EP-4735646-A1 - METHOD FOR DETECTING AND CONFIRMING SHIGA TOXIN-PRODUCING ESCHERICHIA COLI AND/OR ENTEROHAEMORRHAGIC E. COLI

Abstract

The invention relates to a method for detecting and confirming at least one Shiga toxin-producing Escherichia Coli (STEC) which may be present in a sample comprising enterobacteria, comprising the following steps: - performing lysis of the sample, enabling lysis of the STECs in order to obtain a solution comprising the nucleic acids thereof; - bringing the solution of nucleic acids into contact with primers, making it possible to amplify at least the stx1 and/or stx2 gene or gene fragment; - if at least one of the stx1 and/or stx2 genes or gene fragments is amplified, part of the sample is deposited on an agar reaction medium comprising ■ at least one toxin inducer, ■ at least one agglutinating conjugate formed by at least one binding partner specific to the STX1 protein and/or at least one binding partner specific to the STX2 protein, which binding partner(s) is (are) coupled to a nanoparticle; - detecting and confirming the presence of at least one STEC by the appearance of a halo on the agar around the STEC.

Inventors

• CARRET, Justine
• DOMINGOS, Céline
• HAMON, Fabienne
• JUNILLON, Thomas
• MALLEN, Benoit
• ROGER-CARDOSO, Tamara

Assignees

• BIOMERIEUX

Dates

Publication Date

20260506

Application Date

20240625

Claims (1)

1. CLAIMS 1- Method for detecting and confirming at least one Shiga toxin-producing Escherichia Coli (STEC) likely to be present in a sample containing enterobacteria, comprising the following steps: Perform sample lysis to lyse STEC to obtain a solution containing their nucleic acids Bring the nucleic acid solution into contact with primers capable of amplifying at least the stxl and/or stx2 gene or gene fragment. - If at least one of the genes or fragments of the stxl and/or stx2 genes is amplified, part of the sample is placed on an agar reaction medium comprising ■ at least one toxin inducer, ■ at least one agglutinating conjugate formed by at least one specific binding partner of the STX1 protein and/or at least one specific binding partner of the STX2 protein, coupled to a nanoparticle - Detect and confirm the presence of at least one STEC by the appearance of a halo on the agar around said STEC. 2- Method for detecting and confirming at least one Shiga toxin-producing Escherichia Coli (STEC) according to claim 1 in which, - if at least one of the genes or fragments of the stxl and/or stx2 genes is amplified, the nucleic acid solution is brought into contact with primers making it possible to amplify at least the gene or the gene fragment of the serogroups chosen from 026, 045, 080, 091, 0103, 0104, OR I, 0113, 0121, O128ab, 0145, 0146, 0157, 0174 - if at least one of the genes or fragments of the stxl and/or stx2 genes and at least one serogroup chosen from 026, 045, 080, 091, 0103, 0104, OR I, 0113, 0121, O128ab, 0145, 0146, 0157, 0174 are amplified, a portion of the sample is deposited on a reaction medium comprising o at least one toxin inducer and o at least one agglutinating conjugate formed by at least one specific binding partner of the STX1 protein and/or at least one specific binding partner of the STX2 protein, coupled to a nanoparticle. o detecting and confirming the presence of at least one STEC by the appearance of a halo on the agar around said STEC. 3- Method for detecting and confirming at least one Shiga toxin-producing Escherichia Coli (STEC) according to claim 2 in which - if at least one of the genes or fragments of the stxl and/or stx2 genes and at least one gene or gene fragment of at least one serogroup chosen from 026, 045, 080, 091, 0103, 0104, OR I, 0113, 0121, O128ab, 0145, 0146, 0157, 0174. are amplified, a portion of the sample is deposited on a reaction medium comprising o at least one toxin inducer and o at least one agglutinating conjugate formed by at least one specific binding partner of STX1 coupled to a nanoparticle of a first nature producing a halo of a first color when it is agglutinated o and/or at least one agglutinating conjugate formed by at least one specific binding partner of STX2 coupled to a nanoparticle of a first nature producing a halo of a first color when agglutinated o and at least one agglutinating conjugate formed by at least one specific binding partner of said serogroup identified by amplification coupled to a nanoparticle of a second nature producing a halo of a second color when agglutinated - confirm the presence of at least one STEC of a serogroup identified by the appearance of at least one halo of a first colour and at least one halo of a second colour around the same colony or at least one halo resulting from the mixture of a first and a second colour around said colony. 4- Method for detecting and confirming at least one enterohemorrhagic Escherichia Coli (EHEC) likely to be present in a sample comprising enterobacteria, comprising the following steps: - perform a lysis of the sample allowing the lysis of EHEC in order to obtain a solution comprising their nucleic acids - bringing the nucleic acid solution into contact with primers capable of amplifying at least the stxl and/or stx2 and eae gene or gene fragment, - if at least one of the genes or fragments of the stxl and/or stx2 genes and at least one gene or fragment of the eae gene are amplified, a portion of the sample is deposited on an agar medium comprising: o at least one toxin inducer o at least one agglutinating conjugate formed by at least one specific binding partner of STX1 coupled to a nanoparticle of a first nature producing a halo of a first color when agglutinated o and/or at least one agglutinating conjugate formed by at least one specific binding partner of STX2 coupled to a nanoparticle of a first nature producing a halo of a first color when agglutinated o and at least one agglutinating conjugate formed by at least one specific binding partner of EAE coupled to a nanoparticle of a second nature producing a halo of a second color when agglutinated - confirm the presence of at least one EHEC by the appearance on the agar medium: - at least one halo of a first color - and at least one halo of a second color - or at least one halo resulting from the mixing of a first and a third color around said EHEC strain. 5- Method for detecting and confirming at least one EHEC according to claim 4, comprising bringing the nucleic acid solution into contact with primers making it possible to amplify at least one gene or gene fragment of at least one serogroup chosen from 026, 045, 080, 091, 0103, 0104, OR I, 0113, 0121, O128ab, 0145, 0146, 0157, 0174. 6- Detection and confirmation method according to the preceding claims, characterized in that the nucleic acid solution is brought into contact with primers making it possible to amplify the aggR gene or gene fragment. 7- Method according to any one of the preceding claims, characterized in that a step of enrichment of the sample is carried out prior to its lysis. 8- Detection and confirmation method according to any one of the preceding claims, characterized in that a step of immuno-selection of an E. coli of at least one serogroup chosen from 026, 045, 080, 091, 0103, 0104, 0111, 0113, 0121, O128ab, 0145, 0146, 0157, 0174 is carried out before the lysis step. 9- Detection and confirmation method according to any one of the preceding claims, characterized in that a step of immuno-selection of an E. coli of at least one serogroup chosen from 026, 080, 045, 091, 0103, 0104, 0111, 0113, 0121, O128ab, 0145, 0146, 0157, 0174 is carried out before depositing on the agar reaction medium. 10- Detection and confirmation method according to any one of the preceding claims, characterized in that the nanoparticle coupled to the binding partner is a colloidal nanoparticle having optical properties. 11- Detection and confirmation method according to any one of the preceding claims, characterized in that the nanoparticle is chosen from gold, silver, copper. 12- Detection and confirmation method according to any one of the preceding claims, characterized in that the nanoparticles have a size between 10 and 200 nm, preferably between 20 and 90 nm. 13- Detection and confirmation method according to any one of the preceding claims, characterized in that the toxin inducer is an antibiotic. 14- Detection and confirmation method 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. 15- Detection and confirmation method 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.

