Reaction media and method for detecting Shiga toxin-producing Escherichia coli and / or enterohemorrhagic Escherichia coli.

Abstract

The present invention relates to a gelling reaction medium for the detection, identification, and / or isolation of at least one Shiga toxin-producing Escherichia coli strain, comprising: - at least one toxin inducer; - at least one agglutination conjugate containing at least one specific binding partner of STX1 and / or at least one specific binding partner of STX2, conjugated to nanoparticles; and - a concentration gradient of compounds for inhibiting non-target bacteria. The present invention also relates to related methods for detecting and / or isolating Shiga toxin-producing Escherichia coli that are likely to be present in a sample containing enterobacteria.

Description

Technical Field

[0001] The present invention relates to the field of microbiological examination in a broad sense, such as the microbiological examination of samples of industrial or clinical origin. More specifically, the present invention relates to a reaction medium and a method for detecting Shiga toxin-producing Escherichia coli and / or enterohemorrhagic Escherichia coli.

Background Art

[0002] In microbiological tests of samples of various origins, it is necessary to perform techniques that enable detection for the purpose of, for example, identification and / or counting of microorganisms and / or evaluation of biochemical characteristics, and results must be obtained as quickly as possible.

[0003] In the medical field, it is necessary to predict and diagnose the risk of infection. The faster and more accurate the diagnosis, the more efficient the treatment of the patient and the lower the risk of infection. This approach is the same with respect to the health of animals in the veterinary field.

[0004] The same problem exists in the field of agricultural food products. However, it is distinguished in the following points: - Pathogenic microorganisms such as Shiga toxin-producing bacteria (STEC), Salmonella, Listeria, Cronobacter, Bacillus, and Staphylococcus (the search for these microorganisms is applied to starting materials, intermediate products, and the final products sold), - Non-pathogenic microorganisms (used as quality indicators throughout the production process from starting materials to final products), - Technologically interesting bacteria such as fermented products, - Microorganisms serving as pollution indicators.

[0005] By quickly and accurately detecting foods suspected of being contaminated (within a food batch), it becomes possible to control the contamination and take corrective measures promptly. [[ID=​Technically, one of the main challenges is isolating the bacteria in question to identify it. Microbiological analysis is generally carried out in two stages. The first is the detection stage, which may involve several techniques such as culture media, immunoassays, and molecular biology. This may be followed by a confirmation stage, particularly in the agricultural and food sectors, to confirm the presence of the pathogen under consideration and to meet the standards enforced in this field. Thus, the confirmation step requires additional steps, which in this case inevitably include the step of isolating the bacteria in question.

[0007] Therefore, in the case of Shiga toxin-producing Escherichia coli, diagnosis is based on the use of selective and chromogenic agar media that may contain tellurite, such as CT-SMAC (sorbitol MacConkey agar) or CT-RMAC (rhamnose MacConkey agar), to select the growth of specific bacteria and color the target strain. The drawback of these methods is that they are highly specific, and some strains of Shiga toxin-producing E. coli may not be detected. Conversely, some media are not selective enough, and the presence of a small number of Shiga toxin-producing E. coli can be masked by other widely present microorganisms, making isolation difficult. Furthermore, to assess the pathogenicity of these strains, it is necessary to detect the presence of virulence factors. Specifically, these pathogenicities include the expression of several pathogenic genes, particularly six pathogenic genes encoding two types of Shiga toxins: STX1 and STX2. These two toxins act by inhibiting protein synthesis in eukaryotic cells, ultimately causing apoptosis. Currently, performing PCR against the stx gene is essential for accurately identifying Shiga toxin-producing E. coli.

[0008] Therefore, there is a need to develop reliable and rapid culture media and methods for isolating and identifying target bacteria, particularly STEC and EHEC. [Overview of the Initiative]

[0009] The present invention relates to a gelling reaction medium for the detection, identification, and / or isolation of at least one Shiga toxin-producing Escherichia coli strain, - At least one type of toxin inducer, - At least one aggregated conjugate comprising at least one STX1-specific binding partner and / or at least one STX2-specific binding partner bound to a nanoparticle, - Concentration gradient of compounds that inhibit non-target bacteria This relates to gelling reaction media, including those mentioned above.

[0010] All the more advantageously, the culture medium according to the present invention makes it possible to isolate STEC or EHEC from a large number of E. coli.

[0011] Another subject of this invention is as follows: - A step of placing the aggregated conjugate by contacting it with the gelled culture medium. - A step of depositing at least one compound that inhibits non-target bacteria containing tellurite onto a zone of gelled culture medium, wherein the inhibitory compound diffuses and forms an inhibitory concentration gradient around the deposit zone, and the concentration of the tellurite concentration gradient is between 0 μg / ml and 100 μg / ml, preferably between 0 μg / ml and 50 μg / ml, and more preferably between 0 μg / ml and 30 μg / ml, by depositing at least one compound. The present invention relates to the preparation of a reaction medium, including the present invention.

[0012] Another subject of the present invention is a method for detecting and / or isolating Shiga toxin-producing Escherichia coli that may be present in a sample containing intestinal bacteria, the following: - A step to provide a selective gelling culture medium that enables the growth of E. coli containing a concentration gradient of compounds that inhibit non-target bacteria. - Step of depositing the sample onto the gelled culture medium. - A step of incubating the culture medium under conditions that enable the growth of E. coli, - Steps to isolate E. coli - A step to confirm that the E. coli in question is Shiga toxin-producing E. coli. This includes methods.

[0013] Preferably, the culture medium is the culture medium according to the invention.

