reaction medium and associated method for the detection of a target microorganism.

Abstract

The invention relates to a gelled reaction medium for the detection, identification, enumeration and / or isolation of at least one target microorganism in a sample likely to contain it comprising at least one specific binding partner of a component of a target microorganism or of a component from said microorganism, coupled to at least one nanoparticle to form at least one agglutinating conjugate.

Description

Title of the invention: Reaction medium and associated method for the detection of a target microorganism. Technical field of the invention

[0001] The present invention relates to the field of microbiological control in a broad sense, such as the microbiological control of a sample of industrial or clinical origin. More particularly, the present invention relates to a reaction medium and the associated method for the detection, identification, enumeration and / or isolation of a target microorganism. Previous technique

[0002] The microbiological control of samples from various origins requires the implementation of techniques which allow the detection - for example for the purposes of identification and / or enumeration and / or biochemical characterizations - of microorganisms and whose delivery in terms of results must be as rapid as possible.

[0003] In the medical field, it is necessary to anticipate and diagnose the risk of infection: the faster and more accurate the diagnosis, the more effective the patient care and the minimized the risk of transmission. The approach is similar for animal health in the veterinary field.

[0004] In the agri-food sector, the problem is the same. However, it distinguishes:

[0005] - pathogenic microorganisms such as Shiga-producing bacteria toxins (STEC), Salmonella, Listeria, Cronobacter, Bacillus, Staphylococcus, the research for which applies to raw materials, intermediate products, and finished marketed products; non-pathogenic microorganisms, used as quality indicators of the production process, from raw materials to finished products, throughout the entire chain.

[0006] - bacteria of technological interest such as ferments,

[0007] - microorganisms that are markers of contamination.

[0008] Rapid and accurate detection of suspected contamination (within food batches) allows them to be controlled and corrective actions to be taken in a short time.

[0009] Technically, one of the main difficulties is being able to isolate the target bacterium in order to identify it. Generally, microbiological analysis is carried out in two stages. The first is a detection phase that can utilize numerous technologies such as culture media, immunoassays, and molecular biology. It can be followed, particularly in the field In the agri-food sector, a confirmation phase is required to confirm the presence of the pathogen in question and ensure compliance with applicable standards. This confirmation phase necessitates additional steps and, again, requires the isolation of the target bacterium.

[0010] Thus, in the case of Shiga toxin-producing E. coli (STEC), diagnosis relies on the use of selective and chromogenic agars such as SMAC (Sorbitol MacConkey Agar), RMAC (Rhamnose MacConkey Agar), or Rainbow Agar 0157, in order to select for the growth of certain bacteria and to stain the strains of interest. However, none of these agars is sufficiently specific to STEC without culture to isolate and confirm the strain's identity using molecular biology tests such as PCR. Indeed, their pathogenicity involves the expression of several virulence genes, notably stxl and stx2, which encode the two types of Shiga toxins: STX1 and STX2. These two toxins act by inhibiting protein synthesis in eukaryotic cells, which ultimately causes apoptosis.To accurately identify pathogenic STEC, it is essential to perform PCR for the stxl, stx2, or eae genes (another virulence factor). These procedures are therefore lengthy and tedious, and the organism carrying both genes (stx and eae) is not always found in culture.

[0011] Immunological methods also exist. A large number of tests allow the detection of E. coli O157:H7 in food and / or environmental samples. These systems include conventional ELISA microplate tests, one-step immunological systems, and fully automated systems.

[0012] One-step immunological methods are widely used by manufacturers due to their speed and simplicity. Many of these systems are based on the principle of immunochromatography. The device consists of a plastic support containing a membrane impregnated with gold or latex particles coated with antibodies specific to E. coli O157:H7 (i.e., O157 and possibly H7), a well for the sample, and a test and control window. The appearance of a colored line in the test window indicates a positive result, signifying the probable presence of E. coli O157 in the food. An example is the "VIP EHEC" test (BioControl, Montesson, France), which, after several hours of enrichment, allows visualization of whether a given sample is contaminated by E. coli O157. However, this method does not allow for the identification of the E. coli O157 strain in the sample, nor does it reveal its pathogenicity.

[0013] ELISA / ELFA systems are immunological methods that provide results in 2 hours in a microplate after an enrichment phase (most often lasting 24 hours). bioMérieux® has developed ELISA kits (ELFA) automated methods based on VIDAS® technology. These methods have the drawback, in the event of a positive test, of not directly isolating the suspect colony. It will therefore be necessary to culture the sample in order to isolate the positive colony and confirm by PCR that this bacterium is indeed the one that caused the positive test.

[0014] There are also ELISA methods for the detection of non-0157 STEC, based primarily on the detection of Shiga toxin production, possibly after an enrichment step. Some of these methods use monoclonal or polyclonal antibodies directed against the STX toxin, and detection with antibodies bound to alkaline phosphatase. Among the available ELISA kits is the "Premier EHEC" targeting Shiga toxins (Meridian Diagnostics Inc., USA). Kits using the reverse passive agglutination assay (RPLA) technique adapted for the detection of STX toxins have also been developed and marketed. This technique involves antibody-coated beads that interact with Shiga toxins and produce a diffuse layer at the base of the wells containing the culture supernatant. These methods then require the isolation of the positive colony followed by a confirmation step.

