**4.2 Virulome of EPEC**

Since EPEC does not produce Shiga toxin or other enterotoxins, the major feature the pathogenic strains in this pathotype employ is their ability to attach tightly to the host mucosal membrane, destroy microvilli, and induce the formation of lesions (**Table 1**) [115]. In addition, EPEC carries other genes encoding proteins that have been linked to colonization and adherence to host cells [116, 117].

#### *4.2.1 Colonization and adherence*

The defining characteristic of EPEC is the carriage of LEE locus that is essential for inducing A/E lesions, causing localized lesions by attaching closely to the surface of the intestinal epithelial cells. Like some STEC strains, all the EPEC strains carry *eae* and *tir* genes as well as T3SS that is able to inject a large number of effector proteins into the host cell [3, 111]. Studies have shown that the presence of LEE locus in EPEC strains is enough to cause infection in the host even in aEPEC-related infection scenario [118].

The ~80 kb pEAF plasmid that defines tEPEC carries *per* and *bfp* operons (**Table 1**) [119, 120]. The *per* operon (*perABC*) is plasmid-borne and contains *perA* that encodes a regulator that activates the transcription of *bfp* operon that encodes the type IV pili called bundle-forming pilus (BFP) (**Figure 3**) [119, 120]. The *bfpA* gene which encodes the bundling of the major structure of BFP and 13 other genes are carried on the pEAF plasmid [116]. The carriage of this plasmid has been described to be essential in the localized adherence of EPEC to intestinal epithelium in the host [118]. tEPEC strains carry *lifA* gene that encodes lymphocyte inhibitory factor, a large surface protein that is described to promote the intestinal colonization of mice by *Citrobacter rodentium* [3]. Although pEAF plasmid is absent in aEPEC strains, they often carry virulence determinants typical of STEC strains most likely because they share a common ancestor [113]. Afset et al. [117] identified 12 genes that were statistically associated with aEPEC-related diarrhea in children. Of note are *efa1*/*lifA* genes that are located on OI-122, as well as *lpfA* gene previously reported in STEC [121]. Likewise, *astA* gene that encodes EAST1, an ST-like toxin that is present in ETEC is also carried by EPEC, being more prevalent in aEPEC than in tEPEC strains implicated in diarrhea [122, 123].

#### **4.3 Antibiotics resistance in EPEC**

Although EPEC-related infection could resolve itself or simply by oral rehydration therapy that replenishes the lost fluid, the persistence of this infection may necessitate the use of antibiotics. In this case, especially in adults, the recommended antimicrobial is trimethoprim/sulfamethoxazole, norfloxacin, or ciprofloxacin [124]. However, studies on antibiotic resistance of EPEC strains from different sources and countries have shown high resistance of this pathotype to ampicillin, cefpodoxime, nalidixic acid, trimethoprim, and tetracycline [125, 126]. While resistance to the great majority of these antibiotics is reported to be frequent in tEPEC, trimethoprim resistance is more common in aEPEC strains [127].

In a global study of 185 aEPEC isolates collected from healthy and diarrheal children living in seven sites in sub-Saharan Africa and South Asia, at least 55% of the isolates showed phenotypic resistance to ampicillin, trimethoprim, trimethoprim/ sulphamethoxazole, and tetracycline, while streptomycin resistance was reported in 43% of the isolates. Shockingly, more than 50% of the isolates were resistant to three or more of the tested antibiotics [128]. The study also reported point mutations in genes that are associated with resistance to quinolone (*gyrA*, *parC*) and nitrofurantoin (*nfsA*) in addition to over forty different antibiotics resistance genes reported. Equally, more than 50% of the isolates carried at least four resistance determinants that include *bla*TEM (ampicillin), *strA* and *strB* (streptomycin), *sul2* (sulphonamides), and *dfr genes* (trimethoprim/sulfamethoxazole)*.* These resistance determinants were found singly or co-localized on plasmids (pCERC1, pCERC2) or in transposons (Tn*6029*).

#### **4.4 Population structure of EPEC**

The acquisition of LEE and pEAF has been the defining evolutionary phenomenon for EPEC pathotypes [3, 129]. While tEPEC that carries pEAF plasmid is believed to be less diverse, aEPEC is greatly heterogeneous. The loss of pEAF plasmid in aEPEC and its close relatedness with LEE-positive STEC in serotypes, genetic characteristics, virulence properties, and reservoirs make serotype-based lineage definition unreliable [111, 130]. Based on the conventional MLEE and MLST, EPEC strains belong to six clonal lineages (EPEC1–EPEC6) that were represented among the EPEC strains worldwide [129, 131]. The whole genome-based phylogeny reported nine more EPEC lineages designated as EPEC7-EPEC15 [104, 132]. These phylogenomic EPEC lineages belonged to four *E. coli* phylogroups (A, E, B1, and B2) (**Figure 4**), where the great majority were found in B1 and B2 [104, 133], suggesting a clear genetic heterogeneity within this pathotype.

The close relatedness of aEPEC to other pathotypes could play a significant role in the diversity within this pathotype. This EPEC subtype can also include tEPEC that have lost the pEAF plasmid and LEE-positive STEC strains that have lost the Stx encoding bacteriophage during transmission events between hosts, withinhost evolution, interaction with the host microbiota, or selective pressure in the environment [104, 130]. This could be a possible explanation why some aEPEC strains would cluster with other pathotypes. Indeed, phylogenomic analyses of 106 Brazilian and 221 global aEPEC genomes showed that isolates were clustered into the previously reported phylogroups for this pathotype and phylogroup D. Additionally, 42.5% of the isolates belonged to the four previously defined EPEC lineages [129, 131], while the remaining isolates were found in EPEC11-EPEC14 phylogenomic lineages, suggesting a gradual and continuous clonal expansion of this pathotype [132]. Of note, dissemination of the phylogenomic lineages of EPEC pathotype is not restricted by geography. Conversely, in a multicentre study

*The Biology and the Evolutionary Dynamics of Diarrheagenic* Escherichia coli *Pathotypes DOI: http://dx.doi.org/10.5772/intechopen.101567*

involving seven sites in developing countries, EPEC isolates from sub-Saharan countries (The Gambia and Kenya) were clustered into two EPEC lineages (EPEC5 and EPEC10) in phylogroup A [104]. Overall, EPEC represents a pathotype that is still undergoing clonal expansion due to the occurrence of novel phylogenomic lineages with distinct accessory gene content and their pathogenic potential.
