Reaction medium for detecting a target

Abstract

The present invention relates to a gelling reaction medium for detecting, identifying, counting and / or isolating at least one target microorganism in a sample possibly containing the target microorganism, said medium comprising a component of the target microorganism or at least one specific binding partner of a component derived from said microorganism, the present invention relates to a conjugate that is conjugated to at least one nanoparticle to form at least one agglutination conjugate.

Description

Technical Field

[0001] The present invention generally relates to the field of microbial control, such as microbial control of industrial or clinical source samples. More specifically, the present invention relates to a reaction medium and related methods for detecting, identifying, counting and / or isolating target microorganisms. Prior art

[0002] Microbiological control of samples of various origins requires the implementation of technologies that can detect microorganisms, for example for identification and / or enumeration and / or biochemical characterization purposes, and the results must be obtained as quickly as possible.

[0003] In medicine, it is necessary to predict and diagnose infection risks: the faster and more accurate the diagnosis, the more effective the patient can be managed and the lower the risk of transmission. For animal health in veterinary medicine, the approach is similar.

[0004] In the agri-food sector, the issues are the same. However, a distinction is made between the following microorganisms:

[0005] - Pathogenic microorganisms such as Shiga toxin-producing bacteria (STEC), Salmonella, Listeria, Cronobacter, Bacillus, Staphylococcus, the detection of which is applicable to starting materials, intermediate products and finished products for sale,

[0006] - non-pathogenic microorganisms, used as quality indicators in the production process, from the starting raw materials to the finished product, throughout the entire chain,

[0007] - technologically interesting bacteria, such as starter cultures,

[0008] -Microorganisms as markers of contamination.

[0009] Rapid and accurate detection of suspected contamination (within a food batch) allows it to be monitored so that corrective action can be taken quickly.

[0010] Technically, one of the main difficulties is being able to isolate the bacteria of interest in order to identify them. Generally speaking, microbiological analysis is a two-step process. The first step is the detection phase, which may involve many techniques such as culture media, immunoassays and molecular biology. Subsequently, especially in the field of agricultural products, there may be a confirmation phase to confirm the presence of the pathogen of concern and compliance with current standards in the field. Therefore, the confirmation step involves an additional step, which again requires the isolation of the bacteria of interest.

[0011] Thus, in the case of Shiga toxin-producing Escherichia coli (E. coli) (STEC), diagnosis relies on the use of selective chromogenic agars such as SMAC (Sorbitol MacConkey Agar), RMAC (Rhamnose MacConkey Agar) or Rainbow Agar O157 to select for the growth of certain bacteria and to stain the strains of interest. Ultimately, however, none of these agars are sufficiently specific for STEC without culture to isolate and confirm the identity of the strain using PCR-type molecular biology tests. Specifically, their pathogenicity involves the expression of several virulence genes, in particular stx1 and stx2, which encode two Shiga toxins: STX1 and STX2. These two toxins act by inhibiting protein synthesis in eukaryotic cells, ultimately leading to apoptosis. In order to accurately identify pathogenic STEC, PCR must be performed for the genes stx1, stx2 or eae (another virulence factor). Therefore, these processes are time-consuming and laborious, and bacteria carrying both genes (stx and eae) are not systematically found in culture.

[0012] Immunological methods are also available. A large number of tests are available for the detection of E. coli O157:H7 in food and / or environmental samples. These systems include classic microplate ELISA tests, one-step immunological systems, and fully automated systems.

[0013] "One-step" immunological methods are widely used in industry due to their rapid implementation 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 for E. coli O157:H7 (i.e. O157 and possibly H7), a sample well, and test and control windows. The appearance of a colored line in the test window indicates a positive result, indicating the possible presence of E. coli O157 in the food. As an example, one can mention the "VIP EHEC" test (BioControl, Montesson, France), which allows to visualize, after several hours of enrichment, whether a given sample is contaminated with E. coli O157. However, with this method it is not possible to localize E. coli O157 in the sample, nor to determine its pathogenicity.

[0014] The ELISA / ELFA system is an immunological method that provides results within 2 hours in a microplate format after an enrichment phase (usually lasting 24 hours). The company has developed a The disadvantage of these methods is that in the case of a positive test, the suspected colony cannot be directly isolated. Therefore, it is necessary to culture the sample to isolate the positive colony and confirm by PCR that this bacterium is indeed the one that caused the positive test.

[0015] ELISA methods can also be used to detect non-O157 STEC, which are mainly based on the detection of Shiga toxin production, possibly after an enrichment step. Some of these methods use monoclonal or polyclonal antibodies against STX toxins and visualize using alkaline phosphatase-linked antibodies. Among the available ELISA kits, it can be mentioned that "Premier EHEC" (Meridian Diagnostics Inc., USA) for Shiga toxins. A kit using a reverse passive agglutination assay (RPLA) technology suitable for STX toxin detection has also been developed and sold. The technology uses beads coated with antibodies that interact with Shiga toxins to produce a diffuse layer at the bottom of the wells where the culture supernatant is present. These methods then require the separation of positive colonies, followed by a confirmation step.

[0016] The immunomagnetic separation (IMS) method is also suitable for detecting and isolating strains belonging to the five major STEC serogroups. It is performed in liquid culture medium and uses magnetic beads coated with antibodies against antigens O26, O111, O103, and O145. However, strains isolated by this method must be confirmed for the presence of the stx gene to be considered STEC strains.