[0015] The immunomagnetic separation (IMS) method has also been adapted for the detection and isolation of strains belonging to the 5 major serogroups of STEC. It takes place in a liquid medium and is performed using magnetic beads coated with antibodies directed against the O26, OUI, O103, and O145 antigens. However, strains isolated by this method must be confirmed for the presence of the stx gene before being considered as STEC strains.

[0016] There is therefore a real need to develop a reliable and rapid method for isolating and identifying target bacteria, and in particular pathogenic bacteria. Summary of the invention

[0017]

[00011] A first object of the invention relates to a gelled reaction medium for the detection, identification, enumeration and / or isolation of at least one target microorganism in a sample likely to contain it comprising at least one specific binding partner of a component of a target microorganism or of a component derived from said microorganism, coupled to at least one nanoparticle to form at least one agglutinating conjugate.

[0018]

[0012] The component for detecting the target microorganism is a component released by said microorganism into the reaction medium. This may be a constituent of said target microorganism such as a lipopolysaccharide (LPS) or a component produced by the target microorganism such as a toxin.

[0019]

[0013] Advantageously, the medium thus allows localization on The culture medium allows the targeted colony to release the said component. This enables better detection of the target microorganism and saves valuable time in the field of microbiological control, particularly during the confirmation step.

[0020]

[0014] Preferably, the conjugate comprises a colloidal nanoparticle having optical properties.

[0021]

[0015] Advantageously, the medium according to the invention comprises an inductor of toxin. Thus, the medium according to the invention makes it possible in a completely advantageous way to detect Shiga toxin-producing E. coli strains (STEC).

[0022]

[0016] Another object of the invention relates to a detection method identification, enumeration and / or isolation of a target microorganism in a sample likely to contain it, including the following steps:

[0023] - Bring said sample into contact with a reaction medium according to the invention

[0024] - Incubate

[0025] - Detect the presence of said target microorganism.

[0026]

[0017] Advantageously, detection is achieved by the appearance of a halo around of the target microorganism on the reaction medium. DESCRIPTION OF THE FIGURES

[0027]

[0018] Figure 1 is a photograph of a culture medium according to the invention allowing the detection of the E. coli O26 strain, comprising a phage protein coupled to a gold nanoparticle.

[0028]

[0019] Figure 2 is a photograph of a culture medium according to the invention allowing the detection of the E.coli O111 strain, comprising an antibody coupled to a gold nanoparticle.

[0029]

[0020] Figure 3 is a photograph of a culture medium according to the invention allowing the detection of a target bacterium producing the STX1 toxin comprising an anti-STX1 antibody coupled to a gold nanoparticle, in the presence of ciprofloxacin as a toxin inducer.

[0030]

[0021] Figure 4 is a photograph of a culture medium according to the invention allowing the detection of a target bacterium producing the STX1 toxin comprising an anti-STX1 antibody coupled to silver nanoparticles, in the presence of mitomycin as a toxin inducer

[0031]

[0022] Figure 5 is a photograph of a culture medium according to the invention allowing the detection of a target bacterium producing the STX1 and / or STX2 toxin comprising an anti-STX1 antibody coupled to a silver nanoparticle, an anti-STX2 antibody coupled to a gold nanoparticle, in the presence of mitomycin

[0032]

[0023] Figure 6 is a photograph of a culture medium according to the invention allowing the detection of a target bacterium producing STX1 and / or STX2 toxin comprising an anti-STX1 or anti-STX2 antibody coupled to the same gold nanoparticle, in the presence of ciprofloxacin as a toxin inducer. Detailed description of the invention

[0033]

[0024] Certain terms and expressions used in the context of the invention are detailed below.

[0034]

[0025] A first object of the invention relates to a gelled reaction medium for the detection, identification, enumeration and / or isolation of at least one target microorganism in a sample likely to contain it comprising at least one specific binding partner of a component of a target microorganism or of a component derived from said microorganism, coupled to at least one nanoparticle to form at least one agglutinating conjugate.

[0035]

[0026] Quite surprisingly, it was found that it was possible to detect a target microorganism using a gelled reaction medium comprising an agglutinating conjugate.

[0036]

[0027] By “reaction medium”, we mean a medium comprising all the elements These reaction media are necessary for the expression of metabolism and for the survival and / or growth of microorganisms. This reaction medium can be either a microbiological culture medium or a microbiological detection medium. In the latter case, the microorganisms may be cultured 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 top of the culture medium that allows the growth of the target microorganisms. The reaction medium can be added after the incubation of the culture medium. Prior to use, the reaction medium can be dehydrated. The reaction medium may be in pad form.

[0037]

[0028] According to the present invention, the reaction medium is gelled. It is presented in solid or semi-solid form. Agar is the traditional gelling agent used in microbiology for culturing microorganisms, but other gelling agents can be used, such as gelatin, agarose, and other natural or synthetic gelling agents. Thus, surprisingly, the conjugate is able to form a network with the target component in a gelled medium. This network is visually detectable by the formation of a halo without requiring a reduction in the hardness of a conventional semi-solid reaction medium. Therefore, it is not necessary to modify the physicochemical properties of the reaction medium, such as its hardness, to allow both the diffusion of the target components and the reaction with the conjugates.

[0038]

[0029] 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 the basis for the reaction medium according to the invention. The reaction medium may further comprise 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, etc. A "selective agent" is any compound capable of preventing or slowing the growth of a so-called "non-target" microorganism, that is, one 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, that would normally remain unexpressed.