[0014] Therefore, advantageously, the presence of Shiga toxin-producing E. coli can be confirmed by the presence of a halo around the Shiga toxin-producing E. coli in the inhibitory concentration gradient zone.

[0015] This preferred embodiment has the advantage that the STEC or EHEC can be directly detected and isolated on the culture medium, thereby obtaining results quickly.

[0016] In another embodiment, the method enables the detection of STEC or EHEC. According to this embodiment, the detected Shiga toxin-producing Escherichia coli is enterohemorrhagic Escherichia coli.

[0017] This method is particularly advantageous for samples containing accompanying flora, where a large number of colonies on a petri dish can lead to a risk of false negatives. Given the large number of colonies on the dish and the small number of target microorganisms, a person taking colonies for confirmation may miss the target microorganism. Therefore, in the presence of an inhibitory compound gradient, the probability of isolating STEC increases significantly. Accordingly, the present invention proposes a method for detecting and isolating STEC that avoids false negatives and saves considerable time. [Brief explanation of the drawing]

[0018] [Figure 1] This is a photograph of a control culture medium without an inhibitory compound concentration gradient, into which a "Reblochon cheese" sample was seeded due to depletion. [Figure 2] This is a photograph of a control culture without an inhibitory compound gradient, in which the "Reblochon cheese" sample was seeded by depletion after the immunoselection process. [Figure 3] This is a photograph of a control culture medium without an inhibitory compound gradient, into which a "ground beef" sample was seeded due to depletion. The arrows point to the halo around the colonies. [Figure 4]This is a photograph of a control culture without an inhibitory compound gradient seeded with a "ground beef" sample after the immune selection step, due to depletion. [Figure 5] This is a photograph of a culture medium containing a gradient of an inhibitory compound (cefixime - tellurite mixture) seeded with a "ground beef" sample due to depletion. [Figure 6] This is a photograph of a culture medium containing a gradient of an inhibitory compound (cefixime - tellurite mixture) seeded with a pre - immune - selected "ground beef" sample due to depletion. [Figure 7] This is a photograph of a culture medium containing a gradient of an inhibitory compound (cefixime - tellurite mixture) seeded with a pre - immune - selected "rubroshon cheese" sample due to depletion. The arrow points to the halo around the colony. [Figure 8] This is a photograph of a culture medium containing a gradient of an inhibitory compound (cefixime - tellurite mixture) seeded with a pre - immune - selected "rubroshon cheese" sample due to depletion. **DETAILED DESCRIPTION OF THE INVENTION**

[0019] Specific terms and expressions used in the context of the present invention are detailed below.

[0020] A first subject of the present invention is a gelling reaction medium for the detection, identification, and / or isolation of at least one strain of at least one Shiga toxin - producing Escherichia coli, comprising: - at least one toxin inducer, - at least one agglutination conjugate comprising at least one STX1 - specific binding partner and / or at least one STX2 - specific binding partner bound to nanoparticles, - a concentration gradient of a compound that inhibits non - target bacteria The invention relates to a gelling reaction medium comprising the above.

[0021] The term "reaction medium" refers to a medium containing all the elements necessary for the expression, survival, and / or growth of a microbial metabolic state. This reaction medium may be either a microbial culture medium or a microbial detection medium. In the latter case, the microorganism may be pre-cultured in another medium. The reaction medium may also be placed in contact with the agar culture medium. The reaction medium may be placed below or above the culture medium that allows for the growth of the target microorganism. The reaction medium may be added after incubation of the culture medium. Preferably, the reaction medium is a microbial culture medium containing all the elements necessary for the growth of the microbial state.

[0022] According to the present invention, the reaction medium is gelled. The reaction medium is in a semi-solid form. Agar is a conventional gelling agent used in microbiology for culturing microorganisms, but other gelling agents, such as gelatin, agarose, or other natural or artificial gelling agents, can also be used. This conjugate can form a network with the target component in the gelled medium. This network is visually detectable by the formation of a halo without the need to reduce the hardness of the conventional semi-solid reaction medium. Therefore, it is not necessary to modify the physicochemical properties of the reaction medium, such as its hardness, in order to enable, firstly, the diffusion of the target component, and secondly, the reaction with the conjugate.

[0023] For example, many preparations are commercially available, such as Columbia agar, Tripcase soybean agar, MacConkey agar, Mueller-Hinton agar, or more generally, those described in the Handbook of Microbiological Media. These media can serve as the basis for the reaction medium according to the present invention. The reaction medium may also include possible additives such as amino acids, peptones, one or more growth factors, carbohydrates, nucleotides, minerals, vitamins, one or more selective agents, inducers, toxin inducers, and buffers. The term “selective agent” means any compound that can prevent or slow the growth of “non-target” microorganisms, i.e., microorganisms other than one or more target microorganisms. The term “inducer” means a compound that can induce the expression of a compound such as an enzyme or toxin that would normally remain unexpressed. The reaction medium may also include colorants. For example, colorants may include Evans blue, neutral red, sheep's blood, horse's blood, opacifiers such as titanium dioxide, nitroaniline, malachite green, and brilliant green. When the reaction medium according to the present invention also includes an enzyme substrate specific to the enzyme activity of at least one target microorganism, a chromogenic substrate and / or a fluorescent substrate is preferably used. The term "chromogenic substrate and / or fluorescent substrate" means a substrate that enables the detection of the enzyme activity or metabolic activity of the target / desired microorganism using a detectable signal. The reaction medium according to the present invention is sensitive to pH changes induced by substrate consumption and may further include a pH indicator that reveals the metabolism of the target microorganism. The pH indicator may be a chromophore or a fluorophore. Examples of chromophores include bromocresol purple, bromothymol blue, neutral red, aniline blue, and bromocresol blue.

