Genotypic and phenotypic characterization of attaching and effacing Escherichia coli (AEEC) isolated from children with and without diarrhoea in Londrina, Brazil

The genes necessary for AE lesion formation are located on the bacterial chromosome, forming a pathogenicity island called the locus of enterocyte effacement (LEE) (McDaniel et al., 1995). The LEE contains genes encoding a type-III secretion system (sep and esc), a number of secreted proteins [EspA, EspD, EspB and Tir (translocated intimin receptor)] and intimin (eae), a 94 kDa outer-membrane protein that mediates the intimate adhesion between the bacterium and the epithelial cell membrane (Kenny et al., 1997; Nataro & Kaper, 1998). The AE lesion can be observed by the fluorescent actin-staining (FAS) test; it gives rise to intense fluorescence of filamentous actin, which can be observed in the host cell beneath the site of bacterial attachment (Knutton et al., 1989).

Enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) E. coli are often grouped together as AEEC. In addition to their ability to induce AE lesions, EPEC possess the large EPEC adherence factor (EAF) plasmid and the cluster of genes that encodes bundle-forming pili (BFP) (Baldini et al., 1983; Girón et al., 1991). EPEC are a common cause of severe infantile diarrhoea, particularly in developing countries (Nataro & Kaper, 1998). These strains are characterized by a pattern of adhesion to epithelial cells called localized adhesion (LA), and are found among 12 different O-serogroups (O26, O55, O86, O111, O114, O119, O125O128, O142 and O158) (Scaletsky et al., 1984).

EPEC strains carrying the eae gene, but lacking the EAF plasmid and stx (Shiga toxin) genes, were described as atypical EPEC (Kaper, 1996) at the Second International Symposium on EPEC (São Paulo, Brazil, 1995). Atypical EPEC strains were found in several EPEC O-serogroups, but belonged to serotypes different from those of typical EPEC (Pelayo et al., 1999). These strains present an adhesion pattern called localized adhesion-like (LAL), characterized by the presence of less compact microcolonies or clusters of bacteria in a few cells (Scaletsky et al., 1996, 1999).

EHEC are typically characterized by the ability to cause the AE lesion, possession of a 60 MDa plasmid encoding enterohaemolysin and production of the Shiga toxins (Stx1 and/or Stx2), which are responsible for the major symptoms of haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS) (Nataro & Kaper, 1998). Serotype O157 : H7 is more frequently associated with disease in humans and animals, but other serotypes, such as O26 : H11, O111 : H^-, O111 : H8, O103 : H2, O118 : H^- and O118 : H16, have also been implicated (Nataro & Kaper, 1998). In a recent study performed in São Paulo, Brazil, Vieira et al. (2001) identified E. coli associated with infantile diarrhoea that belonged to non-EPEC serogroups, carried the eae gene and lacked the EAF and stx probe sequences.

The eae gene has been cloned and sequenced from several EPEC and EHEC strains isolated from humans and animals, RDEC (a rabbit EPEC-like E. coli strain), Citrobacter freundii and Hafnia alvei (Jerse et al., 1990; Beebakhee et al., 1992; Frankel et al., 1994; Agin & Wolf, 1997). These genes possess a highly conserved N-terminal region, whereas the C-terminal region shows much less similarity (Frankel et al., 1994). Frankel et al. (1995) have demonstrated that the binding activity of intimin is located in the C-terminal region.

Adu-Bobie et al. (1998) used PCR and antiserum against the C-terminal region of intimin to investigate the antigenic variation in this region, and identified four distinct intimin types: intimins α, ß, γ and δ. More recently, Oswald et al. (2000) characterized a new intimin, referred to as intimin , which was present in human and bovine EHEC strains.

The aim of this study was to determine the genotypic and phenotypic characteristics of AEEC isolated from children with and without diarrhoea in Londrina, Brazil. The investigation has been prompted by the lack of data concerning diarrhoea caused by these micro-organisms in children from this region, and by the need for better understanding of their enteropathogenic potential.

