Typical enteroaggregative and atypical enteropathogenic types of Escherichia coli are the most prevalent diarrhea-associated pathotypes among Brazilian children.

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.

Single multiplex assay to identify simultaneously enteropathogenic, enteroaggregative, enterotoxigenic, enteroinvasive and Shiga toxin-producing Escherichia coli strains in Brazilian children.

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.

Heterogeneity among strains of diffusely adherent Escherichia coli isolated in Brazil.

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.

Enteroaggregative Escherichia coli virulence factors are found to be associated with infantile diarrhea in 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 probe-positive 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.

Atypical enteropathogenic Escherichia coli strains: phenotypic and genetic profiling reveals a strong association between enteroaggregative E. coli heat-stable enterotoxin and diarrhea.

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.

HEp-2-adherent Escherichia coli strains associated with acute infantile diarrhea, São Paulo, Brazil.

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).

Comparison of DNA hybridization and PCR assays for detection of putative pathogenic enteroadherent Escherichia coli.

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.

Diffusely adherent Escherichia coli as a cause of acute diarrhea in young children in Northeast Brazil: a case-control study.

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.

Associaçäo de padröes de adesäo de Escherichia coli às células HEp-2 com diarréia aguda e persistente / Association of patterns of Escherichia coli adherence to HEp-2 cells with acute and persistent diarrhea

Cerca de 1/3 das consultas de emergência em crianças menores de 2 anos deve-se a casos de diarréia aguda, dos quais até 30 por cento dos causados por Escherichia coli enteropatogênica evoluem para diarréia persistente. Este estudotem como objetivo analisar a associação entre os diferentes padrões de adesão de E coli às células Hep-2 com diarréia aguda e persistente. Foram analisadas fezes de 34 crianças, internadas para terapia de reidratação nos Hospitais São Paulo e Darcy Vargas, em São Paulo, SP. Como controle, foram utilizadas fezes de 34 crianças pareadas por faixa etária, internadas nos mesmos hospitais, sem apresentarem quaisquer sintomas gastrointestinais nos 30 dias que precederam a coleta de fezes. Foram pesquisados os seguintes enteropatógenos: E coli causadoras de diarréia, Shigella, Salmonella, Yersinia enterocolitica, Campylobacter, Rotavirus, Adenovirus entérico e protoparasitas. Dentre as E coliisoladas foram pesquisados os padrões de adesão (adesão localizada-AL, adesão difusa-AD, adesão agregativa-AA e adesão localizada-like-ALL) às células Hep-2 e homologia com as sondas de DNA para AL (sondas EAF, eaeA), AA (sonda AA) e AD (sondas F184 e AIDA-I). Dos 34 casos, 24 apresentaram diarréia aguda (tempo máximo de duração de 14 dias) e 10, diarréia persistente (tempo de duração acima de 14 dias). Os padrões AL e AA foram detectados nos casos de diarréia aguda e persistente com freqüência semelhante, não sendo observados nos controles. 23,5 por cento das amostras de E coli isoladas foram Escherichia coli enteropatogênicas típicas (AL+, EAF+ e eaeA+); 8,8 por cento foram EAggEC (AA+) e hibridizaram com a sonda AA. As E coli produtoras de AD (DAEC) não hibridizaram com as sondas para AD e sua freqüência foi semelhante tanto em casos, quanto em controles. Neste estudo de caráter preliminar; E coli eaeA+ apresentando o padrão ALL foi mais freqüente em casos do que em controles, sugerindo que este grupo de bactérias possa realmente ter um potencial efeito enteropatogênico.