Servizio Comunitario di Informazione in materia di Ricerca e Sviluppo - CORDIS

Conventional and molecular characterisation of typical and atypical AEEC

Objectives:
- To establish a large collectionof AEEC strains, available to all the partners, characterized by serotype and profile of virulence markers, in particular by typing of their stx and eae genes.

- To characterize possible new intimin types and investigate on their distribution among AEEC strains .

- To identify the different pathogroups of AEEC on the basis of the types and combination of virulence genes.

- To investigate the host specificity of AEEC and possible epidemiological links between the presence of virulence factors and severe human disease.

Major finding during the project:
- Establishment of a culture collection. It included a total of 50 O serogroups and 300 strains, isolated from healthy and diseased humans and from different animals species. Consensus PCR protocols for detection of the main AEEC virulence genes (all variants of stx and eae, EHEC-hlyA, bundle forming pili, flagellar (fliC) genes) have been harmonised.

- Characterization of new intimin types. Partner CR12 identified four eae variant genes in isolates of human origin. Three of the newly discovered genes were designated as eae-eta, jota, and kappa. The fourth one was identical to the recently described eae-zeta. A PCR scheme for amplification and typing of E. coli eae gene was developed and an eae-nomenclature system based on the Greek alphabet has been proposed.

- Distribution of intimin genes and other LEE locus-related genes among AEEC Partners CR10, CR11, and CR12 carried out investigations on the distribution of the main intimin types among AEEC. Four major clones corresponding to the major intimin types a,b,g, and e have been characterized. The three intimin a, g, and e clones include all the major serogroups of AEEC associated with human disease while the intimin b clone appears to be the most ubiquitous type, in that it has been found in both EPEC and EHEC isolates from several animal species: humans, cattle, pigs, rabbits, dogs, and birds. Moreover, it includes important diarrheagenic clones such as human EHEC O26 and EPEC O26:H11, O111:H2, and O128:H2.

Partner CR10 has investigated the presence of four putative PAIs described in the chromosome of EHEC O157 among AEEC. One of these PAIs, termed O#122, was found to be strongly associated with AEEC in particular those firmly associated with human disease. It contains the putative virulence gene efa1/lifA, which was reported to enhance the adhesion. Partner CR10 investigated on the presence of the virulence genes, efa1/lifA and toxB, in a panel of 60 EHEC and 68 EPEC strains isolated from different sources and belonging to different serogroups. The toxB gene was present only in EHEC O157 and most of the O26 isolates, while, efa1/lifA seemed to be associated with the non-O157 EHEC strains analysed.

As for the EPEC strains investigated, efa1/lifA and toxB were present in 41,5% and in 10.3% of isolates respectively. A polymorphism in the sequence of the toxB gene was observed between EHEC O157 and EHEC O26 by restriction with HindIII followed by hybridisation with a toxB gene probe.

Identification of the LEE insertion site. We showed that the LEE is located in selC or in pheU tRNA loci in most of the AEEC clones. Using an original PCR strategy, we showed that the LEE is inserted in pheV in 8 out of the 14 strains which were negative for both SelC and pheU. These included the intimin e-positive EHEC and some b-positive EPEC.

Determination of AEEC pathogroups. Partner CR11 completed the investigation on a group of STEC strains collected from 677 human patients in Germany over a three years period. Sixty-two% of the STEC strains were eae-positive and 37.4% were eae-negative. eae positive STEC were significantly associated with distinct serotypes of strains and with the presence of an stx1 and/or stx2 gene, whereas stx1c, stx2d and stx2e variant genes were only found in eae-negative STEC. EHEC-Hly, a marker of the EHEC-virulence plasmid, was present in 96.2% of eae-positive and in 65.2% of eae-negative STEC. Fourteen new serotypes of eae-positive STEC which were previously not described to occur in humans were detected.

A collaborative study on STEC in sheep showed that STEC isolated from healthy sheep in Norway belonged to a few dominating serotypes and did not carry an eae-gene indicating that sheep are not a major source of eae-positive STEC and EHEC strains.

Informazioni correlate

Reported by

Head of the Unit of Foodborne Zoonoses and Veterinary Epidemiology
Viale Regina Elena 299
00161 Rome
Italy
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