Reaction culture medium 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, the reaction medium comprising:-at least one toxin-inducing agent,-at least one agglutination conjugate, -a nanoparticle comprising at least one specific binding partner of STX1 and / or at least one specific binding partner of STX2 conjugated to the nanoparticle,-a concentration gradient of a compound for inhibiting non-target bacteria. The invention also relates to related methods of detecting and / or isolating Shiga toxin-producing Escherichia coli that may be present in a sample containing intestinal bacteria.

Description

Technical Field

[0001] This invention broadly relates to the field of microbial detection, such as the microbial detection of samples from industrial or clinical sources. More specifically, this invention relates to a reaction medium and method for detecting Shiga toxin-producing Escherichia coli (STEC) and / or enterohemorrhagic Escherichia coli (EHEC).

[0002] Existing technology Microbial detection of samples from multiple sources requires techniques capable of detecting microorganisms (e.g., for identification and / or counting and / or biochemical characterization purposes) and results must be obtained as quickly as possible.

[0003] In the medical field, it is necessary to predict and diagnose infection risks: the faster and more accurate the diagnosis, the better the patient's treatment outcome and the lower the risk of transmission. A similar approach is used in the animal health field of veterinary medicine.

[0004] In the field of agricultural product processing, the underlying issues are the same. However, it is necessary to distinguish between the following types of microorganisms: - Pathogenic microorganisms, such as Shiga toxin-producing bacteria (STEC), Salmonella, Listeria, Cronobacter, Bacillus, and Staphylococcus, can be detected in raw materials, intermediate products, and finished products. - Non-pathogenic microorganisms are used as quality indicators throughout the entire industrial chain production process from raw materials to finished products; - Bacteria with technological value, such as fermenting bacteria; - Indicator microorganisms of contamination.

[0005] Rapid and accurate detection of suspected contamination (in food batches) enables control and swift corrective action.

[0006] From a technical perspective, one of the main challenges is the ability to isolate and identify the target bacteria. Microbiological analysis typically involves two phases. The first phase is the detection phase, which may involve various techniques such as culture medium incubation, immunoassay, and molecular biology techniques. Especially in the agricultural processing sector, a confirmation phase may follow to confirm the presence of the target pathogen and its compliance with current standards in the field. Therefore, the confirmation step requires additional procedures, and in this case, it inevitably involves the isolation of the target bacteria.

[0007] Therefore, the diagnosis of Shiga toxin-producing Escherichia coli relies on the use of selective chromogenic agar media, which may contain tellurite, such as CT-SMAC (sorbitol MacConkey agar) or CT-RMAC (rhamnosyl MacConkey agar), to select specific bacteria for growth and to chromogenic the target strain. The drawback of these methods is their extremely high specificity, meaning that some Shiga toxin-producing E. coli strains cannot be detected. Conversely, some media lack selectivity, and the presence of small numbers of Shiga toxin-producing E. coli may be masked by the presence of other widely distributed microorganisms, leading to isolation difficulties. Furthermore, to assess the pathogenicity of these strains, the presence of virulence factors needs to be detected. Specifically, pathogenicity involves the expression of several virulence genes, particularly six genes encoding two Shiga toxins (STX1 and STX2). These two toxins exert their effects by inhibiting protein synthesis in eukaryotic cells, ultimately leading to apoptosis. Currently, to accurately identify Shiga toxin-producing E. coli, polymerase chain reaction (PCR) detection of the stx genes is necessary.

[0008] Therefore, there is an urgent need to develop a reliable and rapid culture medium and method for isolating and identifying target bacteria, especially Shiga toxin-producing Escherichia coli (STEC) and enterohemorrhagic Escherichia coli (EHEC). Summary of the Invention

[0009] This invention relates to a gel-like reaction culture medium for detecting, identifying, and / or isolating at least one Shiga toxin-producing Escherichia coli strain, comprising: - At least one toxin inducer; - At least one aggregated conjugate comprising at least one STX1-specific binding chaperone and / or at least one STX2-specific binding chaperone, the binding chaperone being coupled to nanoparticles; - Concentration gradient of compounds that inhibit non-target bacteria.

[0010] The culture medium of the present invention has significant advantages in that it can isolate Shiga toxin-producing Escherichia coli (STEC) or enterohemorrhagic Escherichia coli (EHEC) from a large number of Escherichia coli.

[0011] Another subject of the present invention relates to a method for preparing the above-mentioned reaction culture medium, comprising the following steps: - Contact the aggregated conjugate with a gel-like culture medium; - Add at least one tellurite-containing compound that inhibits non-target bacteria to a region of a gel-like culture medium; the inhibitory compound diffuses and forms an inhibitory concentration gradient around the added region; the concentration of the tellurite concentration gradient is from 0 μg / mL to 100 μg / mL, preferably from 0 μg / mL to 50 μg / mL, and more preferably from 0 μg / mL to 30 μg / mL.

[0012] Another subject of the present invention relates to a method for detecting and / or isolating Shiga toxin-producing Escherichia coli that may be present in a sample containing enterobacteria, comprising the following steps: - Provides a selective gel-like culture medium that allows the growth of Escherichia coli, the culture medium containing a concentration gradient of compounds that inhibit non-target bacteria; - Add the sample to the gel-like culture medium; - Incubate the culture medium under conditions that allow for the growth of Escherichia coli; - Isolate Escherichia coli; - The Escherichia coli was confirmed to be a Shiga toxin-producing Escherichia coli.