[0017] Therefore, there is an urgent need to develop a reliable and rapid method to isolate and identify target bacteria, especially pathogenic bacteria. Summary of the invention

[0018] The first subject of the present invention relates to a gelled reaction medium for detecting, identifying, counting and / or isolating at least one target microorganism in a sample that may contain the target microorganism, wherein the reaction medium comprises a component of the target microorganism or at least one specific binding partner derived from a component of the microorganism, wherein the at least one specific binding partner is coupled to at least one nanoparticle to form at least one agglutination conjugate.

[0019] The component used to detect the target microorganism is a component released by the microorganism into the reaction medium. It can be a component of the target microorganism, such as lipopolysaccharide (LPS), or a component produced by the target microorganism, such as a toxin.

[0020] Very advantageously, the culture medium thus makes it possible to localize the target colonies releasing the components on the culture medium. This improves the detection of the target microorganisms and saves very valuable time in the field of microbiological control, in particular during the confirmation step.

[0021] Preferably, the conjugate comprises colloidal nanoparticles having optical properties.

[0022] Advantageously, the culture medium according to the invention comprises a toxin inducing agent.Thus, the culture medium according to the invention provides a very advantageous means for detecting Shiga toxin producing Escherichia coli (STEC) strains.

[0023] Another subject of the present invention relates to a method for detecting, identifying, counting and / or isolating target microorganisms in a sample that may contain the target microorganisms, comprising the following steps:

[0024] - contacting the sample with a reaction medium according to the invention

[0025] - Incubation

[0026] - Detecting the presence of said target microorganism.

[0027] Advantageously, detection is performed by the appearance of a halo around the target microorganism on the reaction medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a photograph of a culture medium for detecting strain Escherichia coli O26 according to the present invention, which contains bacteriophage proteins coupled to gold nanoparticles.

[0029] Figure 2 is a photograph of a culture medium for detecting strain Escherichia coli O111 according to the present invention, which contains antibodies coupled to gold nanoparticles.

[0030] Figure 3 is a photograph of a culture medium for detecting STX1 toxin-producing target bacteria according to the present invention, which contains anti-STX1 antibodies coupled to gold nanoparticles, and in the presence of ciprofloxacin as a toxin inducer.

[0031] Figure 4 is a photograph of a culture medium for detecting STX1 toxin-producing target bacteria according to the present invention, which contains anti-STX1 antibodies coupled to silver nanoparticles, and in the presence of mitomycin as a toxin inducer.

[0032] Figure 5 is a photograph of a culture medium for detecting target bacteria producing STX1 and / or STX2 toxins according to the present invention, which contains anti-STX1 antibodies coupled to silver nanoparticles, anti-STX2 antibodies coupled to gold nanoparticles, and the presence of mitomycin.

[0033] Figure 6 The culture medium for detecting STX1 and / or STX2 toxin-producing target bacteria according to the present invention comprises anti-STX1 antibody or anti-STX2 antibody coupled to the same gold nanoparticles, and ciprofloxacin is present as a toxin inducer. DETAILED DESCRIPTION

[0034] Certain terms and expressions used in the context of the present invention are described in detail below.

[0035] The first subject of the present invention relates to a gelled reaction medium for detecting, identifying, counting and / or isolating at least one target microorganism in a sample that may contain the target microorganism, the reaction medium comprising at least one specific binding partner of a component of the target microorganism or a component derived from the microorganism, coupled to at least one nanoparticle to form at least one agglutination conjugate.

[0036] In other words, the present invention relates to a gelling reaction medium for detecting, identifying, counting and / or isolating at least one target microorganism in a sample that may contain the target microorganism, comprising at least one agglutinating conjugate formed by coupling at least one specific binding partner of a component of the target microorganism or a component derived from the microorganism to at least one nanoparticle. Very surprisingly, it was found that the target microorganism can be detected using a gelling reaction medium comprising an agglutinating conjugate.

[0037] The term "reaction medium" refers to a medium containing all elements required for metabolic expression, survival and / or growth of microorganisms. This reaction medium can be a microbial medium or a microbial visualization medium. In the latter case, the microorganisms can first be cultured in another medium. The reaction medium can also be in contact with an agar medium. It can be placed below or above a medium that enables the target microorganism to grow. The reaction medium can be added after the incubation medium. Before use, the reaction medium can be dehydrated. The reaction medium can be in the form of a pad.

[0038] According to the invention, the reaction medium is gelled. It is in a solid or semi-solid state. Agar is a conventional gelling agent used in microbiology for the cultivation of microorganisms, but other gelling agents may also be used, such as gelatin, agarose and other natural or artificial gelling agents. Thus, contrary to all expectations, the conjugate is able to form a network with the target component in the gelled medium. It is also surprising that this network can be visually detected by the formation of a halo without reducing the hardness of the conventional semi-solid reaction medium. Therefore, it is not necessary to change the physicochemical properties of the reaction medium, such as hardness, to allow diffusion of the target component on the one hand and reaction with the conjugate on the other hand.

[0039] Many products are commercially available, such as Columbia agar, tryptone soy agar, MacConkey agar, Mueller Hinton agar, or more generally, those products described in the Microbiology Culture Medium Handbook. These culture media can be used as the basis of reaction culture media according to the present invention. Reaction culture media can also include 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. The term "selective agent" refers to any compound that can prevent or slow down the growth of "non-target" microorganisms (i.e., microorganisms other than the target microorganism). The term "inducer" refers to a compound that can induce a compound that is usually not expressed, such as an enzyme or toxin expression.

