Reaction medium and associated method for 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 that may contain same, comprising at least one binding partner specific to 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 binding conjugate.

Description

Description Title of the invention: reaction medium and associated method for the detection of a target microorganism. TECHNICAL FIELD The present invention relates to the field of microbiological control in the broad sense, such as the microbiological control of a sample of industrial or clinical origin. More particularly, the present invention relates to a reaction medium and the associated method for the detection, identification, enumeration and / or isolation of a target microorganism. PRIOR TECHNIQUE

[0001] Microbiological control of samples of various origins requires the implementation of techniques which allow the detection - for example for the purposes of identification and / or counting and / or biochemical characterization - of microorganisms and whose results must be rendered as quickly as possible.

[0002] In the medical field, it is necessary to predict and diagnose the risk of infection: the faster and more accurate the diagnosis, the more effective the treatment of patients and the minimized the risk of transmission. The approach is similar for animal health in the veterinary field. In the agri-food sector, the problem is identical. However, it distinguishes: - pathogenic microorganisms such as Shiga toxin-producing bacteria (STEC), Salmonella, Listeria, Cronobacter, Bacillus, Staphylococcus, the research for which applies to raw materials, intermediate products, finished products marketed, - non-pathogenic microorganisms, used as quality indicators of the production process, from raw materials to finished products, throughout the chain, - bacteria of technological interest such as ferments, - microorganisms that are markers of contamination. Rapid and accurate detection of suspected contamination (within food batches) allows them to be controlled and corrective actions to be taken quickly.

[0003] Technically, one of the main difficulties is being able to isolate the bacteria being sought in order to identify it. Generally, microbiological analysis is carried out in two stages. The first is a detection phase which can involve numerous technologies such as culture media, immunoassays, molecular biology. It can be followed, particularly in the agri-food sector, by a confirmation phase in order to confirm the presence of the pathogen sought and meet the standards in force in this field. The confirmation stage therefore requires additional steps and here again, requires a step of isolation of the desired bacteria.

[0004] 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 the growth of certain bacteria and to stain the strains of interest. But ultimately, none of these agars is sufficiently specific to STEC, without a culture allowing the isolation and confirmation of the identity of the strain using PCR-type molecular biology tests. Indeed, their pathogenicity involves the expression of several virulence genes, notably stx1 and stx2 coding for 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, PCR for the stx1, stx2, or eae genes (another virulence factor) is essential. These procedures are therefore long and tedious, and the germ carrying both genes (stx and eae) is not systematically found in culture.

[0005] 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 microplate ELISA tests, one-step immunoassay systems, and fully automated systems.

[0006] One-step immunological methods are widely used by manufacturers due to their speed and simplicity. A large part of these systems is based on the principle of immunochromatography. The device consists of a plastic support containing a membrane impregnated with gold particles or latex 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, indicating the probable presence of E. coli O157 in the food. An example is the "VIP EHEC" test (BioControl, Montesson, France), which allows, after enrichment for several hours, to visualize whether a given sample is contaminated or not by E. coli O157. However, this method does not allow the E. coli O157 strain to be identified in the sample, nor to see its pathogenic nature.

[0007] ELISA / ELFA systems are immunological methods that provide results in 2 hours in microplates after an enrichment phase (usually lasting 24 hours). The bioMérieux® company has developed automated ELISA (ELFA) kits based on VIDAS® technology. These methods have the disadvantage of not directly isolating the suspect colony in the event of a positive test. 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 gave a positive result.

[0008] There are also ELISA methods for the detection of non-0157 STEC, which are primarily based on the detection of Shiga toxin production, possibly after an enrichment step. Some of these use monoclonal or polyclonal antibodies directed against the STX toxin, and detection with antibodies linked to alkaline phosphatase. Among the available ELISA kits, we can mention the "Premier EHEC" targeting Shiga toxins (Meridian Diagnostics Inc, USA). Kits using the "reverse passive agglutination assay" (RPLA) technique adapted to the detection of STX toxins have also been designed 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.