[0013] Preferably, the culture medium is the culture medium described in this invention.

[0014] Therefore, it is advantageous to confirm the presence of Shiga toxin-producing Escherichia coli by the halo formed around Shiga toxin-producing Escherichia coli present in the region of the inhibition concentration gradient.

[0015] The advantage of this preferred embodiment is that it enables the direct detection and isolation of the STEC or EHEC on the culture medium, providing immediate results.

[0016] In another embodiment, the method can detect 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 microbial communities, where a large number of colonies on the petri dish can lead to false negatives. Because of the high colony count and low proportion of the target microorganism on the petri dish, personnel collecting colonies for confirmation may never be able to collect the target microorganism. The probability of isolating Shiga toxin-producing Escherichia coli (STEC) is significantly increased in the presence of an inhibitory compound gradient. Therefore, this invention provides a method for detecting and isolating STEC that avoids false negatives and saves considerable time. Attached Figure Description

[0018] Figure 1 Photograph of a control culture medium without an inhibitory compound concentration gradient, on which Rebroshin cheese samples were inoculated using the streak plate method.

[0019] Figure 2 Photograph of a control culture medium without an inhibitory compound gradient, on which samples of "Rebroshen cheese" that have undergone an immunoselection process were inoculated using the streak plate method.

[0020] Figure 3Photograph of control culture medium without an inhibitory compound gradient, on which "minced beef" samples were inoculated using the streak plating method. Arrows point to the halos around the colonies.

[0021] Figure 4 Photograph of a control culture medium without an inhibitory compound gradient, on which "minced beef" samples that have undergone an immunoselection step are inoculated by streak plating.

[0022] Figure 5 Photograph of a culture medium containing a gradient of inhibitory compounds (cefixime-tellurite mixture), on which "minced beef" samples were inoculated by streak plating.

[0023] Figure 6 Photograph of a culture medium containing a gradient of inhibitory compounds (cefixime-tellurite mixture), on which previously immunized "minced beef" samples were inoculated by streak plating.

[0024] Figure 7 Photograph of a culture medium containing a gradient of inhibitory compounds (cefixime-tellurite mixture), on which a sample of "Rebroshin cheese" was inoculated using the streak plating method. Arrows point to halos around the colonies.

[0025] Figure 8 Photograph of a culture medium containing a gradient of inhibitory compounds (cefixime-tellurite mixture), on which previously immunized "Rebroshen cheese" samples were inoculated by streak plating. Invention Details The following details certain terms and expressions used in this invention.

[0027] The first subject of this invention relates to a gel-like reaction culture medium for detecting, identifying, and / or isolating at least one Shiga toxin-producing Escherichia coli strain, comprising: - At least one toxin inducer; - At least one aggregated conjugate comprising at least one STX1-specific binding chaperone and / or at least one STX2-specific binding chaperone, and coupled to nanoparticles. - Concentration gradient of compounds that inhibit non-target bacteria.

[0028] The term "reaction medium" refers to a culture medium containing all the elements necessary for the metabolic expression, survival, and / or growth of microorganisms. This reaction medium can be a microbial culture medium or a microbial assay medium. In the latter case, the microorganisms may be pre-cultured in another medium. The reaction medium may also be placed in contact with agar medium. It may be placed below or above a medium that allows the target microorganism to grow. The reaction medium may be added after incubation with the culture medium. Preferably, the reaction medium is a microbial culture medium containing all the elements necessary for microbial growth.

[0029] According to the present invention, the reaction medium is gel-like. It is in solid or semi-solid form. Agar is a conventional gelling agent used in microbiology for microbial culture, but other gelling agents, such as gelatin, agarose, and other natural or artificial gelling agents, can also be used. The conjugate is able to form a network with the target component in the gel-like medium. This network can be visually detected by the formation of halos without reducing the stiffness of conventional semi-solid reaction media. Therefore, without modifying the physicochemical properties of the reaction medium (such as stiffness), the diffusion of the target component can be allowed first, and then the reaction with the conjugate can be achieved.

[0030] A variety of preparations are commercially available, such as Columbia agar, trypsin soybean agar, MacConkey agar, Mueller-Hinton agar, or more generally, those described in the *Handbook of Microbial Culture Media*. These media can be used as the basis for the reaction media of this invention. The reaction media may also contain possible 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 inhibit or slow the growth of "non-target" microorganisms (i.e., microorganisms other than the target microorganism). The term "inducer" refers to a compound that can induce the expression of a compound (such as an enzyme or toxin) that is not normally expressed. The reaction media may also contain coloring agents. For example, Evans blue, neutral red, sheep blood, horse blood, opacifiers (such as titanium dioxide), nitroaniline, malachite green, and brilliant green can be mentioned as coloring agents. When the reaction medium of the present invention further contains an enzyme substrate that is specific to the enzyme activity of at least one target microorganism, a chromogenic and / or fluorescent substrate is preferably used. The term "chromogenic and / or fluorescent substrate" refers to a substrate capable of detecting the enzyme activity or metabolic activity of the target / detectable microorganism by means of a detectable signal. The reaction medium of the present invention may additionally contain a pH indicator that is sensitive to pH changes caused by substrate consumption and indicates the metabolic status of the target microorganism. The pH indicator may be a chromophore or a fluorophore. Exemplary chromophores include bromocresol purple, bromothymol blue, neutral red, aniline blue, and bromocresol green.

[0031] Those skilled in the art may also use segmented culture dishes (such as double or triple dishes) to facilitate comparison of several culture media containing different substrates or different selective mixtures, and inoculated with the same biological sample.

