Reaction media for detecting target microorganisms and related methods

Abstract

The present invention relates to a gelled reaction medium for detecting, identifying, enumerating and / or isolating at least one target microorganism in a sample that may contain the target microorganism, the reaction medium comprising at least one binding partner specific for a component of the target microorganism or a component derived from the target microorganism, the binding partner binding to at least one nanoparticle to form at least one binding conjugate.

Description

[Technical field]

[0001] The present invention relates to microbial control in a broad sense, for example for samples of industrial or clinical origin. More specifically, the present invention relates to reaction media and related methods for detecting, identifying, enumerating and / or isolating target microorganisms. [Background technology]

[0002] Microbiological control of samples of various origins requires the implementation of techniques allowing the detection of microorganisms (e.g. for the purposes of identification and / or enumeration and / or biochemical characterization), the results of which must be delivered as quickly as possible.

[0003] In the medical sector, the risk of infection needs to be anticipated and diagnosed: the faster and more accurate the diagnosis, the more effective the patient management and the lower the risk of transmission. This approach is similar for animal health in the veterinary sector.

[0004] In the agri-food sector, the problem is the same, but with the following differences: Detection of pathogenic microorganisms (e.g. Shiga toxin producing organisms (STEC), Salmonella, Listeria, Cronobacter, Bacillus, Staphylococcus) is performed on starting materials, intermediate products and final products to be sold. · Non-pathogenic microorganisms are used as quality indicators of the production process throughout the entire chain, from starting materials to the final product. Bacteria of technical interest, e.g. fermentative bacteria · Microorganisms that are markers of contamination. Immediate and accurate detection of suspected contamination (within a food batch) allows for monitoring of that food batch, thereby enabling corrective action to be taken quickly.

[0005] Technically, one of the main difficulties is to be able to isolate the bacteria of interest in order to identify them. Microbiological analysis is generally a two-step process. The first step is the detection phase, which can involve many techniques, such as culture, immunoassays, and molecular biology. This can be followed, especially in the agri-food sector, by a phase of confirming the presence of the pathogen of interest and its compliance with the standards in force in this sector. This confirmation step therefore involves an additional step, which also requires the isolation of the bacteria of interest.

[0006] Thus, in the case of Shiga toxin-producing E. coli (STEC), diagnosis relies on the use of selective chromogenic agars (e.g., SMAC (sorbitol MacConkey agar), RMAC (rhamnose MacConkey agar), or Rainbow agar O157) to select for the growth of a particular bacterium and stain the strain of interest. Ultimately, however, none of these agars is specific enough for STEC without isolating the strain and culturing it to confirm its identity using a PCR-type molecular biological test. Specifically, their pathogenicity involves the expression of several virulence genes, in particular stx1 and stx2, which code for two Shiga toxins (STX1 and STX2). These two toxins act by inhibiting protein synthesis in eukaryotic cells, ultimately causing apoptosis. To accurately identify pathogenic STEC, it is essential to perform PCR of the genes stx1, stx2, or eae (another virulence factor). This makes these processes time-consuming and laborious, and pathogens carrying both genes (stx and eae) are not systematically found in culture.

[0007] Immunological methods are also available. Many tests are available to detect E. coli O157:H7 in food and / or environmental samples. These systems include traditional microplate ELISA tests, one-step immunological systems, and fully automated systems.

[0008] "One-step" immunological methods are widely used industrially, because they are fast and simple to perform. Most of these systems are based on the immunochromatographic principle. The device consists of a plastic support containing a membrane impregnated with gold or latex particles coated with antibodies specific for E. coli O157:H7 (i.e. O157, sometimes H7), a sample well and a test and control window. The appearance of a colored line in the test window is a positive result, indicating the high probability of the presence of E. coli O157 in the food. We can for example mention the "VIP EHEC" test (BioControl, Montesson, France), which allows to visualize, after several hours of enrichment, whether a given sample is contaminated with E. coli O157. However, this method does not allow to locate the E. coli O157 strain in the sample, nor its pathogenicity.

[0009] The ELISA / ELFA system is an immunological method that gives a result in 2 hours in microplates after an enrichment phase (usually lasting 24 hours). bioMerieux® has developed an automated ELISA (ELFA) kit based on VIDAS® technology. In case of a positive test, these methods have the disadvantage that the suspect colony is not directly isolated. Therefore, in order to isolate a positive colony, it is necessary to culture the sample and confirm by PCR that this bacterium is in fact the one that caused the positive test.

[0010] ELISA methods are also available for the detection of non-O157 STEC, which are essentially based on the detection of Shiga toxin production (possibly after an enrichment step). Some of these methods use monoclonal or polyclonal antibodies against STX toxins and visualization with alkaline phosphatase-conjugated antibodies. Among the available ELISA kits, one can mention "Premier EHEC" that targets Shiga toxins (Meridian Diagnostics Inc., USA). Also kits using the reverse passive agglutination assay (RPLA) technique suitable for STX toxin detection have been developed and marketed. In this technique, beads coated with antibodies that interact with Shiga toxins are used, generating a spreading layer at the bottom of the well where the culture supernatant is present. These methods require subsequent isolation of positive colonies followed by a confirmation step.

[0011] Immunomagnetic separation (IMS) techniques have also been adapted for the detection and isolation of strains belonging to the five major STEC serotypes. This is performed in liquid media using magnetic beads coated with antibodies against the antigens O26, O111, O103, and O145. However, strains isolated by this method must be confirmed for the presence of stx genes before they can be considered STEC strains.

