**2. Enterotoxigenic** *E. coli* **(ETEC)**

#### **2.1 An overview of ETEC**

Enterotoxigenic *E.* coli (ETEC) is a major cause of travelers' diarrhea, with a high prevalence in developing countries and responsible for about 200 million cases of diarrheal illnesses and 100,000 deaths each year [18, 19]. The incidence of ETEC could be recovered from symptomatic and asymptomatic carriers and is most common in younger children with a high mortality rate in this group. In regions such as Africa, South America, and Southeast Asia the incidence of ETEC-related infection is estimated to be at least one to two episodes per year [3] further reinforcing the significance of this pathotype in that region. While ETEC is not limited to humans, it is also a common cause of edema and post-weaning diarrheal diseases in food production animals such as cattle, pigs, and sheep [20]. ETEC are transmitted through the fecal-oral route by contaminated food as well as surface and groundwater in developing countries with limited access to clean water (**Figure 2**) [3, 6].

#### **Figure 2.**

*Dissemination and transmission routes of pathogenic* Escherichia coli *pathotypes. Solid black arrows represent a direct transmission while gray arrows depict an indirect transmission of pathogenic* E. coli *pathotypes. ETEC: Enterotoxigenic* E. coli*; STEC: Shiga toxin-producing* E. coli*; EPEC: Enteropathogenic* E. coli; *EAEC: Enteroaggregative* E. coli*; EIEC: Entero-invasive* E. coli*; DAEC: Diffusely adherent* E. coli.

There is a great genetic diversity in ETEC as more than 100 different O antigens have been reported to be associated with clinical isolates. Among these, the O6 serogroup is most common, and geographically diverse among all ETEC serogroups, and has been implicated in multiple outbreaks in different countries [21]. Additionally, at least 34 H antigens are also associated with this pathotype. Among the serotypes as determined by the combination of O and H antigens, O6:H16 (heatstable (ST) or heat-labile (LT) toxin), O148:H28 (ST), O167:H5, O153:H45 (ST), O169: H41 (ST only) are frequently isolated from humans, animals, environmental matrices, and from outbreaks in developing countries [22–24]. ETEC produces one or more colonization factors that facilitate its attachment to specific receptors on the mucosal layer of the small intestine of humans and animals, and secretes enterotoxins that cause electrolyte imbalance in the intestinal lumen resulting in dehydration, metabolic acidosis, and diarrheal [3, 18]. The ST toxin is a nonimmunogenic small protein molecule, but the LT toxin is structurally homologous and exhibits a similar mechanism of action to cholera toxin produced by *Vibrio cholerae* [23, 25].

### **2.2 Virulome of ETEC**

ETEC employs an array of genetic factors that are either chromosomal or plasmid-borne that mediate colonization and adherence to the intestinal epithelium, proliferation within the host, and evasion of host defense mechanisms (**Table 1**) [26].

#### *2.2.1 Colonization and adhesion*

Colonization and adhesion are the primary and essential steps in the pathogenesis of pathogens. ETEC is not an exception as the colonization of the host intestinal epithelium by this pathotype is mediated by plasmid-borne genes that encode adhesins and one or more colonization factors (CFs) namely, pilus or pilus-related adhesins [23]. Pili are hair-like appendages on the cell surface of bacteria where they mediate the attachment of bacteria to surfaces.

They are composed of protein subunits (pilins) that are structurally polymeric and are almost exclusively plasmid-borne (**Figure 3**) [27]. ETEC CFs are designated as CS (coli surface antigens) followed by a number, except for CFA/I and PCFO71 [27]. Presently, at least 30 CFs have been reported in ETEC of human origin. It is estimated that about 50% of strains in this pathotype carry one or more CFs that are not detectable, suggesting that there could be more CFs that are yet to be discovered and characterized [18, 23]. The co-expression of one or more CFs with toxin-encoding genes has been described. For example, CFA/I + LT, CS7 with LT, CS5 + CS6 with LT + ST, CS2 + CS3 with LT + STh, among others [28, 29]. In the prototypical ETEC strain H10407, the production of CFA/I is mediated by *cfaABCE* operon that is tightly regulated by CfaD, a transcriptional regulator that triggers its expression.

Other plasmid-encoded genetic factors that have been reported to play a significant role in the pathogenesis of ETEC include a class I SPATE (serine protease autotransporters of the Enterobacteriaceae) EatA that digests EtpA secreted by ETEC, thereby promoting the adhesion of flagella to the host receptor [30, 31]. ETEC can invade the host cell with two chromosomally encoded genes *tia* and *tibA*. The former (*tia* gene) is borne on a 46-kb pathogenicity island (PAI). The expression of these genes was reported to be associated with adhesion and invasion of ETEC in host cells [27]. Likewise, a *leoA* gene encoding GTPase is reported to be associated with virulence in ETEC (**Table 1**) [32].