[0040] The reaction medium can also include a colorant. As a guide, Evans blue, neutral red, sheep blood, horse blood, sunscreens such as titanium oxide, nitroaniline, malachite green and brilliant green can be referred to as colorants. When the reaction medium according to the present invention also includes a specific enzyme substrate for the enzymatic reaction of at least one target microorganism, a colorimetric and / or fluorescent substrate is preferably used. The term "colorimetric and / or fluorescent substrate" refers to a substrate that can detect the enzymatic or metabolic activity of the target microorganism / microorganism of interest by a detectable signal. The reaction medium according to the present invention can also include a pH indicator that is sensitive to the pH changes induced by substrate consumption and displays the metabolism of the target microorganism. The pH indicator can be a chromophore or a fluorophore. Examples of chromophores that can be mentioned include bromocresol purple, bromothymol blue, neutral red, aniline blue and bromocresol blue.

[0041] A person skilled in the art can also use a culture dish divided into multiple sections, such as a double-section dish. or three-segment dish Thereby it is possible to easily compare several culture media, comprising different substrates or different selective mixtures, on which the same biological sample is deposited.

[0042] According to the present invention, the reaction medium comprises a specific binding partner of a component of the target microorganism or a component derived from the microorganism, coupled to the nanoparticle. The specific binding partner is selected 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 that is not combined with the component of the target microorganism or the component derived from the microorganism. Only when the culture medium is used, i.e. when the sample is inoculated on the reaction medium, the specific binding partner can be combined with the component of the target microorganism or the component derived from the microorganism.

[0043] Preferably, the antibody is a monoclonal antibody or a monoclonal antibody fragment.

[0044] According to the present invention, the binding partner is specific for a component of the target microorganism. The component of the target microorganism is a component released by the microorganism. Therefore, the component can be an element derived from the surface of the bacteria, such as a protein, lipopolysaccharide (LPS) or flagella. It can also be an element inside the bacteria, such as RNA or an intracellular protein, which can be detected when a part of the bacterial colony dies during the growth process. In a specific embodiment, the binding partner is specific for the RNA of the target microorganism. In this embodiment, the binding partner consists of at least two different primers that complementarily hybridize with the target RNA.

[0045] In another specific embodiment, the binding partner is specific for a component derived from the microorganism. Such a component may be a molecule of interest produced by the target microorganism, such as a protein, an antibiotic, an antimicrobial resistance molecule, a protease-type enzyme, a lipase or a glucosidase.

[0046] According to the invention, the binding partner is coupled to the nanoparticle. This final complex is called a conjugate. In the same reaction medium, there can be conjugates with binding partners of different properties, coupled to nanoparticles that themselves have different properties.

[0047] The term "nanoparticle" refers to nanometer-sized particles. Nanoparticles can be selected from gold, iron, silver, copper, carbon, latex, silicon and aluminum. Preferably, the nanoparticles are colloidal nanoparticles selected for their optical properties, i.e. their ability to be distinguished when forming a network. More preferably, the nanoparticles are selected from gold, silver and copper. Therefore, when the nanoparticles are gold, when they form a network, they change color, for example from red to gray. When they are not aggregated, the wavelength of light absorbed is in the red region of about 530nm. When they aggregate, the wavelength of absorption changes from red to blue / gray of about 600 to 700nm. In a specific embodiment, several nanoparticles of different colors can be used together. The network thus formed can distinguish several components of the target microorganism.

[0048] Preferably, the size of the nanoparticles is between 10 and 200 nm. Preferably, the size of the nanoparticles is between 20 and 90 nm, allowing for better mobility of the conjugate in the reaction medium.

[0049] Advantageously, the nanoparticles make it possible to reduce the amount of binding partner required for agglutination formation. Thus, the concentration of binding partner required for producing a reaction medium according to the invention requires 100-1000 times less binding partner than a medium without nanoparticles.

[0050] Preferably, the amount of binding partner required corresponds to the amount required to cover at least half of the nanoparticle surface, even more preferably between one third and half of the nanoparticle surface. This ratio enables the aggregated conjugate to form a network in the gelling reaction medium.

[0051] Advantageously, the nanoparticles can visualize agglutination around bacterial colonies whose size is not yet visible to the naked eye. Thus, detection is possible earlier. Advantageously, the nanoparticles can visualize agglutination around bacterial colonies whose translucent appearance does not allow detection by an automatic reader. Thus, detection can be facilitated.

[0052] The coupling of the nanoparticle to the binding partner can be carried out by direct or indirect connection. Direct connection refers to connection by adsorption or covalent bond. Indirect connection refers to connection by interaction between ligand / anti-ligand, such as biotin / streptavidin or other pairings known to those skilled in the art. Depending on the selected binding type, those skilled in the art will adjust the physicochemical conditions of the reaction medium, in particular its pH.

[0053] According to the present invention, the conjugate has agglutinative properties, i.e., it leads to the formation of an agglutination network in the presence of a component of the target microorganism or a component derived from the microorganism. Since the component is a multi-epitope, several conjugates will bind to the component and form an agglutination. The term agglutination refers to the result of the interaction between at least one component of the target microorganism or at least one component derived from the microorganism and a binding partner coupled to the nanoparticle. Agglutination reactions include immune reactions, such as antigen-antibody reactions, or more generally, specific interactions between two molecules. Through this interaction, the components and conjugates aggregate, adhere to each other and form a network in the reaction medium. In fact, several parameters affect the agglutination ability of the conjugate in the gelling medium, such as mainly:

[0054] - Porosity of the gelling medium

[0055] - Size of nanoparticles

[0056] -Amount of binding partner

[0057] -The amount of nanoparticles.