[0012] Thus, there is a real need to develop reliable and rapid methods for isolating and identifying target bacteria, especially pathogenic bacteria. Summary of the Invention

[0013] A first subject of the present invention relates to a gelled reaction medium for detecting, identifying, enumerating and / or isolating at least one target microorganism in a sample likely to contain said target microorganism, said gelled reaction medium comprising at least one specific binding partner for a component of said target microorganism or for a component derived from said microorganism, said binding partner binding to at least one nanoparticle to form at least one aggregate conjugate.

[0014] The component used to detect the target microorganism is a component released by the microorganism into the reaction medium, which may be a component of the target microorganism, such as lipopolysaccharide (LPS), or a component produced by the target microorganism, such as a toxin.

[0015] The medium therefore very advantageously allows target colonies releasing the component to be probed on the culture medium, which improves the detectability of the target microorganism and saves invaluable time in the field of microbial control, especially during the confirmation step.

[0016] The conjugates preferentially comprise colloidal nanoparticles that have optical properties.

[0017] Advantageously, the medium according to the invention comprises a toxin inducer, thus providing a highly advantageous means for detecting Shiga toxin producing E. coli (STEC) strains.

[0018] Another subject of the present invention relates to a detection method for identifying, enumerating and / or isolating a target microorganism in a sample possibly containing a microorganism, comprising the steps of: - contacting said sample with a reaction medium according to the invention, Incubating, Detecting the presence of the target microorganism.

[0019] Advantageously, detection is achieved by the appearance of a halo around the target microorganism in the reaction medium. [Brief description of the drawings]

[0020] [Figure 1] 1 shows a photograph of a medium according to the invention for detecting E. coli strain O26, containing phage proteins bound to gold nanoparticles. [Diagram 2] 1 shows a photograph of a medium according to the invention for detecting E. coli O111 strains, comprising antibodies bound to gold nanoparticles. [Diagram 3] 1 shows a photograph of a medium according to the present invention for detecting STX1 toxin-producing target bacteria comprising anti-STX1 antibodies bound to gold nanoparticles in the presence of ciprofloxacin as a toxin inducer. [Figure 4] 1 shows a photograph of a medium according to the present invention for detecting STX1 toxin-producing target bacteria comprising anti-STX1 antibodies bound to silver nanoparticles in the presence of mitomycin as a toxin inducer. [Diagram 5] 1 shows a photograph of a medium according to the present invention for detecting STX1 and / or STX2 toxin-producing target bacteria, comprising anti-STX1 antibodies bound to silver nanoparticles and anti-STX2 antibodies bound to gold nanoparticles, in the presence of mitomycin. [Figure 6] 1 shows a photograph of a medium according to the present invention for detecting STX1 and / or STX2 toxin-producing target bacteria, comprising anti-STX1 or anti-STX2 antibodies bound to the same gold nanoparticles, in the presence of ciprofloxacin as a toxin inducer.

[0021] Detailed Description of the Invention Certain terms and expressions used in the context of the present invention are detailed below.

[0022] A first subject of the present invention relates to a gelled reaction medium for detecting, identifying, enumerating and / or isolating at least one target microorganism in a sample likely to contain said target microorganism, said gelled reaction medium comprising at least one specific binding partner for a component of said target microorganism or for a component derived from said microorganism, said binding partner binding to at least one nanoparticle to form at least one aggregate conjugate.

[0023] In other words, the present invention relates to a gelled reaction medium for detecting, identifying, enumerating and / or isolating at least one target microorganism in a sample likely to contain said target microorganism, said gelled reaction medium comprising at least one agglutination conjugate formed by at least one specific binding partner for a component of the target microorganism or for a component derived from said microorganism, said agglutination conjugate being bound to at least one nanoparticle. Quite surprisingly, it has been found that the gelled reaction medium comprising the agglutination conjugate can be used to detect the target microorganism.

[0024] The term "reaction medium" refers to a medium that contains all the elements necessary for the expression of the metabolism, survival and / or growth of the microorganism. This reaction medium can be either a microbiological culture medium or a microbiological visualization medium. In the latter case, the microorganism may first be cultivated in another medium. The reaction medium may be placed in contact with an agar medium. The reaction medium may be placed below or above the medium that allows the growth of the target microorganism. The reaction medium may be added after incubation of the medium. Before use, the reaction medium may be dehydrated. The reaction medium may be in the form of a pad.

[0025] According to the invention, the reaction medium is gelled. The reaction medium is solid or semi-solid. Agar is the conventional gelling agent used in microbiology for the cultivation of microorganisms, but other gelling agents can be used, such as gelatin, agarose, and other natural or artificial gelling agents. Thus, contrary to all expectations, the conjugates are able to form a network with the target component in the gelled medium. Also surprisingly, this network can be visually detected by the formation of a halo, without the need to reduce the hardness of the conventional semi-solid reaction medium. Thus, it is not necessary to modify the physicochemical properties of the reaction medium, such as the hardness, in order to allow the diffusion of the target component on the one hand and the reaction with the conjugate on the other hand.

[0026] Numerous preparations are commercially available, such as Columbia agar, trypticase soy agar, MacConkey agar, Mueller Hinton agar, or more generally, those described in the Handbook of Microbiological Media. These media may serve as the basis for the reaction medium according to the invention. The reaction medium may also contain optional additives, such as amino acids, peptones, one or more growth factors, carbohydrates, nucleotides, minerals, vitamins, one or more selective agents, inducers, toxin inducers, buffers. The term "selective agent" refers to any compound capable of preventing or slowing down the growth of "non-target" microorganisms, i.e. microorganisms other than the target microorganism. The term "inducer" refers to a compound capable of inducing the expression of a compound (e.g. an enzyme or a toxin) that would normally remain unexpressed.