[0032] According to the present invention, the term "microorganism" has the generally accepted meaning in microbiology, and includes in particular Gram-positive or Gram-negative bacteria, yeasts, molds, and more generally, single-celled organisms that are invisible to the naked eye and can be manipulated and propagated in the laboratory.

[0033] The term "target microorganism" refers to at least one microorganism that needs to be detected and / or identified. According to the present invention, the microorganism is selected from Shiga toxin-producing *Escherichia coli* strains. Enterohemorrhagic *Escherichia coli* is a subgroup of Shiga toxin-producing *Escherichia coli* strains that possess the *eae* gene or other cell adhesion genes (such as the *aggR* gene) and cause hemolytic uremic syndrome. The co-existence of both *stx* and *eae* virulence factors makes this pathogenic strain extremely toxic to humans. In particular, serogroups O26, O45, O80, O103, O111, O121, O145, and O157 are highly toxic.

[0034] Therefore, according to the present invention, the target microorganism is *Escherichia coli* belonging to the Shiga toxin-producing *Escherichia coli* group, or typical enterohemorrhagic *Escherichia coli* carrying the *stx* and *eae* genes, or atypical enterohemorrhagic *Escherichia coli* carrying the *stx* and *aggR* genes. Non-target microorganisms are bacteria grown on culture media outside the concentration gradient region of the inhibitory compound. Specifically, since the sample is multimicrobial, any potentially present STECs or EHECs are present in nonspecific Gram-negative bacteria (such as symbiotic *Escherichia coli*).

[0035] According to the present invention, the reaction medium contains at least one STX1 and / or STX2 specific binding chaperone and is coupled to nanoparticles. STX1 and STX2 are toxins secreted by Shiga toxin-producing Escherichia coli (STEC) strains. Shiga toxins are mainly of two types: STX1 and STX2, each with multiple variants. To date, STX1 has four subtypes: STX1a, STX1c, STX1d, and STX1e, while STX2 has 12 subtypes: STX2a, STX2b, STX2c, STX2d, STX2e, STX2f, STX2g, STX2h, STX2i, STX2j, STX2k, and STX2l.

[0036] The specific binding partner is selected from antibodies, all types of Fab fragments, recombinant proteins, phages, phage proteins, oligonucleotides, aptamers, affinity molecules, or any other ligand or anti-ligand known to those skilled in the art. Preferably, the binding partner is selected from antibodies and phage proteins. More 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. This component can be a derivative of the bacterial surface, such as a protein, lipopolysaccharide (LPS), or flagella. It can also be a component inside the bacteria, such as RNA or intracellular proteins, which can be detected when a portion of the bacterial colony dies during growth. The binding partner can also be specific to a derivative component of the microorganism. This component can be a target molecule produced by the target microorganism, such as a protein, antibiotic, antimicrobial resistance molecule, protease, lipase, or glycosidase. According to the invention, the culture medium contains at least one STX1 protein-specific binding partner and / or at least one STX2 protein-specific binding partner. In another embodiment, the culture medium contains at least one STX1 protein subtype-specific binding chaperone and / or at least one STX2 protein subtype-specific binding chaperone (e.g., STX2a or STX2d). According to the invention, the binding chaperones are coupled to the nanoparticles. This final complex is called a conjugate. Conjugates with different types of binding chaperones, coupled to different types of nanoparticles, can exist in the same reaction medium.

[0037] The term "nanoparticle" refers to particles with a size approximately at the nanometer scale. Nanoparticles can be selected from gold, iron, silver, copper, carbon, latex, silicon, and aluminum. Preferably, the nanoparticles are colloidal nanoparticles, chosen based on their optical properties, i.e., their distinguishability when forming a network. More preferably, the nanoparticles are selected from gold, silver, and copper. Thus, when the nanoparticles are gold, they change color, for example, from red to gray when forming a network. When unaggregated, they absorb light at wavelengths in the red region, approximately 530 nm. When aggregated, the absorption wavelength changes from red to blue / gray, approximately 600 to 700 nm. In certain embodiments, multiple different colored nanoparticles can be used in combination. The resulting network can distinguish several components of the target microorganism.

[0038] Preferably, the size of the nanoparticles is between 10 and 200 nm. More preferably, the size of the nanoparticles is between 20 and 90 nm, so that the conjugate has better flowability in the reaction medium.

[0039] Advantageously, nanoparticles can reduce the number of binding partners required for aggregation. Therefore, the concentration of binding partners required to prepare the reaction medium of the present invention needs to be 100 to 1000 times less than that of a medium without nanoparticles.

[0040] Preferably, the amount of the desired binding partner corresponds to the amount covering at least half of the nanoparticle surface, more preferably covering one-third to one-half of the nanoparticle surface. This ratio allows the aggregated conjugate to form a network in a gel-like reaction medium.

[0041] Advantageously, nanoparticles allow for visualization of aggregation around bacterial colonies before they reach a size visible to the naked eye, enabling earlier detection. Furthermore, nanoparticles can also be used to visualize aggregation around translucent colonies that cannot be detected by automated readers, thus facilitating detection.

[0042] The coupling of nanoparticles with their binding partners can be achieved through direct or indirect binding. Direct binding refers to binding via adsorption or covalent bonding. Indirect binding refers to binding via ligand / anti-ligand interactions, such as biotin / streptavidin or other pairings well known to those skilled in the art (Nicholas G. Weich et al., 2017). Depending on the type of binding chosen, those skilled in the art will adjust the physicochemical conditions of the reaction medium, particularly its pH.

