CHAPITRE 1 - Université de Bourgogne

virulence status means that absence of these pathogenic E. coli strains must be guaranteed to
ensure a safe release of foods. Although E. coli O157:H7 has remained as the prototypic
EHEC strain since the early 1980s (Dobson, 2006; Morgan et al., 1993; Pritchard et al., 2000;
Riley et al., 1983; Upton and Coia, 1994; Vimont et al., 2006), other E. coli serogroups such
as O26, O103, O111 and O145 have also been implicated in outbreaks (Pearce et al., 2006).
Only a few food laboratories screen for these non-O157 STEC, mainly because of the absence
of a specific International Organisation for Standardisation (ISO) method (ISO 2001).
Moreover, non-O157 STEC strains are a heterogeneous group which display a broad range of
both genotypic and phenotypic differences (Schmidt et al., 1999; Bettelheim 2000; 2003;
2007).
In the National Reference Laboratory, the detection of non-O157 STEC in foods is carried out
using a combination of PCR and DNA probe techniques to detect Shiga toxin genes and other
specific base mutations (Willshaw et al., 2001). These tests are not routinely available in
laboratories. However, an immuno magnetic separation method using beads coated with
antibodies specific to E. coli O26, O103, O111 and O145 has been described for the isolation
of these serogroups (Jenkins et al., 2003).
This study was designed to develop and optimize an automated immuno-concentration
method on the VIDAS system for E. coli O157, O26, O103, O111 and O145 (VIDAS ESPT)
with the use of specific recombinant phage proteins for capture. The aim of the method is to
facilitate the isolation of these specific STEC strains from enriched food products.
MATERIALS AND METHODS
This study was designed to demonstrate the feasibility of the immuno-concentration method
and to evaluate its sensitivity and specificity. These various parameters were evaluated on
pure broth suspensions of the specific STEC strains and on artificially inoculated raw beef
samples.
- STEC strains
The strains used (table 1) were isolated from various sources during previous studies and
characterized by PCR and RT-PCR. One PCR assay was used to test for the +93 uidA base
mutation which is specific for O157:H7 (Cebula et al., 1995) and for the O157 rfb gene
(Perelle et al., 2004). For the others strains, wzx (O26), wbdI (O111), ihp1 (O145) (Perelle et
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