Reaction medium and method for detecting shiga toxin-producing e.coli and / or enterohaemorrhagic e.coli

Abstract

The present invention relates to a gelled reaction medium for detecting, identifying, and / or isolating at least one Shiga toxin-producing strain of E. coli, the reaction medium comprising: - at least one toxin inducer, - at least one agglutinating conjugate comprising at least one specific binding partner of STX1 and / or at least one specific binding partner of STX2, coupled to a nanoparticle; - a concentration gradient of a compound for inhibiting non-target bacteria. The present invention also relates to the associated method for detecting and / or isolating Shiga toxin-producing E. coli which is likely to be present in a sample comprising enterobacteria.

Description

[0001] Description

[0002] Title of the invention: reaction medium and method for detecting a Shiga toxin-producing E. coli and / or an enterohemorrhagic E. coli.

[0003] TECHNICAL FIELD

[0004] The present invention relates to the field of microbiological control in the broad sense, such as the microbiological control of a sample of industrial or clinical origin. More particularly, the present invention relates to a reaction medium and the method for detecting a Shiga toxin-producing E. coli and / or an enterohemorrhagic E. coli.

[0005] PRIOR TECHNIQUE

[0006] Microbiological control of samples of various origins requires the implementation of techniques which allow the detection - for example for the purposes of identification and / or counting and / or biochemical characterization - of microorganisms and whose results must be rendered as quickly as possible.

[0007] In the medical field, it is necessary to predict and diagnose the risk of infection: the faster and more accurate the diagnosis, the more effective the treatment of patients and the minimized the risk of transmission. The approach is similar for animal health in the veterinary field.

[0008] In the agri-food sector, the problem is identical. However, it distinguishes:

[0009] - pathogenic microorganisms such as Shiga toxin-producing bacteria (STEC), Salmonella, Listeria, Cronobacter, Bacillus, Staphylococcus, the research for which applies to raw materials, intermediate products, finished products marketed,

[0010] - non-pathogenic microorganisms, used as quality indicators of the production process, from raw materials to finished products, throughout the chain,

[0011] - bacteria of technological interest such as ferments,

[0012] - microorganisms that are markers of contamination.

[0013] Rapid and accurate detection of suspected contamination (within food batches) allows them to be controlled and corrective actions to be taken quickly.

[0014] Technically, one of the main difficulties is being able to isolate the desired bacteria in order to identify it. Generally, microbiological analysis is carried out in two stages. The first is a detection phase that can use numerous technologies such as culture media, immunoassays, and molecular biology. This can be followed, particularly in the agri-food sector, by a confirmation phase in order to confirm the presence of the desired pathogen and meet the standards in force in this field. The confirmation stage therefore requires additional steps and, here again, requires a stage of isolation of the desired bacteria.

[0015] Thus, in the case of Shiga toxin-producing E. coli, diagnosis is based on the use of selective and chromogenic agars that may include tellurite, such as CT-SMAC (Sorbitol MacConkey Agar) or CT-RMAC (Rhamnose MacConkey Agar), in order to select the growth of certain bacteria and to stain the strains of interest. The disadvantage of these methods is that they are very specific and some Shiga toxin-producing E. coli strains are not detected. Conversely, some media are not selective enough; the presence of a small number of Shiga toxin-producing E. coli may be hidden by other widely present microorganisms, making isolation difficult. In addition, to assess the pathogenicity of these strains, it is necessary to detect the presence of virulence factors.Indeed, their pathogenicity involves the expression of several virulence genes, including six encoding the two types of Shiga toxins: STX1 and STX2. These two toxins act by inhibiting protein synthesis in eukaryotic cells, which ultimately causes apoptosis. Currently, in order to accurately identify Shiga toxin-producing E. coli, it is essential to perform PCR for gene six.

[0016] There is therefore a real need to develop a reliable and rapid medium and method for the isolation and identification of target bacteria, particularly STEC and EHEC.

[0017] SUMMARY OF THE INVENTION

[0018] The present invention relates to a gelled reaction medium for the detection, identification, and / or isolation of at least one Shiga toxin-producing E coli strain comprising

[0019] - at least one toxin inducer,

[0020] - at least one agglutinating conjugate comprising at least one specific binding partner of STX1 and / or at least one specific binding partner of STX2, coupled to a nanoparticle

[0021] - a concentration gradient of a compound inhibiting non-target bacteria. In a highly advantageous manner, the medium according to the invention makes it possible to isolate STEC or EHEC from a multitude of E. coli.

[0022] Another subject of the invention relates to the preparation of a reaction medium according to the invention comprising the following steps:

[0023] - contacting an agglutinating conjugate with a gelling culture medium

[0024] - depositing on an area of ​​the gelled culture medium, at least one compound inhibiting non-target bacteria comprising tellurite; said inhibitory compound diffusing and forming an inhibition concentration gradient around the deposition area; the concentration of the tellurite concentration gradient being between 0 pg / ml and 100 pg / ml, preferably between 0 pg / ml and 50 pg / ml, even more preferably between 0 pg / ml and 30 pg / ml.

[0025] Another subject of the invention relates to a method for detecting and / or isolating Shiga toxin-producing E coli likely to be present in a sample comprising enterobacteria, comprising the following steps:

[0026] - have a selective gelled culture medium allowing the growth of E coli comprising a concentration gradient of a compound inhibiting non-target bacteria

[0027] - deposit the sample on said gelled culture medium

[0028] - incubate said medium under conditions allowing the growth of E coli

[0029] - isolate an E coli

[0030] - confirm that the said E coli is a Shiga toxin-producing E coli

[0031] Preferably, the culture medium is the culture medium according to the invention.

