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Detection of Enteroaggregative Escherichia coli in clinical specimens and foods

Project Code: B14002

31/01/2005

Laboratory of Enteric Pathogens, Centre for Infections, Department of Gastrointestinal Infections, Specialist and Reference Microbiology Division
Chart, H ;
Campden BRI
Baylis, C;
HPA Colindale
Smith, H;
Laboratory of Enteric Pathogens, Centre for Infections, Department of Gastrointestinal Infections, Specialist and Reference Microbiology Division
Martin, H; Jenkins, C; Smith, G;
Campden BRI
Green, B; Higgins, J;
Leeds HPA Laboratory
Tompkins, D

A collection of 378 strains of EAggEC was established, including 272 from cases and controls collected during the Infectious Intestinal Disease study and a study carried out at the Queen Elizabeth Hospital in London. The panel of strains also included 106 isolates from the archive collection held by the LEP, and a further 145 strains comprising isolates from other groups of pathogenic and non-pathogenic E. coli.

Initially, 143 strains of EAggEC and 145 strains of non-EAggEC E. coli were characterised using DNA probes to assess the incidence of thirteen genes thought to be associated with the EAggEC phenotype: - (i) aggC (usher gene for aggregative adherence fimbriae I), (ii) aafC (aggregative adherence fimbriae II), (iii) aggR (fimbrial regulator), (iv) aat (anti-aggregation protein transporter), (v) aap (dispersin), (vi) aaiC (formerly aaiA, a chromosomal gene controlled by AggR), (vii) pet (plasmidencoded toxin), (viii) pic (protein involved in colonisation), (ix) astA (Enteroaggregative heat-stable toxin - EAST), (x) irp2 – (Yersiniabactin biosynthesis gene), (xi) shf (cryptic ORF), (xii) tia (the putative invasion determinant gene) and (xiii) aag3C (usher gene for aggregative fimbrial adhesin gene III). From this panel, three genes were targeted for use in a PCR assay – aat, aap and aaiC. E. coli
usually produce β glucuronidase that is encoded by the uidA genes. This activity results in hydrolysis of substrates such as 4 – methylumbelliferyl-β- D glucuronidase
(MUG) yielding 4 – methylumbelliferone/4MU). By including uidA in the PCR, strains of E. coli could be targeted concurrently.

Strains of EAggEC were detected using the above four genes in multiplex and real-time PCR assays. By these assays strains of EAggEC could be detected using bacterial isolates and in ‘spiked’ foods. Five hundred faecal samples were received from patients with symptoms of diarrhoea referred to the Regional Health Protection Agency Laboratory in Leeds. Thirty-nine of these patients were positive for Enteroaggregative E. coli (EAggEC).

Strains of EAggEC have been shown to produce dispersin, a protein used to disaggregate these bacteria, once clumped together. A rabbit serum prepared to purified dispersin was able to detect strains producing dispersin, when analysed by SDS-PAGE and immunoblotting. However, anti-dispersin antibodies were unable to detect dispersin in a simple colony blot.

The EAggEC were not found to have unusual growth patterns and so a growth medium specific for detecting EAggEC was not achieved.

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