The virulence profiles of most atypical enteropathogenic Escherichia coli (EPEC) strains are unknown. A total of 118 typical and atypical strains of EPEC serotypes and non-EPEC serogroups isolated from children with or without acute diarrhea who were from different cities in Brazil were examined for virulence-associated markers and adherence to HEp-2 cells, and also had random amplified polymorphic DNA (RAPD) analysis performed. Atypical strains were identical to typical strains with regard to the virulence factors encoded on the locus of enterocyte effacement (LEE). In contrast with typical EPEC strains, none of the atypical strains reacted with the bfpA probe, and half of the strains hybridized with the perA probe. Most atypical strains presented Tir sequences that correlated with enteropathogenic or enterohemorrhagic E. coli (98%), had LEE inserted in either selC or pheU (88%), and presented a typeable intimin (52%). Eighteen new serotypes were found in the EPEC strains. Atypical and typical EPEC strains belonged to different RAPD clusters. Most atypical strains showed a localized-like adherence pattern (61.5%). Of the non-LEE-encoded virulence factors, enteroaggregative E. coli heat-stable enterotoxin was noted most frequently (45%) and was significantly associated with diarrhea (P=.01). Thus, this virulence marker may be used as an additional tool for the diagnosis of truly atypical pathogenic strains.

A 1-year prospective study was carried out in two large urban centers of São Paulo State, Brazil, to determine the prevalences and roles of the different Escherichia coli pathotypes in children less than 5 years of age with diarrhea presenting to the emergency rooms of public hospitals or visiting private pediatricians' offices. Of the pathotypes sought, typical enteroaggregative and atypical enteropathogenic types of E. coli were isolated for 8.9% and 5.4% of 774 diarrhea cases, respectively, and were found to be dominant and significantly associated with diarrhea.

A multiplex PCR to differentiate typical and atypical enteropathogenic Escherichia coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic (ETEC), enteroinvasive E. coli (EIEC) and Shiga toxin-producing E. coli (STEC) strains was developed and evaluated. The targets selected for each group were eae and bfpA for EPEC, aggR for EAEC, elt and est for ETEC, ipaH for EIEC and stx for STEC isolates. This PCR was specific and sensitive for rapid detection of target isolates in stools. Among 79 children with acute diarrhea, this technique identified 13 (16.4%) with atypical EPEC, four (5%) with EAEC, three (3.8%) with typical EPEC, one (1.3%) with ETEC and one (1.3%) with EIEC.

In a prospective study carried out in two urban centers in northeastern Brazil, 195 HEp-2-adherent Escherichia coli strains were isolated; 110 were identified as the only pathogen in stools of children with diarrhea, and 85 were from controls. Enteropathogenic E. coli isolates were identified in 21 children with diarrhea (8.9%) and 7 children without diarrhea (3.0%), and they were significantly associated with diarrhea (P < 0.01). Enteroaggregative E. coli strains were isolated from 40 children with diarrhea (16.9%) and 38 children without diarrhea (16.4%) and showed no correlation with diarrhea (P > 0.5). In 49 children with diarrhea (20.7%) and 40 children without diarrhea (17.3%), diffusely adherent E. coli (DAEC) isolates were detected and were not found to be associated with diarrhea (P ‫؍‬ 0.41). However, after stratification, for children older than 12 months of age a significant correlation between DAEC infection and diarrhea was detected (P ‫؍‬ 0.01). These results suggest that DAEC isolates should be considered potential pathogens in northeastern Brazil and also confirm the association of DAEC with age-dependent diarrhea.Diarrhea remains an important public health problem for children in developing areas of northeastern Brazil. The bacterial pathogen most commonly associated with endemic forms of childhood diarrhea is Escherichia coli. At least five categories of diarrheagenic E. coli strains are recognized on the basis of distinct epidemiological and clinical features, specific virulence determinants, and an association with certain serotypes: enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), and enteroaggregative E. coli (EAEC) (13). Recently, diffusely adherent E. coli (DAEC) strains have been recognized as the sixth class of diarrheagenic E. coli and appear as a heterogeneous group (13). EPEC, EAEC, and DAEC isolates are characterized by their distinct patterns of adherence to cultured epithelial cells in vitro. EPEC strains bind to host cells in a pattern called localized adherence (LA), in which microcolonies form on the surfaces of the cells (15). EAEC isolates bind in an aggregative adherence (AA) pattern, which is characterized by a stacked brick-like arrangement on the surfaces of the cells as well as those of glass or plastic containers (13). DAEC strains are defined by a pattern of diffuse adherence (DA), in which the bacteria uniformly cover the entire cell surface (15). The implication of DAEC strains in diarrhea remains controversial, since some studies have reported that these strains are found similarly in children with and without diarrhea (6, 8). Tacket et al. (18) were unable to conclusively induce diarrhea with DAEC in adult volunteers but suggested that DAEC may cause disease in immunologically naive or malnourished children. Discrepancies among epidemiological studies could be explained by age-dependent susceptibility to diarrhea or by the use of an inappropriate detection method such as DNA probing ...

