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Journal of Clinical Microbiology, January 2004, p. 398-400, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.398-400.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Reduced Etiological Role for Enteropathogenic Escherichia coli in Cases of Diarrhea in Brazilian Infants

Josias Rodrigues,^* Cristiane M. Thomazini, Alexandra Morelli, and Georgea C. M. de Batista

Departamento de Microbiologia e Imunologia, Instituto de Biociências da UNESP em Botucatu, São Paulo, Brazil

Received 24 June 2003/ Returned for modification 14 August 2003/ Accepted 28 September 2003

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Previously common in Brazil, enteropathogenic Escherichia coli (EPEC) strains of serogroups O55, O111, and O119 are now rare, while enteroadherent strains other than EPEC, belonging to serogroups such as O125, were prevalent among 126 diarrheic infants less than 1 year old who were surveyed. None of these strains had the EPEC bundle-forming pilus (bfpA) gene.

Top Abstract Introduction References

 
A number of organisms, including viral, protozoan, and bacterial agents, are associated with the etiology of infantile infectious diarrhea, a leading cause of infantile mortality and morbidity worldwide. Although representing a common species of the human intestinal microbiota, Escherichia coli is also considered an important bacterial agent of infantile infectious diarrhea, an association first described in the report of John Bray (2), when enteropathogenic E. coli (EPEC) was found as the cause of a diarrhea outbreak originating from a pediatric nursery in London, United Kingdom, in 1945. In addition to EPEC, five other diarrheagenic E. coli (DEC) pathotypes were later described: enterotoxigenic (ETEC), enteroinvasive (EIEC), enterohemorrhagic (EHEC), enteroaggregative (EAEC), and diffusely adhering (DAEC) E. coli. ETEC, EAEC, and EHEC are toxin producers; EPEC, EAEC, and DAEC express characteristic adhesion patterns (localized, aggregative, and diffuse, respectively) to epithelial cells and are thus referred to as enteroadherent E. coli; EIEC is distinguished by the capacity to invade epithelial cells and the inability to produce toxins. Serotyping, screening for pathogenicity factors, and adherence to cultured epithelial cells comprise the main laboratorial diagnosis tools for diarrhea caused by DEC. For a detailed review of DEC, see reference 12.

In a recent survey carried out to investigate DEC prevalence in Botucatu, São Paulo State, Brazil, a region where some of the first epidemiological studies of enteropathogenic enterobacteria in Brazil were conducted (10), EAEC and rotavirus were found to be dominant, while traditional (EPEC, ETEC, EIEC, and EHEC) DEC pathotypes were absent from the stools of diarrheic children less than 13 years old (14). In order to better assess the role of EAEC in diarrhea and confirm the absence of EPEC in this population, we carried out the present study, restricting the survey to patients less than 1 year old, an age group more susceptible than others to EPEC-related diarrhea. The patient group consisted of 126 infants who attended the UNESP Hospital and the Medical Training Center of Botucatu between 1997 and 2000, with a record of at least two daily episodes of fluid stool evacuations, associated or not with vomiting, for 3 days or more. A total of 39 age- and geographically matched controls, randomly chosen at the same time among healthy volunteers in the community, were also surveyed. The stool collection was performed with the consent of the infant's family and the approval of the Hospital Committee on Ethics in Research. According to conventional bacteriological procedures, one to three out of five colonies randomly picked on MacConkey agar stool culture from each patient was identified as E. coli. These isolates were stocked at -80°C in tryptic soy broth supplemented with 20% glycerol. The E. coli collection, encompassing 165 isolates from the 126 diarrheic infants and 66 isolates from the 39 healthy infants, was then characterized by O:H serotyping, adhesion assays, and PCR to detect the EAEC plasmid for aggregative adhesion (pAA), the EPEC bundle-forming pilus structural subunit (bfpA), and the E. coli attaching and effacing (eae) genes.

O and H serotyping was done by slide and tube agglutination tests, respectively, as recommended by Ewing (4). The O antisera (Probac, São Paulo, Brazil) were specific for antigens of EPEC classical O serogroups (O26, O55, O86, O111, O114, O119, O125, O126, O127, O128, O142, and O158) (21). Adhesion assays were performed in HEp-2 cell monolayers for a 3-h incubation period at 37°C (14). Strains that were unable to adhere in these initial assays were submitted to 6-h incubation tests. Primers and mixture components as well as amplification conditions of PCR for eae, bfpA, and pAA are described in references 5, 8, and 16, respectively. EPEC strain E-2348/69 and EAEC strain 17-2 were used as positive controls both in the adhesion tests and in PCR.

