EAEC: Virulence Factors and Clinical Manifestations of Infection

This document provides a comprehensive overview of Enteroaggregative Escherichia coli (EAEC), a significant food-borne pathogen responsible for causing acute and persistent diarrhea, particularly in children and immunocompromised individuals. The report delves into the definition of EAEC, characterized by its aggregative adherence pattern on epithelial cells, and its emergence as a global health concern, especially in developing countries. It explores the general concepts, including the evolution of the term 'aggregative adherence' and the classification of EAEC strains. The document also examines the general epidemiology of EAEC, highlighting its association with both acute and persistent diarrhea, traveler's diarrhea, and outbreaks worldwide, including those linked to contaminated food. Clinical features, such as watery diarrhea, abdominal pain, and fever, are discussed, along with the heterogeneity of symptoms and the role of virulence factors. Furthermore, the document explores histopathological studies, illustrating EAEC's interactions with intestinal cells and its impact on the intestinal mucosa. The heterogeneity of EAEC strains, in terms of serotypes, genetic determinants, and virulence factors, is also addressed, emphasizing the ongoing research to understand the pathogenesis of this complex pathogen.

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Enteroaggregative Escherichia coli (EAEC)
Chapter · October 2016
DOI: 10.1007/978-3-319-45092-6_2
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DOI 10.1007/978-3-319-45092-6_2
Chapter 2
EnteroaggregativeEscherichia coli (EAEC)
Waldir P. Elias and Fernando Navarro-Garcia
Summary EnteroaggregativeEscherichia coli (EAEC) is defi ned by the production
of the characteristic aggregative adherence pattern on cultured epithelial cel
pathotype is a food-borne emerging enteropathogen , responsible for case
and persistent diarrhea in children and immunocompromised patients in deve
countries, as well as in travelers returning from endemic areas. Growth and c
impairment are linked to EAEC infections in children living in developing coun
The pathogenesis of EAEC is characterized by abundant adherence to the in
mucosa,elaborationof enterotoxins/cytotoxins,and inductionof mucosal
infl ammation. Several putative virulence factors associated with these three
have been identifi ed and characterized, but none of them is present in all str
The virulence gene(s) that defi ne virulent strains within this complex heter
is yet to be determined. An increasing attention to this pathotype emerged si
massive outbreak caused by a hybrid hypervirulent Shiga toxin-expressing EA
strain with severe clinical consequences.
W. P.Elias
Laboratório de Bacteriologia, Instituto Butantan, São Paulo, Brazil
e-mail: [e-mail]
F.Navarro-Garcia(* )
Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN
(CINVESTAV-IPN), México, Distrito Federal, Mexico
e-mail: [e-mail]

28
1 General Concepts
1.1 Defi ning EAEC
The term aggregative adherence was coined by Nataro and colleagues
examining the adherence properties ofE. coli strains isolated in an epidemiological
study of childhood diarrhea in the city of Santiago, Chile (Nataro et al.1987 ). The
isolates were tested for interaction with HEp-2 cells, and three adherence pat
were described. In addition to the previously described localized adherence (L
pattern, the term “diffuse adherence” was distinguished into the truly diffuse
sion (DA) and the aggregative adherence (AA). Standard AA is defi ned as ba
adhering to each other, on the surface of epithelial cells as well as on the sur
the coverslip in the absence of cells (Fig.2.1 ). Such confi guration resembles stacked
bricks forming heterogeneous aggregate or distributed in chains (Hebbe
Jensen et al.2014 ).
Strains expressing the AA pattern were then called “enteroadherent-aggr
E. coli, ” but this term was replaced by the current name of enteroaggregativE. coli ,
or EAEC (Estrada-Garcia and Navarro-Garcia2012 ). The AA phenotype issine qua
non to classify anE. coli strain as belonging to the category of EAEC. Nowadays,
most appropriate and updated defi nition of EAEC encompasses strains that p
Fig. 2.1Characteristic aggregative adherence (AA)patternof EAEC on HEp-2 cells. EAEC pro-
totypical strain 042 was incubated with epithelial cells for 3 h and the preparation was sta
May-Grünwald and Giemsa
W.P. Elias and F. Navarro-Garcia

29
the AA pattern on HeLa or HEp-2 cells (3 or 6 h adhesion assay) and are devo
virulence markers that defi ne other types of diarrheagenicE. coli. An exception is for
strains presenting the AA pattern and other EAEC-specifi c genetic markers in
bination with the production of Shiga toxin (Stx) , which defi nes the hybrid
and Shiga toxin-producingE. coli (STEC), discussed below.
Currently, EAEC is considered an emerging enteropathogen, responsib
cases of acute and persistent diarrhea in children and adults worldwide, and
opmental consequences in children living in developing countries (Hebb
Jensen et al.2014 ). An increasing attention to this pathotype has arisen from t
massive outbreak caused by ahybrid hypervirulent EAEC strain (Stx2-expressing
EAEC) with severe sequelae such as the development of hemolytic uremic sy
drome (Navarro-Garcia2014 ).
1.2 General Epidemiology
Since its description in 1987, when EAEC was signifi cantly associated with a
diarrhea in children (Nataro et al.1987 ), numerous epidemiological studies of the
etiology of diarrhea searched for EAEC in an attempt to clarify its role as diarr
agent. In the early years, the association between EAEC and persistent
(≥14 days of duration) in children was well supported (Cravioto et al.1991 ).
However, the association with acute diarrhea in childhood was controversial.
In the following years, a large number of studies have reported the detect
EAEC in cases of acute diarrhea in developing and developed countries, persi
diarrhea in developing countries, and signifi cant outbreaks worldwide. In fac
and colleagues demonstrated by a meta-analysis study of the literature betw
and 2006 that EAEC was statistically associated with acute and persistent dia
developed and developing countries, to diarrhea in HIV-infected patients in d
ing countries, and adults traveler’s diarrhea (Huang et al.2006 ). Another recent
meta-analysis study of published articles between 1989 and 2011 showed as
of EAEC with acute diarrhea in children of South Asian populations (Pabalan e
2013 ). Thus, EAEC has been systematically identifi ed as an emerging enter
gen, globally distributed (Estrada-Garcia and Navarro-Garcia2012 ).
A high rate of asymptomatic young children carrying EAEC is still a
reported in several studies in the last years (Hebbelstrup Jensen et al.2014 ).
Moreover, such persistent colonization has a link with growth impairment in
dren from low socioeconomic status.
The linkage between EAEC and diarrhea in individuals living in developed c
tries became clearer in the last years. In the USA and Europe, EAEC has been
quently isolated from cases of diarrhea from children and adults. In prospecti
EAEC was the major cause of diarrhea in children in the USA (Nataro et al.2006 ).
Traveler’s diarrhea (TD) is the most frequent disease that affects individ
ing in developed countries when visiting middle and low incoming endemic a
EAEC has been systematically found among the most prevalent bacterial age
2 Enteroaggregative Escherichia coli (EAEC)