[0027] The reaction medium may comprise a coloring agent. By way of guide, Evans blue, neutral red, sheep's blood, horse's blood, opacifiers (e.g. titanium dioxide), nitroaniline, malachite green and brilliant green may be mentioned as coloring agents. When the reaction medium according to the invention also comprises an enzyme substrate specific for at least one enzymatic activity of the target microorganism, it is preferred to use a chromogenic and / or fluorogenic substrate. The term "chromogenic and / or fluorogenic substrate" refers to a substrate that allows the detection of the enzymatic or metabolic activity of the target microorganism / microorganism of interest by means of a detectable signal. The reaction medium according to the invention may also comprise a pH indicator that is sensitive to the pH variations induced by the consumption of the substrate and that reveals the metabolism of the target microorganism. The pH indicator may be a chromophore or a fluorescent substance. Examples of chromophores that may be mentioned include bromocresol purple, bromothymol blue, neutral red, aniline blue and bromocresol blue.

[0028] One skilled in the art can also use segmented Petri dishes (e.g., two-dish or three-dish), which facilitates the comparison of several media containing different substrates or different selective mixtures, on which the same biological sample is deposited. According to the invention, the reaction medium comprises a specific binding partner for a component of the target microorganism or for a component derived from said microorganism, which is bound to the nanoparticles. The specific binding partner is selected from antibodies, all kinds of Fab fragments, recombinant proteins, phages, phage proteins, oligonucleotides, aptamers, affimers or any other ligands or antiligands well known to the person skilled in the art. According to the invention, the reaction medium comprises a specific binding partner that is not bound to said component of the target microorganism or to a component derived from said microorganism. Only by using said medium, i.e. by inoculating the reaction medium with a sample, can the specific binding partner bind to the component of the target microorganism or to a component derived from said microorganism.

[0029] Preferentially, the antibody is a monoclonal antibody, or a monoclonal antibody fragment.

[0030] According to the present invention, the binding partner is specific for a component of the target microorganism. The component of the target microorganism is a component released by the microorganism. The component can therefore be an element from the surface of the bacterium, such as a protein, lipopolysaccharide (LPS) or flagella. It can also be an internal element of the bacterium, such as RNA or intracellular protein, which can be detected when part of the bacterial colony dies during growth. In a particular embodiment, the binding partner is specific for the RNA of the target microorganism. In this embodiment, the binding partner is composed of at least two different primers that hybridize complementary to the target RNA.

[0031] In another particular embodiment, the binding partner is specific for a component derived from the microorganism, which may be a molecule of interest produced by the target microorganism, such as a protein, an antibiotic, an antimicrobial resistance molecule, a protease-type enzyme, a lipase or a glucosidase.

[0032] According to the invention, the binding partners are bound to the nanoparticles. This final complex is called a conjugate. In the same reaction medium, it is possible to have conjugates with binding partners of different natures, themselves bound to nanoparticles of different natures.

[0033] The term "nanoparticle" refers to a particle of nanometer size. The nanoparticles may be selected from gold, iron, silver, copper, carbon, latex, silicon and aluminum. The nanoparticles are preferentially colloidal nanoparticles, selected for their optical properties, i.e. their ability to be differentiated when a network is formed. Even more preferentially, the nanoparticles are selected from gold, silver and copper. Thus, if the nanoparticles are 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 region, around 530 nm. When aggregated, the absorbed wavelength changes from red to blue / gray, around 600-700 nm. In certain embodiments, several nanoparticles of different colors may be used together. The network thus formed allows several components of the target microorganism to be differentiated.

[0034] Preferentially, the size of the nanoparticles is between 10 and 200 nm. Preferentially, the size of the nanoparticles is between 20 and 90 nm, which allows better mobility of the conjugates in the reaction medium.

[0035] Advantageously, nanoparticles allow a reduction in the amount of binding partner required for aggregate formation, so that the concentration of binding partner required to produce a reaction medium according to the invention is 100-1000 times less than in a medium without nanoparticles.

[0036] Preferentially, the amount of binding partner required corresponds to the amount required to cover at least half of the nanoparticle surface, and even more preferentially corresponds to the amount required to cover one third to one half of the nanoparticle surface, which ratio allows the aggregated conjugates to form a network in the gelled reaction medium.

[0037] Advantageously, nanoparticles can visualize agglutination around bacterial colonies that are still invisible to the naked eye, thus facilitating detection. Advantageously, nanoparticles can visualize agglutination around bacterial colonies that are not detectable by automated readers due to their translucent appearance, thus facilitating detection.

[0038] The binding of the nanoparticles to the binding partner can occur either by direct or indirect attachment. Direct attachment means attachment either by adsorption or by covalent binding. Indirect attachment means attachment by ligand / antiligand interactions (e.g. biotin / streptavidin or other pairs well known to those skilled in the art). Depending on the type of binding chosen, the skilled person will adapt the physicochemical conditions of the reaction medium, in particular its pH.

