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EP-4735619-A1 - REACTION MEDIUM AND METHOD FOR DETECTING SHIGA TOXIN-PRODUCING E.COLI AND/OR ENTEROHAEMORRHAGIC E.COLI

EP4735619A1EP 4735619 A1EP4735619 A1EP 4735619A1EP-4735619-A1

Abstract

The present invention relates to a gelled reaction medium for detecting, identifying, and/or isolating at least one Shiga toxin-producing strain of E. coli , the reaction medium comprising: - at least one toxin inducer, - at least one agglutinating conjugate comprising at least one specific binding partner of STX1 and/or at least one specific binding partner of STX2, coupled to a nanoparticle; - a concentration gradient of a compound for inhibiting non-target bacteria. The present invention also relates to the associated method for detecting and/or isolating Shiga toxin-producing E. coli which is likely to be present in a sample comprising enterobacteria.

Inventors

  • JUNILLON, Thomas
  • MALLEN, Benoit

Assignees

  • BIOMERIEUX

Dates

Publication Date
20260506
Application Date
20240625

Claims (20)

  1. [Claim 1] Gelled reaction medium for the detection, identification, and/or isolation of at least one strain of E. coli producing shiga toxin comprising - at least one toxin inducer, - at least one agglutinating conjugate comprising at least one specific binding partner of STX1 and/or at least one specific binding partner of STX2, coupled to a nanoparticle - a concentration gradient of a compound inhibiting non-target bacteria
  2. [Claim 2] Gelled reaction medium according to claim 1, characterized in that it is a microbiological culture medium.
  3. [Claim 3] Reaction medium according to any one of the preceding claims, characterized in that the binding partner is chosen from antibodies or phage proteins.
  4. [Claim 4] Medium according to any one of the preceding claims, characterized in that the inhibitory compound comprises tellurite.
  5. [Claim 5] Medium according to the preceding claim, characterized in that the inhibitory compound comprises cefixime.
  6. [Claim 6] Medium according to any one of the preceding claims, characterized in that the tellurite concentration gradient on the medium is between 0 pg/ml and 100 pg/ml, preferably between 0 pg/ml and 50 pg/ml, even more preferably between 0 pg/ml and 30 pg/ml.
  7. [Claim 7] Medium according to any one of the preceding claims, characterized in that the nanoparticle is a colloidal nanoparticle having optical properties.
  8. [Claim 8] Medium according to any one of the preceding claims, characterized in that the nanoparticle is a gold nanoparticle of size between 20 and 90 nm and is present at a concentration of between 10 10 and 10 12 nanoparticles/ml of reaction medium.
  9. [Claim 9] Medium according to any one of the preceding claims, characterized in that the toxin inducer is an antibiotic.
  10. [Claim 10] Medium according to any one of the preceding claims, characterized in that the toxin inducer is ciprofloxacin at a concentration of between 0.005 and 0.030 mg/l.
  11. [Claim 11] Medium according to any one of the preceding claims, characterized in that the toxin inducer is mitomycin C at a concentration of between 0.10 mg/l and 0.50 mg/l.
  12. [Claim 12] Medium according to any one of the preceding claims, characterized in that the deposited inhibitor compound has a volume of between 5 μl and 50 μl.
  13. [Claim 13] Medium according to any one of the preceding claims in which the inhibitory compound is contained in at least one substrate capable of diffusing said inhibitory compound onto said gelled culture medium.
  14. [Claim 14] diagnostic kit allowing the preparation of a reaction medium according to claims 1 to 13 comprising - an agglutinating conjugate comprising at least one specific binding partner of STX1 and/or at least one specific binding partner of STX2, coupled to a nanoparticle - a gelling medium comprising a toxin inducer - a compound that inhibits non-target bacteria.
  15. [Claim 15] Preparation of a reaction medium according to claims 1 to 13 comprising the following steps: - contacting an agglutinating conjugate with a gelling culture medium - depositing on an area of the gelled culture medium, at least one compound inhibiting non-target bacteria comprising tellurite; said inhibitory compound diffusing and forming an inhibition concentration gradient around the deposition area; the concentration of the tellurite concentration gradient being between 0 pg/ml and 100 pg/ml, preferably between 0 pg/ml and 50 pg/ml, even more preferably between 0 pg/ml and 30 pg/ml
  16. [Claim 16] Preparation of a medium according to the preceding claim in which the inhibitory compound is deposited after the deposition of the sample.
  17. [Claim 17] Method for detecting and/or isolating Shiga toxin-producing E. coli likely to be present in a sample comprising enterobacteria comprising the following steps: - have a selective gelled culture medium allowing the growth of E. coli comprising a concentration gradient of a compound inhibiting non-target bacteria - place the sample on said gelled culture medium - incubate said medium in conditions allowing the growth of E. coli - isolate an E. coli - confirm that the said E. coli is a Shiga toxin-producing E. coli
  18. [Claim 18] A method according to any preceding claim wherein the sample is deposited and distributed over the medium by an exhaustion technique;
  19. [Claim 19] Method for detecting and/or isolating E coli producing shiga toxin according to the preceding claim, characterized in that the confirmation method is a molecular biology method or an immunological method.
  20. [Claim 20] Method for detecting and/or isolating Shiga toxin-producing E. coli likely to 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 claims 1 to 13.