30
traveler’s diarrhea since the defi nition of this category. The prevalence of EA
TD varies from 19 to 33 %, depending on the geographic region visited (Moha
et al.2011 ).
Studies linked EAEC with diarrhea in HIV-infected adults and children, a gr
usually susceptible to signifi cant cases of protracted diarrhea. EAEC was isol
the only enteropathogen in symptomatic patients presenting diarrhea for 30
(Polotsky et al.1997 ). In addition, isolation of EAEC was similar in a case/contro
study of HIV patients (Medina et al.2010 ).
Several outbreaks of gastroenteritis caused by EAEC have been reported i
and high-income countries. Some of them involving very impressive numbers
infected children or adults and associated with the consumption of contamina
food. Outbreaks in the UK (Dallman et al.2014 ), Japan (Itoh et al. 1997 ), and Italy
(Scavia et al.2008 ) show the relevance of EAEC in developed countries. In one
the Japanese outbreaks, 2697 schoolchildren were affected after consum
school lunches (Itoh et al.1997). In Italy, the outbreaks were transmitted by unpas-
teurized cheese. EAEC was also responsible for outbreaks in developing coun
(Cobeljic et al.1996 ).
1.3 Clinical Features
The most common symptoms reported in EAEC infection are watery diarrhea
mucoid, with or without blood and abdominal pain, nausea, vomiting, and low
These signs and symptoms are often self-limited but some selected patients
develop persistent diarrhea (≥14 days). The diversity of clinical symptoms in
pathotype infection may be due to heterogeneity between EAEC isolates, infe
dose, genetic susceptibility factors in the host, as well as the immune
(Harrington et al.2006 ).
In order to better characterize the virulent properties of EAEC, studi
human volunteers receiving oral inoculum of different EAEC strains were
formed (Mathewson et al.1986 ; Nataro et al.1992 , 1995 ).
Nataro and colleagues evaluated four different EAEC isolated from diff
geographic regions and different serotypes in volunteers (Nataro et al.1995 ). In this
study, the volunteers ingested dose of 1010 CFU and just EAEC O42 (serotype
O44:H18) caused diarrhea in three out of fi ve volunteers. EAEC 042 was isola
from a case of childhood diarrhea in Peru. The clinical data obtained from the
unteers who developed diarrhea suggested that EAEC 042 caused secretory d
rhea, with short incubation period, absence of fever, and leukocytes or blood
stool. Furthermore, the mucus found in the feces of two patients suggested la
intestinal secretion induced by colonization by EAEC 042. These studies, as w
others that demonstrate the virulence of EAEC for humans, have indica
EAEC is a heterogeneous category of diarrheagenicE. coli , including virulent
strains or not, what seems to depend on factors not yet fully understood.
W.P. Elias and F. Navarro-Garcia

31
1.4 Histopathology
Studies in vitro, in vivo, and ex vivo, evaluating EAEC interactions with intest
cells from animals or humans, have tried to elucidate the pathogenesis
pathotype since its description in 1987. The fi rst study about EAEC pathogen
in animal models employed ligated ileal loop of rabbit and rat intestines (Vial
1988 ), showing that EAEC 042 and 17-2 strongly adhered to the mucosa and
ing shortening of microvilli, hemorrhagic necrosis with edema, and mononucl
infi ltrates in the submucosa. Analysis by transmission electron microscopy re
no bacterial invasion and the microvilli architecture was preserved.
Other studies employing in vitro organ culture (IVOC) models have elucid
the intestinal alterations induced by EAEC in fragments of human biopsies of
denum, ileum, and colon (Hicks et al. 1996 ; Nataro et al. 1996 ). Cytotoxic e
the colon were observed, such as microvilli vesiculation, enlarged crypt open
and increased epithelial cell extrusion (Hicks et al.1996 ). Also using IVOC, Nataro
and colleagues demonstrated strong adhesion of EAEC 042 to jejunal
ileum, and more intensely to the colon, while 042 cured of the pAA2 plasmid
this ability (Nataro et al.1996 ). All together, these data strongly indicated that EA
virulence was probable due to intestinal colonization, mainly to the colonic m
in the characteristic aggregative manner, with bacteria forming strong biofi lm
increased mucus layer, followed by cytotoxic and pro-infl ammatory effects
1.5 Strain Heterogeneity
In the decades that followed its original description, EAEC strains have been
acterized in numerous studies around the world, highlighting the particular h
geneity of this category in terms of serotypes, genetic determinants lin
virulence and phylogenetic groups (Boisen et al. 2012; Chattaway et al. 2014b ;
Czeczulin et al.1999; Jenkins et al.2006 ). This heterogeneity together with the
fact that not all EAEC strains were able to cause diarrhea in experimental infe
of humans raises the idea that only a subset of EAEC strains, carrying a speci
of virulence factors, has the capacity to cause diarrhea. This set of factors ha
been determined.
After demonstrating its pathogenicity in human volunteers, EAEC 042 beca
genetically and phenotypically widely studied and considered the prototype s
for EAEC. Major advances in understanding the pathogenesis of EAEC result
from data obtained with this strain whose genome has been sequenced (Chau
et al.2010 ). Other EAEC strains have been also used as prototype in studies d
ing virulence factors and pathogenic mechanisms not present in EAEC 042. D
the variety of identifi ed virulence factors, such as enterotoxins, cytotoxins, s
proteins, outer membrane proteins, and fi mbriae (Table2.1 ), the pathogenesis of the
diarrhea caused by EAEC remains unclear.
2 Enteroaggregative Escherichia coli (EAEC)

