used for the detection of a beta-glucosidase activity may especially be 4-methylumbelliferyl-beta-Glucoside, 5-Bromo-4-chloro-3-indolyl-beta-glucoside, 5-Bromo-6-chloro 3-indolyl-beta-glucoside, 6-chloro-3-indolyl-beta-glucoside, Alizarin-beta-glucoside, Cyclohexenosculetin-beta-Glucoside,
• inducer is meant a compound inducing an increase in the expression of the targeted metabolic activity, any experimental condition being equal, the metabolic activity is greater when the inducer is at an appropriate concentration than when it is absent or at an inappropriate concentration.
• a concentration of between 100 ng / l and 10 g / l, preferably between 10 mg / l and 3 g / l is particularly suitable for the present invention.
• a glucuronide preferably chosen from Glucuronate, methyl-beta-glucuronide.
• a galactoside preferentially chosen from Lactose, Isopropyl-beta-thio-galactoside
• beta-glucosidase a carbohydrate consisting of a carbohydrate bound in the ⁇ position to glucose or carbohydrate with a ⁇ -glucoside subunit, in particular cellobiose, cellulose, starch, cellotriosis, trehalose. Mention may also be made of Methyl- ⁇ -glucoside, Isopropyl- ⁇ -thio-glucoside, Indoxyl- ⁇ -glucoside or Methyl- ⁇ -thio-glucoside.
• inhibitor is meant a compound inducing a decrease in the expression of the targeted metabolic activity, any experimental condition being equal, the metabolic activity is lower when the inducer is at an appropriate concentration than when it is absent or at an inappropriate concentration.
• a concentration of between 100 ng / l and 30 g / l, preferably between 1 mg / l and 3 g / l is particularly suitable for the present invention.
• biological sample is meant a clinical sample, taken from a sample of biological fluid, or a food sample, derived from any type of food or an environmental sample such as a sample of surface, water, air, ....
• This sample can be liquid or solid and can be cited in a non-limiting manner, a clinical sample blood, plasma, urine, faeces, nose samples, throats, skin, wounds, cerebrospinal fluid, a food sample of water, beverages such as milk, fruits; 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 method for detecting and / or identifying E. coli in a biological sample, preferably urinary according to which a) the sample is seeded, preferentially urinary likely to contain E. coli on a detection medium comprising
• a first substrate selected from a substrate of beta-glucuronidase, beta-galactosidase, alpha-galactosidase, a Lactose acidifying enzyme, beta-ribosidase, phosphatase, L-Alanine aminopeptidase, L-Leucine aminopeptidase,
• a second substrate different from said first substrate, chosen from a substrate of beta-glucuronidase, beta-galactosidase, alpha-galactosidase, a Lactose acidification enzyme, beta-ribosidase, phosphatase, L-Alanine aminopeptidase, L Leucine aminopeptidase for obtaining colonies of bacteria b) colonies reacting with the first substrate and / or the second substrate are identified as E. coli colonies.
• said first and second substrates are at a suitable concentration.
• the seeding of the microorganisms can be carried out by all the seeding techniques known to those skilled in the art.
• An incubation step can be carried out at a temperature for which the enzymatic activity that one wishes to detect is optimal, that the person skilled in the art can easily choose according to the enzymatic activity to be detected.
• the detection / identification can be carried out by visual examination, colorimetry or fluorimetry.
• said first substrate is at a concentration of between 20 and 1000 mg / l and said second substrate is at a concentration of between 20 mg / and 30 g / l.
• said first substrate is a beta-glucuronidase substrate and the second substrate is a beta-galactosidase substrate.
• the substrate of a beta-glucuronidase activity is chosen from 4-methylumbelliferyl-beta-glucuronide, 5-Bromo-4-chloro-3-indolyl-beta-glucuronide, 5-Bromo-6-chloro-3 -indolyl-beta-glucuronide, 6-chloro-3-indolyl-beta-glucuronide, Alizarin-beta-glucuronide, Cyclohexenoesculetin-beta-glucuronide or their salts, at concentrations preferably comprised between 20 and 1000 mg / l .
• the beta-galactosidase substrate is at a low concentration.
• the substrate of a beta-galactosidase activity is chosen from 4-methylumbelliferyl-beta-galactoside, 5-Bromo-4-chloro-3-indolyl-beta-galactoside, and 5-Bromo-6-chloro-3 -indolyl-beta-galactoside, 6-chloro-3-indolyl-beta-galactoside, Alizarin-beta-galactoside, Cyclohexenoesculetin-beta-galactoside or their salts, at a concentration preferably between 10 and 1000 mg / l, preferably between 20 and 500 mg / l.