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

*Legend: ETEC: Enterotoxigenic* E. coli*; STEC: Shiga toxin-producing* E. coli*; EPEC: Enteropathogenic* E. coli*; EAEC: Enteroaggregative* E. coli*; EIEC: Entero-invasive* E. coli*; DAEC: Diffusely adherent* E. coli.

#### **Table 1.**

*Summary of the clinical characteristics and virulence factors of* Escherichia coli *pathotypes.*

#### **Figure 3.**

*Colonization and adherence patterns of diarrheagenic* Escherichia coli *to the host epithelium. Enterotoxigenic*  E. coli *(ETEC) uses colonization factors (CFs) to attach to host intestinal mucosa. Shiga toxin-producing*  E. coli *(STEC) and Enteropathogenic* E. coli *(EPEC) attach to the intestinal epithelial cells and efface microvilli, forming characteristic A/E lesions. EPEC also forms microcolonies using bundle-forming pili (Bfp) resulting in a localized adherence pattern. Enteroaggregative* E. coli *(EAEC) forms a biofilm matrix on the intestinal mucosa that promotes the formation of a "stacked brick" adherence pattern. Enteroinvasive* E. coli *(EIEC)/*Shigella *are intracellular pathogens that penetrate the intestinal epithelium through M cells to gain access to the submucosa. Diffusely adherent* E. coli *(DAEC) is scattered over the surfaces of intestinal cells, resulting in a diffuse adherence pattern.*

#### *2.2.2 Enterotoxin secretion*

One of the salient features that define ETEC is its ability to produce two types of enterotoxins, ST or LT [23]. STs are non-antigenic small enterotoxins that are frequent in human diseases, found in about 80% of ETEC either singly or in

combination with LT [18, 33]. STs are classified into two different classes (STa and STb) based on their structure and function. STa is soluble in methanol and protease-resistant. It is frequent in human diseases and encoded by *estA* genes, whereas STb is insoluble in methanol and sensitive to protease, and causes disease only in animals and is encoded by *estB* gene [34]. Based on host specificity, STa is further designated into two genetic variants namely STp and STh. The former (STp) is 18 amino acids in length and produced by ETEC strains of porcine, bovine, and human origin, while STh is 19 amino acids long and exclusive in ETEC strains of human origin [29]. Recently, six genetic variants of STa encoding gene (*estA*) have been reported, where *estA1*, *estA5,* and *estA6* are common in ETEC strains of porcine origin and *estA2*, *estA3*/4 and *estA7* are frequent in isolates of human origin (STh), while *estA5* gene is described to be frequent in ETEC strains causing disease both in animals and humans, especially traveler's diarrhea in adults [29, 34]. Secretion of STh and STp in the intestinal epithelium of the host requires the efflux protein TolC [35].

Unlike, STs, LTs are hexameric and strongly immunogenic that are encoded by the *eltAB* operon [29]. LTs have two subtypes: LT-I and LT-II, both of which have been reported in ETEC strains causing diarrhea in humans and in different species of post-weaned animals. LT-Is are plasmid-borne and highly similar to cholera toxin produced by *V. cholera* [3, 36]. Conversely, LT-II is chromosomal and has been hypothesized to be prophage encoded [3, 36]. LT-II is classified into LT-IIa, LT-II, and LT-IIc, with LT-IIc being the more frequent in LT-II ETEC strains [36].

LTs promote the adherence of ETEC to host intestinal epithelial cells and evade the host defense mechanisms by inhibiting the expression of antimicrobial peptides produced by the hosts, in addition to the activation of host signaling pathways [3]. Another virulence factor encoding enterotoxin in ETEC strains is enteroaggregative heat-stable toxin (EAST1). EAST1 toxin is heat-stable and 38 amino acids long encoded by *astA* gene that is commonly plasmid-borne [37]. ETEC strains producing EAST1 toxin have been recovered from humans and animals. This toxin was reported to have originated from EAEC but it is prevalent in ETEC [38, 39]. While the role of EAST1 toxin in enteric infection is not clear, there has been evidence and direct associational studies linking this toxin to diarrheal illness [38]. EAST1 toxin is functionally and structurally similar to STa, sharing 50% identity in their functional regions [38]. Overall, enterotoxins secreted by ETEC strains have a similar mechanism of causing diarrheal diseases in the host. ETEC enterotoxins increase cyclic AMP or cyclic GMP levels in the intestinal epithelium of the host. This results in excessive secretion of chloride and reduction in the adsorption of sodium chloride in the intestinal epithelium thereby resulting in electrolyte imbalance, fluid loss and dehydration [29, 40].

