Method for detecting and confirming shiga toxin-producing escherichia coli and / or enterohaemorrhagic e. coli

Abstract

The invention relates to a method for detecting and confirming at least one Shiga toxin-producing Escherichia Coli (STEC) which may be present in a sample comprising enterobacteria, comprising the following steps: - performing lysis of the sample, enabling lysis of the STECs in order to obtain a solution comprising the nucleic acids thereof; - bringing the solution of nucleic acids into contact with primers, making it possible to amplify at least the stx1 and / or stx2 gene or gene fragment; - if at least one of the stx1 and / or stx2 genes or gene fragments is amplified, part of the sample is deposited on an agar reaction medium comprising ■ at least one toxin inducer, ■ at least one agglutinating conjugate formed by at least one binding partner specific to the STX1 protein and / or at least one binding partner specific to the STX2 protein, which binding partner(s) is (are) coupled to a nanoparticle; - detecting and confirming the presence of at least one STEC by the appearance of a halo on the agar around the STEC.

Description

[0001] Description

[0002] METHOD FOR THE DETECTION AND CONFIRMATION OF SHIGATOXIN-PRODUCING ESCHERICHIA COLI AND / OR 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 method for the detection, identification, enumeration and / or isolation of Shiga toxin-producing Escherichia coli and / or 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 E. coli (STEC), Salmonella, Listeria, Cronobacter, Bacillus, Staphylococcus, the research of which applies to raw materials, intermediate products, finished products marketed, - non-pathogenic microorganisms, used as quality indicators of the production process, from raw materials to finished products, throughout the chain,

[0010] - bacteria of technological interest such as ferments,

[0011] - microorganisms that are markers of contamination.

[0012] Rapid and accurate detection of suspected contamination (within food batches) allows them to be controlled and corrective actions to be taken quickly.

[0013] In recent years, many countries have been affected by outbreaks of Shiga toxin-producing E. coli (STEC) or Enterohemorrhagic E. coli (EHEC). Human infections caused by these bacteria are now recognized as a major public health problem worldwide. The vast majority of E. coli bacteria are commensal strains of humans. However, some strains have acquired virulence genes and factors. This allows them to colonize the digestive system, bypass immune defenses, and induce cellular damage, thus causing symptoms in humans. Among these STEC strains that have acquired the stx virulence factor, a particular pathovar is implicated: Enterohemorrhagic E. coli strains. EHEC are strains representing a subgroup of STEC, which have acquired the eae gene and cause hemolytic uremic syndrome.The simultaneous presence of these two virulence factors, stx and eae, makes this pathovar very virulent for humans. Other virulence factors include aggR, LT, ST, afaC, ipaH.

[0014] 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. During this detection stage, it is difficult to know whether the same strain carries one or more virulence factors.

[0015] It may be followed by a confirmation phase to confirm the presence of the pathogen being sought and meet the standards in force in this field. The confirmation stage therefore requires additional steps and an isolation stage for the bacteria being sought.

[0016] Thus, the methods of the prior art require a succession of amplification, immunoconcentration and culture steps on a dish in order to detect and confirm the presence of a virulent Escherichia coli pathovar.

[0017] There is therefore a real need to develop a reliable and rapid method for detecting and confirming virulent E. coli pathovars.

[0018] SUMMARY OF THE INVENTION

[0019] The present invention relates to a method for detecting and confirming at least one Shiga toxin-producing Escherichia Coli (STEC) likely to be present in a sample comprising enterobacteria, comprising the following steps: - Performing a lysis of the sample allowing the lysis of the STEC in order to obtain a solution comprising their nucleic acids - Bringing the nucleic acid solution into contact with primers making it possible to amplify at least the stxl and / or stx2 gene or gene fragment

[0020] - If at least one of the genes or fragments of the stxl and / or stx2 genes is amplified, part of the sample is deposited on an agar reaction medium comprising:

[0021] ■ at least one toxin inducer,

[0022] ■ at least one agglutinating conjugate formed by at least one specific binding partner of the STX1 protein and / or at least one specific binding partner of the STX2 protein, coupled to a nanoparticle.

[0023] - Detect and confirm the presence of at least one STEC by the appearance of a halo on the agar around said STEC.

[0024] The method according to the present invention has the advantage of confirming the presence or absence of a STEC directly on the reaction medium by the formation of a halo around said strain.

[0025] This invention is particularly interesting for facilitating the detection and confirmation of the presence of STEC or EHEC in a polymicrobial sample. Indeed, without the present invention which makes it possible to locate 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. 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. Thus, the present invention makes it possible to avoid false negatives and saves time in carrying out microbiological control. It 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.

[0026] Another embodiment relates to a serogroup detection approach. Thus, another embodiment relates to a method for detecting and confirming at least one Shiga toxin-producing Escherichia Coli (STEC) comprising the following steps:

[0027] - Perform a lysis of the sample allowing the lysis of STEC in order to obtain a solution comprising their nucleic acids

[0028] - Bringing the nucleic acid solution into contact with primers for amplifying at least the stxl and / or stx2 gene or gene fragment - If at least one of the genes or fragments of the stxl and / or stx2 genes is amplified, the nucleic acid solution is brought into contact with primers for amplifying at least the gene or gene fragment of the serogroups chosen from 026, 045, 080, 091, 0103, 0104, OR I, 0113, 0121, O128ab, 0145, 0146, 0157, 0174

[0029] - If at least one of the genes or fragments of the stxl and / or stx2 genes and at least one serogroup chosen from 026, 045, 080, 091, 0103, 0104, OR I, 0113, 0121, O128ab, 0145, 0146, 0157, 0174 are amplified, a portion of the sample is deposited on an agar reaction medium comprising o at least one toxin inducer and o at least one agglutinating conjugate formed by at least one specific binding partner of the STX1 protein and / or at least one specific binding partner of the STX2 protein, coupled to a nanoparticle.

[0030] - Detect and confirm the presence of at least one STEC by the appearance of a halo on the agar around said STEC.