[0032] Thus advantageously, the presence of a Shiga toxin-producing E. coli is confirmed by the presence of a halo around said Shiga toxin-producing E. coli present in the inhibition concentration gradient zone.

[0033] This preferred embodiment has the advantage of being able to detect and isolate said STEC or EHEC directly on the culture medium. This allows for an immediate result.

[0034] In another embodiment, the method allows the detection of a STEC or an EHEC. According to this embodiment, the Shiga toxin-producing E. coli detected is an enterohemorrhagic E. coli. This method is particularly advantageous for samples loaded with additional flora, where the large quantity of colonies on the Petri dishes can lead to a risk of false negatives. In view of the large number of colonies on the dish and the low representation of the target microorganism, the person collecting the colonies for confirmation purposes may never collect the target microorganism. The probability of isolating a STEC is then greatly increased in the presence of the gradient of the inhibitory compound. The present invention therefore provides a method for detecting and isolating STEC which makes it possible to avoid false negatives and allows considerable time savings.

[0035] DESCRIPTION OF FIGURES

[0036] Figure 1 is a photo of a control culture medium without an inhibitory compound concentration gradient on which a “reblochon” sample was inoculated by exhaustion.

[0037] Figure 2 is a photo of a control culture medium without an inhibitory compound gradient on which a “reblochon” sample was inoculated by depletion after an immunoselection step.

[0038] Figure 3 is a photograph of a control culture medium without an inhibitory compound gradient onto which a "ground beef" sample was spiked by depletion. The arrow points to a halo around a colony.

[0039] Figure 4 is a photo of a control culture medium without an inhibitory compound gradient on which a “chopped steak” sample was inoculated by depletion after an immunoselection step.

[0040] Figure 5 is a photograph of a culture medium comprising a gradient of an inhibitor compound (cefixime tellurite mixture) onto which a “chopped steak” sample has been inoculated by exhaustion.

[0041] Figure 6 is a photograph of a culture medium comprising a gradient of an inhibitory compound (cefixime tellurite mixture) onto which a previously immunoselected “ground steak” sample has been inoculated by depletion. Figure 7 is a photograph of a culture medium comprising a gradient of an inhibitory compound (cefixime tellurite mixture) onto which a “reblochon” sample has been inoculated by depletion. The arrows point to the halos around colonies.

[0042] Figure 8 is a photo of a culture medium comprising a gradient of an inhibitory compound (cefixime tellurite mixture) on which a “reblochon” sample which had previously been immunoselected was seeded by exhaustion.

[0043] DETAILED DESCRIPTION OF THE INVENTION

[0044] Certain terms and expressions used in the context of the invention are detailed below.

[0045] A first subject of the invention relates to a gelled reaction medium for the detection, identification, and / or isolation of at least one strain of E. coli producing shiga toxin comprising

[0046] - at least one toxin inducer,

[0047] - at least one agglutinating conjugate comprising at least one specific binding partner of STX1 and / or at least one specific binding partner of STX2, coupled to a nanoparticle

[0048] - a concentration gradient of a compound that inhibits non-target bacteria.

[0049] The term "reaction medium" means a medium comprising all the elements necessary for the expression of a metabolism, the survival and / or the growth of microorganisms. This reaction medium can either be a microbiological culture medium or a microbiological revelation medium. In the latter case, the culture of the microorganisms can be carried out beforehand in another medium. The reaction medium can also be brought into contact with an agar culture medium. It can be placed under or on the culture medium which allows the growth of the target microorganisms. The reaction medium can be added after incubation of the culture medium. Preferably, the reaction medium is a microbiological culture medium comprising all the elements necessary for the growth of the microorganisms.

[0050] According to the present invention, the reaction medium is gelled. It is in solid or semi-solid form. Agar is the traditional gelling agent used in microbiology for the culture of microorganisms, but it is possible to use other gelling agents such as, for example, gelatin, agarose as well as other natural or artificial gelling agents. The conjugate is capable of forming a network with the target component in a gelled medium. This network is visually detectable by the formation of a halo without it being necessary to reduce the hardness of a conventional semi-solid reaction medium. Thus, it is not necessary to modify the physicochemical properties of the reaction medium, such as hardness, to allow on the one hand the diffusion of the targeted components and on the other hand the reaction with the conjugates.

[0051] A number of preparations are commercially available, such as Columbia agar, Trypcase-soy agar, MacConkey agar, Mueller Hinton agar or more generally those described in the Handbook of Microbiological Media. These media can serve as a basis for the reaction medium according to the invention. The reaction medium may further comprise possible additives such as, for example, amino acids, peptones, one or more growth factors, carbohydrates, nucleotides, minerals, vitamins, one or more selective agents, inducers, toxin inducers, buffers, etc. By "selective agent" is meant any compound capable of preventing or slowing the growth of a so-called "non-target" microorganism, i.e. other than the target microorganism(s).The term "inducer" refers to a compound capable of inducing the expression of a compound, such as an enzyme or a toxin, which would normally remain unexpressed. Said reaction medium may also comprise a dye. For information purposes, dyes that may be mentioned include Evans blue, neutral red, sheep blood, horse blood, an opacifier such as titanium oxide, nitroaniline, malachite green, brilliant green. When the reaction medium according to the invention further comprises an enzymatic substrate specific for an enzymatic activity of at least one target microorganism, a chromogenic and / or fluorogenic substrate is preferably used. By "chromogenic and / or fluorogenic substrate" is meant a substrate allowing the detection of an enzymatic or metabolic activity of the target / desired microorganisms using a detectable signal.The reaction medium according to the invention may additionally comprise a pH indicator, sensitive to the pH variation induced by the consumption of the substrate and revealing the metabolism of the target microorganisms. Said pH indicator may be a chromophore or a fluorophore. Examples of chromophores include bromocresol purple, bromothymol blue, neutral red, aniline blue, bromocresol blue.