[0024] Those skilled in the art may also use Petri dishes divided into segments such as two or three, and can easily compare several culture media containing different substrates or different selective mixtures by depositing the same biological sample on them.

[0025] According to the present invention, the term "microorganism" has the same meaning as it is commonly accepted in microbiology, and includes, in particular, Gram-positive or Gram-negative bacteria, yeasts, fungi, and, more generally, uninvisible single-celled organisms that can be manipulated and grown in a laboratory.

[0026] The term "target microorganism" means at least one microorganism that is desirable to detect and / or identify. According to the present invention, the microorganism is selected from among Shiga toxin-producing Escherichia coli strains. Enterohemorrhagic Escherichia coli is a strain representative of a subgroup of Shiga toxin-producing Escherichia coli that possesses the eae gene or other genes involved in cell adhesion, such as the aggR gene, and causes hemolytic uremic syndrome. When these two virulence factors, stx and eae, are present simultaneously, this prototype becomes highly toxic to humans. In particular, it is highly toxic to serogroups O26, O45, O80, O103, O111, O121, O145, and O157.

[0027] Therefore, according to the present invention, the target microorganisms are, for example, E. coli belonging to the group of Shiga toxin-producing E. coli, typical enterohemorrhagic E. coli having the stx gene and eae gene, or atypical enterohemorrhagic E. coli having the stx gene and aggR gene. Non-target microorganisms are bacteria growing on the culture medium outside the concentration gradient region of the inhibitory compound. Specifically, since the sample is polybacterial, any STEC or EHEC that may be present are found among nonspecific Gram-negative bacteria such as commensal E. coli.

[0028] According to the present invention, the reaction medium contains at least one binding partner specific to STX1 and / or STX2, which is bound to nanoparticles. STX1 and STX2 are toxins secreted by the STEC strain. There are mainly two types of Shiga toxins: STX1 and STX2, and these themselves have many variants. To date, STX1 has four subtypes: STX1a, STX1c, STX1d and STX1e, while STX2 has twelve subtypes: STX2a, STX2b, STX2c, STX2d, STX2e, STX2f, STX2g, STX2h, STX2i, STX2j, STX2k and STX2l.

[0029] A specific binding partner is selected from antibodies, Fab fragments of any kind, recombinant proteins, phages, phage proteins, oligonucleotides, aptamers, affimers, or other ligands or antiligands well known to those skilled in the art. Preferably, the binding partner is selected from antibodies and phage proteins. Preferably, the antibody is a monoclonal antibody or a monoclonal antibody fragment. The binding partner is specific to a component of the target microorganism. The component of the target microorganism is a component released by the microorganism. Thus, the component may be an element derived from the surface of the bacterium, such as a protein, lipopolysaccharide (LPS), or flagella. It may also be an element inside the bacterium, such as RNA or intracellular proteins, which can be detected when a portion of the bacterial colony dies during growth. The binding partner may also be specific to a component derived from the microorganism. This component may be a target molecule produced by the target microorganism, such as a protein, antibiotic, antimicrobial resistance molecule, protease enzyme, lipase, or glucidase. According to the present invention, the culture medium contains at least one binding partner specific to the STX1 protein and / or at least one binding partner specific to the STX2 protein. According to another embodiment, the culture medium contains at least one binding partner specific to a subtype of the STX1 protein, and / or at least one binding partner specific to a subtype of the STX2 protein, such as STX2a or STX2d. According to the present invention, the binding partners are bound to nanoparticles. This final complex is called a conjugate. The same reaction medium may have conjugates in which different types of binding partners are themselves bound to different types of nanoparticles.

[0030] The term "nanoparticles" refers to particles approximately 1 nanometer in size. 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 a network is formed. Even more preferably, the nanoparticles are selected from gold, silver, and copper. For example, when the nanoparticles are gold, they change color from red to gray when forming a network. When they are not aggregated, the wavelength of light absorbed is in the red region of approximately 530 nm. When they aggregate, the wavelength of light absorbed changes from red to blue / gray, approximately 600 nm to 700 nm. In certain embodiments, the use of combinations of several nanoparticles of different colors is possible. The network thus formed allows for the distinction of several components of the target microorganism.

[0031] Preferably, the nanoparticles have a size between 10 nm and 200 nm. Preferredly, the nanoparticles have a size between 20 nm and 90 nm to allow for better mobility of the conjugate within the reaction medium.

[0032] Advantageously, nanoparticles allow for a reduction in the amount of binding partners required for aggregation. Therefore, the concentration of binding partners required to prepare the reaction medium according to the present invention is 100 to 1000 times less than that required for a medium without nanoparticles.

[0033] Ideally, the required amount of binding partners should be equivalent to the amount needed to cover at least half of the surface of the nanoparticles, and even more ideally, the amount needed to cover the area between one-third and one-half of the nanoparticle's surface. This ratio allows the aggregated conjugates to form a network in the gelled reaction medium.

[0034] Advantageously, nanoparticles can make it possible to visualize aggregations around bacterial colonies that are too small to be seen with the naked eye. This could lead to earlier detection. Advantageously, nanoparticles can be used to visualize aggregations around bacterial colonies whose translucent appearance would prevent detection by automated readers. In this way, detection can be facilitated.