[0031] The advantage of this method is that the serogroup approach strengthens the presumption that it is a STEC of a target serogroup.

[0032] Preferably, the method for detecting and confirming at least one Shiga toxin-producing Escherichia Coli (STEC) comprises the following step:

[0033] - if at least one of the genes or fragments of the stxl and / or stx2 genes and at least one gene or gene fragment of at least one serogroup chosen from 026, 045, 080, 091, 0103, 0104, 0111, 0113, 0121, O128ab, 0145, 0146, 0157, 0174.are amplified, a portion of the sample is deposited on a reaction medium comprising o at least one toxin inducer and o at least one agglutinating conjugate formed by at least one specific binding partner of STX1 coupled to a nanoparticle of a first nature producing a halo of a first color when it is agglutinated o and / or at least one agglutinating conjugate formed by at least one specific binding partner of STX2 coupled to a nanoparticle of a first nature producing a halo of a first color when it is agglutinated o and at least one agglutinating conjugate formed by at least one specific binding partner of said serogroup identified by amplification coupled to a nanoparticle of a second nature producing a halo of a second color when it is agglutinated.

[0034] - confirm the presence of at least one STEC of a serogroup identified by the appearance of at least one halo of a first color and at least one halo of a second color around the same colony or at least one halo resulting from the mixture of a first and a second color around said colony.

[0035] The advantage of this embodiment is that it allows discrimination of different groups of bacteria by a different colored halo. It is thus possible to visually distinguish

[0036] - a group of microorganisms secreting the toxin STX1 and / or STX2

[0037] - a group of microorganisms selected by 026, 045, 080, 091, 0103, 0104, OR I, 0113, 0121, O128ab, 0145, 0146, 0157, 0174

[0038] - a group of microorganisms selected from 026, 045, 080, 091, 0103, 0104, 0111, 0113, 0121, O128ab, 0145, 0146, 0157, 0174 and secreting the toxin STX1 and / or STX2.

[0039] Thus, by choosing the nanoparticles in an appropriate way for the formation of the agglutinating conjugate, it is possible to obtain halos of different colors and thus to discriminate between different groups of target microorganisms.

[0040] According to another embodiment, the present invention relates to a method for detecting and confirming at least one enterohemorrhagic Escherichia Coli (EHEC) likely to be present in a sample comprising enterobacteria, comprising the following steps:

[0041] - Perform a lysis of the sample allowing the lysis of EHEC in order to obtain a solution comprising their nucleic acids

[0042] - Bring the nucleic acid solution into contact with primers capable of amplifying at least the stxl and / or stx2 and eae gene or gene fragment,

[0043] - if at least one of the genes or fragments of the stxl and / or stx2 genes and at least one eae gene or gene fragment are amplified, a portion of the sample is deposited on an agar medium comprising: o at least one toxin inducer o at least one agglutinating conjugate formed by at least one specific binding partner of STX1 coupled to a nanoparticle of a first nature producing a halo of a first color when agglutinated o and / or at least one agglutinating conjugate formed by at least one specific binding partner of STX2 coupled to a nanoparticle of a first nature producing a halo of a first color when agglutinated o And at least one agglutinating conjugate formed by at least one specific binding partner of eae coupled to a nanoparticle of a second nature producing a halo of a second color when agglutinated

[0044] - confirm the presence of at least one EHEC by the appearance on the agar medium

[0045] - at least one halo of a first color

[0046] - and at least one halo of a second color

[0047] - or at least one halo resulting from the mixing of a first and a third color around said EHEC strain.

[0048] The advantage of the supra method is that it allows discrimination in the same sample between STEC bacteria producing shiga toxin STX1 and / or STX2 and EHEC bacteria expressing the EAE protein.

[0049] DESCRIPTION OF FIGURES

[0050] Figure 1 is a photograph of a culture medium comprising anti-STX1 and anti-STX2 antibodies onto which a “minced meat” sample contaminated with an 080 strain carrying the eae stx2 genes was inoculated.

[0051] Figure 2 is a photograph of a culture medium comprising anti-STXl and anti-STX2 antibodies onto which a “carpaccio” sample contaminated with an 045 strain carrying the eae stxl genes was inoculated.

[0052] DETAILED DESCRIPTION OF THE INVENTION

[0053] Certain terms and expressions used in the context of the invention are detailed below.

[0054] A first subject of the invention relates to a method for detecting and confirming at least one Shiga toxin-producing Escherichia Coli (STEC) likely to be present in a sample comprising enterobacteria, comprising the following steps:

[0055] - Carry out a lysis of the sample allowing the lysis of the STEC in order to obtain a solution comprising their nucleic acids - Bring the nucleic acid solution into contact with primers allowing the amplification of at least the gene or gene fragment stxl and / or stx2

[0056] - If at least one of the genes or fragments of the stxl and / or stx2 genes is detected, part of the sample is deposited on an agar reaction medium comprising

[0057] • at least one toxin inducer,

[0058] • at least one agglutinating conjugate formed by at least one specific binding partner of the STX1 protein and / or at least one specific binding partner of the STX2 protein, coupled to a nanoparticle.

[0059] - detect and confirm the presence of at least one STEC by the appearance of a halo on the agar around said STEC.

[0060] The method according to the present invention has the advantage of confirming the presence or absence of a STEC directly on the reaction medium.

[0061] By "detection" is meant 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 can be carried out using an optical device for fluorogenic substrates, or by the naked eye or using an optical device for chromogenic substrates. Since the culture medium comprises an agglutinating conjugate, the detection can be carried out by the naked eye or using an optical device by observing the appearance of a halo due to the agglutination of the conjugate around the target microorganism.

[0062] By "confirmation" we mean confirming by a second method a presumptive positive result obtained by a first detection method. The two methods may or may not be different. The technologies used, such as molecular biology or immunology, may or may not be different.

[0063] By "isolation" we mean obtaining different colonies spaced apart from each other.