[0058] Therefore, these parameters should be adjusted to ensure satisfactory agglutination during detection. Advantageously, the network formed by the specific reaction is then automatically detected visually or using an optical system. The colonies of the target microorganisms are thereby located. Preferably, the network forms a halo in the gelling reaction medium, which can be detected visually or using an optical system. Thus, the network or halo confines the colonies, which can then be advantageously distinguished and / or identified within the population.

[0059] For the purposes of the present invention, the term "at least one target microorganism" refers to at least one microorganism to be detected and / or identified and / or counted. Preferably, the microorganism is selected from the group consisting of 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 selected from strains of Escherichia coli, preferably from strains of Shiga toxin-producing Escherichia coli (STEC). Enterohemorrhagic Escherichia coli (EHEC) are strains representing a subgroup of Shiga toxin-producing E. coli (STEC) that have acquired the eae gene and cause hemolytic uremic syndrome. The possession of both virulence factors makes this pathotype highly virulent to humans. These specifically include serogroups O26, O45, O80, O103, O111, O121, O145, and O157.

[0060] In a specific embodiment, the reaction medium comprises an inducing agent, which promotes or enhances the expression of a molecule of interest produced by the target microorganism. This molecule of interest can be an albumen, an antibiotic, an antimicrobial resistance molecule, a protease-type enzyme, a lipase or a glucosidase. Therefore, the present invention can characterize or select a microbial strain relative to the ability of the microbial strain to produce these molecules of interest. In a specific embodiment, the present invention can relate to the field of biological production, more particularly to the production of recombinant proteins by genetically modified microorganisms, for which it is necessary to identify production clones. Therefore, the present invention can clearly show that recombinant proteins are being produced. In the presence of a halo, the amount produced can also be estimated. In another specific embodiment, the present invention can detect drug-resistant pathogenic bacteria.

[0061] In a particular embodiment, the reaction medium comprises a toxin inducer. Toxin inducers cause stress in bacteria, triggering the lytic cycle of prophages in bacteria, thereby stimulating the production and release of toxins. 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 types: STX2a, STX2b, STX2c, STX2d, STX2e, STX2f, STX2g, STX2h, STX2i, STX2j, STX2K, STX2l. Advantageously, the toxin inducer is selected from antibiotics or physicochemical stress. As antibiotics, mention may be made of trimethoprim, sulfamethoxazole, norfloxacin, azithromycin, gentamicin, polymyxin B, chloramphenicol, streptomycin, chlortetracycline, oxytetracycline, tylosin, mitomycin C, carbodox, oliquindox, rifampicin, imipenem, ciprofloxacin, cotrimoxazole, penicillin G and lincomycin. In a preferred embodiment, the culture medium according to the invention comprises ciprofloxacin in a concentration between 0.005 and 0.030 mg / l. In another preferred embodiment, the culture medium according to the invention comprises mitomycin C in a concentration between 0.10 and 0.5 mg / l.

[0062] The toxin inducer may also be a physicochemical stress, for example, by the addition of salt or EDTA, or by a pH change or UV stress. Stress may also be induced, for example, by the addition of norepinephrine.

[0063] The target microorganism may be present in a sample. The term "sample" refers to a small portion or a small amount separated from an entity for analysis. The sample can be of industrial origin, such as but not limited to air samples, water samples, samples collected from the surface, manufactured parts or products or food. Food samples that can be mentioned include but are not limited to samples of dairy products (yogurt, cheese, etc.), meat, fish, eggs, fruits, vegetables, water and beverages (milk, juice, carbonated soft drinks, etc.). Finally, food samples can be derived from animal feed, especially animal or vegetable powder. The sample can be of biological origin, either animal or human. Then, it can correspond to a biological fluid sample (feces, urine, whole blood, serum, plasma, cerebrospinal fluid, organic secretions, etc.), an external sample (skin, nose, throat, etc.) or a tissue sample or a separated cell. The sample can be used as is, or before analysis, it can be prepared using methods known to those skilled in the art, such as enrichment, extraction, concentration or purification. When the sample is multi-microorganism or loaded with paraflora, such as food samples such as raw milk cheese, or feces in clinical samples, the reaction medium according to the present invention may be more interesting.

[0064] Another subject of the present invention relates to a diagnostic kit for preparing a reaction medium according to the invention, comprising

[0065] - The agglutination conjugate according to the present invention

[0066] - Gelling medium.

[0067] Advantageously, the reaction medium can be prepared extemporaneously using a kit according to the invention. This enables the reaction medium to be used as a unit and improves its stability.

[0068] Therefore, another subject of the present invention relates to a process for obtaining a reaction medium according to the present invention, comprising a step of contacting the agglutinated conjugate with a gelling medium to form a reaction medium according to the present invention. The conjugate is prepared by coupling the binding partner to the nanoparticles. These coupling methods are well known to those skilled in the art (Nicholas G. Welch et al., 2017). The conjugate is then added to the supercooled gelling medium. The whole is then homogenized and poured into a culture dish.

