Phylogenetic Clades 6 and 8 of Enterohemorrhagic Escherichia coli O157:H7 With Particular stx Subtypes are More Frequently Found in Isolates From Hemolytic Uremic Syndrome Patients Than From Asymptomatic Carriers

Over the last 3 decades, enterohemorrhagic Escherichia coli (EHEC), one of the major categories of diarrheagenic E coli, has been frequently reported to cause severe diseases such as hemorrhagic colitis, hemolytic uremic syndrome (HUS), and encephalopathy [1, 2]. The serogroup O157 (serotype O157:H7/H−) was identified as the major EHEC responsible for sporadic cases and outbreaks in several countries [3–7]. In Japan, more than 3000 annual cases of EHEC infections (including asymptomatic carriers [ACs]) were reported during 2004–2012 [8]. Among the isolates collected during 2008–2012, serogroup O157 predominated (62.2%) followed by O26 (22.5%), O111 (3.25%), and O103 (3%) [8–12]. During this time period, serogroup O157 was found in more than 85% of total HUS cases [8–12]. Shiga toxin (Stx) is the most important and critical virulence factor of EHEC that is known to cause these severe diseases. There are 2 antigenically distinct types of Stx, Stx1 and Stx2. Several studies have suggested that, compared with strains harboring stx1 gene, strains with stx2 are more often associated with HUS [13–15]. These 2 genes have been further divided into several subtypes: subtypes stx1a, stx1c, and stx1d for the stx1 gene, and subtypes stx2a–g for the stx2 gene [16]. The subtypes stx2a and/or stx2c were more often found to be associated with HUS than the other stx types [17, 18]. Genomic diversity of serotype O157:H7 has been extensively studied by using several molecular subtyping methods including pulsed-field gel electrophoresis (PFGE) [19–21], multilocus variable tandem repeat analysis (MLVA) [22–24], octamer-based genome sequencing [25], lineage-specific polymorphism assay-6 (LSPA-6) [26], and single-nucleotide polymorphism (SNP) typing [27]. Although PFGE and MLVA have revealed considerable genetic diversity of O157:H7 strains and have been used for epidemiological analysis, they are not applicable to phylogenetic or population genetic analyses. Octamer-based genome sequencing analysis initially identified 2 distinct lineages of EHEC O157:H7 strains, designated as lineage I and lineage II [25]. Lineage-specific polymorphism assay analysis, which can subtype strains by the amplicon sizes of 6 polymerase chain reaction (PCR) products, showed that most of the strains belonging to lineage I were LSPA type 111111, whereas the strains belonging to lineage II were consisted of LSPA types 222222, 222211, 222212, and 222221 [26]. A microarray-based comparative genome hybridization analysis further identified another lineage, termed lineage I/II, which contains LSPA type 211111 [28]. An SNP-based subtyping method was also used to classify O157:H7 strains into 9 genetic clades (clades 1–9) [27]. The lineage I described above corresponded to clades 1, 2, 3, and 4 [29, 30]; lineage I/II corresponded to clades 6, 7 [30], and 8 [29–31]; and lineage II (LSPA type 222222 and 212111) corresponded to clade 7 and 9 [30]. A detailed epidemiological analysis demonstrated that patients with HUS were more likely to be infected with strains belonging to clade 8 than with strains belonging to other clades [27]. However, only 11 isolates from HUS patients were available for characterization in this study, and thus the association between clade membership and HUS requires confirmation, particularly for strains isolated from different geographic locations. Follow-up studies demonstrated that some of the clade 8 isolates showed elevated expression levels of stx2 and locus of enterocyte effacement genes (LEE, which is responsible for the intimate adhesion of EHEC to the epithelial cells), as well as increased adhesion of these isolates to cultured epithelial cells relative to strains of clades 1–3. However, not all clade 8 isolates screened exhibited the same phenotype, suggesting that genetic variability exists within these clade 8 strains [32–34]. In the present study, we have determined the clade distribution and analyzed the stx subtypes of 656 EHEC O157:H7 isolates, which were collected in Japan during the years 1999–2011 for evaluating their public health significance.