Typing of intimin (eae) genes from enteropathogenic Escherichia coli (EPEC) isolated from children with diarrhoea in Montevideo, Uruguay: identification of two novel intimin variants (μB and ξR/β2B)

INTRODUCTION

Enteropathogenic Escherichia coli (EPEC) was first recognized as a cause of infantile diarrhoea in the 1940s, and was associated with outbreaks in hospitals and nurseries in the UK. During one such outbreak, Bray (1945) prepared antiserum to a strain of E. coli isolated from a patient with diarrhoea and used this antiserum to show that the epidemic strains belonged to the same serogroup, later recognized as E. coli O111. In 1987, the World Health Organization (1987) recognized EPEC serotypes of 12 different O serogroups (O26, O55, O86, O111, O114, O119, O125, O126, O127, O128, O142 and O158). Although large outbreaks of infant diarrhoea due to EPEC have largely disappeared from industrialized countries, EPEC remains an important cause of potentially fatal infant diarrhoea in developing countries (Trabulsi et al., 2002). In Brazil, for example, EPEC strains are recovered from up to 30 % of cases of diarrhoea in infants of low socioeconomic level (Gomes et al., 1996). In Uruguay, diarrhoeagenic E. coli (DEC) are the most frequently identified bacterial agents associated with gastroenteritis of children in low-income population groups. Although rotavirus is increasingly recognized as a frequent cause of these infections, EPEC still accounts for a vast proportion of diarrhoea cases. Shigella flexneri and Campylobacter jejuni are also frequently isolated, especially from episodes of bloody diarrhoea (Torres et al., 2001).

For decades, the mechanisms by which EPEC caused diarrhoea were unknown, and this diarrhoeagenic pathotype could only be identified on the basis of O : H serotyping. However, since 1979, numerous advances in our understanding of the pathogenesis of EPEC diarrhoea have been made through the application of tissue culture and molecular genetic methods (Frankel et al., 1998; Trabulsi et al., 2002; Kaper et al., 2004). One of the central mechanisms of EPEC pathogenesis is the formation of attaching and effacing (A/E) lesions, which is characterized by the intimate attachment of the bacteria to the enterocyte membrane and by the effacement of the microvilli of the enterocyte (Jerse et al., 1990; Kaper et al., 1998). The ability to produce A/E lesions has also been detected in strains of Shiga-toxin-producing E. coli (STEC) and in strains of other bacterial species. STEC and EPEC that cause characteristic A/E lesions in the intestinal mucosa are also classified as attaching and effacing E. coli (AEEC). The genetic determinants for the production of A/E lesions are located on a large chromosomal pathogenicity island, the locus of enterocyte effacement (LEE; Kaper et al., 1998). The central portion of LEE encodes intimin (Eae, a 94–97 kDa outer-membrane protein) and Tir, the intimin receptor, which is translocated into the host cell membrane by a type III secretion system. Differentiation of intimin alleles represents an important tool for EPEC and STEC typing in routine diagnostics as well as in pathogenesis, epidemiological, clonal and immunological studies. The C-terminal end of intimin is responsible for receptor binding, and it has been suggested that different intimins may be responsible for different host tissue cell tropisms (Torres et al., 2005). The 5′ regions of eae genes are conserved, whereas the 3′ regions are heterogeneous. This observation has led to the construction of universal PCR primers and allele-specific PCR primers, which have made it possible to differentiate at present 21 variants of the eae gene encoding 21 different intimin types and subtypes: α1, α2, β1, ξR/β2B, δ/β2O, κ, γ1, γ2, θ, ε1, νR/ε2, ζ, ε1, ε2, ι1, μR/ι2, λ, μB, νB, ξB and o) (Gannon et al., 1993; Adu-Bobie et al., 1998; Oswald et al., 2000; Tarr & Whittam, 2002; Zhang et al., 2002; Blanco et al., 2004a, b, d, 2005, 2006; Garrido et al., 2006).

