Identification of enteropathogenic and enterohaemorrhagic Escherichia coli strains by immunoserological detection of intimin.

AIMS: To evaluate the sensitivity and specificity of polyclonal and monoclonal antibodies (Mabs) against intimin in the detection of enteropathogenic and enterohaemorrhagic Escherichia coli isolates using immunoblotting. METHODS AND RESULTS: Polyclonal and Mabs against the intimin-conserved region were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti-intimin IgG-enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of 1.3x10(-8) mol l(-1), failed in the detection of some of these isolates. CONCLUSION: All employed antibodies showed 100% specificity, not reacting with any of the eae-negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti-intimin IgG-enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. SIGNIFICANCE AND IMPACT OF THE STUDY: The rabbit anti-intimin IgG-enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.

Identification of enteropathogenic and enterohaemorrhagic Escherichia coli strains by immunoserological detection of intimin

To evaluate the sensitivity and specificity of polyclonal and monoclonalantibodies (Mabs) against intimin in the detection of enteropathogenic andenterohaemorrhagic Escherichia coli isolates using immunoblotting.Polyclonal and Mabs against the intimin-conservedregion were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti-intimin IgG-enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of1Æ3 · 10)8 mol l)1, failed in the detection of some of these isolates. All employed antibodies showed 100% specificity, not reacting with any of the eae-negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti-intimin IgG-enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. Significance and Impact of the Study: The rabbit anti-intimin IgG-enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.

Immunization of mice with Lactobacillus casei expressing intimin fragments produces antibodies able to inhibit the adhesion of enteropathogenic Escherichia coli to cultivated epithelial cells

Enteropathogenic Escherichia coli (EPEC) are frequently isolated as a cause of infantile diarrhea in developing countries. Its pathogenicity is distinguished by histopathological alterations at the site of infection, known as attaching and effacing (A/E) lesions, in which bacterial virulence factors and host proteins participate. Intimin, a bacterial adhesin expressed by all EPEC described to date, is responsible for the intimate adherence of the bacteria to host cells and is essential for the formation of A/E lesions. Mucosal vaccination may represent an efficacious intervention to prevent EPEC infection and lower morbidity and mortality rates. Strategies for mucosal vaccinations that use lactic acid bacteria for the delivery of heterologous antigens rely on their safety profile and ability to stimulate the immune system. In the present work, we have constructed Lactobacillus casei strains expressing different fragments of intimin â, a subtype that is frequently expressed by EPEC strains. Mucosal immunization of mice with L. casei expressing intimin fragments induced specific systemic and mucosal antibodies. These antibodies were able to recognize native intimin on the surface of EPEC and to inhibit in vitro EPEC binding to epithelial cells.

An enteroaggregative Escherichia coli strain of serotype O111:H12 damages and invades cultured T84 cells and human colonic mucosa.

The pathogenic mechanisms of enteroaggregative Escherichia coli (EAEC) are not well defined. We investigated the interaction of EAEC strain 236 (serotype O111:H12) with polarised Caco-2 and T84 human intestinal epithelial cells lines, and with human jejunal and colonic mucosa. Strain 236 adhered to both polarised cell lines and to both intestinal tissue types, but caused severe damage and was invasive only in T84 cells and colonic mucosa. In contrast, prototype EAEC strain 042, which also adhered to the cultured intestinal cell lines, did not adhere to or invade jejunal or colonic tissue. These observations suggest a heterogeneity of virulence properties within the EAEC category of diarrhoea-causing E. coli.

Adherence patterns and adherence-related DNA sequences in Escherichia coli isolates from children with and without diarrhea in São Paulo city, Brazil.

The correlation between various adherence patterns and adherence-related DNA sequences in Escherichia coli isolates from 1- to 4-year-old children with and without diarrhea in São Paulo, Brazil, was evaluated. A total of 1,801 isolates obtained from 200 patients and 200 age-matched controls were studied. The adherence patterns found were classified as diffuse, aggregative, aggregative in a 6-h assay, aggregative predominantly in coverslips, localized, localized-like, and noncharacteristic. In general, the DNA sequences used as probes showed excellent specificities (>93%), but their sensitivities varied. Thus, the results of bioassays and assays with DNA probes normally used to search for adherent E. coli did not correlate well, and the best method for the identification of these organisms in the clinical research setting remains controversial. Isolates presenting diffuse adherence or hybridizing with the related daaC probe, or both, were by far the most frequent in patients (31.5, 26.0, and 23.0%, respectively), followed by isolates presenting aggregative adherence or hybridizing with the related EAEC probe, or both (21.5, 13.0, and 10.5%, respectively). None of the different combinations of adherence patterns and adherence-related DNA sequences found were associated with acute diarrhea.

Association between alpha-hemolysin production and HeLa cell-detaching activity in fecal isolates of Escherichia coli.

Escherichia coli isolates that cause detachment of cell monolayers during in vitro adherence assays (cell-detaching E. coli [CDEC]) were recently reported as a potential new group of enteropathogenic bacteria. In the present study, 269 E. coli isolates from feces of children 1 to 5 years of age were identified as CDEC in a detaching assay developed with HeLa cells. The great majority of these isolates were hemolytic within 3 h of growth on blood agar plates and hybridized with a DNA probe for alpha-hemolysin (93.7%), while most of the non-detaching isolates were hemolytic within 24 h (3.6%) or nonhemolytic (94.8%). E. coli isolates that produced alpha-hemolysin were found in 60 (30%) of 200 children with diarrhea and 47 (24%) of 200 age-matched controls. No statistical significance was found for the differences in alpha-hemolysin production among the matched pairs (P = 0.2). These data suggest that CDEC isolates are not associated with diarrhea in the population studied.