[0039]

[0030] Said reaction medium may also include a dye. As Examples of suitable dyes include Evans blue, neutral red, sheep blood, horse blood, opacifiers such as titanium dioxide, nitroaniline, malachite green, and brilliant green. When the reaction medium according to the invention further comprises an enzymatic substrate specific to the enzymatic activity of at least one target microorganism, a chromogenic and / or fluorogenic substrate is preferably used. "Chromogenic and / or fluorogenic substrate" means a substrate that allows the detection of the enzymatic or metabolic activity of the target / sought-after microorganisms by means of a detectable signal. The reaction medium according to the invention may also include a pH indicator, sensitive to the pH change induced by the consumption of the substrate and revealing the metabolism of the target microorganisms. This pH indicator may be a chromophore or a fluorophore.Examples of chromophores include bromocresol purple, bromothymol blue, neutral red, aniline blue, and bromocresol blue.

[0040]

[0031] A person skilled in the art may also use a Petri dish divided into segment, such as a bi-box, or a tri-box, allowing easy comparison of several media, including different substrates or different selective mixtures, on which the same biological sample will have been deposited.

[0041]

[0032] According to the present invention, the reaction medium comprises a partner of Specific binding of a component of a target microorganism or a component derived from said microorganism, coupled to a nanoparticle. The specific binding partner is chosen from among 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 antibody is a monoclonal antibody or a fragment of a monoclonal antibody.

[0042]

[0033] According to the present invention, the bonding partner is specific to a A component of a target microorganism. The target microorganism component is a component released by said microorganism. The component can thus be an element from the surface of the bacterium, such as a protein, a lipopolysaccharide (LPS), or a flagellum. It can also be an internal element of the bacterium, such as RNA, an intracellular protein that can be detected when a portion of the bacterial colony dies during its growth. In a particular embodiment, the binding partner is specific to the RNA of the target microorganism. In this embodiment, the binding partner is composed of at least two different primers that hybridize complementaryly to the target RNA.

[0043]

[0034] In another particular embodiment, the liaison partner is specific to a component 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.

[0044]

[0035] According to the present invention, the linking partner is coupled to a nanoparticle. This final complex is called a conjugate. In the same reaction medium, it is possible to have conjugates with binding partners of different natures coupled to nanoparticles of different natures.

[0045]

[0036] The term “nanoparticle” refers to particles of the order of magnitude of the nanometer. The nanoparticles can be chosen from gold, iron, silver, copper, carbon, latex, silicon, and aluminum. Preferably, the nanoparticles are colloidal nanoparticles chosen for their optical property, namely their ability to stand out when a lattice forms. Even more preferably, the nanoparticles are chosen from gold, silver, and copper. Thus, when the nanoparticles are made of gold, they change color, for example, from red to gray when they form a lattice. When they are not aggregated, the wavelength of the absorbed light is in the red range, around 530 nm. When they are aggregated, the absorbed wavelength changes from red to blue / gray, around 600 to 700 nm. In a particular embodiment, it is possible to use several nanoparticles of different colors together.The networks thus formed allow the distinction of several components of the target microorganism.

[0046]

[0037] Preferably, the nanoparticles have a size between 10 and 200nm. Preferably, nanoparticles have a size between 20 and 90nm, allowing better mobility of conjugates in the reaction medium.

[0047]

[0038] Advantageously, nanoparticles make it possible to reduce the quantity necessary binding partners for the formation of an agglutination. Thus, the concentration of binding partners required to prepare a reaction medium according to the invention requires 100 to 1000 times fewer binding partners than a medium without nanoparticles.

[0048]

[0039] Preferably, the required quantity of linking partners corresponds to the quantity necessary to cover at least half of the nanoparticle surface, and even more preferably to cover between one-third and one-half of the nanoparticle surface. This proportion allows the agglutinating conjugate to form a network in the gelled reaction medium.

[0049]

[0040] Advantageously, nanoparticles can allow visualization of a Agglutination around a bacterial colony is possible when the size of the particles is not yet visible to the naked eye. This allows for earlier detection. Advantageously, nanoparticles can make it possible to visualize agglutination around a bacterial colony whose translucent appearance prevents detection by automated readers. This can thus facilitate detection.

[0050]

[0041] The coupling of the nanoparticle to the binding partner can be done either by The reaction can be achieved through direct or indirect binding. Direct binding refers to binding by adsorption or covalent bonding. Indirect binding refers to binding through the interaction of ligands / antiligands, such as biotin / strepatividin or other pairs well-known to those skilled in the art. Depending on the type of binding chosen, those skilled in the art will adjust the physicochemical conditions of the reaction medium, particularly its pH.

[0051]

[0042] According to the present invention, the conjugate is agglutinating, that is to say that it This process induces the formation of an agglutination network in the presence of a component of a target microorganism or a component derived from said microorganism. Since the component is multi-epitope, several conjugates will bind to this component and form an agglutination. Agglutination is defined as the result of an interaction between at least one component of a target microorganism or at least one component derived from said microorganism and 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 within the reaction medium. In practice, several parameters influence the conjugate's ability to agglutinate in a gelled medium, primarily:

[0052] - the porosity of the gelled medium

[0053] - the size of the nanoparticles

[0054] - the quantity of liaison partners

[0055] - the quantity of nanoparticles

[0056] It will therefore be necessary to adapt these parameters to allow for satisfactory agglutination enabling 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 located. 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.