[0052] A person skilled in the art can also use a Petri dish divided into segments, such as a bi-dish or a tri-dish, making it possible to easily compare several media, comprising different substrates or different selective mixtures, on which the same biological sample has been deposited.

[0053] According to the present invention, the term "microorganism" has the same meaning as that generally accepted in microbiology and includes in particular gram-positive or gram-negative bacteria, yeasts, molds and more generally, unicellular organisms, invisible to the naked eye, which can be manipulated and multiplied in the laboratory.

[0054] By "target microorganism" is meant at least one microorganism that one wishes to detect and / or identify. According to the present invention, the microorganism is chosen from Shiga toxin-producing E. coli strains. Enterohemorrhagic E. coli are strains representing a subgroup of Shiga toxin-producing Escherichia coli, which have the eae gene or other genes for cell adhesion such as the aggR gene and cause hemolytic uremic syndrome. The possession of these two virulence factors stx and eae simultaneously makes this pathovar very virulent for humans. And in particular when it comes to serogroups 026, 045, 080, 0103, OU I, 0121, 0145 and 0157.

[0055] Thus, according to the invention, the target microorganism is an E. coli belonging to the groups of Shiga toxin-producing E. coli or typical enterohemorrhagic E. coli carrying the stx and eae genes or atypical enterohemorrhagic E. coli carrying the stx and aggR genes, for example. Non-target microorganisms are bacteria growing on the culture medium outside the concentration gradient zone of an inhibitory compound. Indeed, since the sample is polymicrobial, the STEC or EHEC potentially present are present among non-specific Gram-negative bacteria such as commensal E. coli.

[0056] According to the present invention, the reaction medium comprises at least one specific binding partner of STX1 and / or STX2, coupled to a nanoparticle. STX1 and STX2 are the toxins secreted by STEC strains. There are in fact two main types of shiga toxins: STX1 and STX2 which themselves have numerous variants. To date, STX1 has 4 subtypes STX1a, STXlc, STXld, STXle, and STX2 has 12: STX2a, STX2b STX2c, STX2d, STX2e, STX2f, STX2g, STX2h, STX2i, STX2j, STX2K, STX21.

[0057] The specific binding partner is chosen from antibodies, all types of Fab fragments, recombinant proteins, phages, phage proteins, oligonucleotides, aptamers, affimers or any other ligand or anti-ligand well known to those skilled in the art. Preferably, the binding partner is chosen from antibodies or phage proteins. Preferably the antibody is a monoclonal antibody or a monoclonal antibody fragment. The binding partner is specific for a component of a target microorganism. The component of the target microorganism is a component released by said microorganism. The component can thus be an element from the surface of the bacteria such as a protein, a Lipopolysaccharide (LPS), a flagellum.It may also be an element internal to the bacterium such as RNA, an intracellular protein which can be detected when a part of the bacterial colony dies during its growth. The binding partner may also be specific for a component derived from said microorganism. This component may be a molecule of interest produced by the target microorganism such as a protein, an antibiotic, a molecule of resistance to antimicrobial agents, enzymes such as proteases, lipases or glusidases. According to the present invention, the medium comprises at least one binding partner specific for the STX1 protein and / or at least one binding partner specific for the STX2 protein. According to another embodiment, the medium comprises at least one binding partner specific for a subtype of the STX1 protein and / or at least one binding partner specific for a subtype of the STX2 protein such as XTX2a or STX2d.According to the present invention, the binding partner is coupled to a nanoparticle. This final complex is called a conjugate. In the same reaction medium, it is possible to have conjugates having binding partners of different nature coupled to nanoparticles themselves of different nature.

[0058] The term "nanoparticle" refers to particles in the order of magnitude of a nanometer. Nanoparticles can be chosen from gold, iron, silver, copper, carbon, latex, silicon, aluminum. Preferably, nanoparticles are colloidal nanoparticles with optical properties, namely their ability to distinguish themselves when a network is formed. Even more preferably, the nanoparticle is chosen from gold, silver, copper. Thus, when the nanoparticles are made of gold, they change color, for example from red to gray when they form a network. When they are not aggregated, the wavelength of the absorbed light is in the red around 530nm. When they are aggregated, the absorbed wavelength changes from red to blue / gray around 600 to 700nm. In a particular embodiment, it is possible to use together several nanoparticles of different colors.The networks thus formed allow the distinction of several components of the target microorganism.

[0059] Preferably, the nanoparticles have a size between 10 and 200nm. Preferably, the nanoparticles have a size between 20 and 90nm, allowing better mobility of the conjugates in the reaction medium.

[0060] Advantageously, the nanoparticles make it possible to reduce the quantity of binding partners required for the formation of agglutination. Thus, the concentration of binding partners required for the production of a reaction medium according to the invention requires 100 to 1000 times fewer binding partners than a medium without nanoparticles.

[0061] Preferably, the quantity of binding partners required corresponds to the quantity required to cover at least half of the surface of the nanoparticle, and even more preferably to cover between a third and a half of the surface of the nanoparticle. This proportion allows the agglutinating conjugate to form a network in the gelled reaction medium.

[0062] Advantageously, nanoparticles can make it possible to visualize agglutination around a bacterial colony whose size does not yet allow it to be visible to the naked eye. Detection can thus be earlier. Advantageously, nanoparticles can make it possible to visualize agglutination around a bacterial colony whose translucent appearance does not allow detection by an automated reading device. Detection can thus be facilitated.