[0064] The sample may be of various origins, for example, food, environmental, veterinary or clinical. Generally speaking, the term "sample" refers to a part or quantity (especially a small part or quantity) taken from one or more entities for the purpose of analysis. This sample may possibly have undergone prior processing, involving for example mixing, dilution or grinding steps, particularly if the starting entity is in the solid state.

[0065] The sample may undergo an enrichment step to grow microorganisms, particularly STEC. The sample may also undergo an immunoselection step to select at least one STEC strain.

[0066] The sample analyzed is, in general, likely to - or suspected of - containing at least one Shiga toxin-producing E. coli or one enterohemorrhagic E. coli that should be detected for health purposes. Enterohemorrhagic E. coli are strains representing a subgroup of Shiga toxin STX-producing E. coli, which have the eae gene and cause hemolytic uremic syndrome. The possession of these two virulence factors simultaneously makes this pathovar very virulent for humans. These include serogroups 026, 045, 080, 091, 0103, 0104, OU I, 0113, 0121, O128ab, 0145, 0146, 0157, 0174.

[0067] Thus, the present method makes it possible to detect target microorganisms which can be the STEC group, the typical EHEC group carrying the stx and eae genes, or the atypical EHEC group carrying the stx and aggR gene for example.

[0068] According to one embodiment of the present invention, an incubation or enrichment step is carried out prior to lysis of the sample. 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 may be 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 of between 225 and 3375 milliliters (mL). At the end of this enrichment step, an aliquot (generally of a volume of between 5 microliters (pL) and 5 mL) is traditionally taken to implement the step of detecting the target microorganisms.

[0069] According to the present invention, the sample of target microorganisms undergoes lysis. Indeed, in order to carry out amplification, it is necessary to extract the DNA from the bacteria. For example, the GENE-UP® lysis kit will be used. Any other lysis method known to those skilled in the art is possible.

[0070] It may be desirable to perform immuno-selection of at least one STEC before the lysis step. For example, the VIDAS® ESPT kit and the applicant's VIDAS® instrument will be used. Immuno-selection makes it possible to increase the proportion of bacteria of interest compared to the additional flora. It is advantageous when the polymicrobial sample has a significant amount of flora, such as dairy products. The presence of a small amount of STEC or EHEC is then difficult to detect. There is a risk of a false negative. According to the present invention, a step of amplification of at least one of the genes or fragments of the stxl and / or stx2 genes is carried out. It is also possible to amplify the subtypes of stxl or stx2 such as stx2a or stx2d.

[0071] Amplification can be carried out by any molecular biology method known to those skilled in the art. Examples include amplification methods such as all PCR methods or isothermal methods.

[0072] A Tissue PCR detection, if a sample is presumptively positive, it is necessary to perform a confirmation. Before inoculation on a reaction medium, it may be desirable to perform an immuno-selection of microorganisms. Immunoselection is advantageous when the sample contains a significant additional flora. Indeed, it allows to reduce the diversity of bacteria that will be visible on the plate and to simplify the discrimination between STEC of the targeted serogroups and non-STEC.

[0073] According to the present invention, the confirmation is carried out from an aliquot of the sample or enrichment or from a sample having undergone a step of immuno-selection of at least one STEC of a targeted serogroup. The confirmation is carried out on a reaction medium comprising at least one toxin inducer and at least one specific binding partner of a component of a Shiga toxin-producing E. coli or of a component derived from the latter, coupled to at least one nanoparticle to form at least one agglutinating conjugate. Advantageously, the culture medium comprises a specific binding partner of STX1 and / or a specific binding partner of STX2. It is also possible to have a specific binding partner of certain subtypes of STX1 or STX2 such as STX2a or STX2d.

[0074] The inoculation is carried out according to conventional microbiology techniques. In a preferred embodiment of the invention, the inoculation is carried out by exhausting the polymicrobial sample on the culture medium. The techniques for inoculating by exhaustion are well known to those skilled in the art. This may involve inoculating in quadrants. The quadrant method consists of dividing a Petri dish in two, then dividing one half in half again in order 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 again a quarter to inoculate the last small quadrant. It can also be a multiple streak exhaustion which consists of spreading the inoculum downwards in tight streaks and then again other streaks starting from the edges of the previous ones edge to edge.

[0075] Following seeding, the reaction medium is incubated under appropriate conditions known to those skilled in the art.

[0076] According to the present invention, the reaction medium is a gelled reaction medium comprising at least one toxin inducer and at least one specific binding partner of STX1 and / or at least one specific binding partner of STX2, coupled to a nanoparticle.

[0077] The term "reaction medium" means a medium comprising all the elements necessary for the expression of a metabolism, the survival and / or 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. The reaction medium is gelled. It is in solid or semi-solid form.Agar is the traditional gelling agent used in microbiology for the cultivation of microorganisms, but other gelling agents such as gelatin, agarose, and other natural or artificial gelling agents can be used. 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.

[0078] 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 example, 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 also 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 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.

[0079] 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.

[0080] According to the present invention, the reaction medium comprises a specific binding partner of a component of a target microorganism or of a component derived from said microorganism, coupled to a nanoparticle. 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 an antibody or a phage protein. 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 derived 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 part of the bacterial colony dies during its growth. In an advantageous embodiment of the invention, 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. Preferably, 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. Preferably, the medium comprises at least one binding partner specific for STX2a and / or STX2d.

[0081] 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. The term "nanoparticle" designates particles of the order of magnitude of a nanometer. The nanoparticles can be chosen from gold, iron, silver, copper, carbon, latex, silicon, aluminum. Preferably, the nanoparticles are colloidal nanoparticles chosen for their optical property, namely their ability to be distinguished 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 not aggregated, the wavelength of the absorbed light is in the red around 530nm. When aggregated, the absorbed wavelength changes from red to blue / grey around 600 to 700nm. In a particular embodiment, it is possible to use together several nanoparticles of different colours. The networks thus formed allow the distinction of several groups of target microorganisms.

[0082] 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.

