The term enteropathogenic Escherichia coli (EPEC) was first used in 1955 to describe a number of serogroup-defined E. coli strains associated with infantile diarrhea. EPEC are now defined as those that produce a characteristic intestinal histopathology known as attaching and effacing (A/E) and do not produce Shiga toxins. EPEC carry the locus for enterocyte effacement (LEE) pathogenicity island, which contains the eae gene that encodes an outer membrane protein called intimin. Typical EPEC (tEPEC) carry a virulence plasmid known as the pEAF (EPEC adhesion factor plasmid) which encodes the bundle-forming pilus (BFP) that mediate localized adherence to epithelial cells, whereas atypical EPEC (aEPEC) do not possess this plasmid. Typical EPEC strains have been associated with severe outbreaks of infant diarrhea in developing countries. Atypical EPEC strains have been linked to diarrhea outbreaks at all ages worldwide. Diarrhea due to aEPEC in children is not as severe as that caused by tEPEC.
- Escherichia coli
- infantile diarrhea
1. History and definition of EPEC
Escherichia coli were first recognized as diarrheal pathogens in 1898, when Lesage demonstrated that serum from diarrhea patients agglutinated strains of E. coli isolated from other patients in the same outbreak but not those of control . In 1945, Bray discovered that E. coli strains of certain serogroups were the predominant cause of summer diarrhea in infants in the United Kingdom . In 1947, Kauffman published a serotyping scheme based on somatic (O), flagellar (H), and capsular (K) antigens, providing a reliable method of typing diarrheagenic E. coli .
The term enteropathogenic Escherichia coli (EPEC) was introduced in 1955 to describe strains of E. coli implicated epidemiologically with infant diarrhea in the 1940s and 1950s . This definition changed as additional serotypes were associated with infantile diarrhea. EPEC were recognized as important causes of infant diarrhea in the 1950s and 1960s in the developed world and subsequently have been shown to be common agents of gastroenteritis in the developing world. The confirmation that EPEC strains were pathogenic came from human volunteer studies carried out by Levine et al. .
The definition of EPEC has changed as various mechanisms of pathogenesis have been discovered. During the late 1960s and early 1970s, two other diarrheagenic E. coli strains were discovered, strains producing the heat-stable enterotoxin (ST) and the heat-labile enterotoxin (LT) were designated enterotoxigenic E. coli (ETEC), and strains demonstrating Shigella-like invasiveness were designated enteroinvasive E. coli (EIEC). At this time, the original definition of EPEC by Neter  has undergone modification considerably. EPEC were then defined as “diarrheagenic E. coli belonging to serogroups epidemiologically incriminated as pathogens but whose pathogenic mechanisms have not been proven to be related either heat-labile (LT) or heat-stable enterotoxins (ST) or to Shigella-like invasiveness” .
In 1979, the first phenotype characteristic other than serotyping associated with EPEC was the observation of Cravioto et al.  that 80% of EPEC strains as defined by serotype could adhere to HEp-2 cells in cell culture, while most non-EPEC strains could not. Later, the adherence pattern of EPEC was described as “localized adherence” (LA), based on the presence of clusters or microcolonies on the surface of HEp-2 cells . Baldini et al.  subsequently showed that the ability of EPEC E2348/69 strain (O127:H6) to adhere in a localized adherence pattern was associated with the presence of a 60-MDa plasmid. EPEC strains representing a variety of serotypes were found to possess highly conserved high molecular weight plasmids associated with localized adherence, the so-called EPEC adherence factor (EAF) plasmids [10, 11]. Subsequently, differences in adherence patterns were discerned by Scaletsky et al.  and Nataro et al. , giving rise to two other categories of diarrheagenic E. coli and diffusely adherent E. coli and enteroaggregative E. coli. Also in the 1980s, a newly recognized clinical syndrome caused by E. coli led to the discovery that some diarrheagenic E. coli produce a potent cytotoxin known as Shiga toxin (Stx).