#### **2.3 Antibiotic resistance in ETEC**

Since the first isolation of ETEC in Kolkata about five decades ago [41], the emergence and increase in multidrug-resistant strains have been reported. A homogenous and high antibiotic susceptibility pattern was observed for ETEC strains at a time but the treatment of travelers' diarrhea with different classes of antimicrobials such as macrolides (erythromycin and azithromycin), fluoroquinolones (norfloxacin, ofloxacin, ciprofloxacin), tetracycline (doxycycline), rifamycin and sulfamethoxazole-trimethoprim that are used to treat other types of infections [22] may have also contributed to the emergence of antimicrobial resistance in this pathotype [22]. Another contributor could be the indiscriminate use of antibiotics for the treatment of diarrheas caused by viral agents that are sometimes misdiagnosed because they present similar symptoms [3].

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

There are several studies from different countries assessing the antibiotic resistance profile and distribution of resistance determinants in ETEC. In a study, the antimicrobial resistance profile among patients with recent travel history to ETEC endemic regions between 2001 and 2004 reported that up to 60% of the ETEC isolates were resistant to sulfamethoxazole-trimethoprim, tetracycline, and/or ampicillin [42]. Ciprofloxacin resistance was reported to markedly increase from 1% to 8% within 10 years (1994–2004) in patients [42] which clearly suggests a rapid emergence of resistance with time in this pathotype. In a recent study on the WGS analyses of eight strains representing the major ETEC lineages that are causing diarrheal diseases in humans around the globe, all the strains showed resistance and carried resistance determinants to at least two of the 14 antibiotics tested, with resistance to penicillin, norfloxacin and chloramphenicol being the most common. In this study, two plasmids designated (pAvM\_E1441\_17 and pAvM\_E2980\_15) carried resistance determinants to mercury (*mer* operon) and multiple antibiotics including streptomycin (*aadA1-like, strA,* and *strB*) and ampicillin (*bla*TEM-1b, *ampC*) [43].

ETEC in animals, however, may be slightly different. In a study of 112 ETEC isolates recovered from pigs in Canada over a two-decade period (1978–2000), *tetB* gene that encodes resistance to tetracycline was the most common and found in 80% of the collection [44]. Another interesting observation from this study was the increase in the determinants encoding resistance to gentamicin *(aac(3)-IV),* kanamycin *(aph(3*′*)-Ia)* and trimethoprim (*dhfrV*)*,* while others appear to be either consistent or decrease over time [44].

## **2.4 Population structure of ETEC**

ETEC strains are epidemiologically and phenotypically diverse and exhibit high genetic diversity. In addition to being polyphyletic, the distribution of ETEC lineages is not restricted by geography [45]. Several reports on the phylogenetic analyses of strains from the human origin using MLEE and MLST, and well as CF-toxin-based phylogeny showed that this pathotype might have evolved multiple times through clonal expansion and probably due to lack of common clonal lineage [46, 47]. In spite of the genomic diversity among strains in this pathotype, Turner and colleagues [48] reported ETEC to be associated with sequence type 10 (ST10). In a broader evolutionary study of a large collection of 1019 ETEC isolates from humans in 13 countries using MLST, 42 clonal groups were observed with evidence for horizontal gene exchange of plasmid-encoded CF genes between the lineages [46]. Since the advent of next-generation sequencing technologies, the study of the population structure of ETEC has improved the understanding of the genetic diversity and evolution of the pathotype [24, 49, 50].

A global collection of ETEC isolates from humans collected over a period of three decades (1980–2011) in 20 countries and representing four continents was assessed for genetic relatedness using WGS-based single nucleotide polymorphism (SNP) [49]. Indeed, ETEC strains are genetically diverse as they were reported to be distributed across different *E. coli* phylogenetic groups (A, B1, B2, D, and E) (**Figure 4**), an observation that is also in accordance with the structure defined by MLST [46, 48, 51]. An interesting finding from the study that could be attributed to the higher resolution of WGS was the identification of ETEC-specific clusters (L1-L14) that clustered geographically diverse strains that were phylogenetic related and associated with specific plasmid-encoded virulence determinants. The L1 and L2 clustered the commonly found ETEC strains expressing O6 antigen and carried similar profiles for CF and LT and ST enterotoxins, suggesting that these plasmid-encoded virulence determinants could be important to understand the evolutionary histories of these clusters [24, 49].

#### **Figure 4.**

*Single nucleotide polymorphism based Phylogenetic tree of* Escherichia coli. *Draft genomes of* E. coli *pathotypes were downloaded from NCBI and core genomes were defined and aligned against* E. coli *K12. SNPs from the core genome alignment were called using Snippy (https://github.com/tseemann/snippy). Concatenated SNPs alignment cured of recombination (https://github.com/sanger-pathogens/gubbins) was used to construct a phylogenetic tree and visualized using iTOL (http://itol.embl.de/). Different colors and shapes depict* E. coli *phylogroups and pathotypes, respectively.*

Similarly, in a local study on the phylogenomic diversity of 94 ETEC isolates from Bangladesh [24], a polyphyletic scenario and a direct correlation between lineages and virulence profiles and CFs were noted. Using comparative genomic tools, the authors identified six novel CF variants. However, the experimental validation of these CFs would be important to decipher their association with other virulence determinants as well as their interaction with the host cells.
