Servicio de Información Comunitario sobre Investigación y Desarrollo - CORDIS

Comparison of AEEC strains belonging to a same serogroup and isolated from humans and animals

- Establishment of a strain collection of 150 AEEC strains;
- Molecular characterization of AEEC from human, animal, and foods;
- Comparison of PFGE with MLST for characterization of AEEC;
- Assessment of the potential risk of animal AEEC to infect humans.

Major finding during the project:
Strain collection. One of the major requirements for the success of this project was the establishment of a strain collection of important AEEC strains of non-O157 serotypes. We have collected AEEC strains expressing defined O-antigens from a number of various sources and countries. The strain collection consists of 150 strains of sero-groups O49, O84, O103, O145, O128, and O156, derived from humans, animals, and food

ASTS/MLST. We have worked out the complete conditions for the molecular typing with MLST/ASTS. To avoid confusion in the literature we have reclassified our work as MLST. We used the adk, arcA, fumC, icd, mtlD, mdh, and pgi houskeeping genes as well as the eae and escD genes from the LEE locus to amplify fragment from the strains and now we have a complete data set of 950 housekeeping and 256 LEE sequences. These data are under analysis and compared with the data of partner CR11. Phylogenetic trees were constructed and phylogenetic relationships were compared with PFGE patterns and virulence patterns (partner CR11).

We have analysed the whole data set of E. coli O103 strains of our strain collection. The paper on O145 strains is in preparation. From these analyses it became clear that there exists typical animal adapted strains that are not found as human pathogens and only some analyzed strains have been found in both humans and animals. We have been able to better characterize serotype O103 and to show that EPEC and STEC in the O103 group are genetically related but represent two distinct clones with different animal reservoirs.

PFGE has been performed on all 150 strains and data were computerized, analysed and compared with MLST/ASTS for their suitability to define (sub)clones of pathogenic AEEC and to investigate the origin of EPEC and STEC in strains belonging to the same serogroup.

Virulence gene typing. PCR-based virulence gene typing was performed on all strains using primers specific for stx, efa, cif, paa, ehlyA, etpD, katP, iutA, espP, cnf, cdt-I, bfpA, lpfA. The data from these experiments have been intergated into the PFGE/MLST data to give a complete strain characterization.

Intimin genes. In a cooperation project with partner CR11, CR10, and CR5 we have identified and characterized new intimin genes. We determined the sequence of three novel variant intimin genes detected in attaching and effacing Escherichia coli isolates of human origin. We compared these sequences with those of published intimin-a, -b, -g, -e, and __q__ alleles. Sequence analysis of these ten intimin alleles confirmed the great genetic diversity of the intimin gene family in E. coli. Phylogenetic analysis indicates that recombination events have played a role in the evolutionary history of intimin genes. We have recommended an eae-nomenclature system based on the Greek alphabet and provide an updated PCR scheme for amplification and typing of E. coli eae.

Intimin genes in porcine EPEC. By cooperation with partner CR10 we have been worked out the serotyping and intimin typing of a group of AEEC strains from healthy and diseased pigs from Hungaria. The results indicate that different groups of AEEC are present in healthy and diarrheic pigs.

E. coli from healthy infants. Twenty-seven AEEC strains have been isolated from fecal samples of 14 children from Melbourne and from 12 children from Berlin. The 27 AEEC strains were classified as enterohemorrhagic E. coli (EHEC), typical enteropathogenic E. coli (EPEC) and atypical EPEC (25 strains negative for Shiga-toxins and EAF-plasmids). The AEEC were divided into 18 different serotypes, O-untypable and O-rough strains.

Typing of their intimin genes revealed the presence intimin a in 6 strains, b in 11 strains, g in 7 strains, z in 2 strains and h in one strain. Analysis of HEp-2 cell adherence showed diffused (DA) or localized like adherence (LLA) patterns in 26 AEEC strains, local adherence (LA) was only found with the EAF-positive strain. Ten AEEC strains showed an attaching and effacing (AE) property with the fluorescent actin staining (FAS)-test. Our findings indicate that atypical EPEC could play a double role as natural immunizing strains to intimin in humans and as a reservoir for new emerging human pathogenic EPEC strains.

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