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
• • 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)
  • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
• • 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 present invention relates to the identification of pathogenic bacteria of the genus Listeria, and more specifically of the species Listeria monocytogenes.
• Listeria monocytogenes The isolation and identification of the bacterium Listeria monocytogenes is a major problem in the surveillance of agro-food hygiene and medical bacteriology. Of the Listeria bacteria, only Listeria monocytogenes is known to be pathogenic to humans. It can cause listeriosis, sometimes fatal (25 to 30% of cases) in immunocompromised people, young children or pregnant women. Other Listeria species are not pathogenic or are only for animals. This is particularly the case of Listeria ivanovii.
• the genus Listeria sp now comprises six species, of which only Listeria monocytogenes is pathogenic for humans.
• Germs are the cause of severe epidemics in humans, as a result of food contamination.
• foods mainly milk and milk products
• two species of Listeria are mainly isolated: nonpathogenic Listeria innocua and pathogenic Listeria monocytogenes. These two species have many common biochemical characters, so it is difficult to differentiate them.
• the beta-hemolysis test is based on the determination of a beta-hemolytic activity related to the production by the bacterium of a substance that causes lysis of red blood cells (listeriolysin). This test is performed on sheep or horse blood agar, but the answer is often discreet. This test is therefore unreliable and if it makes it possible to differentiate the two species mainly encountered, ie L. mono cyto genes (positive response) of L. innocua (negative response), it is not specific for the pathogenic species.
• the CAMP-test is another test, which is associated with the test of beta-hemolysis previously described.
• the test is carried out on tryptic soy agar, containing 5% of washed red blood cells.
• the beta-hemolytic strain of S. aureus CIP 5710 is inoculated into a streak perpendicular to the streaks formed by the culture of the Listeria strain to be tested.
• the reinforcement of the hemolysis of staphylococcus in contact with the two zones indicates a positive reaction.
• the CAMP-test is a heavy technique to implement, and, like hemolysis, is not always very reliable. It is also possible to use culture media or identification media.
• patent EP496680 issued to the applicant, describes a bacteriological analysis method to differentiate the species Listeria monocytogenes from other bacteria of the genus Listeria sp.
• a reaction medium comprising a chromogenic or fluoro -ogenic substrate capable of being hydrolyzed by an enzyme called Glycine aminopeptidase is used.
• Glycine aminopeptidase is used.
• This very interesting technique has the disadvantage that the species Listeria monocytogenes is the only one that does not have enzymatic Glycine aminopeptidase activity.
• the detection of Listeria monocytogenes is carried out by a negative activity, which is not easy, the use of a negative test lacking specificity in case of mutant or if the other species are stressed and do not respond with normal activity .
• Listeria monocytogenes and Listeria ivanovii species can be differentiated from other Listeria species by demonstrating phospholipase C activity specific for phosphatidylinositol (PI-PLC). Indeed, it has been demonstrated that PI-PLC is secreted into the culture medium by certain species of the genus Listeria such as Listeria monocytogenes and Listeria ivanovii (Leiffle-Wachter et al., Mol Microbiol. (1991) 5 (2)). ), pp. 361-366).
• the Ottaviani Agosti Agar medium is a chromogenic medium that uses on the one hand a ⁇ -Glucosidase substrate that allows the detection of all species of the genus Listeria and on the other hand the highlighting of PI-PLC activity.
• a confirmation test of Listeria monocytogenes generally implementing a second culture medium, for which a 24h incubation step is necessary.
• AES ALOAConfirmation
• BBL CHROMagar Identification listeria
• a duration of 48 hours is therefore necessary to identify and confirm the presence of Listeria monocytogenes: use of a first culture medium, which requires 24 hours of incubation, then the second confirmation step which also requires a 24h incubation .
• This duration of 48 hours is long, and it is desirable to shorten this duration to provide a reliable diagnosis as quickly as possible.
• the present invention aims to provide a detection method that allows the differentiation of Listeria monocytogenes species compared to all other species of Listeria spp.
• the invention proposes a new confirmatory test, which is a very rapid biochemical test since it can be implemented in less than 6 hours.
• biochemical test we mean a test allowing the implementation of a biochemical reaction highlighting the presence of bacteria. Such a test may in particular be implemented directly from an inoculum, without implementing a growth step. It is then the preformed enzymes provided by the bacterial suspension that are detected.
• Such tests are notably carried out in the API® range or the Vitek 2 system. These tests, packaged in dehydrated form, are brought into contact, under incubation, with the sample to be analyzed. The desired biochemical reaction is detected by a change in the state of the test (appearance disappearance of a coloration, a fluorescence).