[0035] The binding of nanoparticles to their binding partners can be achieved by either direct or indirect binding. Direct binding refers to binding by adsorption or covalent bonding. Indirect binding refers to binding by ligand / antiligand interaction, such as biotin / streptavidin or other pairs well known to those skilled in the art (Nicholas G. Weich et al., 2017). Depending on the type of binding selected, those skilled in the art will adapt the physicochemical conditions of the reaction medium, particularly its pH.

[0036] According to the present invention, the conjugate aggregates, that is, it causes the formation of an aggregated network in the presence of a component of the target microorganism or a component derived from the microorganism. Since this component is a multiepitope, multiple conjugates adhere to this component and form aggregates. Aggregation means the result of an interaction between at least one component of the target microorganism or at least one component derived from the microorganism and a binding partner bound to a nanoparticle. The agglutination reaction involves an immunological reaction such as an antigen-antibody reaction, or more generally, a specific interaction between two molecules. Through this interaction, the component and conjugate aggregate, adhere to each other, and form a network in the reaction medium.

[0037] In reality, there are several parameters: - Porosity of gelled culture medium - Size of nanoparticles - Comparing our partner - The amount of nanoparticles However, this primarily affects the ability of conjugates to aggregate within the gelling medium.

[0038] Therefore, these parameters must be adjusted to allow sufficient aggregation to enable detection. Advantageously, the network formed by the aforementioned specific reaction is then detected visually or automatically using an optical system. Thus, colonies of the target microorganism are detected. Preferably, the network forms a halo in the gelling reaction medium, which can be detected visually or using an optical system. The network or halo thus surrounds the colony and can then, advantageously, be differentiated and / or identified within the population.

[0039] In certain embodiments, the binding partner is an antibody present in an amount sufficient to cover at least half, preferably at least one-third, of the surface of the nanoparticles.

[0040] Preferably, in these variants, the nanoparticles are gold nanoparticles having a size between 20 nm and 90 nm, and 10 per 1 ml of reaction medium. 10 10 12 This is the nanoparticle concentration between individual particles.

[0041] According to the present invention, the reaction medium comprises a toxin inducer. The toxin inducer stresses the bacteria, which induces a lytic cycle of prophages within the bacteria and thus stimulates the production of toxins that are released. Advantageously, the toxin inducer is selected from antibiotics or physicochemical stressors. Antibiotics mentioned include trimethoprim, sulfamethoxazole, norfloxacin, azithromycin, gentamicin, polymyxin B, chloramphenicol, streptomycin, chlortetracycline, oxytetracycline, tyrosine, mitomycin C, carbodox, orikindox, rifampicin, imipenem, ciprofloxacin, kotrimoxazole, penicillin G, and linromycin. In one preferred embodiment, the medium according to the present invention comprises ciprofloxacin at concentrations between 0.005 mg / l and 0.030 mg / l. In another preferred embodiment, the culture medium according to the present invention contains mitomycin C at a concentration between 0.10 mg / l and 0.50 mg / l.

[0042] The toxin inducer may also be physicochemical stress generated by, for example, adding salt or EDTA, adjusting the pH, or UV stress. Stress may also be induced by, for example, adding norepinephrine.

[0043] The aforementioned target microorganisms tend to be present in the sample. The term "sample" means a small portion or quantity isolated from a certain entity for analysis. Samples may be of industrial origin, i.e., according to a non-exclusive list, air samples, water samples, samples taken from a surface, parts or manufactured products, or food. Samples may also be biofluid samples (e.g., feces, urine, whole blood, serum, plasma, cerebrospinal fluid, biological secretions), external samples (e.g., skin, nose, throat), tissue samples, or isolated cells. Samples may be used as received, or may be prepared before analysis, such as enrichment, extraction, concentration, or purification, according to methods known to those skilled in the art. The reaction medium according to the present invention may be particularly advantageous when the sample is a polymicrobial sample or contains an associated flora, such as a food sample like raw milk cheese or feces in a clinical sample.

[0044] According to the present invention, the reaction medium includes a concentration gradient of compounds that inhibit non-target bacteria. According to the present invention, the inhibitory compounds are selected such that the minimum inhibitory concentration (MIC) of non-target bacteria, such as commensal intestinal bacteria, is sufficiently lower than the MIC of STEC / EHEC.

[0045] The term "inhibitory compound" refers to any compound that inhibits bacterial growth. Inhibitory compounds may be antibiotics, colorants such as brilliant green, salts such as lithium chloride, or bacteriophage endolysins. Preferably, the inhibitory compound is tellurite. According to the present invention, this inhibitory compound promotes the inhibition of non-STEC and non-EHEC strains.

[0046] The term "gradient" means that the concentration of the inhibitory compound is not uniform, but increases or decreases.

[0047] By applying a concentration gradient according to the bacteria present in a multimicrobial sample, it becomes possible to distinguish non-target microorganisms that have a lower MIC than the target microorganism. Therefore, non-target microorganisms are those that can grow on the culture medium without a concentration gradient of the inhibitory compound. Target microorganisms are those possessing the stx gene, i.e., STECs.

[0048] Therefore, non-target bacteria such as commensal intestinal bacteria generally have lower MICs than STECs and EHECs.

[0049] Preferably, when the inhibitory compound contains tellurite, the tellurite gradient in the culture medium is between 0 μg / mL and 100 μg / mL, preferably between 0 μg / mL and 50 μg / mL, and even more preferably between 0 μg / mL and 30 μg / mL. Different strains belonging to STEC and EHEC have different sensitivities to inhibitory compounds such as tellurite. In particular, STEC and EHEC are more resistant to tellurite than other Enterobacteriaceae.