[0057]

[0043] By "at least one target microorganism", we mean, within the meaning of this The invention involves at least one microorganism that one wishes to detect and / or identify and / or enumerate. Preferably, the microorganism is chosen from among Escherichia coli, Shiga toxin-producing Escherichia 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 chosen from E. coli strains, and preferably from Shiga toxin-producing E. coli (STEC) strains. Enterohemorrhagic E. coli (EHEC) are strains representing a subgroup of Shiga toxin-producing Escherichia coli (STEC) that have acquired the eae gene and cause hemolytic uremic syndrome. The simultaneous presence of these two virulence factors makes this pathovar highly virulent to humans.These include serogroups O26, O45, O80, O103, OUI, O121, O145 and O157.

[0058]

[0044] In a particular embodiment, the reaction medium comprises a An inducer that causes or increases the expression of a molecule of interest produced by the target microorganism. This molecule of interest can be a protein, an antibiotic, a resistance molecule to antimicrobial agents, a protease-type enzyme, a lipase, or a carbohydrate. Thus, the present invention allows for the characterization or selection of microbial strains based on their ability to produce these molecules of interest. In a particular embodiment, the present invention can relate to the field of bioproduction, and more specifically to the production of recombinant proteins from a genetically modified microorganism, for which it is necessary to identify the producing clones. The present invention thus makes it possible to clearly see that the recombinant protein is produced. In the presence of a halo, it is also possible to estimate the quantity produced.In another particular embodiment, the present invention enables the detection of resistant pathogenic bacteria.

[0059]

[0045] In a particular embodiment, the reaction medium comprises A toxin inducer. The toxin inducer causes stress in the bacterium, which triggers the lytic cycle of prophages within the bacterium and thus stimulates the production of toxins that will be released. Examples include the toxins secreted by Staphylococcus aureus or the Shiga toxins secreted by STEC strains. There are two main types of Shiga toxin: STX1 and STX2, which themselves have numerous variants. To date, STX1 has four subtypes: STX1a, STX1c, STX1d, and STX1e, and STX2 has twelve: STX2a, STX2b, STX2c, STX2d, STX2e, STX2f, STX2g, STX2h, STX2i, STX2j, STX2K, and STX21. Advantageously, the toxin inducer is chosen from among antibiotics or a physicochemical stress.Examples of antibiotics used include trimethoprim, sulfamethoxazole, norfloxacin, azithromycin, gemtamicin, polymyxin B, chloramphenicol, streptomycin, chlortetracycline, oxytetracycline, tylosin, mitomycin C, carbodox, oliquindox, rifampin, imipenem, ciprofloxacin, cotrimoxazole, penicillin G, and 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 0.5 mg / L.

[0060]

[0046] The toxin inducer can also be a physico-chemical stress effect, For example, by adding salt or EDTA, by changing the pH, or by UV stress. Stress can also be induced, for example, by adding norepinephrine.

[0061] The target microorganism is likely to be present in a sample. A "sample" is defined as a small part or quantity isolated from an entity for analysis. The sample may be of industrial origin, including, but not limited to, an air sample, a water sample, a sample taken from a surface, a part or a manufactured product, or a food product. Examples of food samples include, but are not limited to, samples of dairy products (yogurt, cheese, etc.), meat, fish, eggs, fruit, vegetables, water, and beverages (milk, fruit juice, soda, etc.). A food sample may also be derived from animal feed, such as animal or vegetable meal. The sample may also be of biological origin, either animal or human.It can then correspond to a sample of biological fluid (stool, urine, whole blood, serum, plasma, cerebrospinal fluid, organic secretion, etc.), an external sample (skin, nose, throat, etc.) or tissue, or isolated cells. The sample can be used as is or, prior to analysis, undergo preparation such as enrichment, extraction, concentration, or purification, according to methods known to those skilled in the art. The reaction medium according to the invention can be of particular interest when the sample is a polymicrobial sample or loaded with accessory flora, such as food samples like raw milk cheese or stools in clinical samples.

[0062]

[0047] Another object of the present invention relates to a diagnostic kit enabling the preparation of a reaction medium according to the invention comprising

[0063] - an agglutinating conjugate as described in the present invention

[0064] - a gelling medium.

[0065]

[0048] Advantageously, the reaction medium can be prepared extemporaneously using a kit according to the invention. This allows the unit use of a reaction medium, and increases its stability.

[0066]

[0049] Thus, another object of the present invention relates to the method of obtaining of a reaction medium according to the invention, comprising the steps of contacting an agglutinating conjugate with a gelling medium to form the reaction medium according to the invention. The conjugate is prepared 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 gelling medium. The mixture is then homogenized and poured into a Petri dish.

[0067]

[0050] Another object of the present invention relates to a method of cultivation In vitro microbiological testing, in which microorganisms likely to be present in a sample are inoculated into or onto a culture medium according to the invention. The inoculated culture medium is incubated under suitable conditions known to those skilled in the art. Inoculation is carried out using conventional microbiological techniques. It can also be performed in bulk, i.e., by embedding.

[0068]

[0051] Another object of the present invention relates to a detection method, identification, enumeration and / or isolation of at least one target microorganism in a sample likely to contain it comprising the following steps:

[0069] - -contacting said sample with a reaction medium according to the invention

[0070] - Incubate

[0071] - detect the presence of said target microorganism.

[0072]

[0052] By "detection" is meant detection with the naked eye or with the aid of a device optical detection of the presence of growth of target microorganisms, preferably target bacteria. When the reaction medium from which the target microorganisms are to be detected includes a chromogenic or fluorogenic substrate, 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.