[0039] According to the invention, the conjugates are agglutinating, i.e. in the presence of a component of the target microorganism or a component derived from said microorganism, they cause the formation of an agglutination network. As this component is multi-epitope, several conjugates will bind to it and form an agglutination. The term "agglutination" refers to the result of an interaction between at least one component of the target microorganism or at least one component derived from said microorganism and a binding partner bound to the nanoparticle. The agglutination reaction includes an immunological reaction, such as an antigen-antibody reaction, or more generally, a specific interaction between two molecules. Through this interaction, the component and the conjugates aggregate, adhere to each other and form a network in the reaction medium. In practice, several parameters have an influence on the ability of the conjugates to aggregate in the gelled medium, such as mainly the following parameters: Porosity of the gelled medium Nanoparticle size Amount of binding partner -Nanoparticle quantity. These parameters should therefore be adapted to ensure satisfactory aggregation for detection. Advantageously, the network formed by the particular reaction is then detected visually or automatically using optical systems. Thus, colonies of the target microorganism are sought out. The network preferably forms a halo in the gelling reaction medium that is detectable visually or using optical systems. Thus, the network or halo surrounds the colonies, which can then be advantageously differentiated and / or identified within the population.

[0040] For the purposes of the present invention, the term "at least one target microorganism" refers to at least one microorganism to be detected and / or identified and / or enumerated. The microorganism is preferentially selected from Escherichia coli, Shiga toxin-producing E. coli, Shigella, Salmonella typhimurium, Salmonella enteritidis, Pseudomonas, Bacillus cereus group, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis, Staphylococcus aureus MSSA, Staphylococcus aureus MRSA, and Streptococcus agalactiae. Preferably, the microorganism is selected from E. coli strains, preferentially from Shiga toxin-producing E. coli (STEC) strains. Enterohemorrhagic E. coli (EHEC) is a strain that represents a subgroup of Shiga toxin STX-producing E. coli (STEC) and acquires the eae gene, causing the hemolytic uremic syndrome. The simultaneous possession of these two virulence factors makes this pathogen highly virulent for humans. These include, inter alia, serotypes O26, O45, O80, O103, O111, O121, O145, and O157.

[0041] In a particular embodiment, the reaction medium comprises an inducer that promotes or enhances the expression of a molecule of interest produced by the target microorganism. This molecule of interest may be a protein, an antibiotic, an antimicrobial resistance molecule, a protease-type enzyme, a lipase, or a glucidase. The invention thus allows the characterization or selection of microbial strains with respect to their ability to produce these molecules of interest. In a particular embodiment, the invention may relate to the field of bioproduction, more specifically the production of recombinant proteins from genetically modified microorganisms, where it is desired to identify the producing clones. The invention thus allows a clear indication that a recombinant protein is being produced. In the presence of a halo, it is also possible to estimate the amount produced. In another particular embodiment, the invention allows the detection of resistant pathogenic bacteria.

[0042] In a particular embodiment, the reaction medium comprises a toxin inducer, which causes stress to the bacteria and triggers the lytic cycle of the prophage in the bacteria, stimulating the production of toxins, which are released. By way of example, mention may be made of the toxins secreted by Staphylococcus aureus or the Shiga toxins secreted by STEC strains. There are two main types of Shiga toxins, STX1 and STX2, which themselves have a large number of variants. To date, there are four subtypes of STX1 (STX1a, STX1c, STX1d, STX1e) and 12 subtypes of STX2 (STX2a, STX2b, STX2c, STX2d, STX2e, STX2f, STX2g, STX2h, STX2i, STX2j, STX2K, STX21). Advantageously, the toxin inducer is selected from antibiotics or physicochemical stress. Examples of antibiotics include trimethoprim, sulfamethoxazole, norfloxacin, azithromycin, gentamicin, polymyxin B, chloramphenicol, streptomycin, chlortetracycline, oxytetracycline, tylosin, mitomycin C, carbodox, oliquindox, rifampicin, imipenem, ciprofloxacin, cotrimoxazole, penicillin G and lincomycin. In a preferred embodiment, the medium according to the invention comprises ciprofloxacin in a concentration of 0.005 to 0.030 mg / l. In another preferred embodiment, the medium according to the invention comprises mitomycin C in a concentration of 0.10 to 0.5 mg / l.

[0043] The toxin inducer can be a physicochemical stress, for example caused by the addition of salt or EDTA, or by a pH change or UV stress. Stress can also be induced, for example, by the addition of noradrenaline. The target microorganism may be present in a sample. The term "sample" refers to a small part or a small amount isolated from an entity for analysis. The sample may be of industrial origin, for example but not limited to air samples, water samples, samples collected from surfaces, manufactured parts or products, or food. Food samples that may be mentioned include but are not limited to samples of dairy products (yogurt, cheese, etc.), meat, fish, eggs, fruits, vegetables, water, and beverages (milk, fruit juices, carbonated soft drinks, etc.). Finally, food samples may be derived from animal feed, in particular animal or vegetable diets. The sample may be of either animal or human biology. In this case, it may correspond to a sample of body fluids (faeces, urine, whole blood, serum, plasma, cerebrospinal fluid, organic secretions, etc.), an external sample (skin, nose, throat, etc.), or a tissue sample or isolated cells. The sample may be used as is or may undergo preparation, such as enrichment, extraction, concentration or purification, prior to analysis, using methods known to the skilled person. The reaction medium according to the invention may be even more interesting if the sample is polymicrobial or if the sample is loaded with a complementary flora (e.g. food samples such as raw milk cheese, or faeces in clinical samples).

[0044] Another subject of the invention relates to a diagnostic kit for producing a reaction medium according to the invention, said diagnostic kit comprising: an aggregate conjugate according to the invention, Gelling medium Includes.

[0045] Advantageously, the reaction medium can be prepared extemporaneously using the kit according to the invention, which allows the reaction medium to be used as a unit and increases its stability.

[0046] Another subject of the invention therefore relates to a method for obtaining a reaction medium according to the invention, which method comprises contacting an aggregated conjugate with a gelling medium to form a reaction medium according to the invention. The conjugate is produced by binding a binding partner to nanoparticles. These binding methods are well known to those skilled in the art (Nicholas G. Welch et al., 2017). The conjugate is then added to the supercooled gelling medium. The whole is then homogenized and poured into a Petri dish.