32
Table 2.1EAEC prototype strains and their main virulencefactors
Prototype
strain
(serotype) Virulence factor Genes References
042
(O44:H18)
AggR—aggregative master
regulator
aggR Morin et al.
( 2013 )
Shf—Shigella fl exneri
homologue involved in
biofi lm formation
shf Czeczulin et al.
(1999 )
VirK—Shigella fl exneri
homologue Pet chaperone
virK Tapia-Pastrana
et al. (2012)
CapU—hexosyltransferase
homologue
capU Czeczulin et al.
( 1999 )
ABC transporter system—
dispersin transporter system
aatA Nishi et al.
(2003 )
AAF/II—aggregative
adherence fi mbria II
aafABCD Elias et al.
(1999 )
Pet—plasmid-encoded
toxin
pet Eslava et al.
( 1998 )
Pic—protein involved in
colonization
pic Henderson et al.
( 1999a)
EAST-1—aggregative
heat-stable toxin 1
astA Savarino et al.
( 1991 )
Dispersin—anti-
aggregation protein
aap Sheikh et al.
(2002 )
Type VI secretion systemaaiA-Y Dudley et al.
(2006b )
ETT2 Escherichia colitype
three secretion system 2
eprHIJK, etrA, eivH,
epaOPQRS , eivFGEACIJ
Sheikh et al.
( 2006 )
Air—enteroaggregative
immunoglobulin repeat
protein
air Sheikh et al.
(2006 )
EilA—Salmonella HilA
regulatorhomologue
eilA Sheikh et al.
( 2006 )
Orf61—hypothetical
plasmid-encoded hemolysin
orf61 Chaudhuri et al.
(2010 )
Ag43—phase-variable
antigen 43
agn43 Chaudhuri et al.
(2010 )
Hra1—heat-resistant
agglutinin 1
hra1 Bhargava et al.
(2009 )
17-2 (O3:H2) AAF/I—aggregative
adherence fi mbria I
aggABCD Nataro et al.
( 1992 )
55989
(O104:H4)
AAF/III—aggregative
adherence fi mbria III
agg3ABCD Bernier et al.
( 2002 )
C1010-00
(OR:H1)
AAF/IV—aggregative
adherence fi mbria IV
agg4ABCD Boisen et al.
( 2008 )
(continued)
W.P. Elias and F. Navarro-Garcia

33
Due to the association of AA phenotype with high-molecular weight plasm
carrying large number of plasmid-encoded virulence factors in EAEC, these
mids are called aggregative virulence plasmids or pAA (Harrington et al.2006 ).
From the pAA1 plasmid present in the prototype EAEC 17-2 (serotype O3:H
Baudry et al. ( 1990 ) isolated the CVD432 probe fragment, widely us
molecular diagnosis of EAEC. Plasmid pAA2, present in the EAEC 042, also ha
approximately 100 kb of genetic information and encodes many well-charact
virulence factors (Czeczulin et al.1999 ).
AggR is a transcriptional activator and regulates the expression of various
lence factors present in the chromosome and pAA2 plasmid of EAEC 042, defi
the AggR regulon (Harrington et al.2006 ). At least 44 genes are regulated byaggR ,
including the genes for AAF/II biogenesis, dispersin and its secretion system,
CapU, theaai type VI secretion system, andaagR itself (Morin et al.2013 ; Dudley
et al.2006b ). Not all EAEC strains harbor aggR and, consequently, the pAA p
mid. Thus, the classifi cation of EAEC was proposed into two subgroups, e.g.,
cal and atypical, taking into account the presence or absence ofaggR , respectively
(Harrington et al. 2006 ). This classifi cation defi nes two groups of strains, on
them consisting of typical strains with higher pathogenic potential due to the
ence of the AggR regulon and the pAA virulence plasmid (Estrada-Gar
Navarro-Garcia2012 ). However, atypical EAEC strains are commonly isolat
from cases of diarrhea, as the sole pathogen (Huang et al.2007 ; Jiang et al. 2002 ).
In one epidemiological study on the etiology of acute diarrhea in childr
Espírito Santo (Brazil), atypical strains were more frequent than typical
(Isabel Scaletsky, unpublished data). In addition, at least two outbreaks of dia
were caused by atypical EAEC (Cobeljic et al. 1996 ), one of them affected m
than two thousand children (Itoh et al.1997 ).
Table 2.1(continued)
Prototype
strain
(serotype) Virulence factor Genes References
C338-14
(O55:H19)
AAF/V—aggregative
adherence fi mbria V
Agg5ABCD Jonsson et al.
(2015 )
C1096
(O4:HNT)
Pil—type IV pilus pilLMNOPQRSTUV Dudley et al.
( 2006a)
JM221
(O92:H33)
AAF/I—aggregative
adherence fi mbria I
aggABCD Mathewson et al.
( 1986 )
60A (ND) Hra2—heat-resistant
agglutinin 2
hra2 Mancini et al.
( 2011)
OR O rough,NT non-typable,ND not determined
2 Enteroaggregative Escherichia coli (EAEC)

34
1.6 Pathogenesis in Three Steps
EAEC ability to mediate diarrhea was clearly established through the
study with EAEC strain 042 (Nataro et al.1995 ). However, this study and others
have left clear that the pathogenesis of EAEC is complex, and EAEC strains ar
very heterogeneous.
Data accumulated from several studies have suggested three major featur
EAEC pathogenesis : (1) abundant adherence to the intestinal mucosa, (2) e
tion of enterotoxins and cytotoxins, and (3) induction of mucosal infl am
(Fig.2.2 ). These stages of EAEC pathogenicity have been obtained from stud
in vitro in cell cultures, animal models, and patients infected with EAEC (Hicks
et al. 1996; Navarro-Garcia and Elias2011 ). Heterogeneity is also found in EAEC
colonization, because once ingested, the location of infection in the gastroint
tract has not been well defi ned. Studies done on endoscopic intestinal specim
demonstrate that EAEC can bind to jejunal, ileal, and colonic epithelium (Nata
et al.1996 ). These fi ndings were recently validated in fragments from termina
and colon that were excised from pediatric patients undergoing intestinal sur
and from adult patients that underwent colonoscopy treatment; such intestin
ments were used to defi ne interaction with three EAEC strains. These bacter
nized ileal and colonic mucosa in the typical stacked-brick confi guration. In b
regions, the strains were seen over a great amount of mucus and sometimes
intact epithelium. It was possible to see adhered bacteria to the intestinal sur
with visualization of fi mbrial structures that could be responsible for the adh
process (Andrade et al.2011 ). Although a great diversity of adhesins, toxins, and
proteins involved in EAEC pathogenesis has been described, the prevalence o
virulence factors-encoding genes is highly variable and none of these h
found present in all EAEC strains (Czeczulin et al.1999 ; Jenkins et al.2006 ).
1.6.1 Adherence
Adhesion to the intestinal epithelium is facilitated by fi mbriae and is the fi rs
the bacterial colonization of the gut. Several authors demonstrated that the A
is associated with the presence of fi mbrial and afi mbrial adhesins in EAEC st
(Bhargava et al.2009 ; Boisen et al.2008 ; Czeczulin et al.1997 ; Hicks et al.1996 ).
However, the genes encoding these adhesion factors are found in low prevale
indicating a high diversity of adhesive structures responsible for the AA pattefi mbriae bind to components of the extracellular matrix of intestinal epithelia
(Farfan et al. 2008 ; Izquierdo et al. 2014b ), and the AA pattern is thought t
from binding to the epithelial cell surface and binding to adjacent EAEC bacte
Abundant adherence of EAEC to the intestinal mucosa includes mucoid bi
formation . Biofi lm formation is a potential important contributor in persisten
tion by allowing the bacteria population to evade the local immune system an
W.P. Elias and F. Navarro-Garcia

