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We describe the characterization of 126 atypical enteropathogenic Escherichia coli (aEPEC) isolates from 1,749 Brazilian children. Classic aEPEC strains were more frequently found in children with diarrhea than in controls (P < 0.001), showing their importance as acute diarrhea agents in our country. Only aEPEC strains carrying either the ehxA or paa gene were significantly associated with diarrhea.
Enteropathogenic Escherichia coli (EPEC), one of the six E. coli diarrheagenic pathotypes, produces an adherence factor chromosomally encoded by the eae (EPEC attaching and effacing) gene located within the locus for enterocyte effacement (LEE) pathogenicity island (15, 16, 18).
“Typical” EPEC strains contain, in addition to eae, the EPEC adherence factor (EAF) plasmid (4), which encodes the bundle-forming pili that mediate localized adherence to epithelial cells (9, 26). EPEC strains lacking the EAF plasmid have been designated “atypical” EPEC (aEPEC) (12). Whereas typical EPEC strains express only the virulence factors encoded by the LEE region and the EAF plasmid, aEPEC strains have additional virulence properties (11, 32).
Recently Afset et al. (2) described several virulence genes associated with diarrhea in aEPEC isolates from Norwegian children. In their study, genes belonging to the pathogenicity island OI-122 (efa1/lifA, nleB, nleE, and sen), present in the enterohemorrhagic E. coli (EHEC) reference strain EDL933 (17), and the gene for long polar fimbriae (lpfA) (10), found in EHEC O113 strains, were particularly frequent. Other genes, such as the porcine A/E-associated gene (paa) (5) and the EHEC hemolysin gene (ehxA) (27), were also found to be associated with diarrheal disease.
Other E. coli adhesion factors recently described include the protein ToxB, required for full adherence expression in EHEC O157:H7 (30); Iha, an adherence-conferring protein similar to Vibrio cholerae IrgA (29); Saa, an autoagglutinating adhesin identified in LEE-negative strains (22); and Spf, a sorbitol-fermenting EHEC O157 fimbria (8). In addition, we recently identified a diffuse adherence (lda) locus in an aEPEC strain of the O26 serogroup that codes for adherence to HEp-2 cells (25).
In this report, we describe the prevalence and virulence profile of aEPEC strains isolated from diarrhea patients and control subjects in several cities of Brazil from 1999 through 2004.
Stool specimens from 1,102 children under 2 years of age with diarrhea, presenting to the emergency room of public hospitals in seven cities representing different regions of Brazil, and 647 randomly selected children without any gastrointestinal symptoms from the same hospitals were studied. All specimens were investigated for the presence of enteric pathogens, such as diarrheagenic E. coli, Shigella species, Salmonella species, Yersinia enterocolitica, Campylobacter species, and rotavirus (24).
Atypical EPEC strains were isolated, identified, and serotyped as described elsewhere (11). One isolate per subject was stored at −70°C.
aEPEC strains were screened by colony blot hybridization with 16 different DNA probes representing a panel of toxin, adhesin, and OI-122 genes. Fourteen DNA probes were prepared by PCR amplification from prototype strains. The genes, primers, amplicon size, PCR conditions, and prototype strains are given in Table Table1.1. Two DNA probes were prepared by plasmid extraction using the method of Birnboim and Doly (7) and digestion with appropriate restriction endonucleases. The cdt probe was a 1,357-bp fragment from plasmid pCVD448 (28), and the cnf probe was a 335-bp fragment of pEOSW1 (20). The probes were then purified by gel extraction (23) and labeled with [α-32P]dCTP, and colony hybridization assays were performed as described elsewhere (24).
