The aEPEC group is large and comprised of heterogeneous strains and serotypes. In addition to eae, some aEPEC strains can carry virulence genes associated with other pathogenic E. coli groups, leading to speculations that aEPEC may represent different E. coli pathotypes that have acquired LEE by horizontal gene transfer (Bando et al., 2009). Other aEPEC strains are closely related to typical EPEC strains and are thought to be EPEC that have lost the EAF plasmid. Still, some aEPEC have been found to be more closely related to EHEC and may be strains that have lost the ability to produce Stx. The stx genes are phage encoded and EHEC strains can lose the phage during culturing or infection (Feng et al., 2001; Friedrich et al., 2007), resulting in strains that only have eae. In addition to pathotype differences, there is considerable genetic diversity even within specific serotypes. Analysis of aEPEC strains of serotype O26:H11 and O55:H7 in Brazil showed that although strains within the same serotype clustered in the same sequence type, their PFGE profiles were very distinct (Bando et al., 2009). An exception, however, may be the aEPEC strains of O157:H16 serotype, which have been isolated worldwide. In a previous study, we examined eae-positive and eae-negative O157:H16 strains isolated from the US and France and observed that the XbaI profiles of the French and a few of the US isolates were nearly identical, suggesting that the O157:H16 aEPEC strains are a homogeneous group (Feng et al., 2010). To determine whether these strains are part of a larger, conserved clone that exists worldwide, we examined 45 additional O157:H16 strains isolated from various sources from four other countries. The results obtained were entirely consistent with our previous findings from the 14 US and French O157:H16 strains (Table 1). In both studies, not all O157:H16 strains had eae, but the majority did carry eae and of those, except for a few that had ß-eae, most had the ε-eae allele. None of these strains had other virulence genes, including bfpA, suggesting the absence of the EAF plasmid and therefore they appeared to be aEPEC. Analysis by PFGE showed that the eae-negative O157:H16 strains had diverse XbaI profiles but the eae-positive strains shared similar profiles. For example, strains CB4720 and CB7248 that carried ß and ε-eae allele, respectively, had similar profiles, and some German strains (CB7858) had nearly identical profiles as the US strains (ARS4.2126; Fig. 1). Analysis by MLST showed that the eae-negative strains clustered apart from the eae-positive strains and exhibited greater ST diversity, with most strains being ST344 and one ST83 strain. On the other hand, all the eae-positive strains, regardless of the eae allele carried, had ST171 and formed a highly conserved group. This finding is consistent with the results obtained by a study that examined the distribution of the O157-antigen biosynthesis gene among O157 serogroup strains and observed that the eae-positive and negative O157:H16 strains clustered in separate groups (Iguchi et al., 2011). Phylogenetically, these O157:H16 aEPEC strains are distantly related to the two major EHEC lineages, the four EPEC lineages, and the three Shigella groups (Fig. 2). Therefore, they do not appear to be EPEC strains that have lost the EAF plasmid nor are they EHEC strains that have lost stx. The fact that the O157:H16 strains examined in both studies were isolated from various clinical, animal, and environmental sources from 6 different countries, suggests it is unlikely that the same strain had been broadly disseminated but rather supports the assumption that the O157:H16 aEPEC strains belong to a highly conserved clonal group that exists worldwide.
Many studies report that aEPEC are pathogenic and cause diarrhea (Trabulsi et al., 2002; Gomes et al., 2004; Robins-Browne et al., 2004). Some aEPEC have also been implicated in bloody diarrhea, but many of these also produced enterohemolysin and genetic analysis has shown that these were most likely EHEC strains that had lost stx (Vieira et al., 2001; Bielaszewska et al., 2008). Adherence studies showed that unlike typical EPEC, which exhibit localized adherence on HEp-2 cells, aEPEC strains exhibit a localized adherence-like (LAL) pattern that is also mediated by intimin (Trabulsi et al., 2002). Analysis of O157:H16 aEPEC from Argentina showed that these strains also exhibited LAL on HEp-2 cells (Bentancor et al., 2010). Still, the fact that the aEPEC strains are prevalent in both patients with and without diarrhea (Gomes et al., 2004), and that two of the Norwegian strains we examined, Trh11 and Trh16, were isolated from children without diarrhea, but the third was from a 15-month-old diarrhea patient that also had norovirus (Afset et al., 2008), the pathogenicity of these O157:H16 aEPEC strains we examined remains uncertain. One should also bear in mind that many pathogenic E. coli virulence factors reside on mobile genetic elements and can be transferred. An example is the phage-encoded stx gene, which has been found and expressed in other enteric bacteria. So, it is not entirely inconceivable that some aEPEC strains of O157:H16 serotype, which already carry the ε-eae attachment factor, may acquire the stx phage via transduction and possibly become pathogenic STEC strains.
In conclusion, the O157:H16 serotype contains both eae-positive and eae-negative strains and as a whole is phylogenetically diverse and comprised of strains that have different ST and PFGE profiles. However, the aEPEC strains within the O157:H16 serotype, regardless of the eae allele carried, belong to a highly conserved and homogeneous group of ST-171 strains that are prevalent worldwide.