[0043] According to the present invention, the conjugates possess agglutinative properties, meaning they are capable of forming agglutinative networks in the presence of a component of the target microorganism or a derivative component of said microorganism. Since the component is multi-epitope, multiple conjugates will attach to the component and form agglutinates. Agglutination refers to the result of an interaction between at least one component of the target microorganism or at least one derivative component of said microorganism and a binding chaperone coupled to the nanoparticle. Agglutination reactions include immune reactions, such as antigen-antibody reactions, or more generally, specific interactions between two molecules. Through such interactions, components and conjugates aggregate, adhere to each other, and form networks in the reaction medium.

[0044] In fact, several parameters affect the aggregation ability of conjugates in gel-like culture media, mainly including: - Porosity of gel-like culture media; - The size of the nanoparticles; - The amount of the partner; - Amount of nanoparticles.

[0045] Therefore, these parameters must be adjusted to achieve a satisfactory agglutination effect that can be detected. Advantageously, the network formed by the specific reaction can be automatically detected by the naked eye or using an optical system. Thus, colonies of the target microorganism are detected. Preferably, the network forms a halo in the gel-like reaction medium, which can be detected by the naked eye or using an optical system. This network or halo surrounds the colony, thereby enabling it to be distinguished and / or identified within the population.

[0046] In a particular embodiment, the binding partner is an antibody, and the amount of the antibody present is sufficient to allow coverage of at least half of the nanoparticle surface, preferably at least one-third of the nanoparticle surface.

[0047] In these embodiments, preferably, the nanoparticles are gold nanoparticles with a size of 20 to 90 nm, and the concentration is 10 ppm per milliliter of reaction medium. 10 Up to 10 12 Nanoparticles.

[0048] According to the present invention, the reaction medium contains a toxin inducer. The toxin inducer stresses the bacteria, thereby triggering the lytic cycle of prophages in the bacteria, and thus stimulating the production and release of toxins. Advantageously, the toxin inducer is selected from antibiotics or physicochemical stressors. Exemplary antibiotics include trimethoprim, sulfamethoxazole, norfloxacin, azithromycin, gentamicin, polymyxin B, chloramphenicol, streptomycin, chlortetracycline, oxytetracycline, tylosin, mitomycin C, carbadox, quinethol, rifampin, imipenem, ciprofloxacin, trimethoprim-sulfamethoxazole, penicillin G, and lincomycin. In a preferred embodiment, the culture medium of the present invention contains ciprofloxacin at a concentration of 0.005 to 0.030 mg / L. In another preferred embodiment, the culture medium of the present invention contains mitomycin C at a concentration of 0.10 to 0.50 mg / L.

[0049] Toxin inducers can also be physicochemical stressors, such as those produced by adding salt or EDTA, altering pH, or ultraviolet radiation. Stress can also be induced, for example, by adding norepinephrine.

[0050] The target microorganism may be present in the sample. The term "sample" refers to a small portion or amount of substance isolated from an entity for analysis. Samples can be of industrial origin, including but not limited to air samples, water samples, surface-collected samples, parts or finished products, or food. Samples can also be biological fluid samples (feces, urine, whole blood, serum, plasma, cerebrospinal fluid, biological secretions, etc.), body surface samples (skin, nasal cavity, pharynx, etc.), tissue samples, or isolated cells. Samples can be used directly or prepared prior to analysis according to methods known to those skilled in the art, such as enrichment, extraction, concentration, or purification. The reaction medium of the present invention may be more advantageous when the sample is a multimicrobial sample or contains accompanying microbiota (e.g., food samples such as raw milk cheese or feces in clinical samples).

[0051] According to the present invention, the reaction medium contains a concentration gradient of compounds that inhibit non-target bacteria. According to the present invention, the selected inhibitory compounds should result in a minimum inhibitory concentration (MIC) of non-target bacteria (such as symbiotic enterobacteria) that is significantly lower than the MIC of STEC / EHEC.

[0052] The term "inhibitory compound" refers to any compound that inhibits bacterial growth, and may be an antibiotic, a coloring agent (such as brilliant green), a salt (such as lithium chloride), or a phage lysin. Preferably, the inhibitory compound is a tellurite. According to the present invention, the inhibitory compound helps to inhibit non-STEC and non-EHEC strains.

[0053] The term "gradient" refers to the non-uniform, increasing or decreasing concentration of an inhibitory compound.

[0054] Based on the types of bacteria present in a multimicrobial sample, a concentration gradient can be used to distinguish non-target microorganisms whose minimum inhibitory concentration (MIC) is lower than that of the target microorganism. Therefore, non-target microorganisms are those capable of growing on media without an inhibitory compound concentration gradient. The target microorganism is one carrying the stx gene, i.e., a STEC.

[0055] Therefore, the minimum inhibitory concentration (MIC) of non-target bacteria (such as symbiotic Enterobacteriaceae) is usually lower than that of STEC and EHEC.

[0056] Preferably, when the inhibitory compound contains tellurite, the tellurite gradient in the culture medium is from 0 μg / mL to 100 μg / mL, more preferably from 0 μg / mL to 50 μg / mL, and even more preferably from 0 μg / mL to 30 μg / mL. Different strains belonging to STEC and EHEC have varying sensitivities to inhibitory compounds such as tellurite. Specifically, STEC and EHEC generally exhibit higher resistance to tellurite than other enteric bacteria.