[0069] Another subject of the present invention relates to an in vitro microbial culture method, wherein the microorganisms that may be present in the sample are inoculated into or onto a culture medium according to the present invention. The inoculated culture medium is incubated under suitable conditions known to those skilled in the art. Inoculation is performed using conventional microbiological techniques. It can also be performed in an integral manner, i.e. by inclusion.

[0070] 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 that may contain the target microorganism, comprising the following steps:

[0071] - contacting the sample with a reaction medium according to the invention

[0072] - Incubation

[0073] - Detecting the presence of said target microorganism.

[0074] The term "detection" refers to detecting the presence of growth of a target microorganism (preferably a target bacterium) with the naked eye or with the aid of an optical instrument. When the reaction medium from which the target microorganism is to be detected contains a chromogenic or fluorogenic substrate, detection may be performed with the aid of an optical instrument for a fluorogenic substrate or with the naked eye or with the aid of an optical instrument for a chromogenic substrate.

[0075] The term "identification" refers to determining the genus and / or species and / or group to which the target microorganism belongs.

[0076] The term "enumerating at least one target microorganism" refers to counting / quantifying the number of target microorganisms, such as the number of bacterial colonies when the target microorganisms are bacteria.

[0077] The term "separate" refers to the production of distinct colonies that are separated from each other.

[0078] Microbiological detection corresponds to analyzing a sample in order to detect microorganisms that may be present in said sample. It is therefore very surprising to find that the culture medium according to the invention can detect target bacteria and separate them. Advantageously, the culture medium according to the invention can therefore locate and select the target microorganism from a mixed population present on a non-selective or insufficiently selective reaction medium. Preferably, the method according to the invention can be carried out by the appearance of a halo around the target microorganism on the reaction medium according to the invention. In other words, the detection is carried out by observing the appearance of a halo around the target microorganism on the reaction medium.

[0079] Without limiting, the medium according to the invention appears to be particularly suitable for detecting E. coli. Thus, according to a particular embodiment, the invention relates to a gelling reaction medium for detecting, identifying, counting and / or isolating E. coli strains, comprising a bacteriophage protein specific for the LPS of said strain, coupled to nanoparticles to form a conjugate.

[0080] In this particular embodiment, the amount of LPS-specific bacteriophage protein is such that at least one third of the surface of the nanoparticle is covered.

[0081] Preferably, in this particular embodiment, the nanoparticles are gold, have a size between 20 and 90 nm, and a concentration between 10 10 Up to 10 12 between nanoparticles / ml reaction medium.

[0082] In this embodiment, the reaction medium is formed by a known medium for culturing E. coli strains, such as Applicant's TBX or Coli medium, to which the agglutination conjugate is added.

[0083] In another specific embodiment, the present invention relates to a gelling reaction medium for detecting, identifying, counting and / or isolating Escherichia coli strains, comprising monoclonal antibodies specific to said strains, coupled to nanoparticles to form a conjugate.

[0084] Preferably, in this particular embodiment, the amount of monoclonal antibodies is such that at least half of the surface of the nanoparticle is covered, even more preferably between one third and half of the surface of the nanoparticle is covered.

[0085] Preferably, in this particular embodiment, the nanoparticles are gold, have a size between 20 and 90 nm, and a concentration between 10 10 Up to 10 12 between nanoparticles / ml reaction medium.

[0086] In another specific embodiment, the present invention relates to a gelling reaction medium for detecting, identifying, counting and / or isolating Shiga toxin-producing Escherichia coli strains, comprising at least one toxin inducer, at least one Stx1 and / or Stx2-specific antibody, and the at least one antibody is coupled to nanoparticles to form an agglutination conjugate. In other words, the present invention relates to a gelling reaction medium for detecting, identifying, counting and / or isolating Shiga toxin-producing Escherichia coli strains, comprising at least one toxin inducer, at least one agglutination conjugate, and the agglutination conjugate is formed by coupling at least one Stx1 and / or Stx2-specific antibody to nanoparticles.

[0087] Advantageously, this embodiment allows the localization of Shiga toxin-producing E. coli strains by forming a halo around the strain. Specifically, the conjugate formed by coupling the antibody to the nanoparticles agglutinates around the colonies. Thus, it is easy to visualize the target colonies.

[0088] In this embodiment, the amount of the at least one antibody is such that at least half of the nanoparticle surface can be covered, preferably at least one-third of the nanoparticle surface. In these specific embodiments, the culture medium comprises ciprofloxacin at a concentration between 0.005 and 0.030 mg / l. In the variants of these specific embodiments, the culture medium comprises mitomycin C at a concentration between 0.10 mg / l and 0.50 mg / l.

[0089] Preferably, in these variants, the nanoparticles are gold nanoparticles, have a size between 20 and 90 nm, and a concentration between 10 10 Up to 10 12 between nanoparticles / ml reaction medium.

[0090] The invention is particularly advantageous for distinguishing in the same sample between bacteria producing Shiga toxins STX1 or STX2 and bacteria producing both types of Shiga toxins STX1 and STX2 when the culture medium according to the invention comprises binding partners of several toxins.