In 1996, EPEC strains were defined as intimin-containing DEC isolates that possess the ability to form A/E lesions on intestinal cells and do not possess Shiga toxin (stx) genes (Kaper, 1996). However, EPEC strains can be further classified as typical or atypical. Typical EPEC strains possess a virulence plasmid (EAF plasmid) that includes genes encoding the bundle-forming pilus (Bfp), which is required for localized adherence on cultured epithelial cells; atypical EPEC strains do not possess the EAF plasmid with the bfpA gene (Trabulsi et al., 2002). In industrialized countries, atypical EPEC (eae⁺ bfpA⁻ stx⁻) are more frequently isolated from diarrhoeal cases, whereas typical EPEC (eae⁺ bfpA⁺ stx⁻) dominate in developing countries (Trabulsi et al., 2002).

The aim of this study was to establish the serotypes, intimin types and genetic diversity of typical and atypical EPEC strains isolated from children with diarrhoea in Montevideo, Uruguay.

METHODS

RESULTS

DISCUSSION

Diarrhoeal illness is a major public health problem worldwide, with over 2 million deaths occurring each year, particularly among infants younger than 5 years (). One of the most common causes of infantile diarrhoea is EPEC. Typical EPEC strains belonging to classical EPEC serotypes were associated historically with outbreaks of infantile diarrhoea in industrialized countries, particularly during the 1940s and 1950s, but at present they are very rare (Trabulsi et al., 2002; Jenkins et al., 2003; Blanco et al., 2006). Recently, however, Gerner-Smidt et al. (2003) have reported that classical EPEC is the most common bacterial cause of diarrhoea in children less than 2 years old in Denmark. In industrialized countries, atypical EPEC are more frequently isolated from diarrhoeal cases than typical EPEC. Furthermore, Nguyen et al. (2006) have recently shown that, in contrast to patients infected with other pathogens, patients infected with atypical EPEC are far more likely to experience diarrhoea past 14 days, the point long recognized as a clinical watershed that heralds an increased risk of illness and death. Interestingly, atypical EPEC strains have also been isolated frequently from healthy infants (Afset et al., 2003; Beutin et al., 2003).

The situation in developing countries is not well defined, but several studies in Brazil, Chile, Uruguay and Bangladesh (Albert et al., 1995; Gomes et al., 1996; Levine et al., 1996; Nunes et al., 2003; Vidal et al., 2004) have shown a high frequency of typical EPEC serotypes in stools from children with diarrhoea. However, some recent studies performed in Brazil, Thailand and Vietnam have shown a very low frequency of typical EPEC and a relatively high frequency of atypical EPEC (Pelayo et al., 1999; Vieira et al., 2001; Ratchtrachenchai et al., 2004; Rodrigues et al., 2004; Vu Nguyen et al., 2005). Our results confirm that typical EPEC strains belonging to classical EPEC serotypes O55 : H−, O111 : H2, O111 : H− and O119 : H6 are important pathogens associated with diarrhoea of children in Uruguay (Torres et al., 2001). These serotypes are the most frequent EPEC serotypes implicated in infantile diarrhoea in Brazil, and have also been frequently isolated in other countries (Trabulsi et al., 2002; Nunes et al., 2003; Ratchtrachenchai et al., 2004).