[0009] The immunomagnetic separation (IMS) method has also been adapted for the detection and isolation of strains belonging to the 5 major STEC serogroups. It is carried out in liquid medium and is performed using magnetic beads coated with antibodies directed against the 026, 0111, 0103 and 0145 antigens. However, strains isolated by this method must be confirmed for the presence of the stx gene before being considered as STEC strains.

[0010] There is therefore a real need to develop a reliable and rapid method for isolating and identifying target bacteria, particularly pathogenic bacteria. SUMMARY OF THE INVENTION

[0011] A first subject of the invention relates to a gelled reaction medium for the detection, identification, counting 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.

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

[0013] In a very advantageous way, the medium thus makes it possible to locate on the culture medium the targeted colony releasing said component. This allows for better detection of the target microorganism and a very valuable time saving in the field of microbiological control, particularly during the confirmation stage.

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

[0015] Advantageously, the medium according to the invention comprises a toxin inducer. Thus, the medium according to the invention makes it possible in a very advantageous manner to detect Shiga toxin-producing E. coli strains (STEC).

[0016] Another subject of the invention relates to a method for detecting, identifying, counting and / or isolating a target microorganism in a sample likely to contain it, comprising the following steps: - Bringing said sample into contact with a reaction medium according to the invention - Incubate - Detect the presence of said target microorganism.

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

[0018] Figure 1 is a photograph of a culture medium according to the invention allowing the detection of the E. coli 026 strain, comprising a phage protein coupled to a gold nanoparticle.

[0019] Figure 2 is a photo of a culture medium according to the invention allowing the detection of the E. coli 0111 strain, comprising an antibody coupled to a gold nanoparticle.

[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.

[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 antibody anti STX1 coupled to silver nanoparticles, in the presence of mitomycin as a toxin inducer

[0022] Figure 5 is a photo 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

[0023] Figure 6 is a photo 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

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

[0025] A first subject of the invention relates to a gelled reaction medium for the detection, identification, counting 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.

[0026] In other words, the invention relates to a gelled reaction medium for the detection, identification, counting 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. Quite surprisingly, it has been found that it is possible to detect a target microorganism using a gelled reaction medium comprising an agglutinating conjugate.

[0027] The term "reaction medium" means a medium comprising all the elements necessary for the expression of a metabolism, the survival and / or growth of microorganisms. This reaction medium can either be a microbiological culture medium or a microbiological revelation medium. In the latter case, the culture of the microorganisms can be carried out beforehand in another medium. The reaction medium can also be brought into contact with an agar culture medium. It can be placed under or on the culture medium which allows the growth of the target microorganisms. The reaction medium can be added after incubation of the culture medium. Prior to its use, the reaction medium can be dehydrated. The reaction medium can be in the form of a pad.

[0028] According to the present invention, the reaction medium is gelled. It is in solid or semi-solid form. Agar is the traditional gelling agent used in microbiology for the cultivation of microorganisms, but it is possible to use other gelling agents such as, for example, gelatin, agarose as well as other natural or artificial gelling agents. Thus, against all expectations, the conjugate is capable of forming a network with the target component in a gelled medium. And surprisingly, this network is visually detectable by the formation of a halo without it being necessary to reduce the hardness of a conventional semi-solid reaction medium. Thus it is not necessary to modify the physicochemical properties of the reaction medium, such as hardness, to allow on the one hand the diffusion of the targeted components and on the other hand the reaction with the conjugates.

[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 a basis for the reaction medium according to the invention. The reaction medium may further comprise possible additives such as, for example, amino acids, peptones, one or more growth factors, carbohydrates, nucleotides, minerals, vitamins, one or more selective agents, inducers, toxin inducers, buffers, etc. By "selective agent" is meant any compound capable of preventing or slowing the growth of a so-called "non-target" microorganism, i.e. other than the target microorganism(s).The term "inducer" refers to a compound capable of inducing the expression of a compound, such as an enzyme or toxin, which would normally remain unexpressed.

