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J Clin Microbiol. 2010 January; 48(1): 302–306.
Published online 2009 October 28. doi:  10.1128/JCM.01980-09
PMCID: PMC2812252

Adherence Factors in Atypical Enteropathogenic Escherichia coli Strains Expressing the Localized Adherence-Like Pattern in HEp-2 Cells[down-pointing small open triangle]

Abstract

Although atypical enteropathogenic Escherichia coli (aEPEC) strains are frequently implicated in childhood diarrhea in developing countries, not much is known about their adherence properties. The phenotypic and genotypic characterization of 29 aEPEC strains expressing the localized adherence-like pattern points toward the involvement of E. coli common pilus (ECP), intimins, and other known E. coli adhesins in this pattern.

Atypical enteropathogenic Escherichia coli (aEPEC) strains are increasingly recognized as an emerging pathotype responsible for childhood diarrhea in many countries (2-4, 19, 23). Atypical EPEC strains together with typical EPEC (tEPEC) strains constitute two distinct groups of organisms that have in common the locus of enterocyte effacement (LEE), a pathogenicity island responsible for the development of attaching-effacing (A/E) lesions. This island encodes the type III secretion system with multiple secreted proteins and a bacterial adhesin called “intimin” encoded by the eae gene (12, 16-18). Unlike tEPEC, aEPEC strains do not possess the EPEC adherence factor (EAF) virulence plasmid that contains the bundle-forming pili (BFP) responsible for a localized adherence (LA) pattern on cultured epithelial cells (5, 8, 28), but aEPEC strains display different adherence patterns. The typical EPEC strain exhibits only the LA pattern, while aEPEC strains display LA-like (LAL), diffuse adherence (DA), or aggregative adherence (AA) patterns (1, 10, 26, 32, 33). In addition, aEPEC strains belong to serotypes other than those of tEPEC strains (1, 10, 32, 33).

Despite the clear differences in adherence patterns, only the factors mediating the LA pattern have been extensively studied in tEPEC, and little is known about the factors mediating adherence of aEPEC strains. In a previous study, we identified in an aEPEC strain belonging to the O26 serogroup an adhesin gene designated as lda, for “locus for diffuse adherence,” which encodes a nonfimbrial structure conferring the DA phenotype (27).

Recently, in a collection of 126 aEPEC strains isolated from Brazilian children, we found many putative E. coli adhesin-encoding genes besides lda (25), such as efa1 (enterohemorrhagic E. coli [EHEC] factor adhesion 1) (13, 20), toxB (a plasmidial locus found in EHEC O157:H7 implicated in adhesion) (31), lpfA (long polar fimbriae) (9), iha (IrgA-homologous adhesion) (30), and paa (porcine A/E-associated gene) (6). In the present study, we characterize these strains regarding intimin types, HEp-2 adherence patterns, and ability to promote actin accumulation in vitro.

The eae gene was subtyped according to the restriction fragment length polymorphism assay described by Ramachandran et al. (21). This method permits detection of the intimin types α, β, γ, epsilon, ζ, θ, ι, κ, λ, ν, ξ, ο, ρ, and σ. As can be seen in Table Table1,1, intimins α (22 strains), β (26 strains), and γ (20 strains) were the most frequently found types. Forty-three (34%) strains had a nontypeable intimin. Intimins ξ, ι, κ, λ, and ν were not found among the entire collection of 126 aEPEC strains. In agreement with previous data (10), a close relationship between classic serotypes and intimin types was seen in this study: intimin α was found mainly among O142:H2 strains, and intimin β was detected in O26:H11, O119:H2, and O128:H2 strains, whereas intimin γ was seen in O55 strains.

TABLE 1.
Characteristics of 126 atypical enteropathogenic Escherichia coli strainsa

The HEp-2 adherence patterns of aEPEC strains were determined according to the method described by Scaletsky et al. (28). Infected monolayers were examined after a 3-h incubation period, and when the adherence pattern was weak or negative, a new preparation was made and examined after a 6-h incubation period. Twenty-nine aEPEC strains (23%) showed the LAL pattern, characterized by the presence of loose bacterial clusters in the 3-h assay and compact clusters, identical to LA of tEPEC, in the 6-h assay (Fig. (Fig.1).1). Other less frequently found patterns included aggregative adherence (AA) (three strains) and diffuse adherence (DA) (two strains), with both patterns detected only in the 6-h assay. Eleven strains (8.7%) promoted cell detaching. Thirty-two strains (25.4%) did not adhere to HEp-2 cells in the 3-h assay, but in the 6 h assay showed an indeterminate adherence pattern. Finally, 49 strains (38.9%) were nonadherent after 6 h. Comparable results were reported by others who found LAL to be the most frequent adherence pattern among aEPEC strains, whereas AA and DA patterns were found in lower frequencies (1, 10, 32).