Specific intimin subtypes may be involved in mediating both tissue tropism and host specificity, and may provide information on the association of EPEC and STEC with severe disease and on the nature of the bacterium–host relationship. In addition, host immunity to the surface-exposed proteins produced by one E. coli strain may not provide protection against intestinal colonization by E. coli strains which bear distinct intimin types (Adu-Bobie et al., 1998; Torres et al., 2005). We have identified six novel intimin variant genes that we originally designated β2, ε2, μ, ν, ξ and o when the sequences were submitted to the EMBL Nucleotide Sequence Database (Blanco et al., 2003, 2004b, 2005, 2006), and before knowing the results obtained by Ramachandran et al. (2003). The intimin β2 that we have found in all 18 typical EPEC strains of classical EPEC serotype O119 : H6 (Blanco et al., 2003; this study) is identical to intimin ξ described by Ramachandran et al. (2003) in one bovine strain of serotype ONT : HNT. Thus, in this study, our β2 intimin is referred to as ξR/β2B. We have found the ξR/β2B intimin in another four human strains of serotypes O119 : H−, ONT : H51 and O33 : H6 in the present study, in six human strains of serotypes O56 : H6 (bfpA negative), O110 : H6 (bfpA negative), O113 : H6 (bfpA negative), O137 : H6 (bfpA negative) and O167 : H6 (bfpA positive) isolated in Spain (Blanco et al., 2006), and in two E. coli strains of serotypes O139 : H14 (bfpA negative) and O167 : H6 (bfpA positive) isolated from neotropical non-human primates with diarrhoea in Brazil (Blanco et al., 2004c). The other five intimins described by us (ε2, μ, ν, ξ and o) are different to the existing intimin types and are referred to as ε2, μB, νB, ξB and o, respectively. Interestingly, all six EPEC strains positive for the novel μB intimin belonged to classical EPEC serogroup O55 (serotypes O55 : H51 and O55 : H−). However, we also detected the γ1 (O55 : H−) and θ (O55 : H40) intimin variant genes in human EPEC strains belonging to serogroup O55 characterized in this study. The novel intimin ε2 was identified in one human atypical EPEC strain of serotype ONT : H45 isolated in Spain and in three bovine typical EPEC strains of serotype ONT : H45 isolated in Switzerland (Blanco et al., 2005). The novel intimins νR and o were detected in human atypical EPEC strains of serotypes O10 : H−, O84 : H− and O129 : H− isolated in Spain (Blanco et al., 2006), whereas the intimin ξB was observed in two Spanish bovine STEC strains of serotype O80 : H− (Blanco et al., 2004b).

Our phylogenetic analysis revealed six groups of closely related intimin genes: (i) α1, α2, ζ, νB and o; (ii) λ; (iii) β1, ξR/β2B, δ/β2O and κ; (iv) ε1, ξB, νR/ε2, ε1 and ε2; (v) γ1, μB, γ2 and θ; and (vi) ι1 and μR/ι2. This analysis is in accordance with the finding of Zhang et al. (2002). However, the intimin alleles α2, ξR/β2, ε2, μB, μR/ι2, νB, νR/ε2, ξB and o were not analysed in the study of Zhang et al. (2002) because no nucleotide sequences were available. Ramachandran et al. (2003) classified the 14 intimin genes they analysed into nine distinct phylogenetic families (α1-α2, β1-ξR/β2B, γ1, κ, ε-ε-νR/ε2, ι-μR/ι2, λ, θ and ζ).

In recent years, DNA macrorestriction analysis by PFGE has increasingly been used for the molecular subtyping of a wide range of bacterial pathogens, and is now considered the ‘gold standard’ for the molecular subtyping of many pathogenic organisms (Mora et al., 2004). However, to our knowledge, very few studies are available concerning PFGE typing of EPEC strains. In our study we have analysed 44 EPEC strains by PFGE, including the most prevalent serotypes. None of the strains presented an identical PFGE pattern to another. In the dendrogram produced by the UPGMA algorithm, the isolates were clustered in 12 groups (1–12 strains per group) of 60 % similar strains according to the Dice similarity index. Only 15 strains were clustered in six groups of closely related (similarity >85 %) PFGE patterns, indicating the prevailing clonal diversity among EPEC strains isolated from children with diarrhoea in Montevideo.

In conclusion, we have identified two novel intimin types in typical EPEC of serotypes O55 : H51/H− (eae-μB) and O119 : H6/H− (eae-ξR/β2B) that could become novel targets for vaccine development. Our results indicate that typical EPEC strains of serotypes O55 : H51/H− (eae-μB), O111 : H2/H− (eae-β1) and O119 : H6 (eae-ξR/β2) are important pathogens associated with diarrhoea in children in Uruguay.