[0030] Said reaction medium may also comprise a dye. For information purposes, dyes that may be mentioned include Evans blue, neutral red, sheep blood, horse blood, an opacifier such as titanium oxide, nitroaniline, malachite green, brilliant green. When the reaction medium according to the invention further comprises an enzymatic substrate specific for an enzymatic activity of at least one target microorganism, a chromogenic and / or fluorogenic substrate is preferably used. By "chromogenic and / or fluorogenic substrate" is meant a substrate allowing the detection of an enzymatic or metabolic activity of the target / desired microorganisms using a detectable signal. The reaction medium according to the invention may additionally comprise a pH indicator, sensitive to the variation in pH induced by the consumption of the substrate and revealing the metabolism of the target microorganisms. Said pH indicator may be a chromophore or a fluorophore.Examples of chromophores include bromocresol purple, bromothymol blue, neutral red, aniline blue, bromocresol blue.

[0031] A person skilled in the art can also use a Petri dish divided into segments, such as a bi-dish, or a tri-dish, making it possible to easily compare several media, comprising different substrates or different selective mixtures, on which the same biological sample has been deposited. According to the present invention, the reaction medium comprises a specific binding partner of a component of a target microorganism or of a component derived from said microorganism, coupled to a nanoparticle. The specific binding partner is chosen from antibodies, all types of Fab fragments, recombinant proteins, phages, phage proteins, oligonucleotides, aptamers, affimers or any other ligand or anti-ligand well known to those skilled in the art. According to the present invention, the reaction medium comprises a specific binding partner which is not bound to said component of the target microorganism or the component derived from said microorganism. Only when using said medium, i.e. when seeding the sample onto the reaction medium, can the specific binding partner bind to the component of a target microorganism or a component derived from said microorganism.

[0032] Preferably the antibody is a monoclonal antibody or a monoclonal antibody fragment.

[0033] According to the present invention, the binding partner is specific for a component of a target microorganism. The component of the target microorganism is a component released by said microorganism. The component can thus be an element from the surface of the bacteria such as a protein, a Lipopolysaccharide (LPS), a flagellum. It can also be an element internal to the bacteria such as RNA, an intracellular protein which can be detected during the death of a part of the bacterial colony during its 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 hybridizing in a complementary manner to the targeted RNA.

[0034] In another particular embodiment, the binding partner is specific for a component derived from said microorganism. This component may be a molecule of interest produced by the target microorganism such as a protein, an antibiotic, a molecule of resistance to antimicrobial agents, enzymes such as proteases, lipases or glucoidases.

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

[0036] The term "nanoparticle" refers to particles in the order of magnitude of a nanometer. Nanoparticles can be chosen from gold, iron, silver, copper, carbon, latex, silicon, aluminum. Preferably, the nanoparticles are colloidal nanoparticles chosen for their optical property, namely their ability to distinguish themselves when a network is formed. Even more preferably, the nanoparticle is chosen from gold, silver, copper. Thus, when the nanoparticles are made of gold, they change color, for example from red to gray when they form a network. When they are not aggregated, the wavelength of the absorbed light is in the red around 530nm. When they are aggregated, the absorbed wavelength changes from red to blue / grey around 600 to 700nm. 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.

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

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

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

[0040] Advantageously, nanoparticles can make it possible to visualize agglutination around a bacterial colony whose size does not yet allow it to be visible to the naked eye. Detection can thus be earlier. Advantageously, nanoparticles can make it possible to visualize agglutination around a bacterial colony whose translucent appearance does not allow detection by an automated reading device. Detection can thus be facilitated.

[0041] The coupling of the nanoparticle to the binding partner can be done either by direct attachment or by indirect attachment. Direct attachment means attachment by adsorption or by covalent bonding. Indirect attachment means attachment by the interaction of ligands / anti-ligands such as biotin / strepatividin or other pairs well known to those skilled in the art. Depending on the type of bond chosen, the person skilled in the art will adapt the physicochemical conditions of the reaction medium and in particular its pH.