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...

We have previously shown that enteroaggregative Escherichia coli (EAEC) is an important pathogen among Brazilian infants. Most EAEC strains harbor a plasmid (pAA) from which a DNA fragment has been used as a probe (EAEC probe). To better understand the characteristics of EAEC in Brazil, 109 strains carrying and lacking the EAEC probe sequence were tested for the presence of pAA plasmid-borne and chromosomal factors. Common virulence factors of probe-positive and probe-negative isolates included the presence of the Pet, EAST-1, Shf, Irp2, ShET1/Pic, and Hly virulence markers. The presence of AggR or one other virulence factor (AAF/I, AAF/II, AAF/III, or Aap) was predominantly identified only in probe-positive strains. In EAEC probepositive strains, the virulence marker Aap was found significantly more frequently (P ‫؍‬ 0.023) in isolates from children with diarrhea (22%) than in isolates from controls (3%). EAST-1 and Shf were the markers most frequently detected (61%) in EAEC probe-negative strains and were found to be significantly associated with diarrhea (P ‫؍‬ 0.003 and P ‫؍‬ 0.020, respectively). Furthermore, our data suggest that AggR can be used as an important genetic marker for EAEC probe-positive strains.Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a cause of diarrhea worldwide (18). EAEC is defined by its characteristic "stacked brick" aggregative adherence (AA) pattern of adherence to HEp-2 cell (17). Most EAEC strains harbor a 60-to 65-MDa virulence plasmid (pAA). A 1-kb fragment of pAA, referred to as the EAEC probe or CVD432 (1), has been widely used for epidemiological studies (7,19,25,29). The pAA plasmid also encodes AA fimbriae (AAF) I, II, and III (2, 4, 15); the transcriptional activator AggR (16); enteroaggregative heat-stable enterotoxin 1 (EAST-1) (23); a 104-kDa cytotoxin designated Pet (8); the cryptic secreted protein Shf (5); and a novel antiaggregation protein (dispersin) encoded by the aap gene (formerly known as aspU) (27). In addition to the pAA plasmid, some EAEC strains express putative virulence factors that are encoded on the chromosome, including a 116-kDa secreted mucinase (Pic) (12), yersiniabactin (26), and the E. coli ␣-hemolysin (30). Shigella enterotoxin 1 (ShET1) is encoded by the antisense strand of the pic gene (10, 21). However, none of these factors is consistently found in all EAEC isolates, as determined by hybridization studies.To find a specific virulence marker for the detection of EAEC in epidemiological and clinical studies, we tested 109 strains, isolated from Brazilian children in a previously described study (25), for their abilities to hybridize to eight pAA plasmid-derived and three chromosomal gene probes. MATERIALS AND METHODSBacterial strains. The strains examined in this work were isolated during a study of the epidemiology of acute diarrhea in children less than 2 years old conducted in different regions of Brazil from 1997 to 1999 (25). The children were admitted to public hospitals in the following cities (states) for t...

In this paired case-control study of infants with diarrhea in São Paulo, we examined the association between HEp-2–adherent Escherichia coli strains and diarrhea. We tested isolates from stool specimens of infants with diarrhea and matched controls in an HEp-2 cell adherence assay; we then hybridized isolates with DNA probes and identified enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), and diffusely adherent E. coli (DAEC). From 100 patient-control pairs, we isolated 78 HEp-2–adherent strains; of these, 61 strains were single pathogens identified in stools of infants with diarrhea. While typical EPEC was significantly associated with diarrhea (p<0.001), EAEC was more frequently associated with diarrhea in clinical cases (20%) compared with healthy controls (3%) (p<0.001). Atypical EPEC, showing a localized adherence-like pattern, was also more common in patients than controls (p>0.1). DAEC was isolated with equal frequency from patients and controls (p>0.1).

One hundred twelve diffusely adherent Escherichia coli strains isolated from children in a case control study were evaluated for virulence-associated characteristics, serotyping, antibiotic resistance, and plasmid profiles. Half of the strains hybridized with the probes for icuA (aerobactin) and fimH (type 1 pili); daaE (F1845 fimbriae), afa (afimbrial Dr adhesin), agg-3A (aggregative adhesion fimbria type III fimbriae), pap (P fimbriae), astA (EAST1 toxin), and shET1 (Shigella enterotoxin 1) sequences were present in <20% of the strains. The shET1 gene was noted most frequently in strains isolated from patients. A minority (7%) of the strains produced hemolysin or colicin or showed cytotoxic effects on Vero cells. Forty-five different serotypes were found. The majority (70%) of the strains presented multiple antibiotic resistance. Antibiotic resistance and diffuse adherence were located on the same conjugative plasmids. These results suggest that the transfer of these potential virulence markers could be important in the epidemiology of diffusely adherent E. coli.