[0050] Furthermore, even within the same serogroup, such as EHEC or STEC, different serogroups can exhibit different susceptibility. For example, E. coli O157 does not have the same MIC to cefixime tellurite as E. coli O103. There are differences in susceptibility even within the same serogroup. Therefore, one E. coli O111 strain may not have the same MIC as another E. coli O111 strain.

[0051] Much more advantageously, the culture medium according to the present invention makes it possible to isolate STEC or EHEC from a large number of E. coli.

[0052] In a preferred embodiment, the inhibitory compound is a mixture of cefixime and tellurite. Preferably, the amount of cefixime in the deposited cefixime-tellurite mixture is between 0.20 μg and 0.30 μg, and the amount of tellurite is between 12 μg and 13 μg.

[0053] Preferably, the inhibitor compound deposited on the culture medium will have a volume between 5 μl and 50 μl. The inhibitor compound can be deposited by any means, for example, by pipetting.

[0054] In certain embodiments, the inhibitory compound is contained within at least one substrate suitable for diffusion of the inhibitory compound onto a gelling culture medium. This may be, for example, a blotting paper pad containing the inhibitory compound and suitable for diffusion. In this particular embodiment, the substrate may be positioned to come into contact with the agar medium by placing the substrate at the bottom of the dish before the medium is poured into the dish.

[0055] Another subject of the present invention is a diagnostic kit for preparing a reaction medium according to the present invention, - Aggregated conjugates comprising at least one STX1-specific binding partner and / or at least one STX2-specific binding partner bound to nanoparticles, - Gel culture medium containing toxin inducer, - Compounds that inhibit non-target bacteria, This includes information about diagnostic kits.

[0056] Advantageously, the reaction medium is prepared instantaneously using the kit according to the present invention. This allows for unit-use of the reaction medium and improves its stability. Preferably, once prepared, the medium is used within 24 hours, and even more preferably within 12 hours.

[0057] Another subject of this invention is the following steps: - A step of forming a reaction medium by contacting it with a gelling medium and arranging the agglutinating conjugate, - A step of depositing at least one compound that inhibits non-target bacteria on a zone of gelled culture medium, wherein the inhibitory compound diffuses and forms an inhibitory concentration gradient around the deposit zone, and the concentration gradient of the tellurite is between 0 μg / ml and 100 μg / ml, preferably between 0 μg / ml and 50 μg / ml, and more preferably between 0 μg / ml and 30 μg / ml. The present invention relates to a method for producing a reaction medium, including the present invention.

[0058] Thus, the agglutinating conjugate is placed in contact with the supercooled gelling medium to form the reaction medium according to the present invention. The gelling medium also contains a toxin inducer. The mixture is then homogenized and poured into a petri dish.

[0059] A compound that inhibits non-target bacteria is deposited on a zone of gelled culture medium. The inhibitory compound diffuses around the deposition zone, forming an inhibition gradient. In another embodiment, the inhibitory compound is deposited after the sample has been deposited. In yet another embodiment, the inhibitory compound is a suitable substrate for the diffusion of the inhibitory compound and is placed in the dish before the medium is poured.

[0060] Preferably, the inhibitory compound includes a colorant that allows for the localization of its deposition onto the culture medium, and then deposits the sample at the site of the inhibitory compound.

[0061] Another subject of the present invention relates to an in vitro microbial culture method in which microorganisms likely to be present in a sample are seeded in or on a culture medium according to the present invention. The seeded culture medium is incubated under appropriate conditions known to those skilled in the art. Seeding is performed using conventional microbiological techniques.

[0062] In a preferred embodiment of the present invention, seeding is carried out by depleting a polymicrobial sample on a culture medium. The sample is deposited on the inhibitory compound deposition site such that the sample with the highest microbial load comes into contact with the inhibitory concentration gradient. In particular, in the first isolation zone, the microbial concentration is so high that it is rarely possible to distinguish between target and non-target microorganisms.

[0063] Seeding techniques are well known to those skilled in the art. One such technique is quadrant seeding. The quadrant method involves dividing a petri dish in half to obtain three quadrants of 50%, 25%, and 25%, and then dividing it in half again. A small amount of inoculum is placed in the largest quadrant and spread out. Next, the dish is rotated a quarter turn to spread the bacteria into a smaller quadrant, and then rotated another quarter turn to seed the bacteria into the last small quadrant. In this embodiment, the inhibitory compound and sample are deposited in the same quadrant. Naturally, there are variations of this technique. It may also involve depletion by multiple streaking, which involves spreading the inoculum downwards in narrow streaks, and then performing other streaks from edge to edge of the previous streaks.

[0064] Another subject of the present invention is a method for detecting and / or isolating Shiga toxin-producing Escherichia coli that may be present in a sample containing intestinal bacteria, comprising the following steps: - A step to provide a selective gelling culture medium that enables the growth of E. coli containing a concentration gradient of compounds that inhibit non-target bacteria. - Step of depositing the sample onto the culture medium. - A step of incubating the culture medium under conditions that enable the growth of E. coli, - Steps to isolate E. coli - A step to confirm that the E. coli in question is Shiga toxin-producing E. coli. This includes methods.

[0065] The term "detection" means detecting the presence of growth of a target microorganism, preferably a target bacterium, either visually or using an optical device. When the culture medium in which the target microorganism is detected contains a chromogenic or fluorescent substrate, detection may be performed using an optical device for the luminescent substrate, or visually, or using an optical device for the chromogenic substrate. When the culture medium contains an agglutination conjugate, detection may be performed visually or by observing agglutination around the target microorganism.