[0083] Advantageously, the nanoparticles make it possible to reduce the necessary quantity of binding partners for the formation of agglutination. Thus, the necessary concentration of binding partners for the production of a reaction medium according to the invention requires 100 to 1000 times fewer binding partners than a medium without nanoparticles. Preferably, the necessary quantity of binding partners corresponds to the quantity necessary to cover at least half of the surface of the nanoparticle, and even more preferably to cover between a third and half of the surface of the nanoparticle. This proportion allows the agglutinating conjugate to form a network in the gelled reaction medium. Advantageously, the 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 be used to visualize agglomeration around a bacterial colony whose translucent appearance prevents detection by an automated reading device. Detection can thus be facilitated.

[0084] In a particular embodiment, the binding partner is an antibody present in an amount making it possible to cover at least half of the surface of the nanoparticle and preferably at least one third of the surface of the nanoparticle. Preferably, in these variants the nanoparticle is a gold nanoparticle of size between 20 and 90 nm and at a concentration of between 10 10 and 10 12nanoparticles / ml of reaction medium. 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. Depending on the type of bond chosen, the skilled person will adapt the physicochemical conditions of the reaction medium and in particular its pH.

[0085] 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.In practice, several parameters influence the capacity of the conjugate to be agglutinating in a gelled medium, mainly: - the porosity of the gelled medium.

[0086] - the size of the nanoparticles

[0087] - the quantity of binding partners

[0088] - the quantity of nanoparticles

[0089] 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.

[0090] According to the present invention, the reaction medium 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. Shiga toxins secreted by STEC strains will be mentioned. There are two main types of Shiga toxins: STX1 and STX2 which themselves have many variants. To date, STX1 has 4 subtypes STX1a, STX1c, STX1d, STX1e, and STX2 has 12: STX2a, STX2b STX2c, STX2d, STX2e, STX2f, STX2g, STX2h, STX2i, STX2j, STX2K, STX21. Advantageously, the toxin inducer is chosen from antibiotics or a physicochemical stress.Examples of antibiotics that may be mentioned are trimethoprim, sulfamethoxazole, norfloxacin, azithromycin, gemtamicin, polymyxin B, chloramphenicol, streptomycin, chlortetracycline, oxytetracyline, tylosin, mitomycin C, carbodox, oliquindox, rifampincin, imipenem, ciprofloxacin, cotrimoxazole, penicillin G, linlomycin. 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 0.50 mg / l.

[0091] 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.

[0092] Advantageously, the reaction medium can be manufactured extemporaneously. This allows the unitary use of a reaction medium, and to increase its stability.

[0093] The method for obtaining a reaction medium comprises the steps of bringing an agglutinating conjugate into contact with a gelling medium to form the reaction medium. The preparation of the conjugate is carried out by coupling the binding partner with the nanoparticle. These coupling methods are well known to those skilled in the art (Nicholas G. Weich et al, 2017). The conjugate is then added to the supercooled gelled medium. The whole is then homogenized and poured into the Petri dish.

[0094] According to the present invention, the presence of at least one STEC is detected and confirmed by the appearance of a halo on the agar around said STEC.

[0095] In this embodiment it may also be of interest to detect by molecular biology the subtypes of STX1 and / or STX2 detected on the agar such as STX2a and / or STX2d.

[0096] Another subject of the invention relates to a method for detecting and confirming at least one Shiga toxin-producing E. coli comprising the following steps:

[0097] Perform sample lysis to lyse STEC to obtain a solution containing their nucleic acids

[0098] - Bring the nucleic acid solution into contact with primers capable of amplifying at least the stxl and / or stx2 gene or gene fragment

[0099] - If at least one of the genes or fragments of the stxl and / or stx2 genes is amplified, the nucleic acid solution is brought into contact with primers making it possible to amplify at least one gene or a gene fragment of a serogroup chosen from 026, 080, 045, 091, 0103, 0104, OR I, 0121, O128ab, 0145, 0146, 0113, 0157, 0174. if at least one of the genes or fragments of the stxl and / or stx2 genes and at least one serogroup chosen from 026, 080, 045, 091, 0103, 0104, OR I, 0121, O128ab, 0145, 0146, 0113, 0157, 0174, are amplified, a portion of the sample is deposited on a reaction medium comprising o at least one toxin inducer and o at least one agglutinating conjugate formed by at least one specific binding partner of the STX1 protein and / or at least one specific binding partner of the STX2 protein, coupled to a nanoparticle.

[0100] - detect and confirm the presence of at least one STEC by the appearance of a halo on the agar around said STEC.

[0101] The advantage of this method is that it allows the detection and confirmation of the presence of STEC. In addition, the serogroup approach strengthens the presumption that it is a STEC of a target serogroup. In this embodiment, the serogroup can be confirmed by molecular biology from the isolated colony. The serogroup can also be confirmed by latex particle agglutination test such as SLIDEX® E. coli 026 or E. coli 080 etc.

[0102] Another subject of the invention relates to a method for detecting and confirming at least one Shiga toxin-producing E. coli likely to be present in a sample comprising enterobacteria, comprising the following steps:

[0103] - Perform a lysis of the sample allowing the lysis of STEC in order to obtain a solution comprising their nucleic acids

[0104] - Bring the nucleic acid solution into contact with primers capable of amplifying at least the stxl and / or stx2 gene or gene fragment

[0105] - if at least one of the genes or fragments of the stxl and / or stx2 genes is amplified and at least one gene or gene fragment of at least one serogroup chosen from 026, 045, 080, 091, 0103, 0104, OR I, 0113, 0121, O128ab, 0145, 0146, 0157, 0174 are amplified,a portion of the sample is deposited on a reaction medium comprising: o at least one toxin inducer and o at least one agglutinating conjugate formed by at least one specific binding partner of STX1 coupled to a nanoparticle of a first nature producing a halo of a first color when it is agglutinated o and / or at least one agglutinating conjugate formed by at least one specific binding partner of STX2 coupled to a nanoparticle of a first nature producing a halo of a first color when it is agglutinated o and at least one agglutinating conjugate formed by at least one specific binding partner of said serotype identified by amplification coupled to a nanoparticle of a second nature producing a halo of a second color when it is agglutinated,