[0047] Another subject of the invention relates to an in vitro microbiological culture method, in which microorganisms possibly present in a sample are inoculated into or onto a medium according to the invention. The inoculated medium is incubated under suitable conditions known to the skilled artisan. Inoculation is carried out using conventional microbiological techniques. It can also be carried out in mass, i.e. by inclusion.

[0048] Another subject of the invention relates to a method for detecting, identifying, enumerating and / or isolating at least one target microorganism in a sample possibly containing said target microorganism, said method comprising the following steps: - contacting said sample with a reaction medium according to the invention, Incubating, -detecting the presence of the target microorganism Includes.

[0049] The term "detection" refers to detecting the presence of growth of a target microorganism, preferably a target bacterium, with the naked eye or with an optical device. If the reaction medium in which the target microorganism is to be detected contains a chromogenic or fluorogenic substrate, detection can be performed with the naked eye or with an optical device for a fluorogenic substrate or with an optical device for a chromogenic substrate.

[0050] The term "identification" refers to determining the genus and / or species and / or group to which a target microorganism belongs.

[0051] The term "enumerating at least one target microorganism" refers to counting / quantifying the number of target microorganisms, for example the number of bacterial colonies if the target microorganism is a bacterium.

[0052] The term "isolation" refers to the production of distinct colonies spaced apart from one another.

[0053] Microbiological testing corresponds to the analysis of a sample with the aim of detecting microorganisms that may be present in the sample. It has therefore been found, very surprisingly, that the medium according to the invention makes it possible to detect and isolate a target bacterium. Advantageously, the medium according to the invention therefore makes it possible to search out and select a target microorganism from a mixed population present on a non-selective or poorly selective reaction medium. Preferentially, the method according to the invention allows detection by the appearance of a halo around the target microorganism on the reaction medium according to the invention. In other words, detection is carried out by observing the appearance of a halo around the target microorganism on the reaction medium.

[0054] The medium according to the invention appears to be particularly suitable (but not exclusive) for the detection of E. coli. Thus, according to a particular embodiment, the invention relates to a gelled reaction medium for the detection, identification, enumeration and / or isolation of an E. coli strain, which comprises a phage protein specific for the LPS of said strain, which binds to nanoparticles to form a conjugate.

[0055] In this particular embodiment, the amount of phage protein specific for LPS makes it possible to cover at least one third of the nanoparticle surface.

[0056] Preferentially, in this particular embodiment, the nanoparticles are gold, have a size between 20 and 90 nm and a concentration of 10 nanoparticles per ml of reaction medium. 10 ~10 12 There are 10.

[0057] In this embodiment, the reaction medium is formed from a medium known for the cultivation of E. coli strains (eg Applicant's TBX or chromID® Coli media) to which the flocculation conjugate has been added.

[0058] In another particular embodiment, the present invention relates to a gelled reaction medium for detecting, identifying, enumerating and / or isolating an E. coli strain, the reaction medium comprising a monoclonal antibody specific for said strain that binds to nanoparticles to form a conjugate.

[0059] Preferentially, in this particular embodiment, the amount of monoclonal antibody makes it possible to cover at least half of the nanoparticle surface, and even more preferentially makes it possible to cover between one third and one half of the nanoparticle surface.

[0060] Preferentially, in this particular embodiment, the nanoparticles are gold, have a size between 20 and 90 nm and a concentration of 10 nanoparticles per ml of reaction medium. 10 ~10 12 There are 10.

[0061] In another particular embodiment, the present invention relates to a gelled reaction medium for detecting, identifying, enumerating and / or isolating Shiga toxin-producing E. coli, the reaction medium comprising at least one toxin inducer, at least one antibody specific for Stx1 and / or Stx2, the at least one antibody being bound to nanoparticles to form an aggregate conjugate. In other words, the present invention relates to a gelled reaction medium for detecting, identifying, enumerating and / or isolating Shiga toxin-producing E. coli, the reaction medium comprising at least one toxin inducer, at least one antibody specific for Stx1 and / or Stx2 bound to nanoparticles to form an aggregate conjugate.

[0062] Advantageously, this embodiment allows for the locating of Shiga toxin-producing E. coli strains by forming a halo around the strain, specifically conjugates formed from antibodies bound to nanoparticles that aggregate around the colony, thus making it easier to visualize the target colony.

[0063] In this embodiment, the at least one antibody is present in an amount that allows it to cover at least half of the nanoparticle surface, preferentially one third of the nanoparticle surface. In these particular embodiments, the medium comprises ciprofloxacin in a concentration of 0.005 to 0.030 mg / l. In a variant of these particular embodiments, the medium comprises mitomycin C in a concentration of 0.10 mg / l to 0.50 mg / l.

[0064] Preferentially, in these variants, the nanoparticles are gold nanoparticles with a size between 20 and 90 nm and a concentration of 10 nanoparticles per ml of reaction medium. 10 ~10 12 There are 10.

[0065] When the medium according to the invention contains binding partners for several toxins, the invention is particularly advantageous for distinguishing between bacteria producing Shiga toxins STX1 or STX2 and bacteria producing both Shiga toxins STX1 and STX2 in the same sample.