35
preventing the transport of antibacterial factors, including antibiotics. Secreti
excessive mucus has been described when the gut is colonized with EAEC an
event is followed by the formation of biofi lm (Hicks et al.1996 ; Navarro-Garcia
et al. 2010 ). Biofi lm formation is an important pathogenicity trait of EAEC a
present mainly in the colon and in the small intestine (Hicks et al.1996 ).
Fig. 2.2The 3-steps model of EAEC pathogenesis. Three major features of EAEC pathogene
(1) abundant adherence to the intestinal mucosa, (2) elaboration of enterotoxins and cyto
and (3) induction of mucosal infl ammation
2 Enteroaggregative Escherichia coli (EAEC)

36
1.6.2 Toxins
Once the biofi lm has been established, EAEC produces cytotoxic effects such
microvillus vesiculation, enlarged crypt openings, and increased epithelia
extrusion (Harrington et al.2006 ). It is thought that the secretion of toxins plays a
important role in secretory diarrhea, which is a typical clinical manifest
EAEC infection. Numerous putative EAEC virulence factors, such as Pet, EAST
and ShET1 toxins, and Pic, have been associated to these cytotoxic effects.
1.6.3 Infl ammation
EAEC is an infl ammatory pathogen, as demonstrated both in clinical (Greenb
2002 ) and laboratory (Steiner et al.1998 ) studies. It is clear that multiple factors con
tribute to EAEC-induced infl ammation, and further characterization of the na
EAEC pro-infl ammatory factors will greatly advance the understanding of this
ing pathogen. The initial host infl ammatory response to EAEC infection is dep
on the host innate immune system and the type of EAEC strain causing infect
role of putative virulence genes and clinical outcomes is not well understood;
ever, the presence of several EAEC virulence factors correlate with fi ndings i
ing that levels of fecal cytokines and infl ammatory markers in stools of adult
children with diarrhea are elevated, including interleukin (IL)-1 receptor
IL-1β, IL-8, interferon (INF)-γ, lactoferrin, fecal leukocytes, and occult blood (Ji
et al. 2002 ). The IL-8 infl ammatory response appears to be partially caused
(FliC) in a Caco-2 cell assay, as it was found that an afl agellated mutant of EA
not produce the same infl ammatory response (Steiner et al.2000 ).
Besides the pathogenic EAEC mechanisms, host factors are also determina
EAEC infl ammation. It was found that single nucleotide polymorphisms in the
moter region of the gene encoding the lipopolysaccharide receptor CD14 ar
ated with bacterial diarrhea in US and Canadian travelers to Mexico (Mohame
et al. 2011 ). The CD14 gene encodes a crucial step in the infl ammatory res
bacterial lipopolysaccharide stimulation mediated by the innate immune
Thus, this study found that one SNP in the promoter region of the CD14 gene
associated with an increased risk of EAEC-induced diarrhea. Patients wit
CD14 − 159 TT genotype were signifi cantly associated with EAEC-induced
rhea compared with healthy controls.
1.7 Main Virulence Factors
In the initial stage of EAEC colonization, the role of fi mbrial and afi mbrial ad
is fundamental. Five aggregative adherence fi mbriae (AAF/I–AAF/V) have bee
characterized in EAEC (Boisen et al. 2008 ; Czeczulin et al. 1997 ; Nataro et
Jonsson et al. 2015 ). All AAF fi mbriae are encoded by genes located in the p
W.P. Elias and F. Navarro-Garcia

37
plasmids, which regulated by AggR and their biogenesis follows the usher- ch
pathway. Also, a type IV fi mbrial, calledPil pilus, is responsible for the AA pattern in
cultured epithelial cells and abiotic surface and it’s exhibited by an atypical E
strain isolated in the outbreak in Serbia (Cobeljic et al.1996 ; Dudley et al.2006a ).
Non-fi mbrial adhesins, or outer membrane proteins with molecular w
between 30 and 58 kDa, and associated with AA pattern have been described
various EAEC strains (Bhargava et al.2009 ). In addition, Hra1 and Hra2 are heat-
resistant agglutinins involved in autoaggregation, biofi lm formation, and agg
tive adherence phenotypes (Bhargava et al.2009 ; Mancini et al. 2011 ). The presence
of agn43gene, encoding the autotransporter protein antigen 43, was associate
biofi lm formation and autoaggregation in EAEC 042 (Chaudhuri et al.2010 ). The
multifactorial characteristic of the AA phenotype is clear from studies with str
expressing multiple adhesins. Furthermore, the low prevalence of genes enco
these adhesins highlights the great diversity of adhesive structures in EAEC.
The dispersin protein (anti-aggregation protein) is an important EAEC vir
factor that mediates the dispersion of EAEC along the intestinal mucosa (She
et al.2002 ). Dispersin neutralizes the negative charge on the surface of the ba
cell and allows AAF/II fi mbrial projection , leading to anti-aggregation and d
sal of bacteria in the intestinal wall. This protein requires an ABC-type transpo
system encoded by the aatPABCD operon, which is present in the pAA2 plas
(Nishi et al.2003 ).
As described before, EAEC produces cytotoxic effects evident during in vit
and in vivo studies. Several putative virulence factors associated to those cyt
effects have been identifi ed and characterized in EAEC.
The fi rst toxin described in EAEC was the enteroaggregative heat-stabl
1 (EAST-1) , which is related to the heat-stable toxin (STa) of enterotoxigenic
(Savarino et al.1991 ). EAST-1 activates adenylate cyclase inducing increased cy
GMP levels in enterocytes, generating a secretory response (Savarino et al. 1
EAST-1 is a 38 amino acids peptide (4.1 kDa) encoded by astA gene,
located in pAA2 of EAEC 042 (Czeczulin et al.1999 ). Since STa toxin causes
secretory diarrhea, it was believed que EAST-1 was responsible for this effect
EAEC-induced diarrhea. However, the presence of EAST-1 in EAEC 17-2 was n
suffi cient to provoke diarrhea in volunteers (Nataro et al.1995 ).
Another toxin encoded by a chromosomal gene in EAEC 042 is called ShET
(Shigella enterotoxin 1), which is encoded by setAB located in the antisense str
of pic (Henderson et al.1999a ; Navarro-Garcia and Elias2011). ShET1 is an A:B
type toxin that causes accumulation of fl uid in rabbit ileal loops. The enterot
mechanism of ShET1 is independent of cAMP, cGMP, and calcium. However, t
precise mechanism of ShET1 action remains unclear.
Amongst the virulence factors produced by EAEC, the autotransporter prot
have a relevant role in the EAEC pathogenesis. Initially, two autotransporter p
teins were identifi ed in EAEC, comprising high-molecular weight protein
104 kDa for Pet and 109 kDa for Pic (Eslava et al.1998 ; Henderson et al.1999a ).
Autotransporter proteins are currently assigned to the type 5 secretio
(T5SS), which is described in detail in Chap. 10. Autotransporters contain three
2 Enteroaggregative Escherichia coli (EAEC)