In 1983, Moon et al.  published electron micrographs of pigs and rabbits infected with EPEC and coined the term “attaching and effacing” (A/E) to describe the loss of microvilli, intimate attachment of the bacteria to the host, and formation of pedestals at the sits of bacteria attachment. In 1987, a number of studies clarified the relationship between LA phenotype and A/E lesion, which confirmed earlier reports that LA is associated with the EAF plasmid, and demonstrated for the first time that A/E is encoded on the chromosome .
Originally defined by serotype, EPEC are now defined as those having the ability to cause diarrhea, the ability to produce A/E histopathology on the intestinal epithelium, and the inability to produce Shiga toxins based on pathogenic characteristics . Improvements in techniques allowing a better understanding of the genome and virulence mechanisms among EPEC strains over the years have led to the classification into “typical” and “atypical” subtypes based on the presence or absence of the pEAF plasmid .
2. Atypical versus typical EPEC
Most of the typical EPEC strains belong to the traditional EPEC serogroups O55, O86, O111, O114, O119, O127, and O142, and the most common flagellar antigens are H6 and H2 [16, 17]. A less common EPEC type is H34, and a number of typical EPEC strains are nonmotile in conventional testes and classified as H-. Typical EPEC strains belonging to nonclassic serotypes have also been reported [18, 19]. Currently, more than 180 different O serogroups and more than 60 H antigens are recognized. Atypical EPEC belong to a large diversity of classical and nonclassical serotypes [18, 20]. Based on multilocus enzyme electrophoresis analysis of allelic differences among housekeeping genes, typical EPEC strains have been subtyped into two major lineages, previously designated EPEC1 and EPEC2 [16, 17]. EPEC1 includes serotypes O55:H6 and O119:H6, whereas EPEC2 consists of serotypes O111:H2 and O114:H2. Recently, EPEC strains have been demonstrated to cluster in three main lineages, designated EPEC1, EPEC2, and EPEC4, which probably acquired the locus of enterocyte effacement region (LEE) and pEAF independently . Interestingly, it has been found that 35% of the atypical EPEC strains also belong to the typical EPEC lineages . Thus, it has been hypothesized that at least some atypical EPEC may have originated from typical EPEC strains that lost pEAF in the host or in the environment .
3. Epidemiology of EPEC
The prevalence of EPEC infection varies between epidemiological studies based on differences in study populations, age, distributions, and methods used for detection and diagnosis . Also, geographic region and socioeconomic class may contribute to the epidemiology of EPEC-induced diarrheal disease . Adults and older children with typical EPEC infections are rarely reported; this has been attributed to the loss of specific receptors with age or development of immunity .
For the two last decades, studies conducted worldwide have shown the association of typical EPEC serotypes with diarrhea in children <1 year of age, mainly in poor children of urban centers . This association was particularly strong in infants less than 6 months of age. Between 1977 and 1982, epidemiologic studies in Brazil, Chile, Mexico, and South Africa have shown that 30–40% of infantile diarrhea was caused by typical EPEC serotypes . However, recent studies in these countries have not identified a significant association between typical EPEC and infantile diarrhea. At this time, a change in the epidemiology of EPEC occurred in both developing and developed countries. The proportion of atypical EPEC strains has increased and outnumbered typical EPEC strains, and atypical EPEC strains have also been associated with childhood diarrhea in both developing and developed countries [19, 22, 25]. In Brazil, 92% of EPEC isolates collected from children between 2001 and 2002 were atypical , compared to 38% in a 1998–1999 study [27, 28, 29]. However, other studies still report typical being more prevalent than atypical EPEC as a cause of diarrhea . Recently, a prospective, population-based case-control study involving seven sites in Africa and Asia showed that typical EPEC was significantly associated with moderate to severe diarrhea in children under 2 years of age in Kenya, whereas atypical EPEC was not associated with this type of diarrhea .
