Comparison of DNA Hybridization and PCR Assays for Detection of Putative Pathogenic Enteroadherent Escherichia coli

Abstract

The correlation of the different adherence patterns with DNA probes and PCR primers for the identification of Escherichia coli was analyzed in isolates from children, less than 2 years of age with or without diarrhea, from different regions of Brazil. A total of 1,428 isolates obtained from 338 patients and 322 control children were studied. The enteropathogenic E. coli (EPEC) adherence factor (EAF) probe was shown to be as good as the HEp-2 adhesion assay for the detection of typical EPEC strains. The DNA probes used to detect diffusely adhering E. coli and enteroaggregative E. coli (EAEC) showed low sensitivities (64 and 50%, respectively), and the best method of identifying these organisms in clinical research remains the HEp-2 adherence assay. The "bundle-forming pilus" (BFP) and the EAEC PCR assays could be used instead of the DNA probes as a screening method for typical EPEC and EAEC carrying the EAEC probe sequence in the clinical laboratory. In our study, only typical EPEC strains that carried EAF and BFP were associated with acute diarrhea. The LA exhibited by typical enteropathogenic E. coli (typical EPEC) is mediated by an inducible bundle-forming pilus (BFP), whose expression correlates with the presence of a plasmid designated the EPEC adherence factor (EAF) plasmid (1, 17). EPEC strains also cause attaching and effacing lesions on eukaryotic cells that involve a 94-kDa protein encoded by the chromosomal eae gene (25, 28). The pathogenicity of EPEC strains has been demonstrated in human volunteers, and their role in childhood diarrhea was confirmed in epidemiological studies (9,10,11,15,18,26). Atypical EPEC strains do not carry the EAF plasmid, were found to exhibit LAL, and have been isolated from acute infantile diarrhea in São Paulo (35).The adherence of many enteroaggregative E. coli strains requires the presence of a plasmid that contains genes encoding the AA (38). Epidemiological studies have implicated EAEC as a cause of diarrhea in children in developing countries, and the pathogenic potential of EAEC in human infections was substantiated by challenge studies (5,10,23,27,39).Two factors, F1845 and AIDA-I were found to encode DA in diffusely adhering E. coli (DAEC) (4, 6). Several recent studies have implicated DAEC strains as agents of diarrhea, while other studies have not recovered DAEC strains more frequently from diarrheal patients than from asymptomatic controls (2,15,16,21,24).DNA probes derived from the adherence-related sequences have been constructed (3,4,6,17,25,30) and used in hybridization assays for the detection of the different putative categories of diarrheagenic E. coli in many epidemiologial studies. PCR primers have been also developed for several of the categories of diarrheagenic E. coli (8,14,20,36).In order to optimize screening methods for putative pathogenic enteroadherent E. coli in the clinical laboratory we analyzed the correlation of the different HEp-2 adherence patterns with DNA probes and PCR primers in E. coli isolates from different urban centers of Brazil...

Rapid Categorization of Pathogenic Escherichia coli by Multiplex PCR

“…EAF is a useful marker for EPEC and is linked to the localized adherence (LA) to Hep-2 cells (5,12). In general, both bfpA and EAF-positive strains are typical EPECs that express the LA pattern (12,21,29). However, there are some strains that are EAF-negative and bfpA-positive (Table 3).…”

“…A typical EAggEC possesses CVD432 and aggR that are present in the virulence plasmid pAA. The pAA virulence plasmid of EAggEC contains the fimbriae genes that are necessary for the aggregative adhesion (AA) pattern to Hep-2 cells or HeLa cells (12,21,22). However, there are a few CVD432-negative and aggR-positive strains and vice versa (Table 3).…”

Displacement of bacterial pathogens from mucus and Caco-2 cell surface by lactobacilli

“…The ability of probiotic bacteria to compete with pathogens for adhesion sites on the intestinal mucosal surface is still under investigation. Ability to adhere to the surface of epithelial cells is a key pathogenic factor of intestinal pathogens (Levine, 1987;Alam et al, 1996;Weinstein et al, 1998;Scaletsky et al, 2002). Lactobacilli have been shown to possess surface adhesins similar to those on bacterial pathogens (Neeser et al, 2000).…”

Rapid Detection Method for Enteroaggregative <i>Escherichia coli</i> Using Simple Clump Formation and Aggregative Assay

“…Some researchers believe that a typical EAggEC possesses both aggR and pCVD432 genes that are present in the virulence plasmid pAA [17,18]. Kimata et al [19] identified AA was characterized by the prominent autoagglutination of bacterial cells as well as adherence to glass cover slips without HEp-2 cells.…”