[0106] - confirm the presence of at least one STEC of a serogroup identified by the appearance of at least one halo of a first color and at least one halo of a second color or at least one halo resulting from the mixture of a first and a second color around said strain

[0107] The advantage of the supra serogroup method is that it allows the detection and confirmation of the presence of STEC of a target serogroup. The advantage of this embodiment is that it allows discrimination of different groups of bacteria by a different color halo. It is thus possible to visually distinguish - a group of microorganisms secreting the STX1 and / or STX2 toxin

[0108] - a group of microorganisms selected by 026, 045, 080, 091, 0103, 0104, OR I, 0113, 0121, O128ab, 0145, 0146, 0157, 0174

[0109] - a group of microorganisms selected from 026, 045, 080, 091, 0103, 0104, 0111, 0113, 0121, O128ab, 0145, 0146, 0157, 0174 and secreting the toxin STX1 and / or STX2.

[0110] Thus, by choosing the nanoparticles in an appropriate way for the formation of the agglutinating conjugate, it is possible to obtain halos of different colors and thus to discriminate between different groups of target microorganisms. It is thus possible to envisage another embodiment of the method comprising the following steps:

[0111] - Perform a lysis of the sample allowing the lysis of STEC in order to obtain a solution comprising their nucleic acids

[0112] - Bring the nucleic acid solution into contact with primers capable of amplifying at least the stxl and / or stx2 gene or gene fragment

[0113] - If at least one of the genes or fragments of the stxl and / or stx2 genes is amplified and at least one gene or gene fragment of at least one serogroup chosen from 026, 045, 080, 091, 0103, 0104, OR I, 0113, 0121, O128ab, 0145, 0146, 0157, 0174 are amplified,a portion of the sample is deposited on a reaction medium comprising: o at least one toxin inducer and o at least one agglutinating conjugate formed by at least one specific binding partner of STX1 coupled to a nanoparticle of a first nature producing a halo of a first color when it is agglutinated o and / or at least one agglutinating conjugate formed by at least one specific binding partner of STX2 coupled to a nanoparticle of a second nature producing a halo of a second color when it is agglutinated o and at least one agglutinating conjugate formed by at least one specific binding partner of said serogroup identified by amplification coupled to a nanoparticle of a third nature producing a halo of a third color when it is agglutinated,

[0114] - confirm the presence or absence of at least one STEC of a serogroup identified by the appearance on the agar medium:

[0115] • at least one halo of a first color or second color

[0116] • and at least one halo of a third color • or at least one halo resulting from the mixing of a first and / or a second color and a third color around said strain

[0117] This embodiment allows discrimination of different groups of bacteria by a halo of different colors. It is thus possible to visually distinguish

[0118] - a group of microorganisms secreting the STX1 toxin

[0119] - a group of microorganisms secreting the STX2 toxin

[0120] - a group of microorganisms chosen from 026, 080, 045, 091, 0103, 0104, OR I,

[0121] 0121, O128ab, 0145, 0146, 0113, 0157, 0174.

[0122] - a group of microorganisms chosen from 026, 080, 045, 091, 0103, 0104, OR I, 0121, O128ab, 0145, 0146, 0113, 0157, 0174 and secreting the toxin STX1 and / or STX2.

[0123] According to the present invention, it is also possible to detect and confirm the presence of an enterohemorrhagic Escherichia Coli (EHEC). An EHEC is a STEC having the eae gene. Thus, another subject of the invention relates to a method for detecting and confirming at least one enterohemorrhagic Escherichia Coli (EHEC) likely to be present in a sample comprising enterobacteria, comprising the following steps:

[0124] - incubate the sample in an enrichment medium allowing the growth of EHEC

[0125] - Perform a lysis of the sample allowing the lysis of EHEC in order to obtain a solution comprising their nucleic acids

[0126] - Bring the nucleic acid solution into contact with primers capable of amplifying at least the stxl and / or stx2 and eae gene or gene fragment,

[0127] - if at least one of the genes or fragments of the stxl and / or stx2 genes and at least one eae gene or gene fragment are amplified, depositing a portion of the enriched sample on an agar medium comprising: o at least one toxin inducer o at least one agglutinating conjugate formed by at least one specific binding partner of STX1 coupled to a nanoparticle of a first nature producing a halo of a first color when agglutinated o and / or at least one agglutinating conjugate formed by at least one specific binding partner of STX2 coupled to a nanoparticle of a first nature producing a halo of a first color when agglutinated o And at least one agglutinating conjugate formed by at least one specific binding partner of eae coupled to a nanoparticle of a second nature producing a halo of a second color when agglutinated

[0128] -confirm the presence of at least one EHEC by the appearance on the agar medium

[0129] - at least one halo of a first color

[0130] - and at least one halo of a second color

[0131] - or at least one halo resulting from the mixture of a first and a third color around said EHEC strain

[0132] The advantage of the supra method is that it allows discrimination in the same sample between STEC bacteria producing shiga toxin STX1 and / or STX2 and EHEC bacteria expressing the EAE protein.

