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