[0091] The present invention is particularly advantageous in facilitating the detection of STEC in polymicrobial samples. In fact, without the present invention, which is capable of locating the colonies of interest, the reference method ISO 16136 specifies that up to 50 colonies need to be tested by molecular methods to confirm the presence of STEC. Given the large number of colonies on a culture dish and the poor representation of the target microorganism, a person picking colonies for confirmation purposes may never pick the target microorganism. Thus, the present invention saves time in performing microbial testing. This is particularly advantageous for supplies loaded with paraflora, as a large number of colonies on a culture dish may lead to the risk of false negatives.

[0092] Example

[0093] Example 1: Detection of Escherichia coli O26 using a culture medium according to the present invention comprising bacteriophage proteins coupled to gold nanoparticles

[0094] Preparation of gold nanoparticles

[0095] 40nm nanoparticles are made by reducing gold chloride with sodium citrate (a method described by Turkevich and Frens in 1951). Therefore, 20nm gold nanoparticles are made using a gold chloride solution diluted in distilled water, to which a trisodium citrate solution is added and then boiled. The presence of an absorption peak at 517-519nm makes it possible to confirm the correct particle size. 40nm particles are synthesized from these 20nm particles. To this end, the 20nm particles are diluted in distilled water, and then trisodium citrate and gold chloride are added and boiled. An absorption peak is then observed at 524-526 in the cold state, reflecting the increase in particle size.

[0096] Preparation of conjugates:

[0097] The phage evaluated in this assay was phage O26 BP1, directed against the LPS of E. coli O26. This phage is from the applicant's collection.

[0098] The adsorption of phage proteins on nanoparticles was carried out according to methods known to those skilled in the art (Nicholas G. Welch et al.) taking into account the value of their isoelectric points: the above phages were coupled as follows

[0099] - Dilute 270 μg of phage protein in 3 ml Tris HCl (0.025 M) pH 6

[0100] - Add 30ml of gold with an optical density of 1, i.e. 7.7x10 10 Nanoparticles / ml, pre-adjusted to pH 6 with 0.1M K2CO3 solution

[0101] - Stir for 20 minutes

[0102] - Add 3 ml of 10% BSA for passivation

[0103] - Centrifuge at 8600g for 30 minutes

[0104] - Remove the supernatant and measure the resulting concentrate using a spectrophotometer.

[0105] Preparation of the reaction medium according to the present invention:

[0106] The resulting conjugate was added to supercooled agar (chrom ID coli-ref 42017 bioMérieux) at 50°C in order to obtain, using a spectrophotometer, a nanoparticle concentration of an optical density of 3. 18 ml of culture medium were then poured into each dish. The dishes were then left to dry.

[0107] Vaccination:

[0108] In this example, E. coli O26 strain and E. coli O111 strain (Applicant's collection) were inoculated and incubated at 37°C for 72 h.

[0109] result

[0110] A gray halo was observed around the colonies of LPS O26. Figure 1 ). This graying is due to the network formed between LPS released by the colonies and the conjugates present in the agar.

[0111] Example 2: Detection of Escherichia coli O111 using a culture medium according to the present invention comprising antibodies coupled to gold nanoparticles

[0112] Preparation of gold nanoparticles

[0113] 40 nm gold particles were produced as described in Example 1.

[0114] Preparation of conjugates

[0115] 2H5E9 IgG anti-E. coli O111 monoclonal antibody was from the applicant's collection.

[0116] Adsorb the antibodies to the nanoparticles as follows:

[0117] - Dilute 90 μg of antibody in 3 ml Tris HCl (0.025 M) pH 6.5

[0118] - Add 30 ml of gold at OD 1 adjusted to pH 8 with 0.1 M K2CO3 solution

[0119] - Stir for 20 minutes

[0120] - Add 3 ml of 10% BSA for passivation

[0121] - Centrifuge at 8600g for 30 minutes

[0122] - Remove the supernatant and measure the resulting concentrate using a spectrophotometer.

[0123] Preparation of the reaction medium according to the invention

[0124] The resulting conjugate was added to supercooled agar (chrom ID coli-ref 42017 bioMérieux) at 50°C in order to obtain, using a spectrophotometer, a nanoparticle concentration of an optical density of 3. 18 ml of culture medium were then poured into each dish. The dishes were then left to dry.

[0125] Vaccination

[0126] In this example, E. coli O26 strain and E. coli O111 strain from the applicant's collection were inoculated and incubated at 37°C for 72 h.

[0127] result

[0128] There is no gray halo around colony O26, but it is clearly visible around colony O111 ( Figure 2 ), which allowed the identification of the latter.

[0129] Example 3: Detection of STX1 toxin-producing Escherichia coli using a culture medium according to the present invention comprising an anti-Stx1 antibody coupled to gold nanoparticles and ciprofloxacin as a toxin inducer

[0130] Preparation of gold nanoparticles

[0131] 40 nm gold particles were produced as described in Example 1.

[0132] Preparation of conjugates

[0133] The antibody evaluated in this assay is an IgG antibody against STX1 toxin (reference ATCC 13C4 hybridoma CRL-1794).

[0134] Adsorb the antibodies to the nanoparticles as follows:

[0135] - Dilute 90 μg of antibody in 3 ml Tris HCl (0.025 M) pH 8

[0136] - Add 30 ml of gold at OD 1 adjusted to pH 8 with 0.1 M K2CO3 solution

[0137] - Stir for 20 minutes

[0138] - Add 3 ml of 10% BSA for passivation

[0139] - Centrifuge at 8600g for 30 minutes

[0140] - Remove the supernatant and measure the resulting concentrate using a spectrophotometer.