3.2 Transmission and reservoirs
Typical EPEC transmission follows a fecal-oral process through contaminated surfaces, weaning fluids, and human carriers . EPEC outbreaks among adults, although rare, seem to occur through ingestion of contaminated food and water; however, no specific environmental reservoir has been identified . EPEC outbreaks have been reported to show a seasonal distribution with peaks during the warm months . Humans are the only known reservoir for typical EPEC, with symptomatic and asymptomatic children and asymptomatic adults being the most likely source .
In contrast to the tEPEC, many aEPEC strains have been found in diarrheic as well as in healthy animals and from the environment. Interestingly, animal aEPEC serogroups associated with human diarrhea have been identified (e.g., O26, O103, O119, O128, O142, and O157); however, so far a direct transmission from animals to humans has not been confirmed. In addition, foods including raw meats, pasteurized milk, meat samples, vegetables, and water have been also implicated as vehicles of aEPEC to human infections (reviewed in [34, 35]).
4. EPEC virulence factors and genetics
4.1 Localized adherence
Typical EPEC strains adhere to HeLa, HEp-2, and other cell lines and to organ cultures in vitro in a distinctive pattern of three-dimensional microcolonies so-called localized adherence (LA) pattern within 3 h of infection (Figure 1A) [8, 24]. A similar adherence pattern has been seen in tissue biopsies of EPEC-infected humans . The LA phenotype is mediated by a type IV fimbriae bundle-forming pilus (BFP) associated with the EAF plasmid, which mediates bacterium-to-bacterium adherence, resulting in formation of compact microcolonies .
Atypical EPEC strains may display a variant LA pattern designated LA-like (LAL) pattern, which is characterized by the presence of loose compact microcolonies or clusters of bacteria in few cells observed in tests using prolonged incubation periods (6 h) (Figure 1B) [39, 40]. Interestingly, the LAL pattern is determined in prolonged assays (6 h) of bacteria-cell interaction . LAL is the most common pattern seen among EPEC strains; however, some strains display alternate adherence phenotypes such as diffuse adherence (DA) and aggregative adherence (AA) .
4.2 Attaching and effacing (A/E) lesion
The hallmark of EPEC infection is the ability of the organism to attach intimately to epithelial cells and efface microvilli (Figure 1C). This effect was first described by Staley et al. , although the term attaching and effacing (A/E) was coined by Moon et al. . The A/E characteristic can be observed by electron microscopic examination of cultured epithelial cells exposed to EPEC or of intestinal biopsies from infants or animals infected with EPEC .
Intracellular typical EPEC have been observed both in tissue culture and in small intestinal biopsies from an EPEC-infected infant (Figure 1D) . Fletcher et al.  and Scaletsky et al.  have reported that EPEC O111:NM strains contain plasmid sequences that confer invasiveness upon E. coli K12 strains. However, despite their invasive potential in vitro, most EPEC are considered as noninvasive pathogens .
4.4 Biofilm formation
Typical EPEC have the ability to form biofilms on abiotic surfaces under static conditions, or on a flow through continuous culture system, and a model of EPEC biofilm formation has been proposed . Biofilm formation requires adhesive structures as type 1 pili, antigen 43, BFP, and the EspA filament (see below) as participants in bacterial aggregation during biofilm formation on abiotic surfaces . Atypical EPEC strains have also been shown to adhere to abiotic surfaces (polystyrene and glass) [46, 47]. The non-fimbrial adhesin curli and the T1P were shown to mediate binding to these surfaces in some atypical EPEC at different temperatures [48, 49].