[0063] The coupling of the nanoparticle to the binding partner can be done either by direct attachment or by indirect attachment. Direct attachment means attachment by adsorption or by covalent bonding. Indirect attachment means attachment by the interaction of ligands / anti-ligands such as biotin / strepatividin or other pairs well known to those skilled in the art (Nicholas G. Weich et al, 2017). Depending on the type of bond chosen, the person skilled in the art will adapt the physicochemical conditions of the reaction medium and in particular its pH.

[0064] According to the present invention, the conjugate is agglutinating, that is to say that it causes the formation of an agglutination network in the presence of a component of a target microorganism or a component derived from said microorganism. The component being multi-epitope, several conjugates will bind to this component and form an agglutination. By agglutination, we mean the result of an interaction between at least one component of a target microorganism or at least one component derived from said microorganism with binding partners coupled to a nanoparticle. Agglutination reactions include immunological reactions, such as antigen-antibody reactions or more generally specific interactions between two molecules. Through this interaction, components and conjugates aggregate, adhere to each other and form a network in the reaction medium.

[0065] In practice, several parameters influence the capacity of the conjugate to be agglutinating in a gelled medium, mainly:

[0066] - the porosity of the gelled medium

[0067] - the size of the nanoparticles - the quantity of binding partners

[0068] - the quantity of nanoparticles.

[0069] It will therefore be necessary to adapt these parameters in order to allow satisfactory agglutination allowing its detection. Advantageously, the network formed by said specific reaction is then detected either visually or automatically using an optical system. The colony of the target microorganism is thus identified. Preferably, the network forms a halo in the gelled reaction medium detectable either visually or using an optical system. The network or halo thus circumscribes the colony which can then be advantageously differentiated and / or identified within a population.

[0070] In a particular embodiment, the binding partner is an antibody in an amount making it possible to cover at least half of the surface of the nanoparticle and preferably at least a third of the surface of the nanoparticle.

[0071] Preferably, in these variants the nanoparticle is a gold nanoparticle of size between 20 and 90nm and at a concentration between 1O 10 and 10 12 nanoparticles / ml of reaction medium.

[0072] The reaction medium according to the invention comprises a toxin inducer. The toxin inducer causes stress in the bacteria which will trigger the lytic cycle of the prophages in the bacteria and therefore stimulate the production of toxins which will be released. Advantageously, the toxin inducer is chosen from antibiotics or a physicochemical stress. Examples of antibiotics include trimethoprim, sulfamethoxazole, norfloxacin, azithromycin, gemtamicin, polymyxin B, chloramphenicol, streptomycin, chlortetracycline, oxytetracyline, tylosin, mitomycin C, carbodox, oliquindox, rifampincin, imipenem, ciprofloxacin, cotrimoxazole, penicillin G, linomycin. In a preferred embodiment, the medium according to the invention comprises ciprofloxacin at a concentration of between 0.005 and 0.030 mg / l. In another preferred embodiment, the medium according to the invention comprises mitomycin C at a concentration of between 0.10 and 0.5 mg / l.

[0073] The toxin inducer can also be a physicochemical stress achieved, for example, by the addition of salt or EDTA or by a change in pH or even by UV stress. Stress can also be induced, for example, by the addition of noradrenaline.

[0074] Said target microorganism is likely to be present in a sample. By "sample" is meant a small part or small quantity isolated from an entity for analysis. The sample may be of industrial origin, i.e., according to a non-exhaustive list, an air sample, a water sample, a sample taken from a surface, a part or a manufactured product, a product of food origin. The sample may also correspond to a sample of biological fluid (stools, urine, whole blood, serum, plasma, cerebrospinal fluid, organic secretion, etc.), an external sample (skin, nose, throat, etc.) or tissue or isolated cells. The sample may be used as is or, prior to analysis, undergo preparation such as enrichment, extraction, concentration, purification, according to methods known to those skilled in the art.The reaction medium according to the invention may be all the more interesting when the sample is a polymicrobial sample or one loaded with additional flora, such as for example food samples such as raw milk cheese or stools in clinical samples.

[0075] According to the present invention, the reaction medium comprises a concentration gradient of a compound that inhibits non-target bacteria. According to the present invention, the inhibitory compound is chosen such that the minimum inhibitory concentrations (MICs) of non-target bacteria such as commensal Enterobacteriaceae are sufficiently lower than the MIC of STEC / EHEC.

[0076] The term "inhibitory compound" means any compound that inhibits bacterial growth. It may be an antibiotic, a dye such as brilliant green, a salt such as lithium chloride, or a bacteriophage endolysin. Preferably, the inhibitory compound is tellurite. According to the present invention, the inhibitory compound promotes the inhibition of non-S TEC and non-EHEC strains.

[0077] A gradient is a non-homogeneous, increasing or decreasing concentration of an inhibitory compound.

[0078] Depending on the bacteria present in the polymicrobial sample, applying a concentration gradient allows for the discrimination of non-target microorganisms that have a lower MIC than the target microorganisms. Thus, non-target microorganisms are microorganisms capable of growing on the medium without the concentration gradient of the inhibitory compound. Target microorganisms are microorganisms carrying stx genes, i.e., STEC.

[0079] Thus non-target bacteria such as commensal Enterobacteriaceae generally have a lower MIC than STEC and EHEC.