[0042] According to the present invention, the conjugate is agglutinating, that is to say that it causes the formation of an agglutination network in the presence of a component of a target microorganism or a component originating from said microorganism. The component being multi-epitope, several conjugates will bind to this component and form an agglutination. By agglutination, we mean the result of an interaction between at least one component of a target microorganism or at least one component from said microorganism with binding partners coupled to a nanoparticle. Agglutination reactions include immunological reactions, such as antigen-antibody reactions or more generally specific interactions between two molecules. Through this interaction, components and conjugates aggregate, adhere to each other and form a network in the reaction medium. In practice, several parameters influence the capacity of the conjugate to be agglutinating in a gelled medium, such as mainly: - the porosity of the gelled medium - the size of the nanoparticles - the quantity of binding partners - the quantity of nanoparticles It will therefore be necessary to adapt these parameters in order to allow satisfactory agglutination allowing its detection. Advantageously, the network formed by said specific reaction is then detected either visually or automatically using an optical system. The colony of the target microorganism is thus identified. Preferably, the network forms a halo in the gelled reaction medium detectable either visually or using an optical system. The network or halo thus circumscribes the colony which can then be advantageously differentiated and / or identified within a population.

[0043] For the purposes of the present invention, the term "at least one target microorganism" means at least one microorganism that it is desired to detect and / or identify and / or count. Preferably, the microorganism is chosen from 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, Streptococcus agalactiae. Preferably, the microorganism is chosen from E. coli strains, and preferably from Shiga toxin-producing E. coli strains (STEC). Enterohemorrhagic E. coli (EHEC) are strains representing a subgroup of Shiga toxin STX-producing Escherichia coli (STEC), which have acquired the eae gene and cause hemolytic uremic syndrome.The simultaneous presence of these two virulence factors makes this pathovar very virulent for humans. These include serogroups 026, 045, 080, 0103, 0111, 0121, 0145 and 0157.

[0044] In a particular embodiment, the reaction medium comprises an inducer causing or increasing the expression of a molecule of interest produced by the target microorganism. This molecule of interest may be a protein, an antibiotic, a molecule of resistance to antimicrobial agents, a protease-type enzyme, a lipase or a glucidase. Thus, the present invention allows the characterization or selection of microbial strains with respect to their capacity to produce these molecules of interest. In a particular embodiment, the present invention may relate to the field of bioproduction, and more particularly 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 allows the detection of resistant pathogenic bacteria.

[0045] In a particular embodiment, the reaction medium comprises a toxin inducer. The toxin inducer causes stress in the bacteria which will trigger the lytic cycle of the prophages in the bacteria and therefore stimulate the production of toxins which will be released. For example, toxins secreted by Staphylococcus aureus or shiga toxins secreted by STEC strains can be mentioned. There are two main types of shiga toxins: STX1 and STX2 which themselves have many variants. To date, STX1 has 4 subtypes STX1a, STX1c, STX1d, STX1e, and STX2 has 12: STX2a, STX2b, STX2c, STX2d, STX2e, STX2f, STX2g, STX2h, STX2i, STX2j, STX2K, STX2I. Advantageously, the toxin inducer is chosen from antibiotics or a physicochemical stress.Examples of antibiotics that may be mentioned are trimethoprim, sulfamethoxazole, norfloxacin, azithromycin, gemtamicin, polymyxin B, chloramphenicol, streptomycin, chlortetracycline, oxytetracyline, tylosin, mitomycin C, carbodox, oliquindox, rifampincin, imipenem, ciprofloxacin, cotrimoxazole, penicillin G, linlomycin. In a preferred embodiment, the medium according to the invention comprises ciprofloxacin at a concentration of between 0.005 and 0.030 mg / l. In another preferred embodiment, the medium according to the invention comprises mitomycin C at a concentration of between 0.10 and 0.5 mg / l.