[0066] The present invention is particularly advantageous for facilitating the detection of multiple STEC in a polymicrobial sample. Indeed, without the present invention, which allows the colony of interest to be picked out, the reference method ISO 16136 specifies that up to 50 colonies must be examined by molecular methods to confirm the presence of STEC. Given the high number of colonies on a dish and the poor reproduction of the target microorganism, a person picking a colony for confirmation purposes may never pick the target microorganism. In this way, the present invention saves time in the performance of microbiological tests. It is particularly advantageous for samples loaded with auxiliary microflora, where a large number of colonies on a Petri dish may lead to the risk of false negatives.

[0067] Working Example Example 1: Detection of E. coli O26 with a medium according to the invention comprising phage proteins bound to gold nanoparticles

[0068] Preparation of gold nanoparticles

[0069] Nanoparticles of 40 nm are prepared by reducing gold chloride with sodium citrate (method described by Turkevich and Frens in 1951). Thus, gold nanoparticles of 20 nm are prepared using a solution of gold chloride diluted in distilled water, to which trisodium citrate is added and then boiled. The exact particle size can be confirmed by the presence of an absorbance peak at 517-519 nm. From these 20 nm particles, particles of 40 nm are synthesized. To do this, the 20 nm particles are diluted with distilled water, to which trisodium citrate and gold chloride are then added and boiled. Then, at low temperatures, an absorbance peak is observed at 524-526 nm, which reflects an increase in particle size.

[0070] Preparation of conjugates

[0071] The phage evaluated in this assay is Phage Eco O26 BP1 against the LPS of E. coli O26. This phage is from the Applicant's collection. The adsorption of the phage proteins onto the nanoparticles is carried out by methods known to those skilled in the art, taking into account the value of their isoelectric point (Nicholas G. Welch et al.): the phages are bound as follows: 270 μg of phage protein was diluted in 3 ml of Tris HCl (0.025 M) pH 6. Optical density (OD) of 1 (i.e., 7.7 × 10), previously adjusted to pH 6 with 0.1 M K2CO3 solution 10 Add 30 ml of gold nanoparticles / ml. Stir for 20 minutes Passivation by adding 3 ml of 10% BSA Centrifuge at 8600g for 30 minutes Remove the supernatant and assay the resulting concentrate using a spectrophotometer.

[0072] Preparation of the reaction medium according to the invention: The prepared conjugate is added to a supercooled agar medium (chrom ID coli-ref 42017 bioMerieux) at 50 °C, achieving a concentration of nanoparticles with an optical density of 3 using a spectrophotometer. Then, pour 18 ml of medium per dish. The dishes are then allowed to dry.

[0073] Inoculation: In this example, strains of E. coli O26 and E. coli O111 (from the Applicant's collection) are inoculated and incubated at 37° C. for 72 hours.

[0074] result A grey halo is visually observed around colonies carrying LPS O26 (Figure 1). This grey colour change is due to a network formed between the LPS released by the colony and conjugates present in the agar.

[0075] Example 2: Detection of E. coli O111 with a medium according to the invention comprising antibodies bound to gold nanoparticles

[0076] Preparation of gold nanoparticles 40 nm gold particles are prepared as described in Example 1.

[0077] Preparation of conjugates

[0078] The 2H5E9 IgG anti-E. coli O111 monoclonal antibody is from the applicant's collection.

[0079] Antibodies are adsorbed onto the nanoparticles as follows: 90 μg of antibody diluted in 3 ml of Tris HCl (0.025 M) pH 6.5 Add 30 ml of gold with an OD of 1, pre-adjusted to pH 8 with 0.1 M K2CO3 solution. Stir for 20 minutes Passivation by adding 3 ml of 10% BSA Centrifuge at 8600g for 30 minutes Remove the supernatant and assay the resulting concentrate using a spectrophotometer.

[0080] Preparation of the reaction medium according to the invention

[0081] The prepared conjugate is added to a supercooled agar medium (chrom ID coli-ref 42017 bioMerieux) at 50 °C, achieving a concentration of nanoparticles with an optical density of 3 using a spectrophotometer. Then, pour 18 ml of medium per dish. The dishes are then allowed to dry.

[0082] inoculation

[0083] In this example, E. coli strains O26 and O111 from the Applicant's collection are inoculated and incubated at 37° C. for 72 hours.

[0084] result

[0085] There is no grey halo around colony O26, whereas a grey halo is clearly visible around colony O111 (Figure 2), making it possible to identify the latter.

[0086] Example 3: Detection of STX1 toxin-producing E. coli with a medium according to the invention comprising anti-Stx1 antibodies bound to gold nanoparticles and ciprofloxacin as toxin inducer

[0087] Preparation of gold nanoparticles 40 nm gold particles are prepared as described in Example 1.

[0088] Preparation of conjugates The antibody evaluated in this assay is an IgG antibody against the STX1 toxin (see ATCC 13C4 hybridoma CRL-1794). Antibodies are adsorbed onto the nanoparticles as follows: 90μg of antibody diluted in 3ml of Tris HCl (0.025M) pH 8 Add 30 ml of OD1 gold, pre-adjusted to pH 8 with 0.1M K2CO3 solution Stir for 20 minutes Passivation by adding 3 ml of 10% BSA Centrifuge at 8600g for 30 minutes Remove the supernatant and assay the resulting concentrate using a spectrophotometer. Preparation of the medium according to the invention Agar supplementation: The prepared conjugate is added to supercooled agar (chrom ID coli ref 42017 bioMerieux) at 50 °C containing 10 ng / mL of a toxin inducer (in this case ciprofloxacin) to achieve a nanoparticle concentration with an OD of 3. Then, pour 18 ml of medium per dish. The dishes are then allowed to dry.