[0080] Preferably, when the inhibitory compound comprises tellurite, the tellurite gradient on the medium is between 0 pg / ml and 100 pg / ml, preferably between 0 pg / ml and 50 pg / ml, even more preferably between 0 pg / ml and 30 pg / ml. The different strains belonging to STEC and EHEC have different sensitivities to inhibitory compounds such as tellurite. Indeed, STEC and EHEC are mostly more resistant to tellurite than other enterobacteria.

[0081] In addition, even within the group being tested, such as EHEC or STEC, serogroups may have different susceptibilities. For example, an E. coli 0157 strain will not have the same MIC to Cefixime-Tellurite as an E. coli 0103 strain. There is also different susceptibility within a serogroup. Thus, an E. coli 0111 strain will not have the same MIC as another E. coli 0111 strain.

[0082] In a completely advantageous manner, the medium according to the invention makes it possible to isolate STEC or EHEC from a multitude of E. coli.

[0083] In a preferred embodiment, the inhibitor compound is a mixture of cefixime and tellurite. Preferably, the amount of cefixime in the deposited cefixime tellurite mixture is between 0.20 pg and 0.30 pg and the amount of tellurite is between 12 and 13 pg.

[0084] Preferably, the inhibitory compound deposited on the medium has a volume between 5 μl and 50 μl. The inhibitory compound can be deposited by any means, such as a pipette.

[0085] In a particular embodiment, the inhibitory compound is contained in at least one substrate capable of diffusing the inhibitory compound onto the gelled culture medium. This may, for example, be a blotting paper pellet containing the inhibitory compound and capable of diffusing it. In this particular embodiment, the substrate may be brought into contact with the agar medium by placing said substrate at the bottom of the dish before the medium is poured into the dish.

[0086] Another subject of the present invention relates to a diagnostic kit allowing the preparation of a reaction medium according to the invention comprising

[0087] - an agglutinating conjugate comprising at least one specific binding partner of STX1 and / or at least one specific binding partner of STX2, coupled to a nanoparticle

[0088] - a gelling medium comprising a toxin inducer

[0089] - a compound that inhibits bacteria from non-target bacteria

[0090] Advantageously, the reaction medium is manufactured extemporaneously using a kit according to the invention. This allows the unitary use of a reaction medium, and to increase its stability. Preferably, once manufactured, the medium is used within 24 hours, even more preferably within 12 hours. Another object of the present invention relates to the method for obtaining a reaction medium according to the invention comprising the following steps:

[0091] - bringing an agglutinating conjugate into contact with a gelling medium to form the reaction medium.

[0092] - depositing on an area of ​​the gelled culture medium, at least one compound inhibiting non-target bacteria; said inhibitory compound diffusing and forming an inhibition concentration gradient around the deposition area; the concentration of the tellurite gradient being between 0 pg / ml and 100 pg / ml, preferably between 0 pg / ml and 50 pg / ml, even more preferably between 0 pg / ml and 30 pg / ml.

[0093] Thus the agglutinating conjugate is brought into contact with a supercooled gelling medium to form the reaction medium according to the invention. The gelled medium also comprises a toxin inducer. The whole is then homogenized and poured into the Petri dish.

[0094] A compound that inhibits non-target bacteria is deposited on an area of ​​the gelled culture medium. The inhibitory compound diffuses and forms an inhibition gradient around the deposition area; in another embodiment, the inhibitory compound is deposited after the sample has been deposited. In another embodiment, the inhibitory compound is a substrate capable of diffusing the inhibitory compound and is deposited in the dish before the medium is poured. Preferably, the inhibitory compound comprises a dye that makes it possible to localize its deposition on the culture medium and then allow the sample to be deposited at the level of the inhibitory compound.

[0095] Another subject of the present invention relates to a method of in vitro microbiological culture, in which microorganisms likely to be present in a sample are seeded in or onto a culture medium according to the invention. The seeded culture medium is incubated under appropriate conditions known to those skilled in the art. The seeding is carried out according to conventional microbiology techniques.

[0096] In a preferred embodiment of the invention, the seeding is carried out by depleting the polymicrobial sample on the culture medium. The sample is deposited at the level of the deposition of the inhibitory compound so that the sample with a maximum load of microorganisms is in contact with the inhibition concentration gradient. Indeed, in the first zone of isolation, it is rare to be able to distinguish the target microorganisms from the non-target microorganisms due to a very high concentration of microorganisms.

[0097] Seeding techniques are well known to those skilled in the art. This may involve quadrant seeding. The quadrant method involves dividing a Petri dish in half, then dividing one half in half again to obtain 3 quadrants of 50%, 25% and 25%. A small amount of inoculum is placed on the largest quadrant and then spread out. The dish is then turned a quarter to spread the bacteria on a smaller quadrant, then turned a quarter again to seed the last small quadrant. In this embodiment, the inhibitor compound and the sample are placed in the same quadrant. There are obviously variations to this technique. It may also involve multiple streaking, which involves spreading the inoculum downwards in tight streaks and then again in other streaks starting from the edges of the previous edge-to-edge streaks.

[0098] Another subject of the invention relates to a method for detecting and / or isolating Shiga toxin-producing E. coli likely to be present in a sample comprising enterobacteria, comprising the following steps:

[0099] - have a selective gelled culture medium allowing the growth of E. coli comprising a concentration gradient of a compound inhibiting non-target bacteria

[0100] - place the sample on said culture medium

[0101] - incubate said medium under conditions allowing the growth of E. coli

[0102] - isolate an E. coli

[0103] - confirm that the said E. coli is a Shiga toxin-producing E. coli

[0104] "Detection" means the detection by the naked eye or using an optical device of the existence of growth of the target microorganisms, preferably target bacteria. When the culture medium from which it is desired to detect the target microorganisms comprises a chromogenic or fluorogenic substrate, the detection may be carried out using an optical device for fluorogenic substrates, or by the naked eye or using an optical device for chromogenic substrates. When the culture medium comprises an agglutinating conjugate, the detection may be carried out by the naked eye or using an optical device by observing the agglutination around the target microorganism.

