**3.4 Population structure of STEC**

Genetic relatedness of STEC isolates from different hosts and countries have been studied using different molecular tools that ranged from serotyping, PFGE, conventional MLST, and WGS. Evidence for transmission and dissemination of different STEC serogroups and clones have also been documented. Unlike ETEC that evolved multiple times through clonal expansion, STEC appears to have evolved by parallel evolution. Indeed, phylogenetic analyses of STEC strains have shown that isolates form multiple distinct clonal lineages, where strains with the same serotype and virulence content were nested together in the cluster [95]. STEC strains are spread across *E. coli* phylogroups and the great majority belonged to phylogroup B1 (**Figure 4**) [96]. STEC O157:H7 are further delineated into three lineages, I, I/II, and II [97, 98] that are disseminated globally. Lineage I is predominant among clinical isolates of human origin while lineage II is more prevalent in animals [99]. Intra-lineage diversity is apparent as lineages varied in the adherence and virulence determinant expression, Stx-encoding bacteriophage (Stxϕ) insertion sites, *stx2* expression, and stress resistance [97]. This intra-clonal diversity is hypothesized to have been a consequence of the global spread of a single clone and geographic expansion [97]. Interestingly, a time-dependent clonal replacement and geographical-dependent clonal expansion of lineages and sub-lineages of STEC O157:H7 have been reported [97, 100]. The STEC O157:H7 lineage I/II that was predominant in human infection in the 1980s in the UK declined

and was replaced by sub-lineage Ic in the 1990s. Also, in the past few years, this region has reported the replacement of the dominant sub-lineage (Ic) by sub-lineage IIb, a phenomenon they reported to have been a consequence of the acquisition of prophage encoding *stx2a* [97, 100].

In a recent study using WGS to understand the population dynamics of 757 STEC O157:H7 isolates from humans and animals from four continents, seven clades were reported and designated as A-G [101]. The most recent common ancestor of the isolates in this study was reported to have originated in the Netherlands in the late 19th century (1890) and then spread to other parts of the world. Although isolates were clustered on a geographical basis, there was an admixture of strains from different hosts suggesting transmission events between them [101]. The pangenome analyses of these isolates also showed that STEC O157:H7 from humans and animals differed in phage-related protein content. The molecular epidemiology of non-STEC O157:H7 is equally important especially considering their roles in outbreaks. From 1995 to 2017, a total of 674 outbreaks by non-O157 STEC strains were reported worldwide, where O26:H11 was predominant during this period [102]. Other serogroups implicated in these outbreaks include O26:H11, O45, O103:H25, O104:H4, O111:H8, O121, and O145:NM [102]. MLST-based phylogenetic analysis of 894 non-STEC isolates from patients over a period of 18 years (2001–2018) in Michigan revealed that the great majority of the isolates (95%) belonged to one clade [103]. Although the information on the evolutionary dynamics of STEC is inexhaustible, studies focusing on identifying new genetic factors associated with ecological adaptation of different lineages are elusive. Further studies should focus on this area.