[0089] inoculation Four strains (three of which carry the stx1 gene) from the applicant's in-house collection are inoculated and incubated at 37°C for 24 hours. Conclusion: A grey halo is observed around the STX1 toxin-producing colonies (Figure 3). The STX1 toxin is detectable. This grey colour change is due to a network formed between the toxin released by the colony and conjugates present in the agar.

[0090] Example 4: Detection of Stx1 toxin-producing E. coli in a medium according to the invention comprising anti-Stx1 antibodies bound to silver nanoparticles and mitomycin as toxin inducer

[0091] Preparation of silver nanoparticles

[0092] Silver nanoparticles of 40 nm were purchased from a commercial supplier (Alfa Aesar; reference: J67090.AE).

[0093] Preparation of conjugates The antibody (reference: ATCC 13C4 hybridoma CRL-1794) is adsorbed onto the nanoparticles as described in Example 3.

[0094] Preparation of the medium according to the invention

[0095] The prepared conjugates are added to TBX supercooled agar medium (reference: AEB622817 bioMerieux), which also contains 250 ng / mL of a toxin inducer (in this case mitomycin C), at 50 °C, to achieve a nanoparticle concentration with an OD of 3. The medium is then poured into dishes and allowed to dry.

[0096] inoculation Two strains (one of which has the stx1 gene) from the applicant's in-house collection are inoculated and incubated at 37°C for 24 hours.

[0097] result A grey halo is observed around the STX1 toxin producers (Figure 4, right colony). This grey colour change is due to a network formed between the toxin released by the colony and the conjugates present in the agar.

[0098] Example 5: Detection of Stx1 and / or Stx2 toxin-producing E. coli in a medium according to the invention comprising anti-Stx1 antibodies bound to silver nanoparticles, anti-Stx2 antibodies bound to gold nanoparticles, in the presence of mitomycin C

[0099] Preparation of silver or gold nanoparticles

[0100] Silver nanoparticles of 40 nm were purchased from a commercial supplier (Alfa Aesar; reference: J67090.AE).

[0101] The nanoparticles are prepared according to example 1.

[0102] Preparation of conjugates

[0103] 40 nm gold nanoparticles are conjugated to the anti-stx2 antibody 9E4H11 from the Applicant's collection according to the method described in Example 1 at pH 9.

[0104] The adsorption of the anti-stx1 antibody 13C4 onto silver nanoparticles is carried out according to example 4.

[0105] Preparation of the medium according to the invention

[0106] The conjugates are added to supercooled TBX agar medium (reference AEB622817 bioMerieux) at 50 °C containing 250 ng / ml of a toxin inducer (in this case mitomycin C) to achieve, for each type of nanoparticle, a concentration of OD 3. The medium is then poured into dishes and left to dry.

[0107] Inoculation:

[0108] Two strains from the applicant's collection, one carrying the stx1 gene and one carrying the stx2 gene, are inoculated and incubated at 37° C. for 24 hours.

[0109] result

[0110] The reaction medium with the conjugate has a light red color. A halo is observed around each of the two strains (Figure 5). It is noteworthy that the color of the halo differs depending on the toxin produced. Specifically, in the case of STX2 toxin production, a network is formed between the toxin and the gold nanoparticles, the gold nanoparticles turn from red to gray, and a yellow halo is generated due to the presence of non-aggregated silver nanoparticles (left colony in Figure 5). In the case of STX1 toxin production, a network is formed between the toxin and the silver nanoparticles, the silver nanoparticles turn from yellow to gray, and a stronger red halo (light red + gray) is generated due to the presence of non-aggregated gold nanoparticles (right colony in Figure 5).

[0111] Example 6: Detection of Stx1 and / or Stx2 toxin producing target E. coli in a medium according to the invention, comprising anti-Stx1 or anti-Stx2 antibodies bound to the same gold nanoparticles, in the presence of ciprofloxacin as toxin inducer

[0112] Preparation of nanoparticles

[0113] Nanoparticles of 40 nm are prepared according to example 1.

[0114] Preparation of conjugates

[0115] The antibodies evaluated in this assay are the 13C4 antibody against STX1 toxin and the 9E4H11 antibody against STX2 toxin.

[0116] This mixture is adsorbed onto the nanoparticles at pH 8 according to the previous example.

[0117] Preparation of the medium according to the invention

[0118] The prepared conjugates are added to supercooled TBX agar (reference AEB622817 bioMerieux) at 50 °C containing 10 ng / ml of the toxin inducer (in this case ciprofloxacin) to achieve a nanoparticle concentration with an OD of 3. Then, 18 ml of medium are poured per dish. The dishes are then allowed to dry.

[0119] inoculation

[0120] The five strains (with or without the stx1 and / or stx2 genes) are inoculated and incubated at 37° C. for 24 hours.

[0121] Conclusion: A grey halo is observed around colonies producing STX1 and / or STX2 toxins (Figure 6). This grey colour change is due to a network formed between the toxins released by the colonies and conjugates present in the agar. Example 6: Detection of STX1 toxin-producing E. coli strains by mass inoculation

[0122] Preparation of nanoparticles

[0123] Nanoparticles of 40 nm are prepared according to example 1.

[0124] Preparation of conjugates

[0125] The antibody used in this study is 13C4 against the STX1 toxin of E. coli. Adsorption is performed as described in the previous examples.

[0126] Preparation of the medium according to the invention

[0127] The conjugates produced in 50° C. supercooled agar (TBX) containing a toxin inducer (in this case mitomycin C at 250 ng / mL) are added to achieve a nanoparticle concentration of OD 3.

[0128] inoculation

[0129] In this example, two strains, one that produces STX1 toxin (O24) and one that does not produce STX1 toxin (O25), are inoculated into masses and incubated at 37° C. for 24 hours.