4.5 The EAF plasmid
Typical EPEC strains possess a large virulence plasmid called the EPEC adherence factor (EAF) plasmid , which varies in sequence among different EPEC strains but is somewhat conserved . The EAF plasmid pMAR2 is found among strains of the EPEC1 lineage, whereas pB171 is more common among EPEC2 strains [50, 51]. Two sets of genes located on the EAF plasmid are important for pathogenicity: the bfp gene cluster encoding BFP  and the per locus encoding a transcriptional activator called plasmid-encoded regulator (Per) . Both BFP and PerA have been shown to contribute to virulence in human volunteers . Between pMAR2 and pB171, the bfp and per loci share 99% sequence similarity . Studies of comparison genomics of the EAF plasmids from varied EPEC phylogenomic lineages demonstrated significant plasmid diversity among isolates within the same phylogenomic lineage .
4.6 Bundle-forming pilus (BFP)
Typical EPEC strains produce a type IV pilus, the bundle-forming pili (BFP), which interconnects bacteria within microcolonies, promoting their stabilization and producing the LA phenotype . The BFP is encoded by an operon of 14 genes contained on the EAF plasmid, with bfpA encoding the major structural subunit (bundlin) . These 14 bfp genes are highly conserved among EPEC1 and EPEC2 strains. Some O128:H2 and O119:H2 EPEC strains that contain part of the bfpA gene have the rest of the bfp gene cluster deleted and replaced with an IS66 element [55, 56].
4.7 The locus of enterocyte effacement (LEE) and the type III secretion system (TTSS)
The locus of enterocyte effacement (LEE) is a 35.6-kb pathogenicity island of EPEC containing genes necessary for the formation of the A/E lesion . The EPEC LEE contains at least 41 open reading frames that are organized into five operons (LEE1 to LEE5) [58, 59, 60]. LEE1, LEE2, and LEE3 encode a type III protein secretion system (T3SS) and Ler (LEE-encoded regulator) regulators, such as GrlA (global regulator of LEE activator, formerly called Orf11) and GrlR (Grl repressor, formerly called Orf10) . LEE4 encodes the EPEC-secreted proteins EspA, EspB, and EspD via the type III system. LEE5 encodes intimin and its translocated receptor, Tir . Besides Tir, the EPEC genome contains other six LEE-encoded effector proteins translocated into the cell (Map, EspF, EspG, EspZ, EspH, and EspB), which interfere with different aspects of the cell physiology ([58, 59]; reviewed in ) .
In addition to the LEE effectors, various non-LEE (Nle)-encoded effector genes (cif, espI/nleA, nleB, nleC, nleD, nleE, and nleH) [59, 63] were described, which are located outside the LEE region of EPEC, in at least six chromosomal PAIs, or in prophage elements (reviewed in  and ). Although they are not required for AE lesion formation, it is understood that they contribute to increased bacterial virulence .
The LEE region of some atypical EPEC strains display a genetic organization similar to that found in the typical EPEC prototype E2348/69 strain . Although the T3SS-encoding genes are considerably conserved [66, 67], the effector protein-encoding genes display important differences, and remarkable differences can be detected at the 5′ and 3′ flanking regions of atypical EPEC, suggesting the occurrence of different evolution events .
The expression of LEE genes is controlled by Per, which is encoded on the EAF plasmid present in typical EPEC strains. Per activates Ler, which in turn activates the LEE2, LEE3, LEE4, and LEE5 operons, and the genes espF, espG, and map [58, 59]. The Ler protein is a histone-like nucleoid-structuring protein (H-NS) that responds to an environmental stimulus (temperature). Ler also controls genes located outside the LEE, such as espC and nleA . Additional regulatory system has been shown to control expression of the LEE . The AI-2 (autoinducer-2) quorum-sensing system regulates LEE1 operon, which increases expression of the LEE3 and LEE4 operons via the ler gene product. Two novel LEE-encoded regulators that have roles in ler expression were reported, GrlA (global regulator of LEE activator) and GrlR (Grl repressor) . GrlR and GrlA are positive and negative regulators, respectively, required for the expression of several LEE-encoded genes . Other LEE regulators include the integration host factor (IHF); Bip, a tyrosine-phosphorylated GTPase; Fis (factor for inversion stimulation); and GadX, which is a member of the AraC transcription factor family .