[0057] Furthermore, within a target population (such as STEC or EHEC), different serogroups may exhibit varying susceptibility. For example, the MIC of *E. coli* O157 to cefixime-tellurite differs from that of *E. coli* O103. Differences in susceptibility also exist within the same serogroup. Therefore, the MIC of one *E. coli* O111 strain may differ from that of another *E. coli* O111 strain.

[0058] The culture medium of the present invention has significant advantages, enabling the isolation of STEC or EHEC from large quantities of Escherichia coli.

[0059] In a preferred embodiment, the inhibitory compound is a mixture of cefixime and tellurite. Preferably, the amount of cefixime in the added cefixime-tellurite mixture is 0.20 to 0.30 μg, and the amount of tellurite is 12 to 13 μg.

[0060] Preferably, the volume of the inhibitory compound added to the culture medium is 5 to 50 μl. The inhibitory compound can be added by any means, such as pipetting.

[0061] In a particular embodiment, the inhibitory compound is contained in at least one matrix suitable for diffusing the inhibitory compound onto a gel-like culture medium. For example, this could be an absorbent paper pad containing the inhibitory compound and suitable for diffusion. In this particular embodiment, the matrix can be placed at the bottom of the culture dish, in contact with the agar medium, before the culture medium is poured into it.

[0062] Another subject of the present invention relates to a diagnostic kit for preparing the reaction medium of the present invention, comprising: - Aggregates comprising at least one STX1-specific binding chaperone and / or at least one STX2-specific binding chaperone, and coupled to nanoparticles. - A gel-like culture medium containing toxin inducers; - Compounds that inhibit non-target bacteria.

[0063] Advantageously, the kit of the present invention can be used to prepare reaction media on an immediate basis, thereby enabling single-use of the reaction media and improving its stability. Preferably, the media should be used within 24 hours after preparation, and even more preferably within 12 hours.

[0064] Another subject of the present invention relates to a method for preparing the reaction culture medium of the present invention, comprising the following steps: - The aggregated conjugate is contacted with a gel-like culture medium to form a reaction medium; - Add at least one compound that inhibits non-target bacteria to a region of a gel-like culture medium; the inhibitory compound diffuses and forms an inhibitory concentration gradient around the region of addition; the concentration of the tellurite gradient is from 0 μg / mL to 100 μg / mL, preferably from 0 μg / mL to 50 μg / mL, and more preferably from 0 μg / mL to 30 μg / mL.

[0065] Therefore, the agglomerated conjugate is contacted with an ultracooled gel-like medium to form the reaction medium of the present invention. The gel-like medium also contains a toxin inducer. The mixture is then homogenized and poured into petri dishes.

[0066] A compound that inhibits non-target bacteria is added to a region of a gel-like culture medium. The inhibitory compound diffuses and forms an inhibition gradient around the added region. In another embodiment, the inhibitory compound is added after the sample has been added. In yet another embodiment, the inhibitory compound is a matrix suitable for diffusion and is placed in the petri dish before being poured into the culture medium.

[0067] Preferably, the inhibitory compound contains a colorant that allows for the localization of its addition site on the culture medium, thereby facilitating the addition of the sample at the location of the inhibitory compound.

[0068] Another subject of the invention relates to an in vitro microbial culture method, wherein microorganisms that may be present in a sample are inoculated into or onto the culture medium of the invention. The inoculated culture medium is then incubated under suitable conditions known to those skilled in the art. Inoculation is performed using conventional microbiological techniques.

[0069] In a preferred embodiment of the invention, inoculation is performed by streaking a multimicrobial sample onto a culture medium. The sample is added at the site where the inhibitory compound is added, so that the sample with the highest microbial load comes into contact with the inhibitory concentration gradient. Specifically, in the first separation zone, due to the extremely high microbial concentration, it is usually difficult to distinguish between target and non-target microorganisms.

[0070] Inoculation techniques are well known to those skilled in the art. One inoculation technique is the four-zone streak method. The four-zone streak method involves dividing a petri dish in half, then dividing each half in half again to obtain three zones of 50%, 25%, and 25%, respectively. A small amount of inoculum is placed in the largest zone and spread out. The petri dish is then rotated a quarter turn to spread the bacteria to the smaller zones, and then rotated another quarter turn to inoculate the last small zone. In this embodiment, the inhibitory compound and the sample are added to the same zone. Of course, there are various variations of this technique, such as the multi-strike plate method, where the inoculum is first densely streaked downwards, and then other streaks are made starting from the edge of the previous streaks.

[0071] Another subject of the present invention relates to a method for detecting and / or isolating Shiga toxin-producing Escherichia coli that may be present in a sample containing enteric bacteria, comprising the following steps: - Provides a selective gel-like culture medium that allows the growth of Escherichia coli, the culture medium containing a concentration gradient of compounds that inhibit non-target bacteria; - Add the sample to the culture medium; - Incubate the culture medium under conditions that allow for the growth of Escherichia coli; - Isolate Escherichia coli; - The Escherichia coli was confirmed to be a Shiga toxin-producing Escherichia coli.

[0072] The term "detection" refers to the observation of the growth of a target microorganism (preferably target bacteria) by visual inspection or the use of optical devices. When the culture medium used to detect the target microorganism contains a chromogenic or fluorescent substrate, the fluorescent substrate can be detected by optical devices, while the chromogenic substrate can be detected by visual inspection or optical devices. When the culture medium contains agglutinating conjugates, detection can be performed by visual inspection or by observing agglutination around the target microorganism.