[0066] The term "isolation" refers to the process of obtaining colonies that are separated from each other.

[0067] Thus, quite unexpectedly, it was found that the presence of an inhibitory compound gradient can improve the detection and isolation of target bacteria, such as Shiga toxin-producing Escherichia coli or enterohemorrhagic Escherichia coli, from a large number of Escherichia coli strains.

[0068] The present invention provides a selective medium that enables the growth of E. coli, which includes a concentration gradient of compounds that inhibit non-target bacteria such as commensal intestinal bacteria. Examples of media that enable the growth of E. coli include CHROMID® E. coli, CHROMID® EHEC, TBX, and Rainbow agar. Subsequently, a concentration gradient of compounds that inhibit non-target bacteria needs to be added.

[0069] The inhibitory compound is selected so as to enable the distinction between strains of Shiga toxin-producing coliform bacteria that are highly susceptible to the inhibitory compound and those that are not.

[0070] The inhibitory compound can be deposited into the culture medium by any means, such as a pipette. Once deposited, the inhibitory compound forms an inhibitory concentration gradient around the deposited zone. It can also be a substrate for diffusing the inhibitory compound.

[0071] In another embodiment, the inhibitory compound is deposited after the sample has been deposited.

[0072] According to the present invention, a step is performed to confirm the presence of Shiga toxin-producing Escherichia coli. This step can be performed using conventional confirmation methods, such as molecular biological or immunological methods. In this embodiment, colonies present on a culture medium in contact with a gradient of the inhibitor compound are collected. When Escherichia coli grows on the gradient, it is highly likely to be Shiga toxin-producing Escherichia coli. Latex tests or PCR tests designed for the target microorganism can be performed. Examples mentioned include the SLIDEX® Escherichia coli test, or the GENE-UP® STEC Top 6 or GENE-UP® Escherichia coli O157:H7 test, or the GENE-UP® STEC stx and eae PCR test.

[0073] In certain embodiments, Shiga toxin-producing Escherichia coli is enterohemorrhagic Escherichia coli.

[0074] In a preferred embodiment of the present invention, an incubation or enrichment step is performed on the sample before depositing it onto a gelling culture medium. This enrichment step requires incubation of a mixture formed by at least one biological sample and a culture medium, as well as a medium for a specific purpose, at an optimal temperature that allows for the growth of one or more target microorganisms. Incubation is generally carried out at a temperature in the range of 25–45°C for a predetermined time (e.g., 6–48 hours). This enrichment stage requires the use of selective or non-selective culture media (depending on the desired result), which have the function of promoting the growth of target microorganisms in the sample while simultaneously limiting the growth of non-target flora. Culture media are often placed in sterile plastic bag-type containers in contact with food, clinical, or environmental samples and used for the purpose of resuspending and enriching the microorganisms of interest. As described above, this enrichment step may be particularly necessary to reveal the presence of at least one target microorganism in a highly variable, and potentially very large, sample, ranging from 25 grams to 375 g, diluted in culture medium volumes between 225 milliliters (mL) and 3375 mL. Once this enrichment step is complete, aliquots (generally volumes between 5 microliters (μL) and 5 mL) are typically collected to carry out the detection and / or isolation steps for the target microorganism.

[0075] In an advantageous embodiment of the present invention, the sample is deposited and diffused onto the culture medium by a depletion technique.

[0076] After sowing, the reaction medium is incubated under appropriate conditions known to those skilled in the art.

[0077] In a preferred embodiment of the present invention, the culture medium is a culture medium according to the present invention comprising at least one agglutination conjugate and a gradient of inhibitory compounds. In this preferred embodiment of the present invention, the presence of Shiga toxin-producing Escherichia coli is confirmed by the presence of a halo around the STEC in the inhibitory concentration gradient zone. This preferred embodiment has the advantage that the STEC or EHEC can be directly detected and isolated on the culture medium. This allows for immediate results.

[0078] The present invention is particularly advantageous for facilitating the detection of Shiga toxin-producing Escherichia coli in multimicrobial samples. Specifically, without the present invention, which enables the placement of target colonies, the ISO 16136 standard method requires the examination of up to 50 colonies using molecular methods to confirm the presence of STEC or EHEC. Given the large number of colonies on a plate and the small number of target microorganisms, those sampling colonies for confirmation purposes may miss the target microorganism. The probability of isolating STEC increases significantly in the presence of an inhibitory compound gradient at the sample deposition site. This method is particularly advantageous for samples containing accompanying flora, where a large number of colonies on a petri dish can lead to a risk of false negatives. [Examples]

[0079] Example 1: Preparation of sample, agar plate, and seeding TIFF2026522003000001.tif31170

[0080] Device: - VIDAS biomérieux reference number 4700023 - Densimat Biomérieux reference number 99234 - Light plate - Smasher BioMérieux Reference Number AESAP1064 - Constant temperature incubator 41.5℃

[0081] Matrix used: - Frozen ground beef with onions: EXP 07 / 05 / 21 Batch number 01973658 - Reblochon de Savoie AOC cheese: EXP 06 / 02 / 23 Batch number 125473226

[0082] Strains used: - Escherichia coli O157:H7;STX1+ eae+;CRA 9405024 / ATCC 43890

[0083] Sample preparation: - Weigh 25g of the matrix to be tested into a filter bag. - Add 225 mL of EPT medium. - Mix for 1 minute using Smacher. - Incubate the bags in a constant temperature incubator at 41.5°C for 18 hours. - After incubation, dispense into 9 mL tubes.