4.8 Intimin and Tir
Intimin is a 94-KDa outer membrane adhesin encoded by the eae gene and required for intimate adherence of EPEC to epithelial cells at the sites of A/E lesions . N-Terminal portions are highly conserved, whereas C-terminus portions are highly variable . C-Terminal intimin differences have been used as a basis for classification into several distinct subtypes (represented by the Greek letters to α (alpha) through ζ (zeta) [71, 72]); human EPEC1 strains express subtype α, while EPEC2 strains express subtype β. The N-terminus portion binds intimin in the bacterial outer membrane, whereas the C-terminus portion binds intimin to Tir. The binding of intimin to Tir leads to intimate adherence of the bacterium to the epithelium and pedestal formation beneath adherent bacteria. In addition, Tir inhibits NF-қB activity by targeting tumor necrosis factor alpha (TNF-α) receptor-associated factors .
4.9 Other potential adhesins
In addition to BFP, two other EPEC surface structures, rodlike fimbriae and fibrillae, have been characterized and have been suggested to be involved in the interaction of EPEC with host cells [74, 75]. Additionally, EPEC strains encode a large surface protein, lymphocyte inhibitory factor (LifA), that contributes to epithelial cell adherence in vitro [76, 77] and is required for intestinal colonization of mice by the related A/E pathogen C. rodentium . The lifA gene is more commonly found among typical rather than atypical EPEC strains ; however, atypical EPEC strains harboring lifA have a significant association with diarrhea in children under 5 years of age . A novel gene cluster, designated the locus for diffuse adherence (lda), was found in an atypical EPEC O26 strain that is responsible for mediating DA adherence; its expression is induced by bile salts . The E. coli common pilus (ECP) has also been shown to act as an accessory adherence factor in EPEC, playing a role during cell adherence and/or in bacterium-bacterium interactions .
Flagella has been suggested to be involved in EPEC adherence to epithelial cells . EPEC mutants with transposon insertion in the flagellar gene fliC were deficient in localized adherence, and anti-flagellum antibodies were effective in blocking the adherence of several EPEC serotypes . However, a subsequent study has not confirmed a role of flagella in EPEC adherence .
EspC is a high-molecular-weight secreted protein of EPEC that induces cytopathic effects on epithelial cells, including cytoskeletal damage [24, 85]. EspC is a member of the serine protease autotransporters of the Enterobacteriaceae (SPATE) family of autotransporter proteins that encodes its own transport mechanism. Moreover, espC has been shown to interact with and degrade hemoglobin  and to hydrolyze other proteins such as pepsin, factor V, and spectrin . In addition, EspC confers enhanced lysozyme resistance to EPEC  and serves as a substratum for adherence and biofilm formation as well as to protect bacteria from antimicrobial compounds . EspC is encoded in a 15-kb chromosomal island specific to EPEC1 strains .
6. Other toxins
Scott and Kaper  reported a cytolethal distending toxin (CDT) in an EPEC strain that induces chromatin disruption, which leads to G2/M-phase growth arrest of the target cell and ultimately cell death . A study has suggested that most EPEC strains from diarrhea harbor the CDT gene . Another toxin is the enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) also present in EPEC strains . The use of an EAST1 DNA probe suggests that this toxin is expressed by a number of clinical EPEC isolates [18, 93]. The role of CDT and EAST1 in EPEC pathogenesis remains to be elucidated.
7. Model of EPEC pathogenesis
A three-stage model of EPEC pathogenesis was first described in the early 1990s , Clarke et al. , including localized adherence to the host cell, signal transduction, and intimate attachment with pedestal formation (Figure 2). In the first stage, an attachment of typical EPEC to the surface of the host intestinal epithelium is mediated by the bundle-forming pili (BFP). The filament EspA also promotes attachment, albeit in a less efficient manner, and could mediate adherence of atypical EPEC strains. In the second stage, Tir and effector proteins (EspB, EspD, EspF, EspG, and Map), translocated into the host cells via type III system apparatus, activate cell-signaling pathways, causing alterations in the host cell cytoskeleton and resulting in actin accumulation and loss of microvilli . In the third stage, bacteria intimately adhered to host cell by intimin-Tir interactions amplifies the accumulation of filaments of actin and other cytoskeletal proteins that result in pedestal-like structures [62, 96, 97]. Finally, the translocated effectors disrupt host cell processes, causing loss of tight-junction integrity and mitochondrial function and leading to both electrolyte loss and eventual cell death.