Table 1. Bacterial strains used as positive controls in this study

PCR.
All 919 E. coli isolates were screened for the presence of the genetic sequences of EAF, bfp, eae and stx. Strains positive for eae by PCR were analysed further to identify the intimin type present. Table 2 shows the primer sequences, sizes of amplified DNA fragments and amplification conditions for all sequences studied. Amplification reactions were performed as follows: cultures on tryptic soy agar (Difco) were suspended in 300 µl Milli-Q water (Millipore) and boiled for 10 min to release and denature the bacterial DNA. Amplification of bacterial DNA was carried out in volumes of 25 µl, containing 10 µl bacterial lysate, 200 µM dNTPs, 1.5 mM MgCl₂, 20 pmol each primer and 1.5 U Taq DNA polymerase (all Gibco-BRL). After amplification, products were electrophoresed in 1 % agarose gel (Difco), stained with ethidium bromide (Gibco-BRL) and visualized using UV light.

Table 2. Primer sequences, sizes of amplified DNA fragments and amplification conditions used in this study

HEp-2 cell adherence assay.
The eae-positive strains were tested for adhesion to HEp-2 cells, as described by Cravioto et al. (1979). When the adhesion pattern was not defined after 3 h incubation, the test was repeated with a 6 h incubation period.

DNA hybridization.
E. coli strains carrying eae and displaying an aggregative adhesion (AA) pattern were tested for hybridization with the specific DNA probe EAEC (enteroaggregative E. coli adherence plasmid), a 1 kb EcoRIPstI fragment of pCVD432 (Baudry et al., 1990) labelled with [α-³²P]dATP, in a colony hybridization assay.

FAS test.
The FAS test, which demonstrates the presence of filamentous actin beneath attached bacteria, was performed as described by Knutton et al. (1989). The strains were tested after 3 and 6 h incubation.

Haemolysin assay.
All eae-positive strains were tested for production of haemolysin as described by Beutin (1991). The strains were cultivated on blood agar base (Difco), supplemented with 10 mM CaCl₂ and 5 % defibrinated sheep erythrocytes washed three times in PBS. Plates were examined for zones of haemolysis around bacterial growth after 3 and 24 h incubation at 37 °C.

Serotyping.
Identification of somatic (O) and flagellar (H) antigens was performed in all eae-positive strains in the Enteric section of Instituto Adolfo Lutz, São Paulo, with specific antisera O1O173 and H1H52.

In this study, of 919 E. coli strains isolated from 125 children with diarrhoea and 98 without diarrhoea, 34 strains possessed the eae gene, either alone or associated with the other genes investigated. Twenty-seven of these strains (79.4 %) were isolated from cases of diarrhoea and seven (20.6 %) from controls. A summary of the genotypic and phenotypic characteristics of these strains is shown in Table 3.

Table 3. Genotypic and phenotypic characteristics of E. coli strains possessing the eae gene

Four different genetic profiles were found (Table 3). Strains possessing the eae gene alone (47.1 %) were most frequent, and were found in children both with and without gastrointestinal disease. In contrast, the characteristic genetic profile of typical EPEC (eae, bfp and EAF) was found only in children with diarrhoea.

The stx gene was not detected in any of the 34 strains identified as eae-positive. In Brazil, the incidence of EHEC in humans is very low in contrast with that of EPEC (Giraldi et al., 1990; Gomes et al., 1991; Saridakis, 1994). In fact, it is possible that EPEC infections in early childhood confer cross-reactive protective immunity against EHEC types that share common antigens (such as LPS and intimin) with classical EPEC strains (Beutin et al., 1998). This could explain why human infections with eae-positive EHEC occur less frequently in developing countries where infections of children by EPEC are common.