38
functional domains: the signal sequence, the passenger domain, and the tran
unit. The signal sequence is present at the N-terminal end of the protein and
targeting of the protein to the inner membrane for its further export into the
plasm (Jose and Meyer2007 ). The passenger domain confers the diverse effecto
functions. The translocation unit (also called theβ-domain ), located at the C-terminal
end of the protein, consists of a short linker region with an α-helical
structure and a β-core that adopts a β-barrel tertiary structure when embedd
outer membrane and facilitates translocation of the passenger domain
Meyer2007 ).
The serine protease autotransporters ofEnterobacteriaceae (SPATE) members
constitute a group of exoproteins secreted through the T5SS by pathogenic e
bacteria of the γ-proteobacteria. The passenger domains contain the proteas
(GDSGSP) characteristic of all proteins of the SPATE group (Navarro-Garcia an
Elias2011 ). SPATEs have been divided into class-1 and class-2 based on struc
differences and biological effects; the class-1 SPATEs are related to cytotoxic
on cultured cells, whereas most class-2 SPATEs exhibit a lectin-like activity w
preference to degrade a variety of mucins (Ruiz-Perez and Nataro2014 ).
The plasmid and chromosomal EAEC SPATEs Pet and Pic are membe
class-1 and class-2, respectively. Originally, Pet and Pic were detected in an e
to identify cytotoxins and enterotoxins secreted by EAEC. Analyses of c
supernatants from strains that caused outbreaks of EAEC diarrhea in Mexican
pitals showed two major proteins of 104 and 109 kDa, which are now known a
secreted proteins Pet and Pic, respectively (Eslava et al.1998 ; Henderson et al.
1999a ; Navarro-Garcia et al.1998 ). Interestingly, both proteins are related wi
pathogenic features in the infection by EAEC: cytotoxicity and mucosal coloni
tion, including the bacteria-mucus biofi lm .
It has been shown that Pet (104 kDa) of EAEC protein is required for induc
dilation of crypt openings and rounding and extrusion of enterocytes in huma
sue explants (Henderson et al.1999b). In an ex-vivo model of infection, Pet causes
raises in short-circuit current and decreases in electrical resistance of rat jeju
while mounted in the Ussing chamber, and the enterotoxic effect is accompa
mucosal damage, increased mucus release, exfoliation of cells, and developm
crypt abscesses (Navarro-Garcia et al.1998 ). The use of cultured epithelial cells to
understand the mode of action of this toxin showed that Pet is a cyto
altering toxin, because it induces contraction of the cytoskeleton, loss of acfi bers, and release of focal contacts followed by complete cell rounding and d
ment. Interestingly, Pet cytotoxicity and enterotoxicity depends on Pet serine
ase activity (Navarro-Garcia et al.1999 ). Furthermore, it was found that Pet require
cytokeratin 8 as receptor to enter the eukaryotic cell and that traffi cking thro
vesicular system appears to also be required for the induction of cytopathic e
(Nava-Acosta and Navarro-Garcia2013 ).
Pet is internalized by receptor-mediated endocytosis, through clathrin
vesicles, and this internalization pathway was found to be an essential mecha
because its inhibition prevents Pet internalization, and thereby the Pet-induce
toxic effect (Nava-Acosta and Navarro-Garcia2013 ). An intracellular target,
W.P. Elias and F. Navarro-Garcia

39
α-fodrin (αII-spectrin), has been found for Pet. Pet binds and cleaves (between1198
and V1199) epithelial fodrin in vitro and in vivo, causing fodrin redistribution wit
the cells to form intracellular aggregates as membrane blebs (Canizalez-Rom
Navarro-Garcia2003 ). This mechanism appears to be a new system of cellular d
age identifi ed for bacterial toxins, which includes the internalization of the pr
to fi nally allow specifi c α-fodrin degradation to destroy the cell (Canizalez-Ro
and Navarro-Garcia2003 ). This was the fi rst report showing cleavage of α-fodrin
by a bacterial protease.
Pic was identifi ed as a second SPATE member in EAEC. In contrast to Pet,
encoding gene is localized in the EAEC chromosome (Czeczulin et al.1999 ;
Henderson et al. 1999a ). Thepic gene has a unique characteristic among the auto
transporter proteins since ShET1-encoding genes are oppositely oriented and
dem within thepic gene (Henderson et al.1999a ). Functional analyses of Pic have
shown that its serine active site is involved in mucinase activity, serum resist
and hemagglutination. Phenotypes identifi ed for Pic suggest that it is involve
early stages of pathogenesis and most probably promotes intestinal col
(Henderson et al.1999a ). It has also been shown that Pic does not damage epith
cells (Gutierrez-Jimenez et al.2008 ).
As we mentioned before, a hallmark of EAEC infection is the format
mucoid biofi lm, which comprises a mucus layer with immersed bacteria
intestines of patients. Interestingly, an isogenicpic mutant was unable to cause
mucus hypersecretion, in contrast to the EAEC wild-type strain, which i
hypersecretion of mucus , accompanied by an increase in the number
containing goblet cells. Site-directed mutagenesis of the serine protease cata
residue of Pic showed that, unlike the mucinolytic activity, secretagogue activ
does not depend on this catalytic serine protease motif (Navarro-Garcia et al.2010 ).
Using specifi c mutants in competition with the wild-type strain, the contri
of several putative EAEC virulence factors to intestinal colonization of strepto
treated mice was evaluated (Harrington et al.2009 ). The data suggest that the dis-
persin surface protein and Pic promote colonization of the mouse. Interesting
has been found that Pic targets a broad range of human leukocyte adhesion
such as CD43, CD44, CD45, CD93, CD162, and the surface-attached chemoki
fractalkine, all implicated in leukocyte traffi cking, migration, and infl am
(Ruiz-Perez et al. 2011 ). The ability of Pic to inactivate key proteins of all com
ment pathways is also a new fi nding, demonstrating its function in immune e
Pic signifi cantly reduces complement activation by cleaving C2, C3, C3b, and
This is an important virulence mechanism in the context of systemic disease,
as sepsis and hemolytic uremic syndrome, which may be caused by Pic-produ
E. coli andShigella fl exneri (Abreu et al.2016 ). All these data strongly suggest that
Pic represents a unique immune-modulating bacterial virulence factor.
Besides Pic, another SPATE from class-2 and found in the hybrid EAEC/STE
O104:H4 is SepA (Munera et al.2014 ). Several SepA-hydrolyzed peptides were
described as specifi c substrates for cathepsin G, a serine protease pro
polymorphonuclear leukocytes that was proposed to play a role in infl amma
However, unlike cathepsin G, SepA degraded neither fi bronectin nor angioten
2 Enteroaggregative Escherichia coli (EAEC)