[0105] By "isolation" we mean obtaining colonies spaced apart from each other.

[0106] Thus, quite surprisingly, it was found that the presence of a gradient of an inhibitory compound made it possible to improve the detection and isolation of a target bacterium such as a Shiga toxin-producing E. coli or an enterohemorrhagic E. coli from a multitude of E. coli. According to the present invention, a selective medium is provided for the growth of E. coli comprising a concentration gradient of a compound that inhibits non-target bacteria such as commensal enterobacteria. Examples of mediums for the growth of E. coli include CHROMID® coli, CHROMID® EHEC, TBX or Rainbow agar. It is then appropriate to add a concentration gradient of a compound that inhibits non-target bacteria.

[0107] The inhibitory compound is chosen in such a way that it makes it possible to discriminate within the group of Shiga toxin-producing E. coli strains that are more or less sensitive to the said inhibitory compound.

[0108] The inhibitory compound can be deposited onto the culture medium by any means such as a pipette. Once deposited, the inhibitory compound forms an inhibition concentration gradient around the deposition area. It can also be a substrate diffusing the inhibitory compound.

[0109] In another embodiment, the inhibitor compound is deposited after the sample is deposited.

[0110] According to the present invention, a step of confirming the presence of the Shiga toxin-producing E. coli is carried out. This step can be carried out according to conventional confirmation methods such as a molecular biology method or an immunological method. In this embodiment, the colony, present on the medium in contact with the gradient of the inhibitory compound, is sampled. When an E. coli has grown on the gradient, there is a very high probability that it is a Shiga toxin-producing E. coli. A latex test or a PCR test designed for the target microorganisms can be carried out. Examples include the SLIDEX® E. coli test or the GENE-UP® STEC top 6 PCR test or GENE-UP® E. coli 0157:H7 or GENE-UP® STEC stx and eae.

[0111] In a particular embodiment, the Shiga toxin-producing E. coli is an enterohemorrhagic E. coli.

[0112] According to a preferred embodiment of the invention, a step of incubation or enrichment of the sample is carried out before deposition on the gelled culture medium. This enrichment step requires not only an ad hoc culture medium but also an incubation of the assembly formed at least by the biological sample and the culture medium at an optimal temperature to allow the growth of the target microorganism(s). The incubation is generally carried out at a temperature ranging from 25 to 45°C for a predetermined period of time (for example from 6 hours to 48 hours). This enrichment phase requires the use of culture media, selective or not (depending on the desired goal), which aim to promote the growth of the target microorganisms in the samples, while limiting the growth of non-target flora.Culture media are frequently used in sterile plastic bag-type containers, in which they are brought into contact with food, clinical or environmental samples, for the purpose of resuspension and enrichment of the desired microorganisms. As mentioned above, this enrichment phase is necessary in particular to reveal the presence of at least one target microorganism in a very variable and possibly very large quantity of sample, for example from 25 grams (g) to 375 g diluted in a volume of culture medium between 225 and 3375 milliliters (mL). At the end of this enrichment step, an aliquot (generally of a volume between 5 microliters (pL) and 5 mL) is traditionally taken to implement the step of detection and / or isolation of the target microorganisms.

[0113] In an advantageous embodiment of the invention, the sample is deposited and distributed over the medium by an exhaustion technique.

[0114] Following seeding, the reaction medium is incubated under appropriate conditions known to those skilled in the art.

[0115] In a preferred embodiment of the invention, the culture medium is the culture medium according to the invention comprising at least one agglutinating conjugate and a gradient of an inhibitory compound. In this preferred embodiment of the invention, the presence of a Shiga toxin-producing E. coli is confirmed by the presence of a halo around said STEC present in the zone of the inhibition gradient. This preferred embodiment has the advantage of being able to detect and isolate said STEC or EHEC directly on the culture medium. This allows an immediate result.

[0116] This invention is particularly interesting for facilitating the detection of Shiga toxin-producing E. coli in a polymicrobial sample. Indeed, without the present invention which allows locating the colony of interest, the ISO 16136 reference method specifies that it is necessary to test up to 50 colonies by molecular method to confirm the presence of a STEC or an EHEC. Given the large number of colonies on the dish and the low representation of the target microorganism, the person collecting the colonies for confirmation purposes may never collect the target microorganism. The probability of isolating a STEC is greatly increased in the presence of the gradient of the inhibitor compound at the sample deposition site. This method is particularly advantageous for samples loaded with additional flora, where the large quantity of colonies on the Petri dishes can lead to a risk of false negatives.

[0117] Examples

[0118] Example 1: Preparation of samples, agar plates and inoculation

[0119] Equipment :

[0120] - VIDAS Biomerieux Ref 4700023

[0121] - Densimat Biomerieux Ref 99234

[0122] - Light plate

[0123] - Smasher biomerieux ref AESAP1064

[0124] - Thermostatic oven 41.5°C

[0125] Matrices used:

[0126] - Frozen minced steak with onions: EXP 07 / 05 / 21 Batch No. 01973658

[0127] - Reblochon de Savoie AOP: EXP 06 / 02 / 23 Batch number 125473226

[0128] Strains used:

[0129] - E. coli O157:H7; STX1+ eae+; CRA 9405024 / ATCC 43890

[0130] Sample preparation:

[0131] Weigh 25 g of matrix to be tested in a bag with a filter

[0132] Add 225 ml of EPT medium

[0133] Mix for 1 minute using a Smacher

[0134] Incubate the bag for 18 hours at 41.5°C in a thermostatically controlled incubator. Following incubation, aliquot into a 9 ml tube.

