**6.4 Population structure of EIEC**

EIEC pathotype is diverse and highly specialized due to the carriage of large virulence plasmid, a genetic element that is shared with *Shigella*. The pINV plasmid that is the hallmark of EIEC lacks the ability for autonomous horizontal transfer. Although there is no consensus on the evolution of pINV in Shigella/

EIEC, it has been hypothesized that this genetic element was probably acquired in an ancestral *E. coli* prior to the diversification of the two bacterial species and the emergence of different Shigella/EIEC lineages. Conversely, Shigella/EIEC strains could have evolved from different *E. coli* strains that had acquired the pINV independently from other pINV-carrying Shigella/EIEC or from an unknown donor [172].

A phylogenetic analysis of 32 EIEC strains based on four housekeeping genes (*trpA, trpB, pabB,* and *putP*) revealed four clusters (clusters 4–7) where most of the O antigens were found in a single cluster [173]. EIEC cluster 4 comprised strains with serotype O28, O29, O124, O136, and O164. Serotypes O124, O135, O152, and O164 belonged to cluster 5, and O143 and O167 to cluster 6 while cluster 7 had only O144 [173]. This is similar to a SNP-based phylogeny described in another study [174]. WGS alignment-based phylogeny of 20 EIEC isolates revealed that the great majority of the strains belonged to three distinct EIEC lineages (lineage 1–3) that belonged to three different *E. coli* phylogroups (A, B1, and E) (**Figure 4**). All the EIEC strains in lineage 1 (phylogroup E) were all serotype O143:H26, whereas, in other lineages the serotypes were diverse. EIEC lineage 3 (phylogroup B1) was reported to be globally disseminated as the strains from six different countries were clustered together in this lineage [175].

Insertion sequences were recently reported to contribute to the population structure of EIEC. A recent study [176] on the evolutionary dynamics of *Shigella* and EIEC lineages identified the genetic factors driving strain-to-strain variation within each population and contributing to functional gene loss within and between species. In the study, the author found that all *Shigella* and EIEC lineages had higher IS copy numbers relative to other *E. coli* pathotypes indicative of IS expansion in these lineages. The authors also found that *Shigella* and EIEC lineages carried the same five ISs (IS*1*, IS*2*, IS*4*, IS*911,* and IS*600*) indicative of a parallel expansion of these IS types, although at a high degree in *Shigella.* The data also suggests that *Shigella* and EIEC lineages underwent an expansion of their native IS*1* alleles and that pINV is a potential source for the introduction of other ISs (IS*2*, IS*4*, IS*600,* and IS*911*) that are rare in *E. coli* into *Shigella* and EIEC lineages [176].

In a comparative pangenome analysis of EIEC with *Shigella* and other *E. coli* pathotypes, seven gene clusters were identified to be enriched in EIEC strains but absent in all other *E. coli* pathotypes and *Shigella* strains. These included genes that encode a putative pyruvate kinase, a periplasmic protein, and some uncharacterized proteins. However, when *Shigella* isolates were excluded, the authors identified 96 gene clusters that were present in more than half of the EIEC strains. A total of 87 gene clusters were reported when EIEC and *Shigella* genomes combined were compared to other *E. coli* pathotypes. Among these were plasmid-associated genes encoding a hypothetical toxin-antitoxin system and putative proteins hypothesized to be involved in conjugal transfer [175].

The EIEC lineages were reported to have distinct phenotypic and genotypic features. Lan et al. [173] reported that EIEC strains belonging to cluster 4 lack mucate fermentation ability, whereas strains in cluster 6 were able to utilize acetate and ferment mucate [173]. Likewise, Hazen et al. [175] identified up to 155 gene clusters that were exclusive in EIEC strains belonging to one phylogroup. Additionally, 12–155 gene clusters were also reported to be lineage-specific in the EIEC pathotype [175]. Protein-encoding genes that are linked to transcriptional regulation, metabolism, and transport, and a colicin were exclusive in EIEC lineage 1, whereas genes that encode membrane protein, the aerobactin siderophore receptor, and hypothetical proteins were exclusive for EIEC lineage 2. Genes encoding several transcriptional regulators and hypothetical proteins were limited to EIEC lineage 3 [175].