[0133] Another subject of the invention relates to a method for detecting and confirming at least one enterohemorrhagic Escherichia Coli (EHEC) likely to be present in a sample comprising enterobacteria, comprising the following steps:

[0134] - incubate the sample in an enrichment medium allowing the growth of EHEC

[0135] - Perform lysis of the enriched sample allowing lysis of EHEC in order to obtain a solution comprising their nucleic acids

[0136] - Bring the nucleic acid solution into contact with primers capable of amplifying at least the stxl and / or stx2 and eae gene or gene fragment,

[0137] If at least one of the genes or fragments of the stxl and / or stx2 genes and at least one of the genes or fragments of the eae gene are amplified, deposit a portion of the enriched sample on an agar medium comprising: o at least one toxin inducer o at least one agglutinating conjugate formed by at least one specific binding partner of STX1 coupled to a nanoparticle of a first nature producing a halo of a first color when agglutinated o and / or at least one agglutinating conjugate formed by at least one specific binding partner of STX2 coupled to a nanoparticle of a second nature producing a halo of a second color when agglutinated o And at least one agglutinating conjugate formed by at least one specific binding partner of EAE coupled to a nanoparticle of a third nature producing a halo of a third color when agglutinated

[0138] - confirm or not the presence of at least one EHEC by the appearance on the agar medium:

[0139] - at least one halo of a first color or second color

[0140] - and at least one halo of a third color

[0141] - or at least one halo resulting from the mixture of a first and / or a second color and a third color around said EHEC strain

[0142] The advantage of this embodiment is that it allows discrimination of different groups of bacteria by a halo of different colors. It is thus possible to visually distinguish

[0143] - a group of microorganisms secreting the STX1 toxin

[0144] - a group of microorganisms secreting the STX2 toxin

[0145] - a group of microorganisms expressing the EAE protein

[0146] Another subject of the invention relates to a method for detecting and confirming at least one Shiga toxin-producing Escherichia Coli (STEC) and / or enterohemorrhagic Escherichia Coli (EHEC) comprising the following steps:

[0147] - Perform a lysis of the sample allowing the lysis of STEC or EHEC in order to obtain a solution comprising their nucleic acids

[0148] - Bringing the nucleic acid solution into contact with primers making it possible to amplify at least the gene or gene fragment stxl and / or stx2 and eae as well as at least one gene or gene fragment of at least one serogroup chosen from 026, 045, 080, 091, 0103, 0104, OR I, 0113, 0121, O128ab, 0145, 0146, 0157, 0174

[0149] - if at least one of the genes or fragments of the stxl and / or stx2 genes and an eae gene or gene fragment are amplified, a portion of the sample is deposited on a reaction medium comprising o at least one toxin inducer and o at least one agglutinating conjugate formed by at least one specific binding partner of STX1 coupled to a nanoparticle of a first nature producing a halo of a first color when it is agglutinated o and / or at least one agglutinating conjugate formed by at least one specific binding partner of STX2 coupled to a nanoparticle of a first nature producing a halo of a first color when it is agglutinated o And at least one agglutinating conjugate formed by at least one specific binding partner of eae coupled to a nanoparticle of a second nature producing a halo of a second color when it is agglutinated

[0150] - confirm the presence of at least one STEC by the appearance on the agar medium

[0151] - at least one halo of a first color

[0152] - confirm the presence of at least one EHEC by the appearance on the ugélose medium

[0153] - at least one halo of a first color

[0154] - and at least one halo of a second color

[0155] - or at least one halo resulting from the mixing of a first and a second color around said strain

[0156] The advantage of the supra method is that it allows a serogroup approach in which the presumption of STEC and / or EHEC of a typical serogroup is reinforced.

[0157] In another embodiment of the method, the nucleic acid solution is contacted with primers for amplifying the aggR gene or gene fragment, thereby also detecting enteroaggregative E. coli.

[0158] Another subject of the invention relates to a method for detecting and confirming at least one Shiga toxin-producing Escherichia Coli (STEC) likely to be present in a sample comprising enterobacteria, comprising the following steps:

[0159] - bring the sample into contact with an enrichment medium

[0160] - contact the sample with anti-STXl and anti-STX2 antibodies

[0161] - if the presence of at least one STX1 protein and / or one STX2 protein is detected, part of the sample is deposited on an agar reaction medium comprising

[0162] ■ at least one toxin inducer,

[0163] ■ at least one agglutinating conjugate formed by at least one specific binding partner of the STX1 protein and / or at least one specific binding partner of the STX2 protein, coupled to a nanoparticle.

[0164] - Detect and confirm the presence of at least one STEC by the appearance of a halo on the agar around said STEC.

[0165] Preferably, the sample is brought into contact with a toxin inducer during or after enrichment. The toxin inducer causes stress in the bacteria which will trigger the lytic cycle of prophages in the bacteria and therefore stimulate the production of toxins which will be released. We will mention the shiga toxins secreted by STEC strains. There are two main types of shiga toxins: STX1 and STX2 which themselves have many variants. To date, STX1 has 4 subtypes STXla, STXlc, STXld, STXle, and STX2 has 12: STX2a, STX2b STX2c, STX2d, STX2e, STX2f, STX2g, STX2h, STX2i, STX2j, STX2K, STX21. Advantageously, the toxin inducer is chosen from antibiotics or a physicochemical stress.Examples of antibiotics that may be mentioned are trimethoprim, sulfamethoxazole, norfloxacin, azithromycin, gemtamicin, polymyxin B, chloramphenicol, streptomycin, chlortetracycline, oxytetracyline, tylosin, mitomycin C, carbodox, oliquindox, rifampincin, imipenem, ciprofloxacin, cotrimoxazole, penicillin G, linlomycin. 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 0.50 mg / l.

[0166] 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.

[0167] According to this embodiment of the invention, the sample is brought into contact with anti-STX1 and anti-STX2 antibodies. For example, the applicant's VIDAS® instrument will be used. According to another embodiment, the specific antibodies used are anti-STX1a and / or anti-STX1d antibodies. The sample is then placed on a culture medium as described above.

[0168] The present invention advantageously makes it possible to detect and confirm the presence of a STEC strain and / or an EHEC strain by the formation of a halo around said strain.

[0169] This invention is particularly interesting for facilitating the detection and confirmation of the presence of STEC or EHEC in a polymicrobial sample. Indeed, without the present invention which makes it possible to locate 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. 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. Thus, the present invention makes it possible to avoid false negatives and saves time in carrying out microbiological control. It 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.