• the reading of the test is then done visually or with the aid of a reader, directly or after the addition of a developer reagent.
• the API® range is based on a probabilistic numerical identification method, associating a biochemical test gallery, in cups and a database. It is the judicious combination of tests that ensures the characterization of a group of germs and the distinction of species between them.
• the VITEK 2 system is a fully automated identification and antibiogram system that implements a 64 micro-well card. By eliminating manual steps, automation saves lab time and ensures consistent analysis procedures that improve the reliability of results.
• the identification performed on this system uses a probabilistic method similar to that of API galleries. The automation of the reading allows a kinetic reading of the biochemical tests implemented and thus allows an identification in 4 to 8 hours for the current Gram positive germs as well as for Listeria monocytogenes.
• the biochemical test according to the invention is a confirmation test for the presence of L monocytogenes, comprising a substrate of PI-PLC and a substrate of ⁇ -mannosidase, but may also comprise inorganic salts, peptones, carbohydrates, one or more compounds limiting pH variations, one or more enzyme inducers.
• 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 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 is in particular a phosphatidyl inositol phospholipase C substrate (PIPLC) or an ⁇ -mannosidase substrate.
• PPLC phosphatidyl inositol phospholipase C substrate
• ⁇ -mannosidase substrate an ⁇ -mannosidase substrate
• This substrate may comprise in particular 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 may be chromogenic, fluorogenic, luminescent ... preferably, the substrates used in the present invention are chromogenic.
• substrates based on indoxyl and its derivatives particularly suitable in the case of phosphatidyl inositol phospholipase C (PI-PLC) substrates, substrates based on nitrophenol and derivatives, particularly suitable in the case of substrates of ⁇ -mannosidase, but also the substrates based on hydroxyquinoline or esculetin or alizarin or catechol or aminophenol and naphthol and their derivatives
• PI-PLC phosphatidyl inositol phospholipase C
• substrates based on nitrophenol and derivatives particularly suitable in the case of substrates of ⁇ -mannosidase
• substrates based on hydroxyquinoline or esculetin or alizarin or catechol or aminophenol and naphthol and their derivatives for the fluorogenic markers, mention may be made of coumarin derivatives and especially of umbelliferone, naphthol, resorufin, fluorescein
• the substrate is preferably chosen
• the PIPLC substrate is a chromogenic substrate.
• the PIPLC substrate is an indolyl-phosphatidyl-myo-inositol, preferentially bromo 4-chloro-3-indoxyl myoinositol phosphate (X-indolyl-phosphatidyl-myo-inositol).
• the ⁇ -mannosidase substrate is a chromogenic substrate or fluorogenic.
• the ⁇ -mannosidase substrate is a chromogenic substrate.
• the ⁇ -mannosidase substrate is a mannopyranosidase substrate.
• the mannopyranosidase substrate is nitrophenyl-4- ⁇ D mannopyrano side.
• the substrate concentration of PI-PLC is between 5 and 0.1 g / l, preferably between 1.5 and 0.5 g / l.
• the concentration of ⁇ -mannosidase substrate is between 2 and 0.1 g / l, preferably between 1 and 0.5 g / l.
• reaction medium is meant a medium comprising all the elements necessary for the expression of a metabolism and / or the growth of microorganisms.
• the environment The reaction may be solid, semi-solid or liquid.
• solid medium is meant for example 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, for example 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 may comprise one or more elements in combination, such as amino acids, peptones, carbohydrates, nucleotides, minerals, vitamins, active molecules such as antibiotics, enzymes, surfactants, buffers, phosphate, ammonium, sodium, metal salts, one or more substrates for the detection of enzymatic or metabolic activity ...
• the medium may also comprise a dye.
• dye of Evans blue, neutral red, sheep blood, horse blood, an opacifier such as titanium oxide, nitroaniline, malachite green, brilliant green ...
• biological sample is meant a clinical sample, from a biological fluid sample 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, cerebrospinal fluid, placenta, a food sample of water, drinks such as milk, a fruit juice.
• the invention relates to a biochemical test for confirming the presence of L. monocytogenes, characterized in that it comprises at least one phosphatidyl inositol phospholipase C substrate (PIPLC) and at least one ⁇ -mannosidase substrate.
• PPLC phosphatidyl inositol phospholipase C substrate
• the PIPLC substrate is an indolylphosphatidyl-myo-inositol, preferentially bromo 4 chloro-3 indoxyl myoinositol phosphate.
• the alpha mannosidase substrate is a mannopyranosidase substrate, preferentially nitrophenyl-4- ⁇ D mannopyrano side.
• the test further comprises an activator of PIPLC, or several such as in particular alpha-glycerophosphate of
• PLC is between 5 and 0.1 g / l, preferably between 1.5 and 0.5 g / l.