Traditionally, the identification of EPEC was based on the O:H serotyping, but serotype designation is no longer precise. The identification of EPEC was based on the characteristic of EPEC’s attachment to epithelial cells and may include phenotypic or genotypic tests. The HeLa adherence assay distinguishes EPEC from other E. coli by their ability to adhere in a localized pattern (LA) on the surface of cells . The fluorescent actin-staining (FAS) assay, originally described by Knutton et al. , leads to the identification of the A/E lesion, by detecting actin condensation under EPEC adhesion pedestals. DNA probes and PCR targeting genes responsible for these characteristics were developed. A 1-kb EAF fragment probe was initially developed as a diagnostic DNA probe (the EAF probe) and subsequently refined as an oligonucleotide probe as well as PCR primers [10, 11, 99]. The identification of bfpA, the structural gene encoding BFP, led to the development of more specific and sensitive probe or PCR tests to detect typical EPEC strains [74, 75, 100]. However, some PCR primers may fail to identify all typical EPEC strains since multiple alleles of bfpA have been identified . The eae sequences by DNA probes and PCR primers have been used to detect the presence of LEE encoding A/E lesion .
9. Clinical features of EPEC infection
The most common symptoms reported in EPEC infection are watery diarrhea, dehydration, vomiting, food intolerance, and low-grade fever [24, 97]. In addition, EPEC infection may lead to severe malabsorption of nutrients resulting in nutritional aggravation and persistence of diarrhea . Edema, neutrophil infiltrate, and reduced enzymatic activity in the intestinal mucosa have been also found in EPEC infection . EPEC diarrhea often lasts 1–2 weeks but can become persistent, lasting more than 2 weeks, and may result in severe infection [24, 25, 32, 102]. In a recent case-control study, EPEC infection was associated with a 2.8-fold elevated risk of death among infants in Kenya [24, 31, 97].
Treatment of EPEC diarrhea includes oral rehydration therapy to prevent dehydration by correcting fluid and electrolyte losses. Oral rehydration may be sufficient for cases of self-limited acute diarrhea, but persistent cases of diarrhea may include parenteral rehydration, and more severe cases may require total parental nutrition and use of antimicrobials . Multidrug resistance has been reported in EPEC strains from diverse parts of the world [27, 28, 29, 44, 104, 105]. Alternative therapies, employing the use of bismuth subsalicylate, specific bovine anti-EPEC milk immunoglobulins, and also zinc, have been proven useful for treatment and prevention of EPEC diarrhea .
There are no currently available vaccines to prevent EPEC infection. However, a recent study has used bacterial ghosts devoid of cytoplasmic contents but expressing all EPEC surface components in vaccination challenge experiments with mice, and the results showed 84–90% protection in control mice . Interestingly, protective effect of breast-feeding was shown to provide excellent protection against EPEC infection. Several investigators have shown that breast milk provides protection against EPEC O antigens and outer membrane proteins [108, 109]. Furthermore, IgA antibodies against BFP, intimin, EspA, and EspB proteins were identified in maternal colostrum and serum samples [110, 111, 112, 113, 114, 115, 116, 117, 118].
Much progress has been made in the last 20 years toward understanding the pathogenesis of EPEC. It has been shown that typical EPEC are still important pathogens associated with severe outbreaks of infant diarrhea, and atypical EPEC are emerging pathogens associated with sporadic outbreaks at all ages worldwide.