40
I and had no effect on the aggregation of human platelets. It was found that t
encoding the autotransporter protease SepA was most strongly associated w
rhea among the EAEC strains as part of a case-control study of moderate to s
acute diarrhea among children in Mali (Boisen et al.2012 ).
SigA is another SPATE of class-1 also detected in EAEC/STEC O104:H
An initial report showed that theshe PAI, which contains thepic ( she ) gene, also
contains a gene encoding a second IgA protease-like homologue,sigA . Functional
analysis showed that SigA is a secreted temperature-regulated serine proteas
ble of degrading casein . Performing similar experiments to those used
revealed that SigA is cytopathic for HEp-2 cells, suggesting that it may be a c
altering toxin with a role in the pathogenesis ofShigella infections . Indeed, it was
found that SigA binds specifi cally to HEp-2 cells and degrades recombinant h
αII spectrin (α-fodrin) in vitro and also cleaves intracellular fodrin in situ, caus
its redistribution within cells (Al-Hasani et al.2009 ).
Some pathogenicity islands (PAI) were identifi ed in 042 EAEC strain harbo
distinct putative virulence factors (Chaudhuri et al.2010 ). The presence of such
mobile genetic elements emphasizes the characteristic mosaic of the EAEC g
(Chattaway et al.2014b ). The EAEC 042pheU PAI encodes an important virulence
component, the type 6 secretion system (T6SS), extensively studied in many
gens in the last years. Type 6 secretion system in EAEC 042 is encoded by thaai
cluster (AggR-activated island), which is composed of 25 genes (Dudley
2006b ). In EAEC 042, the T6SS secretes AaiC (Dudley et al.2006b ). The role of
the T6SS in the pathogenesis of EAEC has not been established. Also, the g
encoding ShET1 (setAB) and the mucinase Pic ( pic) are contained in a PAI called
she , which was initially identifi ed in Shigella (Henderson et al.1999a ).
Analysis of the genome of EAEC strain 042 indicated the presence o
encoding a type 3 secretion system atglyU tRNA locus (Chaudhuri et al.2010 ),
which in other pathogens is associated with the translocation of effectors pro
directly into the cytoplasm of eukaryotic cells (see Chap.10 ). This system is named
ETT2 ( Escherichia coli type 3 secretion system 2) and presents high similarit
that found in enterohemorrhagic E. coli andSalmonella . The possible ETT2 effec-
tors are located atselC tRNA locus, which harbors theeipBCD , eicA, andair genes.
The bacterial surface protein Air is involved in adherence and aggregation of
042 (Sheikh et al.2006 ). The functionality of ETT2 and the role of possible trans
located effectors in EAEC pathogenesis remain elusive.
1.8 Diagnostic
The AA pattern demonstrated in adhesion assays using HeLa or HEp-2 cells
considered the gold standard test for the identifi cation of EAEC since a comm
genetic determinant for all strains of this pathotype has not yet found (Hebbe
Jensen et al.2014 ). However, this technique is costly, time consuming, and requ
infrastructure restricted to reference laboratories. Therefore, molecular diagn
W.P. Elias and F. Navarro-Garcia

41
techniques have been developed as an alternative to detection of the AA pat
which started with the development of a DNA probe (CVD432), initially descri
as a cryptic fragment of the pAA1 plasmid of EAEC strain 17-2. This probe has
been widely used in diagnosis and epidemiological studies showing high spec
but variable sensitivity (20–89 %) when compared to the detection of the AA
tern. Later, CVD432 was characterized as part of theaatA gene, which is part of the
dispersin-secretion system operon (Nishi et al.2003 ). Thus, CVD432 probe is prop-
erly called aatA or AA probe. As aatA is a pAA-borne gene, its detection exc
the majority of atypical EAEC.
There are several reports in the literature on the development of multiplex
for simultaneously detection of all diarrheagenicE. coli pathotypes. However, those
detecting EAEC use only one or two pAA markers, excluding the detection of
cal EAEC (Panchalingam et al.2012 ). Some authors have proposed the diagnosis o
EAEC only based on multiplex PCR, detecting either the plasmid or plasmid an
chromosomal markers (Andrade et al.2014a ). Recently, the detection ofaatA aggR
andaaiG/aaiC has been proposed as an alternative for sensitive and specifi c
ular detection of EAEC, covering the detection of both typical and atypical su
groups (Andrade et al. 2014a ; Boisen et al.2012 ; Panchalingam et al.2012 ).
1.9 Transmission
There is no evidence for an animal reservoir of EAEC; therefore, humans are
ally accepted as the reservoir. The transmission of EAEC is often associated w
food-borne sources or by contaminated water (Jiang et al.2002 ). Food-borne out-
breaks have been described (Itoh et al.1997 ). Risk factors for EAEC infection
include travel to developing countries, ingestion of contaminated food and wa
poor hygiene, host susceptibility, and possibly immunosuppression (Estrada-G
and Navarro-Garcia2012 ). Another route of transmission of EAEC is food han-
dling. A study of Mexican tabletop sauces identifi ed 44 % of sauces co
viable EAEC (Adachi et al.2002 ).
1.10 Treatment
The oral rehydration therapy is the prime recommendation for the cas
limited acute diarrhea caused by EAEC. Antibiotic therapy is indicated only in
of persistent diarrhea, and should be considered case by case. When required
microbial sensitivity evaluation tests are indicated since multidrug resistance
been often reported in EAEC strains isolated from patients from different geo
regions (Hebbelstrup Jensen et al.2014 ). Alternative treatments, employing the use
of zinc and nitazoxanide, have been recently proposed for treatment and pre
of diarrhea based on clinical and laboratorial evidences (Bolick et al.2013 ).
2 Enteroaggregative Escherichia coli (EAEC)