Five distinct adhesion patterns were observed (Table 3): LA, AA, LAL, DA (diffuse adhesion) and IS (isolated bacteria). Two strains were non-adherent (NA). LA was the most frequent pattern of adhesion (44.1 %), followed by AA (23.5 %), LAL (14.7 %), IS (8.8 %) and DA (2.9 %). Strains with AA, LAL and IS were found in children both with and without diarrhoea. The only strain that presented the DA pattern was isolated from a child without diarrhoea. All strains that presented the genetic profile of typical EPEC showed the LA pattern. Our results show a strong association between strains that presented the LA pattern and diarrhoea (one from the control group and 14 from patients). The strains that displayed the AA pattern did not hybridize with the EAEC probe.

Of all the eae-positive strains, 10 (29.4 %) were FAS-negative after incubation for 3 or 6 h. The majority of the eae-positive, FAS-negative strains possessed the eae gene alone (80 %). Vieira et al. (2001) studied 59 E. coli strains that carried eae but lacked the EAF plasmid probe sequence and the stx genes, and found that 25.9 % of the strains were FAS-negative. It is possible that eae-positive, FAS-negative strains possess some deficiency in expression of the genes encoding the type-III secretion system proteins and/or the secreted LEE proteins. Two eae-positive, FAS-negative strains were unable to attach to HEp-2 cells. According to Cantey & Moseley (1991), non-adherent, eae-positive strains can possess adhesins capable of recognizing the intestinal mucosal surface, but not the cultured cells used in laboratory assays, for adhesion.

Fourteen different serogroups were detected. Forty-seven per cent of the strains belonged to classical O-serogroups of EPEC (O26, O86, O111, O114, O119, O127 and O142). The most frequent serotype found in our study was O119 : H6 (14.7 %). All strains of serotype O119 : H6 presented the LA pattern, were FAS-positive and were isolated from children with diarrhoea. Eleven strains were non-O-typable, and two were non-H-typable.

Adu-Bobie et al. (1998) revealed the existence of four distinct intimin types: intimins α, ß, γ and . Oswald et al. (2000) typed the intimin of the serotype O86 : H34 EPEC strain ICC95 as type ß, although Adu-Bobie et al. (1998) had previously classified this intimin as type δ. Restriction analysis of the PCR product obtained from strain ICC95 with ß-specific primers as described by Oswald et al. (2000) showed a digestion profile, termed ß2, that differed from the ß1 pattern shared by all other intimin ß-positive strains. According to Oswald et al. (2000), these data confirm that there are differences between the eae DNA sequences of serogroup O86 EPEC and the other ß-type strains, and indicate that the ß2 subtype corresponds to the δ type of Adu-Bobie et al. (1998). Oswald et al. (2000) also revealed the existence of a new intimin type, referred to as intimin .

It is interesting to note that intimin ß was detected in seven strains in this work, four of which belong to serotype O119 : H6 (a classical EPEC serotype), and all of which were isolated from children with diarrhoea. According to the literature, intimin ß is the most common subtype among human strains of E. coli, and it seems that it is also the most common in bovine E. coli (Aidar et al., 2000; Vieira et al., 2001). On the other hand, intimin was found in two strains of serotype O111 : H38 and one of serotype ONT : H19, isolated from children without diarrhoea. Oswald et al. (2000) detected intimin in human and bovine EHEC strains of serogroups O8, O11, O45, O103, O121 and O165. To our knowledge, this is the first report of the occurrence of intimin in strains isolated from humans in Brazil and the first report of eae⁺, stx^- strains in the literature. The majority of strains studied (70.6 %) produced an intimin that could not be typed.

Haemolysin expression on blood agar was observed for only five strains (four after 24 h incubation and one after 3 h), all of which were isolated from cases of diarrhoea. The low frequency of haemolysin expression among the eae-positive strains did not support a major role in enteropathogenicity.

The results obtained in this study indicate that AEEC are a heterogeneous group of organisms. However, typical EPEC continue to be important pathogens associated with diarrhoea in Brazilian children. EAF^-, bfp^-, eae⁺ E. coli strains have the potential, as a new enteropathogenic group, to become an important cause of diarrhoea in children.