[0084] Different types of matrices were tested: ground beef and Reblochon cheese.

[0085] Preparation of agar gel: - Preparation of gold nanoparticles 40 nm nanoparticles are produced by reducing gold chloride with sodium citrate (a method described by Turkevich and Frens in 1951). Similarly, 20 nm gold nanoparticles are produced using a solution of chloroauric acid diluted with distilled water, to which trisodium citrate is added and then boiled. The presence of an absorbance peak at 517–519 nm confirms that the particles are of the correct size. 40 nm particles are synthesized using these 20 nm particles. To do this, the 20 nm particles are diluted with distilled water, trisodium citrate and gold chloride are added, and the mixture is boiled. Subsequently, an absorbance peak is observed at 524–526 nm at low temperature, indicating an increase in particle size. - Preparation of conjugates The antibodies used are antibody 13C4 against STX1 toxin (reference: hybridoma ATCC CRL-1794) and antibody 9E4H11 against STX2 toxin. This mixture is adsorbed onto nanoparticles at pH 8. - Preparation of culture medium: A conjugate was added to TBX agar supercooled at 50°C, containing 250 ng / mL of the toxin inducer mitomycin C (Sigma M4287), to obtain the OD3 nanoparticle concentration. The culture medium was then poured into a plate and dried.

[0086] Strain preparation: - On day 1, inoculate 9 mL of EPT with CRA strain 9405024. - Incubate in a constant temperature incubator at 41.5°C for 18 hours. - Use a densimeter to calibrate the strain to 0.4 McFarland. - Perform serial dilutions directly into the dispensed matrix tubes.

[0087] Plate sowing: - 10 of the target strain 5 Take the cfu / mL dilution into a tube. - A portion of the tubes uses the VIDAS ESPT2 parameter, which is an immunoselection parameter that allows for the selection of target strains, namely E. coli O26, O45, O103, O111, O121, O145, and O157. The immunoselected samples are then seeded onto STEC agar by depleting 30 μl of the sample. - For the remaining areas, 10 μl is deposited onto STEC agar, and then seeding is carried out by direct depletion. - Incubate the plates in a constant temperature incubator at 37°C for 18-22 hours.

[0088] Example 2: Isolation of target strain (Escherichia coli O157:H7, STX1+eae+, ATCC:43890) in beef mince matrix and reblochon cheese matrix on culture medium without gradient. The culture medium, rubrochon cheese matrix, and inoculum were prepared in the same manner as in Example 1. In the case of a rubrochon cheese matrix that strongly charges the accompanying flora, Figures 1 and 2 show that it is difficult to isolate characteristic colonies using a halo. Not all colonies in the first quadrant can be identified because they are obscured by the accompanying flora. This applies to direct isolation from enrichment (Figure 1), but also after the immunoselection step with VIDAS (ESP2) (Figure 2).

[0089] In the case of the "meat" matrix, which has fewer associated flora than the "Reblochon cheese" matrix, characteristic colonies with halos were observed after direct isolation (Figure 3), and multiple characteristic colonies were observed after the immunoselection process (Figure 4).

[0090] Example 3: Isolation of target strain (Escherichia coli O157:H7, STX1+eae+, ATCC:43890) from ground beef matrix using the method according to the present invention. - Preparation of culture medium according to the present invention: The culture medium and the "ground beef" matrix are prepared according to Example 1. After the final drying step of the culture medium, perform the following steps: - Mark the zone where the CT inhibitor compound will be deposited on the back of the dish with dots using a marker. This zone is located at the edge of the agar. - Rehydrate the CT supplement bottle with 400 μl of sterile deionized water. - Deposit 10 μl of this CT supplement onto the agar at the previously defined point. - Then, let the plate dry for 10 minutes so that it can be used for isolation.

[0091] The plate is seeded in the same manner as in Example 1. The enriched ground beef sample is deposited in the CT supplement deposition zone.

[0092] The plate incubation is the same as that of Example 1.

[0093] Figure 5 shows the large inhibition diameter around the deposition point. Therefore, isolated colonies are observed, rather than as clumps as in the adjacent quadrant. Colonies with a halo characteristic of the target strain are observed in the inhibition concentration gradient zone. By applying this inhibitory compound that forms the gradient, characteristic colonies can be isolated.

[0094] In the case of immunoselected "ground beef" samples on a gradient plate, Figure 6 shows that characteristic bacterial strains can be detected, similar to the case of immunoselected "ground beef" samples on a non-gradient plate (Figure 4). The decrease in CT does not have an adverse effect. Therefore, this method is highly versatile and can be used regardless of the matrix.

[0095] Example 4: Isolation of the target strain (Escherichia coli O157:H7, STX1+eae+, ATCC:43890) in a reblochon cheese matrix using the method according to the present invention. Preparation of culture medium according to the present invention: Prepare the agar in the same manner as in Example 1. After the final drying step, carry out the following steps: - Mark the zone where the CT supplement will be deposited on the back of the plate with dots using a marker. This zone will be located at the edge of the agar layer. - Rehydrate the CT supplement bottle with 400 μl of sterile deionized water. - Deposit 10 μl of this CT onto the agar at the previously defined point. - Then, let the plate dry for 10 minutes so that it can be used for isolation.

[0096] The plates are seeded according to Example 1. A sample of enriched reblochon cheese is deposited in the CT supplement deposition zone.

[0097] The plate incubation is the same as that of Example 1.

[0098] Figure 7 shows the inhibition diameter around the deposition point. While isolated colonies are observed, their number is much greater than in the adjacent quadrant, where the colonies are clustered together. The presence of a concentration gradient, unlike the center of the plate in Figure 1 where there is no gradient, allows for the acquisition of characteristic isolated colonies.