[0141] Preparation of the culture medium according to the invention

[0142] -Agar Supplement:

[0143] The resulting conjugate is added to supercooled agar (chrom ID coli ref 42017 bioMérieux) at 50°C containing a toxin inducer, in this case 10 ng / mL of ciprofloxacin, in order to obtain a nanoparticle concentration of OD 3. 18 ml of culture medium are then poured per dish. The dishes are then left to dry.

[0144] Vaccination

[0145] Four strains from the applicant's in-house collection, three of which had the stx1 gene, were inoculated and incubated at 37°C for 24 hours.

[0146] in conclusion:

[0147] A gray halo was observed around colonies producing STX1 toxin ( Figure 3 ). STX1 toxin was detected. This graying is due to the network formed between the toxin released by the colonies and the conjugate present in the agar.

[0148] Example 4: Detection of STX1 toxin-producing Escherichia coli in a culture medium according to the present invention comprising anti-Stx1 antibodies coupled to silver nanoparticles and mitomycin as a toxin inducer

[0149] Preparation of silver nanoparticles

[0150] 40 nm silver nanoparticles were purchased from a supplier (Alfa Aesar; ref: J67090.AE).

[0151] Preparation of conjugates

[0152] The antibodies (reference ATCC 13C4 hybridoma CRL-1794) were adsorbed onto the nanoparticles as described in Example 3.

[0153] Preparation of the culture medium according to the invention

[0154] The resulting conjugate was added to TBX supercooled agar (ref AEB622817 bioMérieux) at 50°C, also containing the toxin inducer, in this case 250 ng / mL of mitomycin C, in order to obtain a nanoparticle concentration of OD 3. The medium was then poured into dishes and allowed to dry.

[0155] Vaccination

[0156] Two strains from the applicant's in-house collection, one of which possessed the stx1 gene, were inoculated and incubated at 37°C for 24 hours.

[0157] result

[0158] A gray halo was observed around strains producing STX1 toxin ( Figure 4 , right colony). This graying is due to the network formed between the toxin released by the colony and the conjugate present in the agar.

[0159] Example 5: In the presence of mitomycin C, in a cell culture medium containing anti-Stx1 antibody coupled to silver nanoparticles, Detection of Stx1 and / or Stx2 toxin-producing Escherichia coli in a culture medium according to the present invention using anti-Stx2 antibodies coupled to rice grains

[0160] Preparation of silver or gold nanoparticles

[0161] 40 nm silver nanoparticles were purchased from a supplier (Alfa Aesar; ref: J67090.AE).

[0162] Nanoparticles were produced according to Example 1.

[0163] Preparation of conjugates

[0164] According to the method described in Example 1, 40 nm gold nanoparticles were coupled to the anti-stx2 antibody 9E4H11 from the applicant's collection at pH 9.

[0165] Adsorption of the anti-stx1 antibody 13C4 onto silver nanoparticles was performed according to Example 4.

[0166] Preparation of the culture medium according to the present invention:

[0167] The conjugates were added to supercooled TBX agar (ref AEB622817 bioMérieux) at 50°C containing a toxin inducer, in this case 250 ng / mL of mitomycin C, in order to obtain a concentration of each type of nanoparticles of OD 3. The medium was then poured into dishes and left to dry.

[0168] Vaccination:

[0169] Two strains collected by the applicant, one having the stx1 gene and the other having the stx2 gene, were inoculated and incubated at 37°C for 24 hours.

[0170] result

[0171] The medium with the conjugate was reddish. A halo could be observed around each of the two strains ( Figure 5). Notably, the color of the halo changes depending on the toxin produced. Specifically, in the case of STX2 toxin production, a network forms between the toxin and the gold nanoparticles, causing the gold nanoparticles to change from red to gray, and a yellow halo to form due to the presence of unaggregated silver nanoparticles ( Figure 5 In the case of STX1 toxin production, a network was formed between the toxin and the silver nanoparticles, causing the silver nanoparticles to change from yellow to gray and produce a more intense red halo (light red + gray) due to the presence of unaggregated gold nanoparticles ( Figure 5 colony on the right in the middle).

[0172] Example 6: In the presence of ciprofloxacin as a toxin inducer, Anti-Stx1 or anti-Stx2 antibodies are used to detect the production of Stx1 and / or Stx2 toxins in the culture medium of the present invention. bacteria

[0173] Preparation of nanoparticles

[0174] According to Example 1, 40 nm nanoparticles were produced.

[0175] Preparation of conjugates

[0176] The antibodies evaluated in this assay were the 13C4 antibody directed against STX1 toxin and the 9E4H11 antibody directed against STX2 toxin.

[0177] According to the above embodiment, the mixture is adsorbed onto the nanoparticles at a pH of 8.

[0178] Preparation of the culture medium according to the present invention:

[0179] The resulting conjugate was added to supercooled TBX agar (ref AEB622817 bioMérieux) at 50°C containing a toxin inducer, in this case 10 ng / mL of ciprofloxacin, in order to obtain a nanoparticle concentration of OD 3. 18 ml of culture medium was then poured per dish. The dishes were then left to dry.

[0180] Vaccination

[0181] Five strains with or without stx1 and / or stx2 genes were inoculated and incubated at 37°C for 24 hours.

[0182] Conclusion: Gray halos were observed around colonies producing STX1 and / or STX2 toxins ( Figure 6 ). This graying is due to the network formed between the toxin released by the colonies and the conjugates present in the agar.