[0073]

[0053] By "identification" is meant the determination of the genus and / or species and / or a group to which a target microorganism belongs.

[0074]

[0054] By "enumeration of at least one target microorganism", we mean the the act of counting / quantifying the number of target microorganisms, for example the number of bacterial colonies when the target microorganism is a bacterium.

[0075]

[0055] By "isolation" we mean obtaining different, spaced-out colonies from each other.

[0076]

[0056] Microbiological control corresponds to the analysis of a sample in The aim is to detect microorganisms that may be present in the sample. Surprisingly, it was found that a medium according to the invention made it possible to detect and isolate a target bacterium. Advantageously, the medium according to the invention can thus identify and select a target microorganism from a mixed population present on a non-selective or insufficiently selective reaction medium. Preferably, 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.

[0077]

[0057] Without being limiting, it appears that the medium according to the invention is particularly suitable for the detection of E. coli. Thus, according to a particular embodiment, the present invention relates to a gelled reaction medium for the detection, identification, enumeration and / or isolation of an E. coli strain, comprising a phage protein specific to the LPS of said strain coupled to a nanoparticle to form a conjugate.

[0078]

[0058] In this particular embodiment, the quantity of phage protein specific for LPS makes it possible to cover at least a third of the surface of the nanoparticle.

[0079]

[0059] Preferably, in this particular embodiment, the nanoparticle is gold, of size between 20 and 90 nm and at a concentration between 1010 and 1012 nanoparticles / ml of reaction medium.

[0080]

[0060] In this embodiment, the reaction medium is formed from a culture medium known for the culture of E. coli strains such as the applicant's TBX or chromID® Coli media, to which agglutinating conjugates have been added.

[0081]

[0061] In another particular embodiment, the present invention relates to a gelled reaction medium for the detection, identification, enumeration and / or isolation of an E. coli strain, comprising a monoclonal antibody specific to said strain coupled to a nanoparticle to form a conjugate.

[0082]

[0062] Preferably, in this particular embodiment, the quantity in monoclonal antibodies allow for the coverage of at least half of the surface of the nanoparticle, and even more preferentially to cover between one-third and one-half of the surface of the nanoparticle.

[0083]

[0063] Preferably, in this particular embodiment, the nanoparticle is gold, of size between 20 and 90 nm and at a concentration between 1010 and 1012 nanoparticles / ml of reaction medium.

[0084]

[0064] In another particular embodiment, the present invention relates to a gelled reaction medium for the detection, identification, enumeration and / or isolation of a Shiga toxin-producing E. coli strain comprising at least one toxin inducer, at least one antibody specific to Stxl and / or Stx2, said at least one antibody being coupled to a nanoparticle to form an agglutinating conjugate.

[0085]

[0065] This embodiment advantageously allows for the identification of an E. strain. Shiga toxin-producing E. coli is identified by the formation of a halo around the strain. This occurs because the antibody conjugates, coupled to nanoparticles, have agglutinated around the colony. This makes it easy to visualize the target colony.

[0086]

[0066] In this embodiment, said at least one antibody has a quantity allowing for the coating of at least half of the nanoparticle surface and preferably at least one-third of the nanoparticle surface. In these particular embodiments, the medium comprises ciprofloxacin at a concentration between 0.005 and 0.030 mg / L. In a variant of these particular embodiments, the medium comprises mitomycin C at a concentration between 0.10 mg / L and 0.50 mg / L.

[0087]

[0067] Preferably, in these variants the nanoparticle is a nanoparticle gold particles of size between 20 and 90nm and at a concentration between 1010 and 1012 nanoparticles / ml of reaction medium.

[0088]

[0068] When the medium according to the invention comprises bonding partners of several toxins, this invention is particularly advantageous for discriminating in the same sample bacteria producing Shiga toxin STX1 or STX2 from bacteria producing both types of Shiga toxin STX1 and STX2.

[0089]

[0069] This invention is particularly interesting for facilitating detection STEC in a polymicrobial sample. Indeed, without the present invention, which allows for the localization of the colony of interest, the ISO 16136 reference method specifies that it is necessary to test up to 50 colonies using a molecular method to confirm the presence of an STEC. Given the large number of colonies on the plate and the low representation of the target microorganism, the person collecting colonies for confirmation may never actually collect the target microorganism. Thus, the present invention saves time in performing the control. microbiological. It is particularly advantageous for samples loaded with secondary flora, where the large quantity of colonies on Petri dishes can lead to a risk of false negatives. Examples

[0090]

[0070] Example 1: Detection of E. coli O26 with a medium according to the invention including a phage protein coupled to a gold nanoparticle

[0091]

[0071] Preparation of gold nanoparticles

[0092]

[0072] The 40 nm nanoparticles are produced by the reduction of gold chloride by sodium citrate (a method described by Turkevich and Frens in 1951). Thus, 20 nm gold nanoparticles are produced using a solution of gold chloride diluted in distilled water, to which trisodium citrate is added and then boiled. The presence of an absorbance peak at 517-519 nm confirms the correct particle size. From these 20 nm particles, 40 nm particles are synthesized. To do this, the 20 nm particles are diluted in distilled water and then boiled with the addition of trisodium citrate and gold chloride. The absorbance peak is then observed at 524-526 nm at room temperature, indicating the increase in particle size.

