Phenotypic and genotypic analysis of enteroaggregative Escherichia coli sequence types associated with disease or carriage

Enteroaggregative E. coli (EAEC) are emerging pathogens most commonly associated with acute and persistent paediatric diarrhoea and growth retardation in developing nations (1-3). In addition, EAEC are a major cause of acute diarrhoea in travellers to developing countries (4, 5) and persistent enteric infection in HIV/AIDS patients (6, 7). EAEC are a heterogenous group and have been traditionally defined by their characteristic “stacked brick”-like aggregative adherence (AA) to HEp-2 cells (8, 9). Pathogenesis involves adherence to intestinal epithelium, biofilm formation, release of toxins and mucosal inflammation, and several putative virulence factors involved in these processes have been identified (10-12). These include the aggregative adherence fimbriae (AAF) of which five major variants have been described so far (AAF/I to AAF/V) (13-17). AAF are encoded on the pAA virulence plasmid which also contains the major transcriptional regulator AggR and the surface protein dispersin which contributes to spreading of the AAFs (18-21). In addition, other adhesins such as the E. coli common pilus (ECP) can mediate aggregative adherence, particularly in the absence of AAF (22). Other potential virulence factors of EAEC include the cytotoxins Pet, EAST-1, HlyE and the mucinase Pic which promote epithelial damage and intestinal colonisation (10). Despite the identification of multiple EAEC virulence candidates, not all EAEC strains harbouring these factors cause human disease, and genotypic studies have failed to consistently associate a single gene or combination of genes with EAEC virulence (10, 23, 24). To elucidate the population structure of EAEC and determine if certain lineages were more strongly associated with disease, Chattaway and colleagues recently analysed 564 EAEC isolates from three case-control studies in Bangladesh, Nigeria and the UK by Multi Locus Sequence Typing (25). Of the 17 identified sequence type (ST) complexes, ST10 and ST40 were significantly associated with disease, whilst ST31 was significantly associated with carriage. To determine whether the ability of EAEC to cause disease was associated with specific virulence phenotypes in vitro, eight strains of ST40 (disease) and ST31 (carriage) were tested for aggregative adherence (AA) to intestinal epithelium, biofilm formation and stimulation of an inflammatory response. In addition, all strains were sequenced, and bioinformatic analysis was used to identify putative virulence genes associated with disease potential.