[0183] Example 6: Detection of STX1 toxin-producing Escherichia coli by large-scale inoculation

[0184] Preparation of nanoparticles

[0185] According to Example 1, 40 nm nanoparticles were produced.

[0186] Preparation of conjugates

[0187] The antibody used in this study was 13C4 directed against the E. coli STX1 toxin. Adsorption was performed as described in the previous examples.

[0188] Preparation of the culture medium according to the invention

[0189] The resulting conjugate was added to supercooled agar (TBX) at 50°C containing a toxin inducer, in this case 250 ng / mL of mitomycin C, in order to obtain a nanoparticle concentration of OD 3.

[0190] Vaccination

[0191] In this example, two strains, one producing STX1 toxin (O24) and the other not producing (O25), were inoculated on a large scale and incubated at 37°C for 24 hours.

[0192] in conclusion:

[0193] In the case of colony O24, a grey halo was clearly present. This colour change is due to the network formed between the STX1 toxin released by the colony and the conjugate present in the agar.

[0194] References

[0195] 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;

[0196] Turkevich et al., "A study of the nucleation and growth processes in the synthesis of colloidal gold", 1951.

Claims

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

2. The gelling reaction medium according to claim 1, Features It is a microbial culture medium.

3. The gelling reaction medium according to claim 1, Features It is a microbial visualization medium.

4. The reaction medium according to any one of the preceding claims, Features The binding partner is selected from the group consisting of antibodies, aptamers, phage proteins and primers.

5. The culture medium according to any one of the preceding claims, Features The nanoparticles are colloidal nanoparticles having optical properties.

6. The culture medium according to the preceding claim, Features The nanoparticles are selected from the group consisting of gold, silver and copper.

7. The culture medium according to any one of the preceding claims, Features The size of the nanoparticles is between 10 and 200 nm, preferably between 20 and 90 nm.

8. The culture medium according to any one of the preceding claims, wherein the reaction medium is contacted with an agar medium.

9. The culture medium according to any one of the preceding claims, Features The culture medium comprises an inducer derived from a component of the microorganism.

10. The culture medium according to claim 9, Features The culture medium contains a toxin-inducing agent.

11. The culture medium according to claim 10, Features The toxin inducing agent is an antibiotic.

12. The culture medium according to claim 11, Features The toxin inducing agent is ciprofloxacin at a concentration between 0.005 and 0.030 mg / l.

13. The culture medium according to claim 11, Features The toxin inducer is mitomycin C at a concentration between 0.10 mg / l and 0.50 mg / l.

14. The reaction medium according to any of the preceding claims, for detecting, identifying, enumerating and / or isolating at least one target microorganism selected from the group consisting of: 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. A diagnostic kit for preparing a reaction medium according to any one of the preceding claims, comprising - Agglutination conjugate - Gelling medium.

16. The preparation of the reaction medium of claims 1 to 15, comprising the step of contacting the agglutination conjugate with a gelling medium to form the reaction medium.

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

18. The gelling reaction medium according to any one of claims 1 to 14, for detecting, identifying, counting and / or isolating an Escherichia coli strain, comprising a bacteriophage protein specific for the LPS of the strain, the bacteriophage protein being coupled to nanoparticles to form an agglutination conjugate.

19. The gelling reaction medium according to claim 18, Features The amount of LPS-specific phage protein is such that at least half of the nanoparticle surface is covered.

20. The gelling reaction medium according to claim 18 or 19, Features The nanoparticles are made of gold, have a size between 20 and 90 nm, and a concentration of 10 10 Up to 10 12 between nanoparticles / ml reaction medium.

21. The gelling reaction medium according to any one of claims 1 to 14, for detecting, identifying, counting and / or isolating Escherichia coli strains, comprising a monoclonal antibody specific to the strain, the antibody being coupled to nanoparticles to form an agglutination conjugate.

22. The gelling reaction medium of claim 21, for detecting, identifying, counting and / or isolating at least one Shiga toxin-producing Escherichia coli strain, comprising at least one toxin inducer, at least one STX1-specific antibody and / or at least one STX2-specific antibody, wherein the at least one antibody is coupled to nanoparticles to form an agglutination conjugate.

23. The gelling reaction medium of claim 22, for detecting, identifying, enumerating and / or isolating at least one Shiga toxin-producing Escherichia coli, comprising ciprofloxacin at a concentration between 0.005 and 0.030 mg / l.

24. The gelling reaction medium of claim 22, for detecting, identifying, enumerating and / or isolating at least one Shiga toxin-producing Escherichia coli, comprising mitomycin C at a concentration between 0.10 mg / l and 0.50 mg / l.

25. The gelling reaction medium according to any one of claims 21 to 24, for detecting, identifying, enumerating and / or isolating at least one Shiga toxin-producing Escherichia coli, Features The nanoparticles are gold nanoparticles with a size between 20 and 90 nm and a concentration of 10 10 Up to 10 12 between nanoparticles / ml reaction medium.

26. A method for detecting, identifying, enumerating and / or isolating a target microorganism in a sample that may contain the target microorganism, The following steps are involved: - contacting the sample with a reaction medium according to any one of claims 1 to 14 or 18 to 25; - incubation; - Detecting the presence of said target microorganism.

27. The method of the preceding claims, wherein detection is performed by the appearance of a halo around the target microorganism on the reaction medium of any one of claims 1 to 14 or 18 to 25.

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