[0135] Different types of matrix are tested: minced steaks, reblochon. Preparation of the agars:

[0136] - Preparation of gold nanoparticles

[0137] The 40 nm nanoparticles are manufactured by reducing gold chloride with sodium citrate (method described by Turkevich and Frens in 1951). Thus, the 20 nm gold nanoparticles are manufactured using a solution of gold chloride diluted in distilled water, to which trisodium citrate is added and then brought to a boil. The presence of an absorbance peak at 517-519 nm ensures the correct particle size. From these 20 nm particles, 40 nm particles are synthesized. For this, the 20 nm particles are diluted in distilled water and then brought to a boil with the addition of trisodium citrate and gold chloride. The absorbance peak is then observed at 524-526 nm when cold, indicating the increase in particle size.

[0138] - Preparation of the conjugate

[0139] The antibodies used are the 13C4 antibody (hybridoma ref ATCC CRL-1794) directed against the STX1 toxin and the 9E4H11 antibody directed against the STX2 toxin.

[0140] This mixture is adsorbed on the nanoparticles at a pH of 8

[0141] - Preparation of the culture medium:

[0142] The conjugate is added to the TBX agar supercooled at 50°C containing the toxin inducer, namely mitomycin C (Sigma M4287) at 250 ng / mL, in order to obtain a nanoparticle concentration of OD 3. The medium is then poured into the dishes and dried.

[0143] Preparation of the strain:

[0144] At Jl, replant the CRA 9405024 strain in 9 ml of EPT. Incubate for 18 hours at 41.5°C in a thermostatically controlled incubator. Calibrate the strain to 0.4 McFarland using a densimat. Carry out successive dilutions directly in the aliquoted matrix tubes.

[0145] Seeding the boxes:

[0146] Take the tube representing the 10 dilution 5 CFU / ml of the strain of interest

[0147] For a portion of the tube, use the VIDAS ESPT2 parameter. This is an immunoselection parameter allowing the selection of the strains of interest, namely E.coli 026, 045, 0103, 111, 0121, 0145 and 0157. The immunoselected sample is then inoculated by exhausting the 30 μl on the STEC agar.

[0148] For the other part, carry out direct inoculation by exhaustion after depositing 10 μl on the STEC Agar.

[0149] Incubate the dishes for 18-22 hours at 37°C in a thermostatically controlled incubator.

[0150] Example 2: Isolation of the targeted strain (E. coli O157:H7, STX1+ eae+, ATCC: 43890) within a minced steak and Reblochon matrix on a culture medium without gradient

[0151] The culture medium, the "reblochon" matrix and the inoculation are carried out according to example 1. In the case of the Reblochon matrix which has a very loaded additional flora, we observe in Figure 1 and Figure 2 that it is difficult to isolate the characteristic colonies having a halo. Not all the colonies present in the first quadrants are identifiable because they are drowned in the middle of the additional flora. This is valid for direct isolation coming from enrichment (Figure 1), but also after an immuno-selection step with a VIDAS (ESP2) (Figure 2).

[0152] In the case of the "meat" matrix, presenting less additional flora than a "reblochon" matrix, we observe a characteristic colony by the presence of a halo (Figure 3) after direct isolation and multiple characteristic colonies with the immuno-selection step (Figure 4).

[0153] Example 3: Isolation of the targeted strain (E. coli 0157:117, STX1+ eae+, ATCC: 43890) within a minced steak matrix using the method according to the invention

[0154] - Preparation of the culture medium according to the invention:

[0155] The culture medium and the “minced steak” matrix are produced according to example 1.

[0156] After the last step of drying the culture medium, the following steps are carried out:

[0157] - Mark the intended deposition area for the CT inhibitor compound with a marker on the back of the dish. This area is located at the edge of the agar.

[0158] Rehydrate the CT supplement bottle with 400 µl of sterile demineralized water. Place 10 µl of this CT supplement on the agar at the previously defined point.

[0159] Leave to dry for 10 minutes so that the box can then be used for isolation. The boxes are seeded according to example 1. The enriched minced steak sample is placed in the CT supplement deposition area.

[0160] The incubation of the boxes is identical to example 1.

[0161] Figure 5 shows a significant diameter of inhibition around the deposition point. This shows isolated colonies, not masses as in the following quadrant. Colonies with halos characteristic of the targeted strains are visible in the area of ​​the inhibition concentration gradient. The application of this inhibitory compound, which forms a gradient, therefore makes it possible to isolate the characteristic colonies.

[0162] In the case of an immuno-selected "minced steak" sample on a plate with a gradient, we observe in Figure 6 as good detection of the characteristic strains as with the plate without a gradient with an immuno-selected "minced steak" sample (Figure 4). The drop of CT has no negative effect. The method is therefore versatile and can be used regardless of the matrix.

[0163] Example 4: Isolation of the targeted strain (E. coli O157:H7, STX1+ eae+, ATCC: 43890) within a reblochon matrix using the method according to the invention

[0164] Preparation of the culture medium according to the invention:

[0165] The preparation of agar is carried out according to example 1. After the last drying step, the following steps are carried out:

[0166] - Mark the area intended for the CT supplement with a marker on the back of the box. This area is located at the edge of the agar.

[0167] Rehydrate the CT supplement bottle with 400 µl of sterile demineralized water. Place 10 µl of this supplement on the agar at the previously defined point.