[0046] The toxin inducer may also be a physicochemical stress achieved, for example, by the addition of salt or EDTA or by a change in pH or by UV stress. Stress may also be, for example, induced by the addition of noradrenaline. Said target microorganism is likely to be present in a sample. A "sample" means a small part or small quantity isolated from an entity for analysis. The sample may be of industrial origin, i.e., according to a non-exhaustive list, an air sample, a water sample, a sample taken from a surface, a part or a manufactured product, a product of food origin. Samples of food origin include, but are not limited to, a sample of products dairy products (yogurts, cheeses, etc.), meat, fish, eggs, fruits, vegetables, water, drinks (milk, fruit juice, soda, etc.). A food sample can finally come from feed intended for animals, such as animal or vegetable flours. The sample can be of biological origin, either animal or human. It can then correspond to a sample of biological fluid (stools, urine, whole blood, serum, plasma, cerebrospinal fluid, organic secretion, etc.), an external sample (skin, nose, throat, etc.) or tissue or isolated cells. The sample can be used as is or, prior to analysis, undergo preparation such as enrichment, extraction, concentration, purification, according to methods known to those skilled in the art.The reaction medium according to the invention can be all the more interesting when the sample is a polymicrobial sample or one loaded with additional flora, such as for example food samples such as raw milk cheese or stools in clinical samples.

[0047] Another subject of the present invention relates to a diagnostic kit allowing the preparation of a reaction medium according to the invention comprising - an agglutinating conjugate as described in the present invention - a gelling medium.

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

[0049] Thus, another subject of the present invention relates to the method for obtaining a reaction medium according to the invention comprising the steps of bringing an agglutinating conjugate into contact with a gelling medium to form the reaction medium according to the invention. The preparation of the conjugate is carried out by coupling the binding partner with the nanoparticle. These coupling methods are well known to those skilled in the art (Nicholas G. Weich et al, 2017). The conjugate is then added to the supercooled gelled medium. The whole is then homogenized and poured into the Petri dish.

[0050] Another subject of the present invention relates to a method of in vitro microbiological culture, in which microorganisms likely to be present in a sample are seeded in or onto a culture medium according to the invention. The seeded culture medium is incubated under appropriate conditions known to those skilled in the art. The seeding is carried out according to conventional microbiology techniques. It can also be carried out in the mass, in other words by inclusion.

[0051] Another subject of the present invention relates to a method for detecting, identifying, counting and / or isolating at least one target microorganism in a sample likely to contain it, comprising the following steps: - -bringing said sample into contact with a reaction medium according to the invention - Incubate - detect the presence of said target microorganism.

[0052] By "detection" is meant the detection by the naked eye or using an optical device of the existence of growth of the target microorganisms, preferably target bacteria. When the reaction medium from which it is desired to detect the target microorganisms comprises a chromogenic or fluorogenic substrate, the detection may be carried out using an optical device for fluorogenic substrates, or by the naked eye or using an optical device for chromogenic substrates.

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

[0054] “Counting at least one target microorganism” means counting / quantifying the number of target microorganisms, for example the number of bacterial colonies when the target microorganism is a bacterium.

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

[0056] Microbiological control corresponds to the analysis of a sample with the aim of detecting microorganisms likely to be present within said sample. Thus, quite surprisingly, it was found that a medium according to the invention made it possible to detect a target bacterium and to isolate it. Advantageously, the medium according to the invention can thus make it possible to 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 said 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.

[0057] Without this being limiting, it turns out 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, counting and / or isolation of a strain of E. coli, comprising a phage protein specific for the LPS of said strain coupled to a nanoparticle to form a conjugate.

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

[0059] Preferably, in this particular embodiment, the nanoparticle is made of gold, of a size between 20 and 90 nm and at a concentration between 10 10 and 10 12 nanoparticles / ml of reaction medium.

[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 the agglutinating conjugates have been added.

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

[0062] Preferably, in this particular embodiment, the quantity of monoclonal antibody makes it possible to cover at least half of the surface of the nanoparticle, and even more preferably to cover between a third and half of the surface of the nanoparticle.

[0063] Preferably, in this particular embodiment, the nanoparticle is made of gold, of a size between 20 and 90 nm and at a concentration between 10 10 and 10 12 nanoparticles / ml of reaction medium.

[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 for Stx1 and / or Stx2, said at least one antibody being coupled to a nanoparticle to form an agglutinating conjugate. In other words, 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 agglutinating conjugate formed by at least one antibody specific for Stx1 and / or Stx2 coupled to a nanoparticle.