• the detection medium further comprises a third substrate, preferably chosen from a beta-glucosidase, beta-lactosidase, N-acetyl-hexosaminidase, esterase, sulfatase and betaxylosidase substrate.
• a third substrate preferably chosen from a beta-glucosidase, beta-lactosidase, N-acetyl-hexosaminidase, esterase, sulfatase and betaxylosidase substrate.
• phospholipase alpha-mannosidase, beta-mannosidase, beta-cellobiosidase, alpha-glucosidase, tryptophanase, deaminase, oxidase, pigment synthesis, peptidases (beta-Alanine-aminopeptidase, elastase, ).
• said third substrate is a beta glucosidase substrate.
• the substrate of a beta- glucosidase activity is chosen from 4-methylumbelliferyl-beta-glucoside, 5-Bromo-4-chloro-3-indolyl-beta-glucoside and 5-bromo-6-chloro-3.
• the detection medium further comprises an inductor.
• the inducer is at a concentration of between 100 ng / l and 10 g / l.
• the inductor is preferably:
• a glucuronide preferably chosen from Glucuronate, methyl-beta-glucuronide.
• a galactoside preferentially chosen from Lactose, Isopropyl-beta-thio-galactoside
• beta-glucosidase a carbohydrate consisting of a carbohydrate bound in the ⁇ position to glucose or carbohydrate with a ⁇ -glucoside subunit, in particular cellobiose, cellulose, starch, cellotriosis, trehalose. Mention may also be made of Methyl- ⁇ -glucoside, Isopropyl- ⁇ -thio-glucoside, Indoxyl- ⁇ -glucoside or Methyl- ⁇ -thio-glucoside.
• the inducer is cellobiose, at a concentration preferably between 100ng / l and 10g / l.
• the detection medium further comprises an inhibitor.
• the inhibitor is at a concentration of between 100 ng and 30 g / l.
• the inhibitor is preferentially:
• beta-galactosidase 2-Deoxy-galactose, Cellobiose, D-Galactose, D-Glucose
• CPS ID 3 bioMerieux
• 3-indolyl- ⁇ -glucuronide 0-0.1-0.15 and 0.20 g / l
• 5-Bromo-6-chloro-3-indolyl- ⁇ -galactoside 0-0.0.025-0.05 and 0, lg / l
• These media also comprise 5-Bromo-4-chloro-3-indolyl- ⁇ -glucoside at 50 mg / l. They are distributed at a rate of 20ml per petri dish.
• CoIi ID medium (bioMérieux) which associates a substrate of ⁇ -glucuronidase (6-chloro-3-indolyl- ⁇ -glucuronide) and a substrate of ⁇ -galactosidase (5-Bromo-3-indolyl- ⁇ -galactoside), intended the detection and enumeration of E. coli and coliforms in food samples is tested in parallel.
• Microorganisms frequently isolated from urinary samples and from the plaintiff's collection are inoculated on these media by semi-quantitative isolation of 10 ⁇ l of a 0.5 McFarland suspension diluted to 20 e .
• the dishes are incubated at 37 ° C. for 20 hours, and the colonies formed are examined visually. The coloration of these colonies is noted.
• Table 1 The results are shown in Table 1 below:
• the test below can be implemented to define the concentration of said first and second substrates according to the invention, which is variable depending on the substrates employed and more generally on the formulation of the reaction medium. To aid its understanding, this test is carried out below in the case of a combination ⁇ -glucuronidase, and ⁇ -galactosidase, from a kit of strains of microorganisms, including E. coli strains. coli, including strains that do not express or weakly or late positive activity and possibly other microorganisms. This test can be implemented for other types of substrates.
• Two reaction media comprising either a suitable substrate concentration of ⁇ -glucuronidase or no ⁇ -glucuronidase substrate are used to prepare a range of ⁇ -galactosidase substrate including a zero concentration, at least one concentration to obtain a positive reaction with the strains of E. coli expressing ⁇ -galactosidase, as well as intermediate concentrations.
• Each of the media is aliquoted so that each strain of microorganism can be seeded in pure culture on each of the media.
• the media are examined so as to select the medium comprising a combination of ⁇ -galactosidase substrates and ⁇ -glucuronidase to reveal the largest number of E. coli strains coli while differentiating them from the larger number of strains of other microorganisms. It may be necessary to repeat the experiment by adjusting the concentrations in each of the substrates as well as the strain kit. It may be advantageous that the reaction media further comprise inducers and / or inhibitors of ⁇ -galactosidase and / or ⁇ -glucuronidase.

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• 2007
  • 2007-02-08 FR FR0753148A patent/FR2912424A1/fr not_active Withdrawn
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