42
2 Recent Advances
2.1 Intestinal Infl ammation
Several lines of evidence suggest that EAEC infection is mildly infl ammato
nature. Epidemiological reports have documented elevated fecal lactoferrin, I
and IL-1β among infected infants in developing countries and adult trav
India and Mexico (Steiner et al.1998 ; Greenberg et al.2002 ). Travelers to Mexico
who developed symptomatic illness due to EAEC infection excreted high conc
trations of fecal IL-8, compared to travelers who did not develop diarrhea due
EAEC infection (Jiang et al. 2003 ). Steiner and colleagues have shown that E
strain 042 induces IL-8 release from non-polarized Caco-2 intestinal epithelia
(IECs); the pAA plasmid was required for the full infl ammatory effect (Steiner
1998 ). These investigators also reported that a mutation in the gene encodin
lin (fl iC ) abrogated IL-8 release, implicating fl agellin as the major pro- infl am
stimulus (Steiner et al.2000 ). However, it has been reported that signifi cantly m
IL-8 was detected in feces of travelers infected with EAEC strains harboring th
plasmid-borneaggRor aafA genes, compared with those infected with virulence
factor-negative EAEC (Jiang et al.2002 ). In a search to identify additional factors
that could account for the IL-8 release from epithelial cells infected with EAEC
strain 042, it was found that polarized T84 intestinal cells release IL-8, even w
infected with 042 mutated in the major fl agellar subunit FliC. IL-8 release fro
polarized T84 cells was found to require the AggR activator and the AAF fi mb
and IL-8 release was signifi cantly less when cells were infected with mutants
AafB, the minor pilin subunit of AAF/II (Harrington et al. 2005 ). In addition to
IL-8, intestinal epithelial cells infected with EAEC 042 have been shown to upr
late the following genes: IL-6, tumor necrosis factor (TNF)-α, growth-related g
product (GRO)-α , GRO-γ, intercellular adhesion molecule (ICAM)- 1 , granul
macrophagecolony-stimulatingfactor(GM-CSF) ,and IL-1α. Thesecellular
responses are primarily mediated by fl agellin (FliC) of EAEC (Harrington et al
2005 ). It is clear that multiple factors contribute to EAEC-induced infl amma
and further characterization of the nature of EAEC pro-infl ammatory factors w
greatly advance the understanding of this emerging pathogen.
2.2 Growth Impairment in Developing Countries
EAEC persistent diarrhea has been linked to malnutrition and decreased phy
and cognitive development in children (Guerrant et al.2008 ). Interestingly, in this
study the population, even asymptomatic patients infected with EAEC, exhibi
growth retardation compared to uninfected controls (Steiner et al.1998 ). Thus, it
seems that EAEC effects on growth shortfalls in children could also be due to
W.P. Elias and F. Navarro-Garcia

43
persistence in human intestine, with subclinical symptoms, inducing chronic i
mation in the absence of diarrheal disease (Steiner et al.1998 ). Pathogenic EAEC
infection is characterized by release of cytokines from the intestinal mucosa a
lactoferrin, revealing the infl ammatory potential of these strains in damaging
intestinal epithelium and reducing its absorptive function, leading to nutrient
tion and malnutrition. In turn, malnutrition further facilitates infection and pe
ates the cycle of infection, malnutrition, and developmental defi cits, increasi
burden of the disease (Guerrant et al.2008 ). Given the high rate of asymptomatic
excretion of EAEC in a large number of countries in the developing world, und
standing its potential role in malnutrition and growth retardation is a high p
2.3 Animal Models for EAEC Pathogenesis
Researchers have been trying to develop animal models to reproduce human
and immunological responses at the site of EAEC infection. Development of s
els could allow the evaluation of pathophysiology, treatment, and prototype v
Adult CD-1, Balb/c, and C3H/HeJ mice, treated with streptomycin, have bee
used to study intestinal colonization (Harrington et al.2009 ). These murine models
are suitable to quantify intestinal colonization, verify histological alterations,
determine immunological mediators in fecal contents although mice do not d
enteritis symptoms. Using these models, Harrington and colleagues demonst
the role of autotransporter Pic in promoting mucus secretion and colon
(Harrington et al.2009 ).
A murine model to assess EAEC infection malnutrition cycle was developed
using neonatal and weaned C57BL/6 mice (Roche et al.2010 ). Although mild
histological effects in the colonic epithelium were seen, growth impairment c
be assessed in both groups and in weaned mice, infection was persiste
stool shedding. Undernutrition in both groups of mice intensifi ed infection an
growth impairment was dependent on bacterial burden and challenge d
However, this model has some limitations, due to the early age of the animal
For that reason, the same approach has been applied to infect nourished and
nourished weaned 24-day-old C57Bl/6 mice and similar results were obt
(Bolick et al. 2013 ). This model was adapted to investigate the role of zinc in
preventing EAEC pathogenesis. Weight loss, stool shedding, mucus productio
and, more importantly, diarrhea, were observed when mice fed a zinc-free an
protein source defi ned diet, pretreated with antibiotics and then orally challe
with EAEC 042.
A novel model for studying EAEC disease has been developed with human
intestinal xenografts in severe-combined immunodefi cient (SCID) mice (Bo
2012 ). The successful transplants allowed the study of intact and functional
intestinal tissue infected with EAEC 042, showing severe tissue damage and i
tration of polymorphonuclear cells, with pathogenesis strongly related to AAF
2 Enteroaggregative Escherichia coli (EAEC)

44
expression . This model can address the investigation of the role of specifi c
lence factors and the interaction of EAEC with human intestinal mucosa. How
some limitations of the model include the restriction to fetal human tissue, th
of intestinal microbiota in the xenografts, and interaction of innate and adapt
immunity.
2.4 Urinary Tract Infection
In the last years, a growing number of reports have shown a linkage between
and urinary tract infection (UTI). Some studies investigated the presence of ty
virulence factors of EAEC in strains causing extraintestinal infections (Abe et
2008 ; Herzog et al.2014 ). Others demonstrated the presence of uropathogenic
E. coli (UPEC) markers in EAEC collections (Chattaway et al.2014a ). These fi ndings
clearly indicate the potential for some EAEC strains to cause UTI.
Indeed, Olesen and colleagues reported a community-acquired UTI ou
caused by EAEC (Olesen et al.2012 ), which does not fulfi ll the extraintestinal E.
coli (ExPEC) criteria . This multiresistant strain was characterized as belongi
the serotype O78:H10, multilocus sequence type ST10, and the phylogenetic
A (Olesen et al.2012 ). This was the fi rst time that EAEC was implicated as an a
of an outbreak of extraintestinal disease. Subsequently, the uropathogenic pr
ties of this EAEC O78:H10 strain were investigated showing that these proper
were conferred by specifi c virulence factors of the EAEC, such as the AAF/I fi
(Boll et al.2013 ). Recently, EAEC was also implicated as a causative agent of o
case of urosepsis (Herzog et al.2014 ).
2.5 The Hybrid O104:H4 STEC/EAEC Strain
An outbreak caused byE. coli of serotype O104:H4 spread throughout Germany in
May 2011 (Navarro-Garcia2014 ). This was the largest outbreak by a pathogenicE.
coli strain, with 3128 cases of acute gastroenteritis, 782 cases of hemolytic
syndrome (HUS) , and 46 deaths. All these cases were offi cially attributed to
clone of STEC and most of the victims became infected in Germany or France
phenotypic and genotypic characterization of theE. coli O104:H4 indicated that the
isolates from the French and German outbreaks were common to both incide
Fenugreek seeds imported from Egypt, from which sprouts were grown,
implicated as a common source. However, there is still much uncertain
whether this is truly the common cause of the infections, as tests on the seed
not allow the detection of anyE. coli isolate of serotype O104:H4.
This large outbreak was caused by an unusual STEC strain, which is more
to an EAEC of serotype O104:H4. A signifi cant difference, however, is the pre
of a prophage encoding the Shiga toxin, which is characteristic of STEC
W.P. Elias and F. Navarro-Garcia

