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  • 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
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• Reaction medium for the detection and / or identification of bacteria of the genus Legionella Reaction medium for the detection and / or identification of bacteria of the genus Legionella
• the present invention relates to a reaction medium for Legionella, as well as the use of such a medium, and a method implementing this medium.
• Legionellae are bacteria in the water environment (natural ecosystems and water distribution networks) that can cause infections, sometimes fatal in humans.
• Several species of the genus Legionella have been identified in humans, but the most important is Legionella pneumophila (L. pneumophila), responsible for about 90% of legionella cases.
• Other species such as L.jordanis are usually isolated from immunocompromised individuals.
• Legionnaires' disease or legionnaire's disease is a respiratory disease characterized by severe acute pneumonitis, which is acquired by inhalation of Legionella-contaminated aerosols such as cooling towers, air conditioning systems, thermal spas , showers, etc. After an incubation period of 2 to 10 days, patients have a flu-like syndrome.
• legionellosis The diagnosis of legionellosis is very complex because the disease is atypical: classic pneumopathy, it has nothing specific. The mortality is 20% of the cases on average, rather low in the community cases but on the other hand, important in nosocomial cases. Early diagnosis is essential to provide the patient with appropriate treatment.
• the diagnosis can be made according to the following methods: - identification by a direct immunofluorescence method. Carried out with antibodies from the main serogroups of L. pneumophila, this method allows the detection of soluble antigens.
• the methods used mainly are ELISA (Enzyme Linked ImmunoSorbant Assay), the RIA (Radiolmmunology Assay) or the ICM (Membrane ImmunoChromatography).
• the major disadvantage of these techniques is that they only detect the possible presence of certain serogroups of L. pneumophila. serological diagnosis of anti-LPS Antigen O antibodies by indirect immunofluorescence. This method only allows us to identify
• L. pneumophila serogroup 1 presents the risk of many false positives due to cross reactions with mycobacteria, leptospires, Chlamydia, Mycoplasma, Pseudomonas, Flavobacterium ... bacteriological identification by the method of culture.
• the growth of legionella is difficult. Indeed, they do not grow on blood agar, the pH must be strictly controlled (6.9 +/- 0.2) and the requirements of the bacteria are met.
• the cultures are generally carried out on Buffered Charcoal Yeast Extract (BCYE) agar supplemented with ACES, L-Cysteine and Iron buffer; these last two elements being essential growth factors; or GVPC.
• BCYE Buffered Charcoal Yeast Extract
• Legionella GVPC medium bioMérieux
• Bacillus GVPC medium Bacillus GVPC medium
• Bacillus GVPC medium Bacillus GVPC medium
• This medium allows growth inhibition of Gram-positive bacteria, most Gram-negative bacteria, yeasts and molds, thanks to an optimized mixture of three antibiotics.
• the current culture media contain activated charcoal to adsorb toxic compounds produced by Legionella, or present in the medium (yeast extract, agar ...), or by autoclaving the culture medium that generates the formation free radicals, also toxic to legionella.
• the invention proposes to solve the problems of the state of the art by presenting a new reaction medium for bacteria of the genus Legionella. Surprisingly, the inventors have demonstrated that the use of silicon compounds in a reaction medium allows a quick and easy detection of Legionella.
• reaction medium a medium comprising all the elements necessary for the expression of a metabolism and / or the growth of microorganisms such as Legionella.
• the reaction medium 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 gelrite, gelatin, agarose or other natural or artificial gelling agents.
• the reaction medium according to the invention must allow the growth of Legionella.
• the reaction medium may comprise one or more elements in combination, such as amino acids, carbohydrates, nucleotides, minerals, vitamins, etc.
• the medium may also include a dye.
• a dye of Evans blue, of neutral red, an opacifier such as titanium oxide, nitroaniline, malachite green, brilliant green, one or more metabolic indicators, one or more metabolic regulators ...
• the reaction medium can be a culture medium, a revealing medium or a culture and revelation medium.
• a revelation medium the culture of the microorganisms is carried out before seeding, and in the second case, the revelation medium also constitutes the culture medium.
• a bi-box which makes it easy to compare two media, comprising different substrates or different selective mixtures, on which a same biological sample has been deposited.
• selective mixing is meant a mixture of compounds allowing the selective cultivation of particular microorganisms.
• the selective mixture may allow the selective culture of Legionella.
• a medium comprises in particular the following compounds: glycine, polymyxin B sulphate, Vancomycin or cefamandole, Cycloheximide or Anisomycin or natamycin, alone or in combination.
• the selective medium may also allow the selective cultivation of certain legionella species such as L. pneumophila.
• the selective mixture comprises in particular the following compounds: Glycine, polymyxin B sulphate, Vancomycin or cefamandole, Cycloheximide, Anisomycin, natamycin, Bromocresol purple, Bromothymol blue alone or in combination.