[0093]

[0073] Conjugation preparation:

[0094]

[0074] The phage evaluated in this trial is Phage Eco 026 BPI, directed against the Escherichia coli LPS 026. This phage comes from the Applicant's collection.

[0095] The adsorption of the phage protein onto the nanoparticles is carried out taking into account the value of its isoelectric point and according to methods known to those skilled in the art (Nicholas G. Welch et al): The above phage is coupled in the following manner:

[0096] - Dilution of 270 pg of phage protein in 3 ml of Tris HCl (0.025M) pH 6

[0097] - Addition of 30 ml of gold at an optical density of 1, i.e., 7.7 x 10⁻¹⁰ nanoparticles / ml previously adjusted to pH 6 with a 0.1M K2CO3 solution

[0098] - Agitation for 20 minutes

[0099] - Passivation by adding 3 ml of 10% BSA

[0100] - Centrifugation at 8600 g for 30 min

[0101] - Removal of the supernatant and determination of the concentrate obtained by spectrophotometer.

[0102]

[0075] Preparation of the reaction medium according to the invention:

[0103] The conjugate produced is added to supercooled agar (chrom ID coli-ref 42017 bioMerieux) at 50°C to obtain a nanoparticle concentration with an optical density of 3 using a spectrophotometer. 18 ml of medium per plate are then poured. The plates are then dried.

[0104]

[0076] Seeding:

[0105] In this example, an E. coli 026 strain and an E. coli 0111 strain (Applicant's collection) are inoculated and incubated for 72 hours at 37°C.

[0106]

[0077] Results

[0107] A grey halo is visually observed around the colony possessing LPS 026 ([Fig.1]). This grey color change originates from a network formed between the LPS released by the colonies and the conjugates present in the agar.

[0108]

[0078] Example 2: Detection of E. coli YES with a medium according to the invention including an antibody coupled to a gold nanoparticle

[0109]

[0079] Preparation of gold nanoparticles

[0110] The 40nm gold particles are manufactured as shown in Example 1. [YES]

[0080] Conjugation preparation

[0112]

[0081] The 2H5E9 IgG anti-E. coli monoclonal antibody 0111 is from the collection of the Plaintiff.

[0113]

[0082] The adsorption of the antibody onto the nanoparticles is carried out in the manner next:

[0114] - Dilution of 90 pg of antibody in 3 ml of Tris HCl (0.025M) pH 6.5

[0115] - Addition of 30 ml of gold to an OD 1 previously adjusted to pH 8 with a solution K2CO3 0.1M

[0116] - Stirring for 20 minutes

[0117] - Passivation by adding 3 ml of 10% BSA

[0118] - Centrifugation at 8600 g for 30 min

[0119] - Removal of the supernatant and determination of the concentrate obtained by spectrophotometer

[0120]

[0083] Preparation of the reaction medium according to the invention

[0121]

[0084] The conjugate produced is added to supercooled agar (chrom ID coli- (ref 42017 bioMerieux) at 50°C to obtain a nanoparticle concentration with an optical density of 3 using a spectrophotometer. 18 ml of medium per plate are then poured. The plates are then dried.

[0122]

[0085] Seeding

[0123]

[0086] In this example, an E. coli O26 strain and an E. coli O111 strain taken from the Applicant's collection, are inoculated and incubated for 72 hours at 37°C.

[0124]

[0087] Result

[0125]

[0088] No grey halo is observed around colony 026, although it is indeed visible around colony 0111 ([Fig.2]), allowing the identification of the latter.

[0126]

[0089] Example 3: Detection of an E. coli producing STX1 toxin with a medium according to the invention comprising an anti-Stxl antibody coupled to a gold nanoparticle and ciprofloxacin as a toxin inducer

[0127]

[0090] Preparation of gold nanoparticles

[0128] The 40 nm nanoparticles are manufactured as described in Example 1.

[0129]

[0091] Conjugation preparation

[0130] The antibody evaluated in this assay is the IgG antibody (ref ATCC 13C4 hybridoma CRL-1794) directed against STXL toxin

[0131] The adsorption of the antibody onto the nanoparticles occurs in the following manner:

[0132] - Dilution of 90 pg of antibody in 3 ml of Tris HCl (0.025M) pH 8

[0133] - Addition of 30 ml of gold to an OD 1 previously adjusted to pH 8 with a solution K2CO3 0.1M

[0134] - Agitation for 20 minutes

[0135] - Passivation by adding 3 ml of 10% BSA

[0136] - Centrifugation at 8600 g for 30 min

[0137] - Removal of the supernatant and determination of the concentrate obtained by spectrophotometer

[0138] Preparation of the medium according to the invention

[0139] - Agar Supplementation:

[0140] - The conjugate produced is added to supercooled agar (chrom ID coli ref 42017 bioMerieux) at 50°C containing the toxin inducer, here ciprofloxacin at 10 ng / mL, to obtain a nanoparticle concentration of OD 3. 18 ml of medium per plate are then poured. The plates are then dried.

[0141]

[0092] Seeding

[0142] 4 internal collection strains of the Applicant, three of which possess the stxl gene are inoculated and incubated for 24 hours at 37°C.

[0143] Conclusion:

[0144] A grey halo is observed around the STX1 toxin-producing colonies ([Fig. 3]). STX1 toxin is detected. This grey color change results from a network formed between the toxins released by the colonies and the conjugates present in the agar.