[0073] The term "isolation" refers to the process of obtaining colonies of bacteria that are separated from each other.

[0074] Surprisingly, the presence of an inhibitory compound gradient has been found to improve the detection and isolation of target bacteria (such as Shiga toxin-producing Escherichia coli or enterohemorrhagic Escherichia coli) from large numbers of Escherichia coli.

[0075] According to the present invention, a selective culture medium that allows the growth of *Escherichia coli* is provided, comprising a concentration gradient of compounds that inhibit non-target bacteria (such as symbiotic enterobacteria). Examples of culture media that allow the growth of *E. coli* include CHROMID® coli, CHROMID® EHEC, TBX, and Rainbow Agar. The concentration gradient of compounds that inhibit non-target bacteria should then be added.

[0076] Inhibitory compounds were selected to allow for the differentiation of strains with varying sensitivities to the inhibitory compounds within a population of Shiga toxin-producing Escherichia coli.

[0077] Inhibitory compounds can be added to the culture medium by any means (such as pipetting). After addition, the inhibitory compound forms an inhibitory concentration gradient around the area where it is added. Alternatively, the inhibitory compound can be added to a matrix that diffuses.

[0078] In another embodiment, an inhibitory compound is added after the sample is added.

[0079] According to the present invention, a confirmation step is performed to identify the presence of Shiga toxin-producing Escherichia coli. This step can be performed using conventional confirmation methods, such as molecular biological methods or immunological methods. In this embodiment, colonies in contact with a gradient of inhibitory compounds are collected on a culture medium. When E. coli grows on the gradient, it is highly likely to be Shiga toxin-producing E. coli. Latex tests or polymerase chain reaction (PCR) tests targeting the target microorganism can be performed, such as the SLIDEX® E. coli test, or the GENE-UP® STEC top 6 test, or the GENE-UP® E. coli O157:H7 test, or the GENE-UP® STEC stx and eae polymerase chain reaction (PCR) test.

[0080] In a specific implementation, the Shiga toxin-producing Escherichia coli is enterohemorrhagic Escherichia coli.

[0081] According to a preferred embodiment of the invention, the sample undergoes an incubation or enrichment step before being added to a gel-like culture medium. This enrichment step requires not only a dedicated culture medium but also incubation of at least the biological sample and the mixture formed by the culture medium at an optimal temperature to allow the growth of the target microorganism. Incubation is typically carried out for a predetermined time (e.g., 6 to 48 hours) within a temperature range of 25 to 45°C. This enrichment stage requires the use of selective or non-selective culture media (depending on the expected results) that function to promote the growth of the target microorganism in the sample while restricting the growth of non-target microbiota. The culture medium is typically used in sterile plastic bag-type containers, in contact with the food, clinical, or environmental sample within the container to resuspend and enrich the target microorganism. As mentioned above, this enrichment stage may be necessary, particularly to demonstrate the presence of at least one target microorganism in highly variable and potentially very large sample volumes (e.g., 25 to 375 grams, diluted in 225 to 3375 mL of culture medium). After the enrichment step, aliquots of the sample (typically 5 μL to 5 mL) are typically collected for detection and / or isolation steps of the target microorganism.

[0082] In an advantageous embodiment of the invention, the sample is added and spread on the culture medium using a streak plate technique.

[0083] After inoculation, the reaction medium is incubated under appropriate conditions known to those skilled in the art.

[0084] In a preferred embodiment of the invention, the culture medium is the one described herein containing at least one agglutinating conjugate and a gradient of inhibitory compounds. In this preferred embodiment, the presence of Shiga toxin-producing Escherichia coli is confirmed by the halo formed around the Shiga toxin-producing Escherichia coli within the inhibitory gradient region. The advantage of this preferred embodiment is that it allows for the direct detection and isolation of the Shiga toxin-producing Escherichia coli (STEC) or enterohemorrhagic Escherichia coli (EHEC) on the culture medium. This provides immediate results.

[0085] This invention is particularly advantageous for facilitating the detection of Shiga toxin-producing Escherichia coli (STEC) in multimicrobial samples. Specifically, without this invention (which allows for the localization of target colonies), the ISO 16136 reference method specifies that a maximum of 50 colonies must be detected by molecular methods to confirm the presence of Shiga toxin-producing Escherichia coli (STEC) or enterohemorrhagic Escherichia coli (EHEC). Due to the large number of colonies on the plate and the low proportion of the target microorganism, personnel collecting colonies for confirmation may never be able to collect the target microorganism. The probability of isolating Shiga toxin-producing Escherichia coli (STEC) is significantly increased when an inhibitory compound gradient is present at the sample addition site. This method is particularly advantageous for samples containing accompanying microbiota, in which a large number of colonies on the petri dish may lead to a risk of false negatives.

[0086] Example Example 1: Sample preparation, agar plate preparation and inoculation

[0087] equipment: - VIDAS bioMérieux (study number 4700023) - Densimat bioMérieux (species number 99234) - Plain board - Smasher bioMérieux (reference number AESAP1064) - 41.5℃ constant temperature incubator Matrix used: - Frozen ground beef with onions: Expiry date May 7, 2021, batch number 01973658 - Rebrosian cheese from Savoie (AOC): Expiry date February 6, 2023, batch number 125473226 Strains used: - Escherichia coli O157:H7; STX1+ eae+; CRA9405024 / ATCC43890 Sample preparation: - Weigh 25 grams of the test matrix into a bag with a filter. Add 225 ml of EPT medium - Mix with Shmasher for 1 minute - Incubate the bag at 41.5℃ for 18 hours in a constant temperature incubator. - After incubation, dispense into 9 ml test tubes. Tested different types of matrices: ground beef, Rebrozien cheese.