[0065] This embodiment advantageously makes it possible to identify a strain of E. coli producing Shiga toxin by the formation of a halo around said strain. Indeed, the conjugates formed from antibodies coupled to the nanoparticles have agglutinated around said colony. It is thus easy to visualize the target colony.

[0066] In this embodiment, said at least one antibody has an amount making it possible to cover at least half of the surface of the nanoparticle and preferably at least one third of the surface of the nanoparticle. In these particular embodiments, the medium comprises ciprofloxacin, at a concentration of between 0.005 and 0.030 mg / l. In a variant of these particular embodiments, the medium comprises mitomycin C, at a concentration of between 0.10 mg / l and 0.50 mg / l.

[0067] Preferably, in these variants the nanoparticle is a gold nanoparticle of size between 20 and 90nm and at a concentration between 10 10 and 10 12 nanoparticles / ml of reaction medium.

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

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

[0070] Example 1: Detection of E. coli 026 with a medium according to the invention comprising a phage protein coupled to a gold nanoparticle

[0071] Preparation of gold nanoparticles

[0072] The 40 nm nanoparticles are manufactured by reducing gold chloride with sodium citrate (method described by Turkevich and Frens in 1951). Thus, the 20 nm gold nanoparticles are manufactured using a solution of gold chloride diluted in distilled water, to which trisodium citrate is added and then brought to a boil. The presence of an absorbance peak at 517-519 nm ensures the correct particle size. From these 20 nm particles, 40 nm particles are synthesized. For this, the 20 nm particles are diluted in distilled water and then brought to a boil with the addition of trisodium citrate and gold chloride. The absorbance peak is then observed at 524-526 nm when cold, indicating the increase in particle size.

[0073] Preparation of the conjugate:

[0074] The phage evaluated in this test is Phage Eco 026 BP1, directed against the LPS of Escherichia coli 026. This phage comes from the Applicant's collection. The adsorption of the phage protein on the nanoparticles is carried out taking into account the value of its isoelectric point and according to the methods known to those skilled in the art (Nicholas G. Welch et al): The above phage is coupled in the following manner - Dilution of 270 pg of phage proteins in 3 ml of Tris HCL (0.025M) pH 6 - Addition of 30 ml of gold at an optical density of 1 or 7.7x10 10 nanoparticles / ml previously adjusted to pH 6 with 0.1 M K2CO3 solution - Stirring for 20 minutes - Passivation by adding 3 ml of BSA 10% - Centrifugation at 8600 g for 30 min - Removal of the supernatant and measurement of the concentrate obtained using a spectrophotometer.

[0075] Preparation of the reaction medium according to the invention: The produced conjugate 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 dish is then poured. The dishes are then dried.

[0076] Seeding: In this example, a strain of E. coli 026 and a strain of E. coli 0111 (applicant's collection) are inoculated and incubated for 72 hours at 37°C.

[0077] Results A gray halo is visually observed around the colony containing LPS 026 (Figure 1). This gray change results from a network formed between the LPS released by the colonies and the conjugates present in the agar.

[0078] Example 2: Detection of E. coli 0111 with a medium according to the invention comprising an antibody coupled to a gold nanoparticle

[0079] Preparation of gold nanoparticles The 40nm gold particles are fabricated as shown in Example 1.

[0080] Preparation of the conjugate

[0081] The monoclonal antibody 2H5E9 IgG anti-Eco / / 0111 comes from the Applicant's collection.

[0082] The adsorption of the antibody on the nanoparticles is done as follows: - Dilution of 90 pg of antibody in 3 ml of Tris HCL (0.025M) pH 6.5 - Addition of 30 ml of gold to a DO 1 previously adjusted to pH 8 with a 0.1 M K2CO3 solution - Stirring for 20 minutes - Passivation by adding 3 ml of BSA 10% - Centrifugation at 8600 g for 30 min - Removal of the supernatant and measurement of the concentrate obtained using a spectrophotometer

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

[0084] The produced conjugate 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 dish is then poured. The dishes are then dried.

[0085] Seeding

[0086] In this example, a strain of E. coli 026 and a strain of E. coli 0111 from the Applicant's collection are inoculated and incubated for 72 hours at 37°C.