Strains from 123 diarrheic and 39 healthy infants were tested for adhesion to HEp-2 cells, and the following phenotypes were identified: aggregative adhesion (AA), diffuse adhesion (DA), and localized-like adhesion (LLA) (Fig. 1). AA⁺ strains were found in 45 (36.6%) diarrheic and 17 (43.6%) healthy infants, and DA⁺ strains were found in 34 (27.6%) diarrheic and 14 (35.9%) healthy infants. Two LLA⁺ strains were identified, one in each of the surveyed groups. Regardless of the adhesion phenotype, strains displaying characteristic adhesion patterns were detected in higher frequencies in healthy (82%) than in diarrheic (65%) infants (P 0.05). Still-higher figures for healthy infants' strains were observed in regard to the EPEC and EAEC genetic markers. The percentage of eae⁺ strains found in controls (12.8%) was over twice as high as that of eae⁺ strains found in patients (5.6%). In the case of pAA, the percentage of positive strains in healthy infants was about six times that found in patients (38.5 and 6.3%, respectively). Yet, only the difference corresponding to the prevalence of pAA in healthy subjects proved to be statistically significant (P 0.001). PCR amplification products of eae⁺ and pAA⁺ strains are shown in Fig. 2. Twenty-two strains were pAA⁺: 12 of them displayed AA, 5 displayed DA, and 5 were not adherent. Only five of the pAA⁺ strains were detected in patients. The gene combination eae-bfpA was found in only one strain, which was, however, unable to adhere to HEp-2 cells and thus not considered a typical EPEC strain; this strain was isolated from a healthy infant. Strains of the classical O serogroups were found in 26 (20.6%) of the 126 diarrheic and 6 (15.4%) of the 39 healthy infants (Table 1). The most common serogroup was O125, with a total of seven strains detected, six of them in patients. The three adherent O125 strains showed the AA phenotype, but none of them had the pAA plasmid.

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FIG. 1. Adhesion phenotypes to HEp-2 cells found among E. coli strains isolated from 123 diarrheic and 39 healthy infants. (A) AA; (B) DA; (C) LLA. AA+ and DA+ strains were detected after both 3 and 6 h of incubation; LLA+ strains were detected only after 6 h of incubation. Arrows show LLA bacterial microcolonies, usually less dense and compact than the LA clusters of typical EPEC.

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FIG. 2. PCR-amplified DNA resolved by agarose gel electrophoresis of a pAA+ (lane 2) and an eae+ (lane 4) E. coli strain. The positive-control strains 17-2 and E-2348/69 are shown in lanes 1 and 3, respectively. M, 100-bp DNA ladder.

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TABLE 1. Adhesion phenotypes and genetic pathogenicity markers of E. coli strains in relation to O:H serotypes found among 126 diarrheic and 39 nondiarrheic infants

EPEC has been considered the main cause of infantile diarrhea in developing countries (1) and in some cases has been found in up to 30% of infants (6, 17). Data from the 1960s show EPEC of the O111 serogroup as representing 70% of the E. coli isolates from diarrheic children living in Botucatu (11). In 1996, the EPEC concept was refined in order to accommodate molecular and genetic pathogenesis features uncovered in previous years (9). The bfpA and eae genes associated with the capacity to display localized adhesion (LA) and to induce the attaching and effacing lesions in epithelial cells and with failure to produce Shiga cytotoxins are the differential markers of EPEC strains (9). Since not all of the classical O serogroups have strains with these features, the number of recognized EPEC serogroups was reduced. It was also observed that some of the classical O serogroups might include more than one DEC pathotype, which occasionally can be identified by its O:H type (3, 7, 15). For example, serotypes O55:H6, O86:H34, O111:H2, and O119:H6 usually mark EPEC strains (19), whereas O111:H10 and O111:H12 are EAEC (3). In this survey, strains of serogroups O55, O111, O114, O119, and O142 as well as six out of the seven O125 strains were isolated only from diarrheic infants (Table 1). Yet, none of them showed the typical EPEC markers, i.e., LA, eae, and bfpA. Most of these strains showed an AA usually not correlated with the presence of pAA (Table 1).

In the past, the most prevalent EPEC serogroups in Brazil were O55, O111, and O119 (20). O111 strains were dominant particularly among the population that we have studied (11). Though in those times E. coli isolates were not screened for the presence of EPEC pathogenicity markers, comparative (13) and epidemiological (18) analyses demonstrate that these serogroups include a high proportion of EPEC strains. In this survey, we detected a low number of strains of serogroups O55, O111, and O119, which either were nonmotile or did not react with antisera specific for the flagellar antigens of common EPEC serotypes, such as O55:H6, O111:H2, and O119:H6 (Table 1). Thus, by employing currently recognized diagnosis assays, our results reveal an evident decline in the incidence of EPEC strains and the prevalence of a heterogeneous group of AA⁺ and DA⁺ enteroadherent E. coli strains in Botucatu. Since they were found at a higher frequency in healthy infants than in patients, it is likely that at least some of them may belong to normal microbiota clones sharing the adhesion ability and the pAA marker of pathogenic DAEC and EAEC. On the other hand, the specific association of some serogroups with diarrheic patients is suggestive of an enteropathogenic role for some enteroadherent strains of particular serogroups, such as O125. In order to better evaluate the extent of the involvement of these strains with diarrhea and to know whether they are emerging DEC pathotypes that have replaced previously common EPEC serogroups, new studies aiming to characterize putative new virulence factors and to identify enteroadherent E. coli clones are necessary.

 
This work was supported by FAPESP grant no. 1997/07733-9 awarded to J.R.

 
* Corresponding author. Mailing address: Departamento de Microbiologia e Imunologia, IB-UNESP, Campus de Rubião Júnior, CEP 18.618-000, Botucatu, São Paulo, Brazil. Phone: 55 (14) 3811-6058. Fax: 55 (14) 3815-3744. E-mail: josias{at}ibb.unesp.br.

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Journal of Clinical Microbiology, January 2004, p. 398-400, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.398-400.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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