• silicon compound any compound containing the silicon element. Mention may be made in a nonlimiting manner of natural or synthetic zeolites, clays, preferably illite, montmorillonite and preferably sepiolite, or even apolar silicas.
• apolar silica is meant one or more silica particles onto which are grafted apolar chemical functions most often alkyl chains having at least 2 carbon atoms. Preferably, they have 4 to 24 carbon atoms, even more preferably 18 carbon atoms (octadecyl), 8 (octyl) or 4 carbon atoms (butyl).
• this apolar silica is an octadecyl-silica or an octyl-silica.
• enzymatic substrate is meant a molecule that can be metabolized by an enzyme, into a product allowing the direct or indirect detection of a microorganism. It can be natural or synthetic substrate.
• the metabolism of the substrate will cause a variation of the physicochemical properties of the reaction medium or the cells of organisms. This variation can be detected by physico-chemical methods, in particular optical methods by the eye of the operator or by means of instruments, spectrometric, electrical, magnetic, ... Preferably, this will be a change in optical properties, such as a change in absorption, fluorescence or luminescence.
• fluorogenic or chromogenic enzymatic substrate is meant a molecule whose metabolism generates a product having a fluorescence or a different color of the substrate.
• the enzymatic substrate is preferably selected from the oxidoreductase substrates of hydrolase.
• this substrate is a substrate of nitroreductase, oxido-reductase, acetoacetyl-CoA reductase, dehydrogenase, superoxide dismutase, osidase, peptidase, nuclease, esterase.
• chromogenic substrate mention may be made in particular of those based on alizarin, the substrates of alpha-glucosidase and especially alizarin-alpha-glucoside.
• fluorogenic substrate mention may be made of those based on coumarin, those described in patent EP 0641351 ("Enzymatic analysis using substrates that yield fluorescent precipitates" HAUGLAND et al.) Or in patent application FR07 / 55371, the reductase substrates and in particular those of nitroreductase and more preferably those described in patent application FR07 / 55373 or in patent EP 1124986.
• a substrate for the enzymatic activity of oxido-reductase mention may be made more particularly of the reductase substrates and preferentially the substrates of nitroreductase.
• Biological sample means a clinical specimen derived from a bronchial, tracheal or pulmonary aspiration sample, pleural fluid, bronchoalveolar lavage, sputum, blood or lung biopsy and more rarely joint or pericardial fluid; a food sample.
• This sample may thus be liquid or solid and may be mentioned in a nonlimiting manner, a clinical sample of blood, plasma, urine, feces, nose samples, throats, skin, wounds, cerebrospinal fluid, a food sample of water (drinking water).
• sample of the environment is meant a water withdrawal, and may be mentioned in a non-limiting manner: water supply network, air conditioning system, air-cooling tower, vaporizer, fogger.
• the invention relates firstly to a reaction medium for culturing and / or the detection and / or identification of bacteria of the genus Legionella, comprising at least one apolar silica.
• said apolar silica is octadecyl-silica.
• said apolar silica is in combination with a clay, preferentially sepiolite.
• octadecyl-silica is in combination with sepiolite.
• said reaction medium further comprises an enzymatic substrate.
• said enzymatic substrate is a fluorogenic or chromogenic enzymatic substrate, preferentially chromogenic.
• said fluorogenic or chromogenic enzymatic substrate is an oxidoreductase substrate or a hydrolase substrate.
• the concentration of apolar silica is between 0.01 and 100 g / l, preferably between 1 and 5 g / l.
• the clay concentration is also between 0.01 and 100 g / l, preferably between 1 and 5 g / l.
• the reaction medium further comprises a selective mixture allowing selective cultivation of bacteria of the genus Legionella.
• this selective mixture comprises glycine, polymyxin sulphate
• the medium according to the invention may also comprise a selective mixture allowing the selective cultivation of particular species of Legionella such as Legionella pneumophila.
• This selective mixture then preferably comprises glycine, polymyxin B sulphate, Vancomycin, a quinolone or a fluoroquinolone, cycloheximide, anisomycin, bromocresol purple or bromothymol blue alone or in combination.
• the difference in selectivity can also be obtained by varying the Glycine concentration from 2.5g / l to 7.5g / l (selective Glycine-containing medium for isolation of Legionellaceae from environmental specimens, Robert M. Wadowski and
• the invention also relates to the use of a reaction medium comprising at least one silicon compound, for the cultivation, detection and / or identification of bacteria of the genus Legionella.
• said silicon compound is an apolar silica and / or a clay.
• said apolar silica is octadecyl-silica.
• said clay is sepiolite.
• the concentration of silicon compound in the reaction medium used is between 0.01 and 100 g / l, preferably between 1 and 5 g / l.
• the concentration of each of the compounds is between 0.01 and 100 g / l, preferably between 1 and 5 g / l.