[0087] Result

[0088] No gray halo is observed around colony 026, whereas it is clearly visible around colony 0111 (figure 2), allowing the latter to be identified.

[0089] Example 3: Detection of an E. coli producing an STX1 toxin with a medium according to the invention comprising an anti-Stx1 antibody coupled to a gold nanoparticle and ciprofloxacin as a toxin inducer

[0090] Preparation of gold nanoparticles The 40 nm nanoparticles are manufactured as described in Example 1.

[0091] Preparation of the conjugate The antibody evaluated in this assay is the IgG antibody (ref ATCC 13C4 hybridoma CRL-1794) directed against the STX1 toxin. The adsorption of the antibody on the nanoparticles is done as follows: - Dilution of 90 pg of antibody in 3 ml of Tris HCL (0.025M) pH 8 - Addition of 30 ml of gold to a DO 1 previously adjusted to pH 8 with a 0.1M K2CO3 solution - Stirring for 20 minutes - Passivation by adding 3 ml of BSA 10% - Centrifugation at 8600 g for 30 min - Removal of the supernatant and measurement of the concentrate obtained using a spectrophotometer Preparation of the medium according to the invention Agar Supplementation: - The produced conjugate is added to supercooled agar (chrom ID coli ref 42017 bioMerieux) at 50°C containing the toxin inducer, here ciprofloxacin at 10 ng / mL, in order to obtain a nanoparticle concentration of OD 3. 18 ml of medium per dish are then poured. The dishes are then dried.

[0092] Seeding 4 strains from the Applicant's internal collection, three of which possess the stx1 gene, are inoculated and incubated for 24 hours at 37°C. Conclusion : A gray halo is observed around the STX1 toxin-producing colonies (Figure 3). STX1 toxin is detected. This gray change results from a network formed between the toxins released by the colonies and the conjugates present in the agar.

[0093] Example 4: Detection of an E. coli producing STX1 toxin in a medium according to the invention comprising an anti-Stx1 antibody coupled to silver nanoparticles and mitomycin as a toxin inducer

[0094] Preparation of silver nanoparticles

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

[0096] Preparation of the conjugate The adsorption of the antibody (ref ATCC 13C4 hybridoma CRL-1794) on the nanoparticles is carried out as described in example 3.

[0097] Preparation of the medium according to the invention

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

[0099] Seeding Two strains from the Applicant's internal collection, one of which has the stx1 gene, are inoculated and incubated for 24 hours at 37°C.

[0100] Results A gray halo is observed around the strain producing the STX1 toxin (Figure 4, right colony). This gray change results from a network formed between the toxin released by the colonies and the conjugates present in the agar.

[0101] Example 5: Detection of an E. coli producing Stx1 and / or Stx2 toxins in a medium according to the invention comprising an anti-Stx1 antibody coupled to a silver nanoparticle, an anti-Stx2 antibody coupled to a gold nanoparticle, in the presence of mitomycin C

[0102] Preparation of silver or gold nanoparticles

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

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

[0105] Preparation of the conjugate

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

[0107] The adsorption of the 13C4 anti-stx1 antibody on the silver nanoparticles is carried out according to example 4:

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

[0109] The conjugates are added to 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 nanoparticles of an OD 3. The medium is then poured into the dishes and dried.

[0110] Seeding:

[0111] Two strains from the Applicant's collection, one possessing the stx1 gene and the other the stx2 gene, are inoculated and incubated for 24 hours at 37°C.

[0112] Results

[0113] The reaction medium with the conjugates has a light red color. We observe the presence of a halo around each of the 2 strains (Figure 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, we have the formation of a network between the toxin and the gold nanoparticles, causing the gold nanoparticles to turn from red to gray and causing a yellow halo due to the presence of silver nanoparticles that have not aggregated (colony on the left in Figure 5). In the case of STX1 toxin production, a network is formed between the toxin and the silver nanoparticles, causing the silver nanoparticles to turn from yellow to gray and causing a more intense red halo (light red + gray) due to the presence of gold nanoparticles that have not aggregated (colony on the right in Figure 5).