[0099] Figure 8 shows characteristic isolated colonies from a sample that underwent an immunoselection process. The presence of a gradient provides characteristic isolated colonies, unlike the medium on the plate in Figure 2, which does not have a gradient.

[0100] Conclusion: By using a concentration gradient of compounds that inhibit non-target bacteria, it becomes possible to inhibit these non-target bacteria in the presence of a complex matrix containing the accompanying flora, thereby isolating more resistant target colonies.

Claims

1. A gelling reaction medium for the detection, identification, and / or isolation of at least one strain of Shiga toxin-producing Escherichia coli, - At least one type of toxin inducer, - At least one aggregated conjugate comprising at least one STX1-specific binding partner and / or at least one STX2-specific binding partner bound to a nanoparticle, - Concentration gradient of compounds that inhibit non-target bacteria A gelling reaction medium containing the following:

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

3. The gel reaction medium according to claim 1 or 2, characterized in that the binding partner is selected from antibodies or phage proteins.

4. A gelling reaction medium according to any one of claims 1 to 3, characterized in that the inhibitory compound contains tellurite.

5. The gelling reaction medium according to claim 4, characterized in that the inhibitory compound contains cefixime.

6. The gel reaction medium according to any one of claims 1 to 5, characterized in that the concentration gradient of tellurite in the gel reaction medium is between 0 μg / mL and 100 μg / mL, preferably between 0 μg / mL and 50 μg / mL, and more preferably between 0 μg / mL and 30 μg / mL.

7. A gelling reaction medium according to any one of claims 1 to 6, characterized in that the nanoparticles are colloidal nanoparticles having optical properties.

8. The nanoparticles are gold nanoparticles having a size between 20 nm and 90 nm, and 10 per 1 ml of reaction medium. １０ 1 and 10 １２ A gelling reaction medium according to any one of claims 1 to 7, characterized in that it exists at a nanoparticle concentration between 1 and 1.

9. A gelling reaction medium according to any one of claims 1 to 8, characterized in that the toxin inducer is an antibiotic.

10. The gel reaction medium according to any one of claims 1 to 9, characterized in that the toxin inducer is ciprofloxacin at a concentration between 0.005 mg / l and 0.030 mg / l.

11. The gel reaction medium according to any one of claims 1 to 10, characterized in that the toxin inducer is mitomycin C at a concentration between 0.10 mg / l and 0.50 mg / l.

12. The gelling reaction medium according to any one of claims 1 to 11, characterized in that the deposited inhibitor compound has a volume between 5 μl and 50 μl.

13. The gelling reaction medium according to any one of claims 1 to 12, wherein the inhibitory compound is contained in at least one substrate suitable for the diffusion of the inhibitory compound onto the gelling culture medium.

14. A diagnostic kit for preparing a reaction medium according to any one of claims 1 to 13, - Aggregated conjugates comprising at least one STX1-specific binding partner and / or at least one STX2-specific binding partner bound to nanoparticles, - Gel culture medium containing toxin inducer, - Compounds that inhibit non-target bacteria, A diagnostic kit that includes this.

15. The following steps: - A step of placing the aggregated conjugate by bringing it into contact with the gelled culture medium. - A step of depositing at least one compound that inhibits non-target bacteria, containing tellurite, onto a zone of gelled culture medium, wherein the inhibitory compound diffuses and forms an inhibitory concentration gradient around the deposit zone, and the concentration of the tellurite concentration gradient is between 0 μg / ml and 100 μg / ml, more preferably between 0 μg / ml and 50 μg / ml, and even more preferably between 0 μg / ml and 30 μg / ml. Preparation of a reaction medium according to any one of claims 1 to 13, including

16. Preparation of the reaction medium according to claim 15, wherein the inhibitory compound is deposited after the sample has been deposited.

17. A method for detecting and / or isolating Shiga toxin-producing Escherichia coli that may be present in a sample containing intestinal bacteria, comprising the following steps: - A step of providing a selective gelling culture medium that enables the growth of E. coli, which includes a concentration gradient of compounds that inhibit non-target bacteria. - Step of depositing the sample onto the gelled culture medium. - A step of incubating the culture medium under conditions that enable the growth of E. coli, - Process for isolating E. coli - Step to confirm that the E. coli is Shiga toxin-producing E. coli. Methods that include...

18. The method according to any one of claims 1 to 17, wherein the sample is deposited and diffused onto a culture medium by depletion technology.

19. A method for detecting and / or isolating Shiga toxin-producing Escherichia coli as described in claim 18, characterized in that the confirmation method is a molecular biological method or an immunological method.

20. A method for detecting and / or isolating Shiga toxin-producing Escherichia coli that may be present in a sample, according to any one of claims 17 to 19, characterized in that the culture medium is the culture medium according to any one of claims 1 to 13.

21. A method for detecting and / or isolating Shiga toxin-producing Escherichia coli that may be present in a sample, according to any one of claims 17 to 20, wherein the sample is incubated in an enrichment medium that allows for the growth of Shiga toxin-producing Escherichia coli prior to deposition on a culture medium.

22. The method according to claim 20 or 21, wherein the presence of Shiga toxin-producing Escherichia coli is confirmed by the presence of a halo around the Shiga toxin-producing Escherichia coli in the inhibitory concentration gradient zone.

23. The method according to any one of claims 17 to 22, wherein the Shiga toxin-producing Escherichia coli is enterohemorrhagic Escherichia coli.

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