• the concentration of ⁇ -mannosidase substrate is between 2 and 0.1 g / l, preferably between 1 and 0.5 g / l.
• the invention also relates to the use of a biochemical test as described above, ie a biochemical confirmation test comprising a phosphatidyl inositol phospholipase C substrate (PIPLC) and at least one alpha mannosidase substrate, such as previously defined, to confirm the presence of L. monocytogenes.
• a biochemical confirmation test comprising a phosphatidyl inositol phospholipase C substrate (PIPLC) and at least one alpha mannosidase substrate, such as previously defined, to confirm the presence of L. monocytogenes.
• the test according to the invention is particularly suitable for unitary use or combined with a series of biochemical tests so as to give rise to an identification device API® range or Vitek® system.
• identification of L monocytogenes on API or Vitek system uses a reaction profile established by the concatenation of positive or negative responses of several unit biochemical tests.
• the test according to the invention may advantageously be integrated with such devices so as to reduce the number of tests.
• the invention also relates to a method of identifying Listeria monocytogenes, characterized in that it comprises the following steps: a) having a reaction medium for detecting the genus Listeria, b) seeding the medium with a biological sample to be tested (c) incubating (d) revealing the presence of the Listeria genus; (e) confirming the presence of L monocytogenes by a biochemical test according to the invention, comprising a phosphatidyl inositol phospholipase C (PIPLC) substrate and at least one alpha mannosidase substrate, as defined above.
• a biochemical test comprising a phosphatidyl inositol phospholipase C (PIPLC) substrate and at least one alpha mannosidase substrate, as defined above.
• 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.
• Step d) can be carried out by visual examination, colorimetry or fluorimetry.
• OAA medium bioMerieux, ref 43641 which allows the detection of species of the genus Listeria by colony blue staining (beta-glucosidase activity).
• the invention also relates to a method for identifying Listeria monocytogenes, characterized in that it comprises the following steps: a) having a biochemical test or a combination of biochemical tests to detect the genus Listeria, b) revealing the presence of the Listeria genus c) confirming the presence of L monocytogenes by a biochemical test according to the invention, comprising a phosphatidyl inositol phospholipase C substrate (PIPLC) and at least one alpha mannosidase substrate, as defined above.
• a biochemical test comprising a phosphatidyl inositol phospholipase C substrate (PIPLC) and at least one alpha mannosidase substrate, as defined above.
• PPLC phosphatidyl inositol phospholipase C substrate
• a biochemical test according to the invention comprises the following elements in g / 1:
• the test was evaluated to obtain a reaction in 4 to 6 hours at 37 ° C. +/- 20 ° C. after rehydration with 50 ⁇ l of demineralized water and addition of a colony or rehydration with 50 ⁇ l of a bacterial suspension. opacity equivalent to McF 0.5.
• test staining is read and interpreted according to the following grid:
• the test according to the invention was evaluated, initially using an inoculum of 0.5 McFarland, on 90 pure strains of the genus Listeria previously grown on OAA medium (Ref 43641).
• the 6 species of the genus Listeria sp were distributed as follows: L. monocytogenes (30 strains), L. ivanovii (20) (including 10 strains of L. ivanovii spp londoniensis and L. ivanovii spp ivanovii subspecies), L. grayii (10), L. seeligeri (10), L. welshimeri (10), L. innocua (10). 4H, 80% of L.
• the test according to the invention confirmed the belonging of the 6 presumed strains, to the species
• L. monocytogenes in 4H The L. monocytogenes identification of the 6 strains was confirmed by other identification tools (API Listeria gallery, Vitek GP maps and 16S sequencing).
• the other 5 matrices were contaminated with Listeria spp. Other than L. monocytogenes or other germs. In all these cases, the test according to the invention implemented on a colony of each of these matrices gave a negative response.
• test according to the invention was also validated on 250 pure strains tested with the following distribution:
• the 50 strains of Listeria sp developed on OAA medium The 50 strains of Listeria sp developed on OAA medium.

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Analytical Chemistry (AREA)
• Toxicology (AREA)
• Immunology (AREA)
• Microbiology (AREA)
• Molecular Biology (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Biotechnology (AREA)
• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=39590766

Family Applications (1)

Country Status (6)

Families Citing this family (3)

Citations (1)

Family Cites Families (5)

• 2007
  • 2007-10-30 FR FR0758666A patent/FR2922896B1/fr not_active Expired - Fee Related
• 2008
  • 2008-10-29 JP JP2010531564A patent/JP2011500097A/ja active Pending
  • 2008-10-29 WO PCT/FR2008/051946 patent/WO2009056762A2/fr active Application Filing
  • 2008-10-29 US US12/681,014 patent/US20100240091A1/en not_active Abandoned
  • 2008-10-29 EP EP08844892A patent/EP2205976A2/de not_active Withdrawn
  • 2008-10-29 CN CN200880113864A patent/CN101842706A/zh active Pending

Patent Citations (1)

Also Published As

Similar Documents

Legal Events