[0114] Example 6: Detection of a target E. coli producing Stx1 and / or Stx2 toxin in a medium according to the invention comprising an anti-Stx1 or anti-Stx2 antibody coupled to the same gold nanoparticle in the presence of ciprofloxacin as a toxin inducer

[0115] Preparation of nanoparticles

[0116] The 40 nm nanoparticles according to example 1

[0117] Preparation of conjugates

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

[0119] This mixture is adsorbed on the nanoparticles according to the previous examples at a pH of 8

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

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

[0122] Seeding

[0123] 5 strains possessing or not the stx1 and / or stx2 genes are inoculated and incubated for 24 hours at 37°C.

[0124] Conclusion: A gray halo is observed around colonies producing STX1 and / or STX2 toxins (Figure 6). This gray change comes from a network formed between the toxins released by the colonies and the conjugates present in the agar. Example 6: Detection of an E. coli strain producing the STX1 toxin by seeding into the mass

[0125] Preparation of nanoparticles

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

[0127] Preparation of conjugates

[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

[0129] Preparation of the medium according to the invention

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

[0131] Seeding

[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.

[0133] Conclusion : In the case of colony 024, a gray halo is present. This color change comes from a network formed between the STX1 toxins released by the colonies and the conjugates present in the agar. BIBLIOGRAPHICAL REFERENCES

[0134] 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;

[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 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.

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

3. Gelled reaction medium according to claim 1 characterized in that it is a microbiological detection medium.

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 / l and 0.50 mg / l.

14. Reaction medium according to any one of the preceding claims for the detection, identification, enumeration and / or isolation of at least one target microorganism selected from 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, Streptococcus agalactiae.

15. Diagnostic kit for preparing a reaction medium according to any one of the preceding claims comprising - an agglutinating conjugation - a gelling medium

16. Preparation of a reaction medium according to claims 1 to 15 comprising the step of contacting an agglutinating conjugate with a gelling medium to form the reaction medium.

17. In vitro microbiological culture method, wherein microorganisms likely to be present in a sample are inoculated into or onto a culture medium according to any one of claims 1 to 14.

18. Gelled reaction medium according to any one of claims 1 to 14 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 an agglutinating conjugate.

19. Gelled reaction medium according to claim 18 characterized in that the quantity of LPS-specific phage protein covers at least half of the surface of the nanoparticle.

20. A gelled reaction medium according to claim 18 or 19 characterized in that the nanoparticle is gold, of size between 20 and 90 nm and at a concentration between 10 10 and 10 12 nanoparticles / ml of reaction medium.

21. Gelled reaction medium according to any one of claims 1 to 14 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 an agglutinating conjugate.

22. A gelled reaction medium according to claim 21 for the detection, identification, enumeration and / or isolation of at least one Shiga toxin-producing E. coli strain comprising at least one toxin inducer, at least one STX1-specific antibody and / or at least one antibody specific to STX2, said at least antibody being coupled to a nanoparticle to form an agglutinating conjugate.

23. Gelled reaction medium according to claim 22 for the detection, identification, enumeration and / or isolation of at least one Shiga toxin-producing E. coli comprising ciprofloxacin, at a concentration between 0.005 and 0.030 mg / l.

24. Gelled reaction medium according to claim 22 for the detection, identification, enumeration and / or isolation of at least one Shiga toxin-producing E. coli comprising mitomycin C, at a concentration between 0.10 mg / l and 0.50 mg / l.

25. A gelled reaction medium according to any one of claims 21 to 24 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 between 10 10 and 10 12 nanoparticles / ml of reaction medium.

26. A method for detecting, identifying, enumerating and / or isolating a target microorganism in a sample likely to contain it, comprising the following steps: - To bring said sample into contact with a reaction medium according to any one of claims 1 to 14 or 18 to 25. - Incubate - Detect the presence of said target microorganism.

27. ​​Method according to the preceding claim wherein detection is achieved by the appearance of a halo around the target microorganism on the reaction medium according to any one of claims 1 to 14 or 18 to 25. Tl

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