[0088] Preparation of agarose gel: - Preparation of gold nanoparticles 40 nm gold nanoparticles were prepared by reducing gold chloride with sodium citrate (using the method described by Turkevich and Frens in 1951). Therefore, a gold chloride solution was diluted in distilled water, sodium citrate was added, and the mixture was boiled to prepare 20 nm gold nanoparticles. An absorption peak appeared at 517–519 nm, confirming the correct particle size. These 20 nm particles were used to synthesize 40 nm particles. For this purpose, the 20 nm particles were diluted in distilled water, boiled, and sodium citrate and gold chloride were added. After cooling, an absorption peak was observed at 524–526 nm, indicating an increase in particle size.

[0089] - Preparation of conjugates The antibodies used were 13C4 antibody (hybridoma number ATCC CRL-1794) against STX1 toxin and 9E4H11 antibody against STX2 toxin.

[0090] The mixture was adsorbed onto nanoparticles at pH 8.

[0091] - Preparation of culture medium The conjugate was added to supercooled TBX agar at 50°C, the agar containing 250 ng / mL of the toxin inducer mitomycin C (Sigma M4287) to achieve an OD3 concentration of nanoparticles. The culture medium was then poured into plates and dried.

[0092] Preparation of strains: - On day 1, CRA9405024 strain was inoculated into 9 ml of EPT medium.

[0093] - Incubate at 41.5℃ for 18 hours in a constant temperature incubator.

[0094] - Use a densitometer to calibrate the strain to 0.4 McFarland turbidity.

[0095] - Perform continuous dilution directly in dispensed matrix test tubes.

[0096] Plate inoculation: - Take the target strain at a dilution of 10. 5 test tubes with cfu / mL - Take a portion of the test tube and use the VIDAS ESPT2 parameter. This is an immunoselection parameter that allows selection of target strains, namely Escherichia coli O26, O45, O103, O111, O121, O145, and O157. Then, take 30 μL of the immunoselected sample and streak it onto STEC agar.

[0097] - Another portion of the sample was taken in 10 μL and directly streaked onto STEC agar.

[0098] - Incubate the plates at 37°C in a constant temperature incubator for 18-22 hours.

[0099] Example 2: Isolation of the target strain (Escherichia coli O157:H7, STX1+ eae+, ATCC:43890) from ground beef and Rebrosense cheese matrix on a non-gradient medium. The culture medium, Rebrostan cheese substrate, and inoculation were all performed according to Example 1. For the Rebrostan cheese substrate with high accompanying microbial content, Figure 1 and Figure 2It was difficult to isolate colonies with halo characteristics. All colonies in the first quadrant were unidentifiable due to being masked by accompanying flora. This was true regardless of whether they were isolated directly from the enrichment. Figure 1 ), or after the VIDAS (ESP2) immune selection step ( Figure 2 This is true for all of them.

[0100] For the "meat" matrix with a lower bacterial content than the Rebrosche cheese matrix, colonies with halo characteristics were observed after direct isolation. Figure 3 After the immune selection step, multiple characteristic colonies were observed. Figure 4 ).

[0101] Example 3: Isolation of the target strain (Escherichia coli O157:H7, STX1+ eae+, ATCC:43890) from minced beef matrix using the method of the present invention. - Preparation of the culture medium of this invention: The culture medium and "minced beef" matrix were prepared according to Example 1.

[0102] After the final drying step of the culture medium, the following steps are performed: - Use a marker to mark the intended area for adding the CT inhibitory compound on the back of the petri dish with dots. This area is located at the edge of the agar.

[0103] - Reconstitute the CT supplement bottle with 400 μL of sterile deionized water.

[0104] - Add 10 microliters of this supplement to the previously marked agar site.

[0105] - Dry for 10 minutes for subsequent separation.

[0106] Plate inoculation was performed according to Example 1. The enriched minced beef sample was added to the CT supplement addition area.

[0107] The plate incubation was the same as in Example 1.

[0108] Figure 5 A large inhibition diameter is observed around the application point. Therefore, isolated colonies are observed, rather than dense colonies in the next quadrant. Halo-shaped colonies characteristic of the target strain are visible within the inhibition concentration gradient region. Therefore, applying this gradient-forming inhibitory compound allows for the isolation of characteristic colonies.

[0109] For the "minced beef" sample subjected to immune selection on gradient plates Figure 6 The detection efficacy of characteristic strains is shown compared to that of "minced beef" samples subjected to immunoselection on non-gradient plates. Figure 4It works just as well. CT droplets have no negative impact. Therefore, this method is versatile and applicable to any substrate.

[0110] Example 4: Isolation of the target strain (Escherichia coli O157:H7, STX1+eae+, ATCC:43890) from Rebrosense cheese matrix using the method of the present invention. - Preparation of the culture medium of this invention: Agar was prepared according to Example 1. After the final drying step, the following steps were performed: - Use a marker to mark the intended area for adding CT supplement on the back of the petri dish with dots. This area is located at the edge of the agar.

[0111] - Reconstitute the CT supplement bottle with 400 μL of sterile deionized water.

[0112] - Add 10 microliters of this supplement to the previously marked agar site.

[0113] - Dry for 10 minutes so that it can be separated subsequently using the plate.