[0168] Leave to dry for 10 minutes so that the box can then be used for isolation.

[0169] The boxes are seeded according to example 1. The enriched reblochon sample is placed in the CT supplement deposition area.

[0170] The incubation of the boxes is identical to example 1.

[0171] Figure 7 shows an inhibition diameter around the deposition point. This shows isolated colonies, not massed colonies as in the following quadrant where there are many more colonies. The presence of the concentration gradient allows characteristic isolated colonies to be obtained, unlike the middle of the box in Figure 1, which does not have the gradient.

[0172] Figure 8 shows characteristic isolated colonies for a sample that has undergone the immunoselection step. The presence of the gradient makes it possible to obtain characteristic isolated colonies, unlike the middle of the box in Figure 2, which does not have the gradient.

[0173] CONCLUSION: The use of a concentration gradient for inhibiting non-target bacteria, in the presence of a complex matrix loaded with additional flora, allows the inhibition of said non-target bacteria in order to isolate target colonies which are more resistant.

Claims

CLAIMS

1. Gelled reaction medium for the detection, identification, and / or isolation of at least one Shiga toxin-producing strain of E. coli comprising - at least one toxin inducer, - at least one agglutinating conjugate comprising at least one specific binding partner of STX1 and / or at least one specific binding partner of STX2, coupled to a nanoparticle - a concentration gradient of a compound that inhibits non-target bacteria

2. Gelled reaction medium according to claim 1 characterized in that it is a microbiological culture medium.

3. Reaction medium according to any one of the preceding claims, characterized in that the binding partner is chosen from antibodies or phage proteins.

4. Medium according to any one of the preceding claims, characterized in that the inhibitory compound comprises tellurite.

5. Medium according to the preceding claim, characterized in that the inhibitory compound comprises cefixime.

6. Medium according to any one of the preceding claims, characterized in that the concentration gradient of tellurite on the medium is between 0 pg / ml and 100 pg / ml, preferably between 0 pg / ml and 50 pg / ml, even more preferably between 0 pg / ml and 30 pg / ml.

7. Medium according to any one of the preceding claims, characterized in that the nanoparticle is a colloidal nanoparticle having optical properties.

8. Medium according to any one of the preceding claims, characterized in that the nanoparticle is a gold nanoparticle of size between 20 and 90 nm and is present at a concentration of between 10 10 and 10 12 nanoparticles / ml of reaction medium.

9. Medium according to any one of the preceding claims, characterized in that the toxin inducer is an antibiotic.

10. Medium according to any one of the preceding claims, characterized in that the toxin inducer is ciprofloxacin at a concentration of between 0.005 and 0.030 mg / l.

11. Medium according to any one of the preceding claims, characterized in that the toxin inducer is mitomycin C at a concentration of between 0.10 mg / l and 0.50 mg / l.

12. Medium according to any one of the preceding claims, characterized in that the deposited inhibitor compound has a volume of between 5 μl and 50 μl.

13. Medium according to any one of the preceding claims in which the inhibitory compound is contained in at least one substrate capable of diffusing said inhibitory compound onto said gelled culture medium.

14. diagnostic kit allowing the preparation of a reaction medium according to claims 1 to 13 comprising - an agglutinating conjugate comprising at least one specific binding partner of STX1 and / or at least one specific binding partner of STX2, coupled to a nanoparticle - a gelling medium comprising a toxin inducer - a compound that inhibits non-target bacteria.

15. Preparation of a reaction medium according to claims 1 to 13 comprising the following steps: - contacting an agglutinating conjugate with a gelling culture medium - depositing on an area of ​​the gelled culture medium, at least one compound inhibiting non-target bacteria comprising tellurite; said inhibitory compound diffusing and forming an inhibition concentration gradient around the deposition area; the concentration of the tellurite concentration gradient being between 0 pg / ml and 100 pg / ml, preferably between 0 pg / ml and 50 pg / ml, even more preferably between 0 pg / ml and 30 pg / ml

16. Preparation of a medium according to the preceding claim in which the inhibitory compound is deposited after the deposition of the sample.

17. A method for detecting and / or isolating Shiga toxin-producing E. coli likely to be present in a sample comprising Enterobacteria comprising the following steps: - have a selective gelled culture medium allowing the growth of E. coli comprising a concentration gradient of a compound inhibiting non-target bacteria - deposit the sample on said gelled culture medium - incubate said medium under conditions allowing the growth of E. coli - isolate an E. coli - confirm that the said E. coli is a Shiga toxin-producing E. coli

18. A method according to any preceding claim wherein the sample is deposited and distributed over the medium by an exhaustion technique;

19. Method for detecting and / or isolating E coli producing shiga toxin according to the preceding claim, characterized in that the confirmation method is a molecular biology method or an immunological method.

20. Method for detecting and / or isolating Shiga toxin-producing E. coli likely to be present in a sample according to any one of claims 17 to 19, characterized in that the culture medium is the culture medium according to claims 1 to 13.

21. Method for detecting and / or isolating Shiga toxin-producing E. coli likely to be present in a sample according to any one of the preceding claims in which the sample, prior to its deposit on the medium, is incubated in an enrichment medium allowing the growth of Shiga toxin-producing E. coli.

22. A method according to any one of claims 20 or 21 wherein the presence of a Shiga toxin-producing E. coli is confirmed by the presence of a halo around said Shiga toxin-producing E. coli present in the region of the inhibition concentration gradient.

23. A method according to any one of claims 17 to 22 wherein the Shiga toxin-producing E. coli is an enterohemorrhagic E. coli.

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