[0145]

[0093] Example 4: Detection of an STX1 toxin-producing E. coli in a medium according to the invention comprising an anti-Stxl antibody coupled to silver nanoparticles and mitomycin as a toxin inducer

[0146]

[0094] Preparation of silver nanoparticles

[0147]

[0095] The 40 nm silver nanoparticles were purchased from a supplier (Alfa Aesar; ref: J67090.AE).

[0148]

[0096] Conjugation preparation

[0149] Adsorption of the antibody (ref ATCC 13C4 hybridoma CRL-1794) onto the nanoparticles is carried out as described in example 3.

[0150]

[0097] Preparation of the medium according to the invention

[0151]

[0098] The conjugate produced is added to supercooled TBX agar (ref AEB622817 bioMerieux) at 50°C also containing a toxin inducer, here the mitomycin C at 250 ng / mL, in order to obtain a nanoparticle concentration of an OD 3. The medium is then poured into the plates and dried.

[0152]

[0099] Seeding

[0153] Two internal collection strains from the Applicant, one of which possesses the stxl gene, are inoculated and incubated for 24 hours at 37°C

[0154]

[00100] Results

[0155] A grey halo is observed around the strain producing the STX1 toxin ([Fig.4], right-hand colony). This grey color change results from a network formed between the toxin released by the colonies and the conjugates present in the agar.

[0156]

[0101] Example 5: Detection of an E. coli producing Stxl and / or Stx2 toxins in a medium according to the invention comprising an anti-Stxl antibody coupled to a silver nanoparticle, an anti-Stx2 antibody coupled to a gold nanoparticle, in the presence of mitomycin C

[0157]

[0102] Preparation of silver or gold nanoparticles

[0158]

[0103] The 40 nm silver nanoparticles were purchased from a supplier (Alfa Aesar; ref: J67090.AE).

[0159]

[0104] The nanoparticles are manufactured according to example 1.

[0160]

[0105] Conjugation preparation

[0161]

[0106] The 40nm gold nanoparticles are coupled to the 9E4H11 anti antibody stx2, the claimant's collection, according to the method described in example 1 with a pH of 9.

[0162]

[0107] The adsorption of the 13C4 anti-stxl antibody onto the silver nanoparticles Done according to example 4:

[0163]

[0108] Preparation of the medium according to the invention:

[0164]

[0109] The conjugates are added to the supercooled TBX agar (ref AEB622817 bioMerieux) at 50°C containing the toxin inducer, here mitomycin C at 250 ng / mL, in order to obtain a concentration for each type of nanoparticle of an OD 3. The medium is then poured into the plates and dried.

[0165]

[0110] Seeding:

[0166]

[0111] Two strains from the Applicant's collection, one possessing The stxl gene and the stx2 gene are inoculated and incubated for 24 hours at 37°C.

[0167]

[0112] Results

[0168]

[0113] The reaction medium with the conjugates has a light red color. We observe the presence of a halo around each of the two strains ([Fig. 5]). What we notice is that the color of the halo varies depending on the toxin produced. Indeed, in the case of STX2 toxin production, a network forms between the toxin and the gold nanoparticles, causing the gold nanoparticles to change color from red to gray and resulting in a yellow halo due to the presence of the nanoparticles. of silver that have not aggregated (colony on the left in [Fig.5]). In the case of STX1 toxin production, a network forms between the toxin and the silver nanoparticles, causing the silver nanoparticles to change color from yellow to grey and resulting in a more intense red halo (light red + grey) due to the presence of the gold nanoparticles that have not aggregated (colony on the right in [Fig.5]).

[0169]

[0114] Example 6: Detection of a target E. coli producing Stxl toxin and / or Stx2 in a medium according to the invention comprising an anti-Stxl or anti-Stx2 antibody coupled to the same gold nanoparticle in the presence of ciprofloxacin as a toxin inducer

[0170]

[0115] Preparation of nanoparticles

[0171]

[0116] The 40 nm nanoparticles according to example 1

[0172]

[0117] Preparation of conjugates

[0173]

[0118] The antibodies evaluated in this assay are the 13C4 antibody directed against the STX1 toxin and the 9E4H11 antibody directed against STX2 toxin.

[0174]

[0119] This mixture is adsorbed onto the nanoparticles according to the preceding examples to a pH of 8

[0175]

[0120] Preparation of the medium according to the invention:

[0176]

[0121] The conjugate produced is added to supercooled TBX agar (ref AEB622817 bioMerieux) at 50°C containing the toxin inducer, in this case ciprofloxacin at 10 ng / mL, to obtain a nanoparticle concentration of OD 3. 18 ml of medium per plate are then poured. The plates are then dried.

[0177]

[0122] Seeding

[0178]

[0123] 5 strains possessing or not the stxl and / or stx2 genes are inoculated and incubated for 24 hours at 37°C.

[0179]

[0124] Conclusion: A grey halo is observed around the colonies producing the STX1 and / or STX2 toxins ([Fig.6]). This grey color change comes from a network formed between the toxins released by the colonies and the conjugates present in the agar.

[0180] Example 6: Detection of an STX1 toxin-producing E. coli strain by bulk seeding

[0181]

[0125] Preparation of nanoparticles

[0182]

[0126] The 40 nm nanoparticles are manufactured according to example 1.