[0114] Plate inoculation was performed according to Example 1. Enriched Rebroshin cheese samples were added to the CT supplement addition area.

[0115] The plate incubation was the same as in Example 1.

[0116] Figure 7 The addition point showed an inhibition diameter. Therefore, isolated colonies were observed, rather than the denser colonies in the next quadrant, where the number of colonies was much greater. Figure 1 Unlike plates without a gradient, the presence of a concentration gradient allows for the acquisition of characteristic isolated colonies.

[0117] Figure 8 The sample, after the immunoselective step, exhibits characteristic isolated colonies. (Compared to...) Figure 2 Unlike non-gradient plate culture media, the presence of a gradient allows for the acquisition of characteristic isolated colonies.

[0118] in conclusion: In a complex matrix containing accompanying bacterial communities, a concentration gradient of compounds that inhibit non-target bacteria is used to suppress the non-target bacteria, thereby isolating target colonies with stronger resistance.

Claims

1. A gel-like reaction medium for detecting, identifying, and / or isolating at least one Shiga toxin-producing Escherichia coli strain, comprising: - At least one toxin inducer; - At least one aggregated conjugate comprising at least one STX1-specific binding chaperone and / or at least one STX2-specific binding chaperone, the binding chaperone being coupled to nanoparticles; - Concentration gradient of compounds that inhibit non-target bacteria.

2. The gel-like reaction culture medium according to claim 1, characterized in that, The gel-like reaction medium is a microbial culture medium.

3. The reaction culture medium according to any one of the preceding claims, characterized in that, The binding partner is selected from antibodies or phage proteins.

4. The culture medium according to any one of the preceding claims, characterized in that, The inhibitory compound contains tellurite.

5. The culture medium according to the preceding claims, characterized in that, The inhibitory compound contains cefixime.

6. The culture medium according to any one of the preceding claims, characterized in that, The concentration gradient of tellurite in the culture medium is from 0 μg / mL to 100 μg / mL, preferably from 0 μg / mL to 50 μg / mL, and more preferably from 0 μg / mL to 30 μg / mL.

7. The culture medium according to any one of the preceding claims, characterized in that, The nanoparticles are colloidal nanoparticles with optical properties.

8. The culture medium according to any one of the preceding claims, characterized in that, The nanoparticles are gold nanoparticles with a size of 20 to 90 nm, and the concentration is 10 ppm per milliliter of reaction culture medium. 10 Up to 10 12 Nanoparticles.

9. The culture medium according to any one of the preceding claims, characterized in that, The toxin inducer is an antibiotic.

10. The culture medium according to any one of the preceding claims, characterized in that, The toxin inducer is ciprofloxacin at a concentration of 0.005 to 0.030 mg / L.

11. The culture medium according to any one of the preceding claims, characterized in that, The toxin inducer is mitomycin C at a concentration of 0.10 to 0.50 mg / L.

12. The culture medium according to any one of the preceding claims, characterized in that, The volume of the added inhibitory compound was 5 to 50 μl.

13. The culture medium according to any of the preceding claims, wherein the inhibitory compound is contained in at least one matrix suitable for diffusing the inhibitory compound onto the gel-like culture medium.

14. A diagnostic kit for preparing the reaction medium according to any one of claims 1 to 13, comprising: - Aggregates comprising at least one STX1-specific binding chaperone and / or at least one STX2-specific binding chaperone, wherein the binding chaperone is coupled to nanoparticles. - A gel-like culture medium containing toxin inducers; - Compounds that inhibit non-target bacteria.

15. A method for preparing the reaction medium according to any one of claims 1 to 13, comprising the following steps: - Contact the aggregated conjugate with a gel-like culture medium; - Add at least one tellurite-containing compound that inhibits non-target bacteria to a region of a gel-like culture medium; the inhibitory compound diffuses and forms an inhibitory concentration gradient around the added region; the concentration of the tellurite concentration gradient is from 0 μg / mL to 100 μg / mL, more preferably from 0 μg / mL to 50 μg / mL, and even more preferably from 0 μg / mL to 30 μg / mL.

16. The method for preparing the culture medium according to the preceding claims, wherein the inhibitory compound is added after the sample is added.

17. A method for detecting and / or isolating Shiga toxin-producing Escherichia coli that may be present in a sample containing enteric bacteria, comprising the following steps: - Provides a selective gel-like culture medium that allows the growth of Escherichia coli, the culture medium containing a concentration gradient of compounds that inhibit non-target bacteria; - Add the sample to the gel-like culture medium; - Incubate the culture medium under conditions that allow for the growth of Escherichia coli; - Isolate Escherichia coli; - The Escherichia coli was confirmed to be a Shiga toxin-producing Escherichia coli.

18. The method according to any of the preceding claims, wherein the sample is added and spread on the culture medium by streak plating.

19. The method for detecting and / or isolating Shiga toxin-producing Escherichia coli according to the preceding claims, characterized in that, The confirmation method is a molecular biology method or an immunology method.

20. The 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 any one of claims 1 to 13.

21. The method for detecting and / or isolating Shiga toxin-producing Escherichia coli that may be present in a sample according to any of the preceding claims, wherein the sample is incubated in an enrichment medium that allows the growth of Shiga toxin-producing Escherichia coli before being added to the culture medium.

22. The method of claim 20 or 21, wherein the presence of Shiga toxin-producing Escherichia coli is confirmed by the halo surrounding the Shiga toxin-producing Escherichia coli present in the region of the inhibition concentration gradient.

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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