Description

Description Title of the invention: reaction medium and method for detecting a shiga toxin-producing E. coli and/or an enterohemorrhagic E. coli. TECHNICAL AREA The present invention relates to the field of microbiological control in the broad sense, such as the microbiological control of a sample of industrial or clinical origin. More particularly, the present invention relates to a reaction medium and the method for detecting a Shiga toxin-producing E. coli and/or an enterohemorrhagic E. coli. PRIOR TECHNIQUE Microbiological control of samples of various origins requires the implementation of techniques which allow the detection - for example for the purposes of identification and/or counting and/or biochemical characterization - of microorganisms and the rendering of results must be as rapid as possible. In the medical field, it is necessary to predict and diagnose the risk of infection: the faster and more accurate the diagnosis, the more effective the care of patients and the risk of transmission minimized. The approach is similar for animal health in the veterinary field. In the agri-food sector, the problem is identical. However, it distinguishes: - pathogenic microorganisms such as Shiga toxin-producing bacteria (STEC), Salmonella, Listeria, Cronobacter, Bacillus, Staphylococcus, the research for which applies to raw materials, intermediate products, finished products marketed, - non-pathogenic microorganisms, used as quality indicators of the production process, from raw materials to finished products, throughout the chain, - bacteria of technological interest such as ferments, - microorganisms that are markers of contamination. Rapid and precise detection of suspected contamination (in food batches) makes it possible to control them and thus initiate corrective actions quickly. Technically, one of the main difficulties is to be able to isolate the bacteria sought in order to be able to identify it. Generally, microbiological analysis is done in two stages. The first is a detection phase which can use many technologies such as culture media, immunoassays, molecular biology. It can be followed, particularly in the agri-food sector, a confirmation phase in order to confirm the presence of the pathogen sought and meet the standards in force in this field. The confirmation stage therefore requires additional steps and here again, requires a step of isolation of the bacteria sought. Thus, in the case of E. coli producing shiga toxins, the diagnosis is based on the use of selective and chromogenic agars that can include tellurite such as CT-SMAC (Sorbitol MacConkey Agar) or CT-RMAC (Rhamnose MacConkey Agar), in order to select the growth of certain bacteria and to stain the strains of interest. The disadvantage of these methods is that they are very specific and certain strains of E. coli producing shiga toxins are not detected. Conversely, some media are not selective enough, the presence of a small number of E. coli producing shiga toxins can be hidden by the other microorganisms that are very widely present, isolation is then difficult. In addition, to assess the pathogenicity of these strains, it is necessary to detect the presence of virulence factors. Indeed, their pathogenicity involves the expression of several virulence genes, including six coding for the two types of shiga toxins: STX1 and STX2. These two toxins act by inhibiting protein synthesis in eukaryotic cells, which ultimately causes apoptosis. Currently, in order to accurately identify shiga toxin-producing E. coli, it is essential to perform a PCR for gene six. There is therefore a real need to develop a reliable and rapid medium and method for the isolation and identification of target bacteria, particularly STEC and EHEC. SUMMARY OF THE INVENTION The present invention relates to a gelled reaction medium for the detection, identification, and/or isolation of at least one strain of E coli producing shiga toxin comprising - at least one toxin inducer, - at least one agglutinating conjugate comprising at least one specific binding partner of STX1 and/or at least one specific binding partner of STX2, coupled to a nanoparticle - a concentration gradient of a compound inhibiting non-target bacteria. In a highly advantageous manner, the medium according to the invention makes it possible to isolate STEC or EHEC from a multitude of E. coli. Another object of the invention relates to the preparation of a reaction medium according to the invention comprising the following steps: - contacting an agglutinating conjugate with a gelling culture medium - depositing on an area of the gelled culture medium, at least one compound inhibiting non-target bacteria comprising tellurite; said inhibitory compound diffusing and forming an inhibition concentration gradient around the deposition area; the concentration of the tellurite concentration gradient being between 0 pg/ml and 100 pg/ml, preferably between 0 pg/ml and 50 pg/ml, even more preferably bet