Description

Description METHOD FOR THE DETECTION AND CONFIRMATION OF SHIGATOXIN-PRODUCING ESCHERICHIA COLI AND/OR 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 method for the detection, identification, enumeration and/or isolation of Shiga toxin-producing Escherichia 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 E. coli (STEC), Salmonella, Listeria, Cronobacter, Bacillus, Staphylococcus, the research of 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. In recent years, many countries have been affected by outbreaks of Shiga toxin-producing E. coli (STEC) or Enterohemorrhagic E. coli (EHEC). Human infections caused by these bacteria are now recognized as a major public health problem worldwide. The vast majority of E. coli bacteria are commensal strains of humans. However, some strains have acquired virulence genes and factors. This allows them to colonize the digestive system, bypass immune defenses and induce cellular damage, thus causing symptoms in humans. Among these STEC strains that have acquired the virulence factor stx, a particular pathovar is implicated: that of enterohemorrhagic E. coli strains. EHEC are strains representing a subgroup of STEC, which have acquired the eae gene and cause hemolytic uremic syndrome. The possession of these two virulence factors simultaneously, stx and eae, makes this pathovar very virulent for humans. There are other virulence factors such as aggR, LT, ST, afaC, ipaH. Generally, microbiological analysis is done in two stages. The first is a detection phase that can use many technologies such as culture media, immunoassays, molecular biology. During this detection stage, it is difficult to know if the same strain carries one or more virulence factors. It may be followed by a confirmation phase in order to confirm the presence of the pathogen sought and meet the standards in force in this field. The confirmation stage therefore requires additional steps and requires a step of isolation of the bacteria sought. Thus, the methods of the prior art require a succession of amplification, immunoconcentration and culture steps on a dish in order to detect and confirm the presence of a virulent Escherichia coli pathovar. There is therefore a real need to develop a reliable and rapid method for the detection and confirmation of virulent E. coli pathovars. SUMMARY OF THE INVENTION The present invention relates to a method for detecting and confirming at least one Shiga toxin-producing Escherichia Coli (STEC) likely to be present in a sample comprising enterobacteria, comprising the following steps: - Performing a lysis of the sample allowing the lysis of the STEC in order to obtain a solution comprising their nucleic acids - Bring the nucleic acid solution into contact with primers capable of amplifying at least the stxl and/or stx2 gene or gene fragment. - If at least one of the genes or fragments of the stxl and/or stx2 genes is amplified, part of the sample is placed on an agar reaction medium comprising: ■ at least one toxin inducer, ■ at least one agglutinating conjugate formed by at least one specific binding partner of the STX1 protein and/or at least one specific binding partner of the STX2 protein, coupled to a nanoparticle. - Detect and confirm the presence of at least one STEC by the appearance of a halo on the agar around said STEC. The method according to the present invention has the advantage of confirming the presence or absence of a STEC directly on the reaction medium by the formation of a halo around said strain. This invention is particularly interesting for facilitating the detection and confirmation of t