[0130] conclusion In the case of O24 colonies, a grey halo is clearly present: this colour transition is due to a network formed between the STX1 toxin released by the colony and the conjugates present in the agar.

[0131] Bibliography Nicholas G. Welch et al., “Orientation and characterization of immobilized antibodies for improved immunoassays (Review)”; Biointerphases 12, 02D301 (2017); https: / / doi.org / 10.1116 / 1.4978435; Turkevich et al., “A study of the nucleation and growth processes in the synthesis of colloidal gold”, 1951

Claims

1. A gelled reaction medium for detecting, identifying, counting and / or isolating a target microorganism in a sample that may contain at least one target microorganism, comprising at least one specific binding partner to a component of the target microorganism or to a component derived from the target microorganism, wherein the binding partner binds to at least one nanoparticle to form at least one aggregated conjugate.

2. The gelled reaction medium according to claim 1, characterized in that it is a microbial culture medium.

3. The gelled reaction medium according to claim 1, characterized in that it is a microbiological visualization medium.

4. The gelled reaction medium according to claim 1, characterized in that the binding partner is selected from antibodies, aptamers, phage proteins, and primers.

5. The gelled reaction medium according to claim 1, characterized in that the nanoparticles are colloidal nanoparticles having optical properties.

6. The gelled reaction medium according to claim 1, characterized in that the nanoparticles are selected from gold, silver, and copper.

7. The gelled reaction medium according to claim 1, characterized in that the nanoparticles have a size of 10 to 200 nm, preferably 20 to 90 nm.

8. The gelled reaction medium according to claim 1, characterized in that the reaction medium is in contact with an agar medium.

9. The gelled reaction medium according to claim 1, characterized by containing an inducer of a component derived from the microorganism.

10. The gelled reaction medium according to claim 9, characterized by containing a toxin inducer.

11. The gelled reaction medium according to claim 10, characterized in that the toxin inducer is an antibiotic.

12. The gelled reaction medium according to claim 11, characterized in that the toxin inducer is ciprofloxacin at a concentration of 0.005 to 0.030 mg / l.

13. The gelled reaction medium according to claim 11, characterized in that the toxin inducer is mitomycin C at a concentration of 0.10 mg / l to 0.50 mg / l.

14. A gelled reaction medium according to claim 1 for detecting, identifying, counting and / or isolating at least one target microorganism selected from Escherichia coli, Shiga toxin-producing Escherichia coli, Shigella, Salmonella tiphimuria, Salmonella enteritidis, Pseudomonas, Bacillus cereus group, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis, Staphylococcus aureus MSSA, Staphylococcus aureus MRSA, and Streptococcus agalactie.

15. A diagnostic kit for producing a gelled reaction medium according to any one of claims 1 to 14, • Aggregated conjugates • Gelation medium A diagnostic kit that includes this.

16. A method for producing a gelled reaction medium according to any one of claims 1 to 14, A method comprising the step of contacting an aggregated conjugate with a gelling medium to form a reaction medium.

17. An in vitro microbial culture method, wherein microorganisms that may be present in a sample are inoculated into or onto a culture medium according to any one of claims 1 to 14.

18. A gelled reaction medium according to claim 1 for detecting, identifying, counting and / or isolating Escherichia coli strains, comprising a phage protein specific to the LPS of the strain, wherein the phage protein binds to nanoparticles to form an aggregated conjugate.

19. The gelled reaction medium according to claim 18, characterized in that the amount of LPS-specific phage protein makes it possible to cover at least half of the surface of the nanoparticles.

20. The nanoparticles are made of gold, have a size of 20-90 nm, and have a concentration of 10 nanoparticles per 1 ml of reaction medium. １０ ~10 １２ A gelled reaction medium according to claim 18, characterized in that it is individual.

21. A gelled reaction medium according to claim 1 for detecting, identifying, counting and / or isolating Escherichia coli strains, comprising a monoclonal antibody specific to the strain, wherein the monoclonal antibody binds to nanoparticles to form an agglutination conjugate.

22. A gelled reaction medium according to claim 21 for detecting, identifying, counting and / or isolating at least one Shiga toxin-producing Escherichia coli, comprising at least one toxin inducer, at least one antibody specific to STX1, and / or at least one antibody specific to STX2, wherein the at least one antibody binds to nanoparticles to form an agglutination conjugate.

23. A gelled reaction medium according to claim 22 for detecting, identifying, counting and / or isolating at least one Shiga toxin-producing Escherichia coli, comprising ciprofloxacin at a concentration of 0.005 to 0.030 mg / l.

24. A gelled reaction medium according to claim 22 for detecting, identifying, counting and / or isolating at least one Shiga toxin-producing Escherichia coli, comprising mitomycin C at a concentration of 0.10 mg / l to 0.50 mg / l.

25. A gelled reaction medium according to claim 21 for detecting, identifying, counting and / or isolating at least one Shiga toxin-producing Escherichia coli, wherein the nanoparticles are gold nanoparticles having a size of 20 to 90 nm, and the concentration is 10 nanoparticles per 1 ml of reaction medium. １０ ~10 １２ A gelled reaction medium characterized by being individual.

26. A method for detecting, identifying, counting, and / or isolating a target microorganism in a sample that may contain such a microorganism, comprising the following steps: - A step of bringing the sample into contact with the gelled reaction medium described in any one of claims 1 to 14 or 18 to 25. • Incubation process, - Steps to detect the presence of the target microorganism. Methods that include...

27. The method according to claim 26, wherein detection is performed by the appearance of a halo around the target microorganism in the gelled reaction medium.

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