[0183]

[0127] Preparation of conjugates

[0184]

[0128] The antibody used in this study is 13C4 directed against the STX1 toxin of Escherichia coli. Adsorption is carried out as indicated in the previous examples

[0185]

[0129] Preparation of the medium according to the invention

[0186]

[0130] Add the product conjugates to supercooled agar (TBX) at 50°C containing the toxin inducer here mitomycin C at 250 ng / mL in order to obtain a nanoparticle concentration of an OD 3.

[0187]

[0131] Seeding

[0188]

[0132] In this example, two strains, one producing the STX1 toxin (024), the other not producing it (025), are inoculated into the mass and incubated for 24 hours at 37°C.

[0189]

[0133] Conclusion:

[0190] In the case of colony 024, a grey halo is clearly present. This color change originates from a network formed between the STX1 toxins released by the colonies and the conjugates present in the agar. BIBLIOGRAPHICAL REFERENCES

[0191]

[0134] Nicholas G. Welch et al; “Orientation and characterization of immobilized antibodies for improved immunoassays (Review)”; Biointerphases 12, 02D301 (2017); https: / / doi.org / 10.! 116 / 1.4978435;

[0192]

[0135] Turkevich et al, “A study of the nucleation and growth processes in the synthesis of colloidal gold”, 1951

Claims

Demands

1. Gelled reaction medium for the detection, identification, enumeration and / or isolation of at least one target microorganism in a sample likely to contain it comprising at least one agglutinating conjugate formed by at least one specific binding partner of a component of a target microorganism or of a component derived from said microorganism, coupled to at least one nanoparticle.

2. Gelled reaction medium according to claim 1 characterized in that it is a microbiological culture medium comprising the elements necessary for the culture of said target microorganism.

3. Gelled reaction medium according to claim 1 characterized in that it is a microbiological detection medium comprising the elements necessary for the detection of said target microorganism.

4. Reaction medium according to any one of the preceding claims characterized in that the binding partner is selected from antibodies, aptamers, phage proteins, primers.

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

6. Medium according to the preceding claim characterized in that the nanoparticle is selected from gold, silver, copper.

7. Medium 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.

8. Medium according to any one of the preceding claims in which the reaction medium is in contact with an agar culture medium.

9. Medium according to any one of the preceding claims characterized in that the medium comprises an inducer of the component derived from said microorganism.

10. Medium according to claim 9 characterized in that the medium comprises a toxin inducer.

11. Medium according to claim 10 characterized in that the toxin inducer is an antibiotic.

12. Medium according to claim 11 characterized in that the toxin inducer is ciprofloxacin at a concentration between 0.005 and 0.030 mg / l.

13. Medium according to claim 11 characterized in that the toxin inducer is mitomycin C at a concentration between 0.10 mg / 1 and 0.50 mg / 1.

14. Gelled reaction medium according to any one of claims 1 to 13 for the detection, identification, enumeration and / or isolation of an E. coli strain characterized in that it comprises a phage protein specific to the LPS of said strain coupled to a nanoparticle to form an agglutinating conjugate.

15. Gelled reaction medium according to claim 14 characterized in that LPS-specific phage proteins cover at least half of the surface of the nanoparticle.

16. Gelled reaction medium according to claim 14 or 15 characterized in that the nanoparticle is gold, of size between 20 and 90 nm and at a concentration between 1010 and 1012 nanoparticles / ml of reaction medium.

17. Gelled reaction medium according to any one of claims 1 to 13 for the detection, identification, enumeration and / or isolation of an E. coli strain characterized in that it comprises a monoclonal antibody specific to said strain coupled to a nanoparticle to form an agglutinating conjugate.

18. Gelled reaction medium according to any one of claims 1 to 13 for the detection, identification, enumeration and / or isolation of at least one Shiga toxin-producing E. coli strain characterized in that it comprises at least one toxin inducer, at least one STX1-specific antibody and / or at least one STX2-specific antibody, said at least one antibody being coupled to a nanoparticle to form an agglutinating conjugate.

19. Gelled reaction medium according to claim 18 for the detection, identification, enumeration and / or isolation of at least one Shiga toxin-producing E. coli characterized in that the toxin inducer is ciprofloxacin, at a concentration between 0.005 and 0.030 mg / 1.

20. A gelled reaction medium according to claim 18 for the detection, identification, enumeration and / or isolation of at least one Shiga toxin-producing E. coli, characterized in that the inducer the toxin is mitomycin C, at a concentration between 0.10 mg / l and 0.50 mg / l.

21. Gelled reaction medium according to any one of claims 18 to 20 for the detection, identification, enumeration and / or isolation of at least one Shiga toxin-producing E. coli characterized in that the nanoparticle is a gold nanoparticle of size between 20 and 90 nm and at a concentration of between 1010 and 1012 nanoparticles / ml of reaction medium.

22. A diagnostic kit for preparing a reaction medium according to any one of the preceding claims, comprising: - a binding conjugate - a gelling medium

23. Preparation of a reaction medium according to claims 1 to 13 comprising the step of contacting an agglutinating conjugate with a gelling medium to form the reaction medium.

24. In vitro microbiological culture method, wherein microorganisms likely to be present in a sample are inoculated into or onto a culture medium as defined in claim 2.

25. Method for detecting, identifying, enumerating and / or isolating a target microorganism in a sample likely to contain it, comprising the following steps: - Contacting said sample with a reaction medium according to any one of claims 1 to 21. - Incubating - Detecting the presence of said target microorganism.

26. Method according to the preceding claim characterized in that the detection is done by observing the appearance of a halo around the target microorganism on the reaction medium according to any one of claims 1 to 21.

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