[0170] Examples

[0171] Example 1- Preparation of samples and culture medium

[0172] - Preparation of contaminated “minced meat” samples:

[0173] A 25g sample of minced beef with vegetable protein (Carrefour Ref., lot 671579) was artificially contaminated by equilibration with a bacterial suspension of the E. coli serogroup 080 strain possessing the virulence markers eae stx2 (Robert Debré Hospital Center Paris REF H15-66.3). Equilibration was carried out for 48 / 72 hours at 2-8°C. The inoculation rates for the generated samples were 1.5 and 3 cfu / 25g, respectively. A negative control was also carried out: 25g of minced beef with vegetable protein that was not artificially contaminated.

[0174] The artificially contaminated samples were then diluted 1 / 10 with buffered peptone water (BPW Dilubag 5L ref AEB910305 / 2, bioMérieux) in an enrichment bag. Homogenization was carried out using a paddle mixer (Seward Stomacher), then the bags were placed in an oven at 41.5°C for 8-24 hours.

[0175] - Preparation of contaminated “carpaccio” samples:

[0176] A 25g sample of Carpaccio marinade with basil (Charal, lot 71394005) was artificially contaminated by equilibration with a bacterial suspension of the strain Escherichia coli serogroup 045 (ATCC BAA-2198) carrying the eae and stxl genes. Equilibration was carried out 48 / 72h at 2-8°C. The inoculation rate for the generated sample was 1.1 cfu / 25g. A negative control was also carried out: 25g of uncontaminated carpaccio.

[0177] The artificially contaminated samples were then diluted 1 / 10 with buffered peptone water (BPW Dilubag 5L ref AEB910305 / 2, bioMérieux) in an enrichment bag. Homogenization was carried out using a paddle mixer, then the bags were placed in an oven at 41.5°C for 8-24 hours.

[0178] - Preparation of agar culture media:

[0179] Preparation of gold nanoparticles:

[0180] 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.

[0181] Preparation of the conjugate:

[0182] 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.

[0183] This mixture is adsorbed on the nanoparticles at a pH of 8

[0184] Preparation of the culture medium:

[0185] The conjugate is added to the agar (bioMerieux TBX ref AEB522819) 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.

[0186] Example 2- Detection and confirmation of the presence of a Shiga toxin-producing E. coli 080 within a matrix comprising enterobacteria according to the invention

[0187] A sample of the contaminated "minced meat" was taken from each of the enrichment bags and 20 μL was transferred into a GENE-UP lysis kit tube (Ref 414057, bioMérieux) and shaken for 5 minutes using the Talboys Troemner Vortex Mixer (161111001, Talboys). A PCR was performed by transferring 100 μL of this lysate into a GENE-UP STEC stx & eae 2 PCR tube (Ref 423109, bioMerieux). In case of a positive result, a second PCR was performed by transferring 100 μL of lysate into a tube containing a PCR reaction mixture containing primers and probes specific for serogroup 080 in lyophilized form.

[0188] PCR results:

[0189] PCRs performed on the non-artificially contaminated “minced meat” sample were negative. PCRs performed on the two artificially contaminated enrichments detected the virulence genes stxl and / or stx2 and eae, as well as the serogroup 080 marker.

[0190] Since the result is positive, the sample is inoculated onto a medium as described in Example 1.

[0191] Isolation is carried out on the environment.

[0192] After incubating the dishes at 37°C for 24 hours, a gray halo is observed around certain colonies (Figure 1). The presence of a halo confirms that it is a STEC.

[0193] A PCR is performed on this colony using a PCR reaction mix containing primers and probes specific for the eae, stxl, and stx2 genes or gene segments. The PCR gives a positive result for eae and stx2. A second PCR using a PCR reaction mix containing primers and probes specific for serogroup 080 is also performed.

[0194] The PCR carried out with primers and probes specific to serogroup 080 carried out on the colony presenting a halo gives a positive result for 080. The overall method presented therefore allowed the detection and confirmation of the 080 strain (REF H15-66.3; CH Robert Debré Paris) with which the sample had been artificially contaminated. Example 3 - Detection and confirmation of the presence of a Shiga toxin-producing E. coli 045 within a matrix comprising enterobacteria according to the invention

[0195] A sample of the "contaminated carpaccio" was then taken from each of the enrichment bags. Lysis was performed using the VIDAS ESPT kit, ESP1 program (Ref 8300900, bioMérieux). PCR was performed by transferring 100L of this lysate into a GENE-UP STEC stx 1 & eae PCR tube (Ref 423109, bioMerieux).

[0196] In case of a positive result, a second PCR was performed by transferring 100 ml of lysate into a tube containing a PCR reaction mixture containing primers and probes specific for serogroup O45 in lyophilized form.

[0197] PCRs performed on the non-artificially contaminated basil marinade carpaccio sample were negative. PCR performed on the artificially contaminated enrichment detected the virulence genes stxl and / or 2 and eae, as well as the serogroup 045 marker.

[0198] Since the result is positive, the sample undergoes immunoselection with anti-045 antibodies. The sample is inoculated onto a medium as described in Example 1.

[0199] Incubation of these dishes at 37°C for 24 hours allows the growth of colonies. A gray halo is observed around certain colonies (Figure 2). A PCR is carried out on a colony showing a halo using a PCR reaction mixture containing primers and probes specific to the genes or portions of genes eae, stxl, stx2. A second PCR using a PCR reaction mixture containing primers and probes specific to serogroup 045 is also carried out.

[0200] The PCR carried out allows us to confirm the presence of the stx gene and the eae gene on the colony showing a gray halo.

[0201] A second PCR allowing the detection of serogroup 045 is carried out.

[0202] PCR performed with primers and probes specific to serogroup 045 carried out on the colony presenting a halo gives a positive result for 045.

[0203] The method presented therefore enabled the detection and confirmation of strain 045 (ATCC BAA-2198) with which the sample had been artificially contaminated.