FIG. 1.
Localized adherence-like (LAL) patterns in HEp-2 cells of representative classic aEPEC strains. (A and B) LAL patterns of strain HDV133-1 in the 3-h assay and 6-h assay, respectively. (C to E) LAL patterns of strains AMB6-3-3 (C), HSP23-5 (D), and SC241-1 ...

The ability of adherent strains to cause A/E lesions was evaluated by the fluorescent-actin staining (FAS) test (15). All but two adherent aEPEC strains were able to cause the A/E lesions, indicating the functionality of the LEE region (16). The strains presenting LAL resulted in areas of discrete and intense fluorescence in the FAS test after 6 h of incubation (data not shown). The strains presenting AA or DA were also able to accumulate actin in the adherence site. The ability to aggregate actin in HEp-2 cells by LAL, AA, or DA has also been reported by others (1, 23).

The origins and properties of the 29 aEPEC strains expressing LAL are presented in Table Table2.2. Most of the strains were isolated from patients with diarrhea, belonged to the classical EPEC serotypes, and, as previously reported, carried at least one known E. coli adhesin gene (25). As can be seen in Table Table2,2, common genetic profiles, represented in boldface, could be observed among strains belonging to the same serotype. These common genetic profiles led us to evaluate the presence of common plasmids in the 29 aEPEC strains. One or two high-molecular-mass plasmid bands (between 23.9 and 98 MDa) were found after DNA extraction by the alkaline lysis method (7) in all strains (Fig. (Fig.2),2), but no common plasmid profile could be detected. Interestingly, a plasmid band of 60 to 65 MDa was observed in almost all 29 strains, although none of them had the EAF plasmid (data not shown).

FIG. 2.
Plasmid contents of aEPEC strains expressing the LAL pattern to HEp-2 cells. Lanes: 1, strain HDV133-1; 2, strain RN216-5; 3, strain AMB6-3; 4, strain RP60-3; 5, strain HSP23-5; 6, strain HSP28-8; 7, strain HSP11-1; 8, strain AMB118-1; 9, strain HSP35-9; ...
TABLE 2.
Origins and properties of 29 atypical EPEC strains presenting a localized adherence pattern to HEp-2 cellsa

The involvement of the recently described E. coli common pilus (ECP) in the adherence properties of the 29 aEPEC strains displaying the LAL pattern was sought by PCR (22). All strains were positive for ecpA, the pilin subunit of ECP. Although this pilus is widespread among E. coli strains, including nonpathogenic strains, evidence is accumulating that it may also contribute to epithelial cell adherence of commensal and pathogenic E. coli strains, including EHEC (22). In addition, ECP was shown to be an accessory factor contributing to the multifactorial complex interaction of tEPEC, in association with BFP and other adhesins (24). Unlike the LA pattern of tEPEC which is mediated by multiple factors, including BFP, intimin, ECP, and possibly other adhesins, the mechanism involved in the LAL pattern is still unknown.

It is possible that ECP, in association with other adhesins, is able to compensate for the absence of BFP and permits bacteria to adhere with a localized pattern to cultured cells in a prolonged assay. All of the 29 aEPEC strains displaying the LAL pattern carried the ecpA gene, and all but two carried at least one of the known E. coli adhesion genes. In addition, almost all of the 29 aEPEC strains had some type of intimin. Recently, Hernandes et al. (11) showed that the compact microcolony formation of one aEPEC ONT strain was mediated by intimin ο (omicron).

In summary, the results obtained in this study suggest that the LAL pattern represents a virulence property of aEPEC strains, particularly of classic aEPEC strains. This pattern has been referred to as “poor LA” by Knutton et al. (14), “LA” by Scotland et al. (29), and “LA6” by Vieira et al. (34). Our data point toward an involvement of ECP, intimins, and other known E. coli adhesins in the LAL pattern. However, we cannot rule out the existence of an adhesive structure not yet identified involved with the LAL pattern. Further studies are under way to address these questions.

Acknowledgments

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).

Footnotes

[down-pointing small open triangle]Published ahead of print on 28 October 2009.

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