• the invention finally relates to a method for detecting and / or identifying bacteria of the genus Legionella, characterized in that it comprises the following steps: a) bringing into contact a sample that may contain bacteria of the genus
• Legionella with a reaction medium as defined above b) incubate, and, c) detect the presence of Legionella bacteria
• the incubation is preferably carried out at a temperature of between 30 ° C. and 50 ° C., and more preferably between 36 ° C. and 42 ° C.
• Legionellae are preferentially detected by an alpha-glucosidase or oxido-reductase activity which makes it possible to obtain colored or fluorescent colonies. Colonies of other genera, if uninhibited, appear either colorless or of a different color or color or fluorescence than Legionella.
• the incubation time allows the growth of Legionella bacteria to allow detection. Without being limiting, an incubation of 48-72h is well suited, but a shorter incubation is possible.
• legionellae are preferably detected by an oxidoreductase activity which makes it possible to obtain colored or fluorescent colonies.
• the other bacteria are either inhibited, or colorless, of a different color or fluorescence, or of a color or fluorescence identical to that of Legionella.
• composition in g / l yeast extract: 10 g / l
• BCYE medium (Feeley et al., J. Clin Microbiol., 1979 10: 437-41) was a growth control medium.
• the media were divided into Petri dishes. Inoculation of the different strains of Legionella was carried out by isolation in three dials. The plates were incubated for 96h at 37 ° C. in the presence of CO 2 . Readings are taken after 72 and 96 hours of incubation.
• the media containing apolar-like silicas allowed the growth of all the strains tested, growth superior to the BCYE medium.
• composition in g / l yeast extract: 10 g / l
• the media were divided into Petri dishes. Inoculation of different strains of
• Legionella was performed by isolation in three dials. The plates were incubated for 96h at 37 ° C. in the presence of CO 2 .
• Readings are taken after 72 and 96 hours of incubation.
• the media containing sepiolite at concentrations of between 5 and 10 g / l allowed growth of all Legionella pneumophila, growth substantially identical to the control medium.
• composition in g / l yeast extract: 10 g / 1 alpha-ketoglutaric acid: 1 g / l
• Glutathione 5 g / l Octadecyl-silica: lg / 1 to this medium were added 17 g / l of agar (T medium), 17 g / l of gelrite (Tl medium) and 5 g / l of sepiolite and 17 g agar (medium T2).
• the media were divided into Petri dishes. Inoculation of the different strains of Legionella was carried out by isolation in three dials. The plates were incubated for 96h at 37 ° C. in the presence of CO 2 .
• Readings are taken after 72 and 96 hours of incubation.
• composition in g / l yeast extract: 10 g / 1 alpha-ketoglutaric acid: 1 g / l
• a stock solution of fluorogenic substrate 2- (5'-fluoro-2'-nitrophenyl) -benzothiazole was carried out in a solvent type DMSO at a rate of 50 g / l.
• a volume corresponding to a final concentration of 5 mg / l of substrate was added to said supercooled medium.
• the media were cast in Petri dishes with a diameter of 55 mm and Legionella strains were seeded by isolation in three dials from 0.5McFarland suspensions. The dishes were incubated at 37 ° C for 5 days.
• the colonies formed were examined visually after 72, 96 and 120 hours of incubation. Fluorescence (read under UV lamp at 366 nm) as well as intensity were noted.
• 0 indicates either the absence of isolated colonies or the absence of fluorescence.
• the fluorescence intensities are read on a scale from 0 (no fluorescence) to 4 (very intense fluorescence). 4. INTERPRETATION
• a medium according to the invention comprising a fluorogenic substrate, allowed the growth of all the strains tested, and easy detection of Legionella.
• Example 5 Various strains of the genus Legionella were tested on a medium according to the invention comprising a chromogenic substrate of alpha-glucosidase, alizarin-alpha-glucoside. The reading of the boxes is then carried out after 72h and 96h of incubation.
• composition in g / l yeast extract: 10 g / 1 alpha-ketoglutaric acid: 1 g / l
• a stock solution of chromogenic substrate (alizarin-alpha-glucoside) was carried out in a solvent type DMSO at a rate of 40 g / l. A volume corresponding to a final concentration of 50 mg / l of substrate was added to said supercooled medium. In the same way, a stock solution of iron citrate was made in osmosis water, then the solution was filtered before being added in the medium at a rate of 100 mg / l. 2.
• the media were cast in Petri dishes with a diameter of 55 mm and Legionella strains were seeded by isolation in three dials from 0.5McFarland suspensions. The plates were incubated at 37 ° C for 4 days.
• 0 indicates either no growth, no isolated colonies, or no staining.
• the color intensities are read on a scale from 0 (no staining) to 4 (very intense intensity). The colors vary from pink (noted R) to violet (Vi) through Rvi (pink / purple).
• a medium according to the invention comprising a chromogenic substrate, allowed the growth of 8/10 strains tested, and easy detection of legionella via the appearance of a pink to purple coloration of the mass and colonies.

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