[0204] Tl

Claims

CLAIMS 1- Method for detecting and confirming at least one Shiga toxin-producing Escherichia Coli (STEC) likely to be present in a sample comprising enterobacteria, comprising the following steps: Perform sample lysis to lyse STEC to obtain a solution containing their nucleic acids Bring the nucleic acid solution into contact with primers capable of amplifying at least the stxl and / or stx2 gene or gene fragment - If at least one of the genes or fragments of the stxl and / or stx2 genes is amplified, part of the sample is deposited on an agar reaction medium comprising ■ at least one toxin inducer, ■ at least one agglutinating conjugate formed by at least one specific binding partner of the STX1 protein and / or at least one specific binding partner of the STX2 protein, coupled to a nanoparticle - Detect and confirm the presence of at least one STEC by the appearance of a halo on the agar around said STEC. 2- Method for detecting and confirming at least one Shiga toxin-producing Escherichia Coli (STEC) according to claim 1 in which, - if at least one of the genes or fragments of the stxl and / or stx2 genes is amplified, the nucleic acid solution is brought into contact with primers making it possible to amplify at least the gene or gene fragment of the serogroups chosen from 026, 045, 080, 091, 0103, 0104, OR I, 0113, 0121, O128ab, 0145, 0146, 0157, 0174 - if at least one of the genes or fragments of the stxl and / or stx2 genes and at least one serogroup chosen from 026, 045, 080, 091, 0103, 0104, OR I, 0113, 0121, O128ab, 0145, 0146, 0157, 0174 are amplified, a part of the sample is deposited on a reaction medium comprising o at least one toxin inducer and o at least one agglutinating conjugate formed by at least one specific binding partner of the STX1 protein and / or at least one specific binding partner of the STX2 protein, coupled to a nanoparticle. o detecting and confirming the presence of at least one STEC by the appearance of a halo on the agar around said STEC. 3- Method for detecting and confirming at least one Shiga toxin-producing Escherichia Coli (STEC) according to claim 2 in which - if at least one of the genes or fragments of the stxl and / or stx2 genes and at least one gene or gene fragment of at least one serogroup chosen from 026, 045, 080, 091, 0103, 0104, OR I, 0113, 0121, O128ab, 0145, 0146, 0157, 0174.are amplified, a portion of the sample is deposited on a reaction medium comprising o at least one toxin inducer and o at least one agglutinating conjugate formed by at least one specific binding partner of STX1 coupled to a nanoparticle of a first nature producing a halo of a first color when it is agglutinated o and / or at least one agglutinating conjugate formed by at least one specific binding partner of STX2 coupled to a nanoparticle of a first nature producing a halo of a first color when it is agglutinated o and at least one agglutinating conjugate formed by at least one specific binding partner of said serogroup identified by amplification coupled to a nanoparticle of a second nature producing a halo of a second color when it is agglutinated. - confirm the presence of at least one STEC of a serogroup identified by the appearance of at least one halo of a first color and at least one halo of a second color around the same colony or at least one halo resulting from the mixture of a first and a second color around said colony. 4- Method for detecting and confirming at least one enterohemorrhagic Escherichia Coli (EHEC) likely to be present in a sample comprising enterobacteria, comprising the following steps: - carry out a lysis of the sample allowing the lysis of EHEC in order to obtain a solution comprising their nucleic acids - bringing the nucleic acid solution into contact with primers capable of amplifying at least the stxl and / or stx2 and eae gene or gene fragment, - if at least one of the genes or fragments of the stxl and / or stx2 genes and at least one eae gene or gene fragment are amplified, a portion of the sample is deposited on an agar medium comprising: o at least one toxin inducer o at least one agglutinating conjugate formed by at least one specific binding partner of STX1 coupled to a nanoparticle of a first nature producing a halo of a first color when agglutinated o and / or at least one agglutinating conjugate formed by at least one specific binding partner of STX2 coupled to a nanoparticle of a first nature producing a halo of a first color when agglutinated o and at least one agglutinating conjugate formed by at least one specific binding partner of EAE coupled to a nanoparticle of a second nature producing a halo of a second color when agglutinated - confirm the presence of at least one EHEC by the appearance on the agar medium: - at least one halo of a first color - and at least one halo of a second color - or at least one halo resulting from the mixing of a first and a third color around said EHEC strain. 5- Method for detecting and confirming at least one EHEC according to claim 4, comprising bringing the nucleic acid solution into contact with primers making it possible to amplify at least one gene or gene fragment of at least one serogroup chosen from 026, 045, 080, 091, 0103, 0104, OR I, 0113, 0121, O128ab, 0145, 0146, 0157, 0174. 6- Detection and confirmation method according to the preceding claims, characterized in that the nucleic acid solution is brought into contact with primers making it possible to amplify the aggR gene or gene fragment. 7- Method according to any one of the preceding claims, characterized in that a step of enriching the sample is carried out prior to its lysis. 8- Detection and confirmation method according to any one of the preceding claims, characterized in that a step of immuno-selection of an E. coli of at least one serogroup chosen from 026, 045, 080, 091, 0103, 0104, 0111, 0113, 0121, O128ab, 0145, 0146, 0157, 0174 is carried out before the lysis step. 9- Detection and confirmation method according to any one of the preceding claims, characterized in that a step of immuno-selection of an E. coli of at least one serogroup chosen from 026, 080, 045, 091, 0103, 0104, 0111, 0113, 0121, O128ab, 0145, 0146, 0157, 0174 is carried out before depositing on the agar reaction medium. 10- Detection and confirmation method according to any one of the preceding claims, characterized in that the nanoparticle coupled to the binding partner is a colloidal nanoparticle having optical properties. 11- Detection and confirmation method according to any one of the preceding claims, characterized in that the nanoparticle is chosen from gold, silver, copper. 12- Detection and confirmation method according to any one of the preceding claims, characterized in that the nanoparticles have a size between 10 and 200 nm, preferably between 20 and 90 nm. 13- Detection and confirmation method according to any one of the preceding claims, characterized in that the toxin inducer is an antibiotic. 14- Detection and confirmation method 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. 15- Detection and confirmation method 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.

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