Bacteriology
Genotyping of enteroaggregative Escherichia coli and identification of target genes for the detection of both typical and atypical strains

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Abstract

Enteroaggregative Escherichia coli (EAggEC) is an important cause of diarrhea worldwide, and there is a need for better detection methods in diagnostic laboratories. The aims of this study were i) to characterize strains of EAggEC by assigning each isolate a genotypic profile and (ii) to determine target genes for the detection of both typical and atypical EAggEC. The heterogeneity of the EAggEC group makes selection of a single target gene difficult. The plasmid-encoded genes, aat, aggR, and aap, are all appropriate targets for the detection of typical EAggEC. Of the chromosomally encoded genes, aaiA would be the most suitable target gene to identify typical and atypical EAggEC. The astA gene, encoding the enteroaggregative heat stable toxin, although not specific for EAggEC, may be used effectively in combination with other specific EAggEC genes. A polymerase chain reaction test based on the detection of characteristic EAggEC virulence genes, such as aat, astA, and aaiA, would improve EAggEC diagnosis.

Introduction

Enteroaggregative Escherichia coli (EAggEC), first described by Nataro et al. (1987), is an important cause of persistent diarrhea in developing countries (Bhan et al., 1989, Fang et al., 1995), although their association with acute diarrhea is less consistent (Nataro et al., 1995, Okeke et al., 2000). There is evidence that EAggEC cause diarrhea in developed countries (Tompkins et al., 1999, Knutton et al., 2001, Pabst et al., 2003) and they have been responsible for outbreaks of diarrheal disease, notably in the UK (Smith et al., 1997, Spencer et al., 1999), Serbia (Cobeljic et al., 1996), and Japan (Itoh et al., 1997). The pathogenesis of EAggEC infection is thought to involve initial adherence to the host intestinal mucosal epithelium by aggregative adherence fimbriae and subsequent destruction of the mucosa, possibly by toxins (Nataro et al., 1994a, Cerna et al., 2003). However, the precise mechanisms by which EAggEC cause diarrhea, and the role of the various pathogenicity factors, are poorly understood.

EAggEC is characterized by their ability to adhere to HEp-2 cells in an aggregative adherence (AA) pattern, described as a “stacked brick” formation (Nataro et al., 1994a, Law and Chart, 1998). Certain strains carry a high-molecular-weight plasmid (pAA) associated with AA (Law and Chart, 1998, Vial et al., 1988), on which a number of virulence genes are located. These include the AA fimbrial genes, AAFI (aagA) (Nataro et al., 1992), AAFII (aafA) (Czeczulin et al., 1997), and AAFIII (agg3A), and their corresponding usher genes, aagC, aafC, and aag3C, respectively (Bernier et al., 2002). Also located on the plasmid are the transcriptional activator gene (aggR) (Nataro et al., 1994a, Nataro et al., 1994b), antiaggregation protein (dispersin) gene (aap, previously called aspU) (Sheikh et al., 2002), and an antiaggregation protein transporter gene (aat) (previously called CVD432 or the AA probe) (Baudry et al., 1990). In addition, the plasmid-encoded toxin (pet) (Eslava et al., 1998) and enteroaggregative heat-stable toxin (EAST) genes (astA) (Savarino et al., 1991) and a cryptic open reading frame known as shf (Czeczulin et al., 1999) are also plasmid encoded. Strains that carry pAA represent an important subgroup (Cerna et al., 2003, Elias et al., 2002) and may be regarded as “typical EAggEC”. However, EAggEC is a heterogeneous group of E. coli (Czeczulin et al., 1999) and certain strains, although still capable of causing diarrheal disease, do not carry the AA plasmid (Cobeljic et al., 1996, Itoh et al., 1997, Elias et al., 2002, Gioppo et al., 2000) and may be called “atypical” EAggEC. These strains adhere to HE-p2 cells with a stacked-brick formation, but do not hybridize to the CVD432 or the AA probe (Baudry et al., 1990). Certain atypical strains carry putative EAggEC virulence genes in a variety of combinations and others carry none of these genes (Elias et al., 2002, Gioppo et al., 2000).

Other putative EAggEC pathogenicity genes, all located on the chromosome, include pic (Henderson et al., 1999), a protein involved in colonization, irp2, a protein involved in the expression of yersiniabactin (Schubert et al., 1998), aaiA, which is located on a pathogenicity island and is thought to be under control of aggR (E.G. Dudley and J.P., unpublished data), and the putative invasion determinant gene, tia (Fleckenstein et al., 1996).

Interpretation of the HEp-2 cell adhesion assay (Scotland et al., 1991) for the identification of EAggEC can be subjective. A polymerase chain reaction (PCR) test based on the detection of a combination of characteristic EAggEC virulence genes would greatly improve EAggEC diagnosis. Strains of EAggEC, with and without the AA plasmid, may share other genes either located on the chromosome or on other plasmids. The aims of this study were i) to characterize strains of EAggEC in the culture collection at the Laboratory of Enteric Pathogens by assigning each isolate a genotypic profile and ii) to determine target genes for the detection of both typical and atypical EAggEC.

Section snippets

Bacterial strains

The strains of E. coli characterized in this study belonged to 3 groups. Group 1 (archive strains) comprised 86 strains of EAggEC from the culture collection held by the Laboratory of Enteric Pathogens (LEP). These isolates showed the AA pattern with HEp-2 cells and harbored the aat gene, and were from various sources including sporadic cases of diarrhea (Spencer et al., 1999, Smith et al., 1994, Knutton et al., 1991) and outbreaks (Spencer et al., 1999, Smith et al., 1994, Kleanthous et al.,

Serotyping

The 86 strains in group 1 (archive strains—comprising typical EAggEC only) consisted of at least 39 different O serogroups and included 15 strains that could not be serogrouped using the current serotyping scheme (designated O? or O-rough) (Table 1). The most common serotypes in the archive group were O126:H27 (5), O44:H18 (6), and O111ab:H21 (8). Strains with the same serotype often had different genotypic profiles. The serotyping results of the strains in group 2 (QEH strains—comprising of

Discussion

Historically, the HEp-2 cell adhesion assay and the AA probe have been used to identify EAggEC (Nataro et al., 1994a, Law and Chart, 1998). Many studies have shown that the majority of EAggEC strains, phenotypically characterized as having the AA HEp-2 pattern, also have the aat gene (AA probe-positive) (Cerna et al., 2003, Baudry et al., 1990), although this is not always the case (Gioppo et al., 2000). It was recognized that not all strains adhering to HEp-2 cells in an AA pattern reacted

Acknowledgments

We thank Dr Edward Dudley and Prof Jim for providing us with the primers for the aaiA gene and for all their help and advice during this study. We would also like to thank Dr Stuart Knutton and Dr Alan Phillips for allowing us to include data on the Queen Elizabeth Hospital strains. This study was funded by the Food Standards Agency.

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    Current address: Microbiology Department, Royal Free Hospital, Pond Street, NW3 2QG London, UK.

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