A total of 126 aEPEC strains were isolated as the only pathogen in stool specimens from 92/1,102 (8.3%) children with diarrhea and 34/647 (5.2%) controls (P < 0.05). Forty-nine (38.9%) strains belonged to classical EPEC serotypes (O26, O55, O111, O119, O127, or O142), 35 (27.8%) were classified as non-EPEC serotypes, and 42 (33.3%) were untypeable (ONT) (Table (Table2).2). EPEC serotype strains were isolated significantly more often from patients than from controls (41 [3.7%] versus 8 [1.1%]; P < 0.001), while strains of non-EPEC serotypes and ONT occurred at similar frequencies in cases and controls (51 [4.6%] versus 26 [4.0%], respectively; P = 0.656).
Among the 126 aEPEC strains, positive hybridization was detected with 13 of the 16 virulence genes tested (Table (Table3).3). Most of the strains belonging to the O26, O55, O108, O119, O127, O142, and O153 serogroups carried the OI-122 genes and various combinations of adhesins, and some also carried the astA gene. The ehxA gene was found in O15 and O26 strains, and the cdt gene was found in two ONT strains. Curiously, iha occurred in 21% of the isolated aEPEC strains, lda and afa were found in very few strains, and spf and saa were not found at all. As far as we know, this is the first report of iha detection in aEPEC strains.
Table Table44 presents the frequencies of the 16 virulence genes in strains from patients and controls. Only the ehxA and paa genes were found to be significantly associated with diarrhea (P = 0.027 and 0.022, respectively).
In a previous study encompassing 65 aEPEC isolates, the virulence marker astA was significantly associated with diarrhea (11). However, in this study, with a larger number of isolates, the former observation was not confirmed.
Several aEPEC strains presented the OI-122 virulence gene nleB, nleE, efa1/lifA, or sen. Both the nleB and nleE genes, coding for effector proteins, were found in 26 aEPEC strains belonging to the serogroups O26, O55, O119, O127, and O142. Among these, 22 also carried the efa1/lifA lymphostatin gene and 19 had the putative enterotoxin gene sen. The presence of the OI-122 virulence genes in classical aEPEC strains was previously reported by Morabito et al. (17), and in a more recent study, they were also detected in untypeable or nonclassic EPEC serotypes associated with diarrhea (1).
In our study, the plasmid-encoded enterohemolysin (encoded by ehxA) of EHEC O157:H7 was significantly associated with diarrhea, in agreement with the data of Afset et al. for Norwegian children (2).
The identification of genes usually linked to the EHEC pathotype in a considerable proportion of our classic aEPEC strains is consistent with previous evidence from epidemiological and experimental studies showing that aEPEC may convert to, or be a conversion from, the EHEC pathotype by either acquisition or loss of stx genes (3, 6, 33). Several studies have revealed the genetic relatedness of O26 EHEC to O26 aEPEC by comparison of their core genomes (6) and housekeeping genes (33) and the presence of OI-122 genes and the high-pathogenicity island (3). It seems possible that other classic aEPEC strains genetically related to EHEC may have subsequently acquired additional virulence factors by horizontal transfer.
In agreement with data from Norwegian children, paa was much more frequent in aEPEC strains isolated from Brazilian children with diarrhea than in those from controls (P < 0.05). On the other hand, the positive association between lpfAO113 and diarrhea recently found in Norwegian children (2) was not observed in our study.
Based on our results, we show that in Brazil aEPEC strains could be classified into two subgroups: those belonging to classic aEPEC serotypes, which were associated with diarrhea, and those that are either untypeable or belong to nonclassic serotypes. This view is supported by our observation that OI-122 genes were most frequently found among the classic serotypes.
Recently, Afset et al. (2) suggested that aEPEC could be classified into two main virulence groups based only on the presence of OI-122 genes, regardless of serotype. In contrast to our results, in Norway most diarrhea-associated aEPEC strains carrying OI-122 genes either were untypeable or belonged to nonclassic serotypes (1).
In conclusion, we have shown that classic aEPEC strains are important agents of acute diarrhea among Brazilian children. Furthermore, we have identified two virulence markers, the ehxA and paa genes, that could be useful in the detection of truly enteropathogenic aEPEC.
This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
Published ahead of print on 16 September 2009.