45
(Rasko et al.2011 ). This unique combination of genomic features, associating c
teristics from both EAEC and STEC, represents a new pathotype. Since typica
is isolated primarily from humans, the origin of this outbreak may not be zoon
Due to the special features and high pathogenicity of this hybrid clone, on
tive question is what makes this E. coli O104:H4 outbreak strain so dangero
explanation is that this hybrid strain with EAEC features but with a phage cod
Shiga toxin type 2 is a better colonizer of the gut due to its aggregative phen
enhanced adherence of this strain to intestinal epithelial cells might facilitate
absorption of Shiga toxin and could explain the high frequency of cases progr
HUS. Thereby, it is believed that EAEC of serotype O104:H4 is by itself an em
serovar that has acquired an original set of virulence factors (Frank et al.2011 ).
EAEC strains of serotype O104:H4 contain a large set of virulence-associat
genes regulated by AggR. These include the pAA plasmid genes encoding the
which anchor the bacterium to the intestinal mucosa and induce infl ammatio
well as a protein-coat secretion system (Aat), that secretes the protein
(Navarro-Garcia2014 ). Thus, a switch of pAA together with the type of the aggr
gative adherence fi mbriae could be an additional explanation for the higher
lence of this outbreak strain. Indeed, the outbreak STEC O104:H4 strain is sim
to the EAEC O104:H4 strain 55989, isolated in the late 1990s, from a patient
Central African Republic with persistent diarrhea, and to the EAEC O104:H4 s
HUSEC041 that was associated in 2001 with very few HUS cases in Ge
Interestingly, the EAEC O104:H4 strain HUSEC041 carries the plasmid encodin
AAF/III fi mbriae (also present in the EAEC strain 55989). In contrast, outbreak
EAEC O104:H4 isolates acquired a new plasmid, encoding AAF/I fi mbriae, and
lost the plasmid encoding AAF/III fi mbriae (Rasko et al.2011 ).
Other interesting features that might also contribute to the higher virulenc
this outbreak strain include that EAEC strains of serotype O104:H4 produce a
able number of SPATEs implicated in mucosal damage and colonization. This
serovar contains Pic, SigA, and SepA (Rasko et al. 2011 ). Rasko and colleagu
speculate that the combined activity of these SPATEs together with other EA
virulence factors accounts for the increased uptake of Shiga toxin into the sy
circulation, resulting in the high rates of HUS (Rasko et al.2011 ). Indeed, a recent
study showed that SPATEs but not pAA are critical for rabbit colonization by S
toxin-producingE. coli O104:H4 (Munera et al.2014 ). However, it has been also
shown recently that the presence of the pAA plasmid in the EAEC/STEC O104
strain promotes the translocation of Stx2a across an epithelial cell mon
(Boisen et al.2014 ). Interestingly, Ec55989 contains three copies ofpic , which are
conserved in the German outbreak strain. In addition, there is a fourthpic gene pres-
ent in the EAEC plasmid of the outbreak strain. The outbreak strain also enco
SigA that cleaves the cytoskeletal protein spectrin, inducing rounding and ex
tion of enterocytes (Al-Hasani et al.2009 ), and SepA, associated with increasedS.
fl exneri virulence, but with unknown function in EAEC.
The ability of STEC to cause severe disease in humans is mainly associated
with the production of one of the two Shiga toxin groups, Stx1 and Stx2, with
similar biological activity but different immunogenicity. The EAEC/STEC hybrid
2 Enteroaggregative Escherichia coli (EAEC)

46
clone produces the Shiga toxin 2 (Stx2). An interesting fi nding that hig
Stx2 is its epidemiological association with severe diseases in humans (Frie
et al.2002 ).
Two remarkable features of EAEC O104:H4 have to be highlighted: it is the
agent of a massive outbreak and the high proportion of cases developing HUS
complication was diagnosed in a 22 % of the cases, while historical rates of H
after O157:H7 infection typically range from 6 to 15 % (Frank et al.2011 ). It is
challenging to know why the outbreak strains are so virulent, given that EAEC
human-specifi c pathogens and that few animal infection models mimic
disease. Interestingly, the lack of the pAA plasmid abolished the capaci
outbreak strain (C227-11) to adhere to viable colonic tissue harvested
cynomolgus monkeyMacaca fascicularis(Boisen et al.2014 ). However, C227-11
adhered in an aggregative manner and forms heavy biofi lms with a thick m
layer upon interaction with the monkey colonic tissue.
2.6 Vaccine Development
A few studies in the last years tried to use aggregative adherence fi mbriae
candidate vaccine antigens. AAF/I and AAF/II structural genes (aafA andaggA ,
respectively) were coupled to the gene encoding nontoxic B subunit of Shiga
(StxB). The recombinant polypeptides elicited immune response in subcutane
immunized Balb/C, and the antibodies generated inhibited the adherence of p
type EAEC strains to HeLa cells and protected the immunized mice against a
dose of Shiga toxin (Oloomi et al. 2009 ).
The use of the polysaccharide part of LPS has been evaluated as a vaccine
against EAEC belonging to the serogroup O111. However, LPS is not appropri
for human use due to its high level of toxicity. As an alternative, polysacchari
O111 LPS was conjugated with cytochrome C or recombinant B subunit of the
labile toxin (LT). These two different approaches induced systemic and mucos
antibodies in rabbit and in mice, which were able to inhibit the adhesion of al
egories of O111 E. coli to HEp-2 cells serogroups (Andrade et al. 2014b ).
this approach protects against one O-antigen, it does not represent the vast d
of circulating EAEC serogroups.
A combined formalin-killed whole-cell vaccine candidate, consisting of a m
ture of EAEC (strain 17-2), EPEC, STEC, enterotoxigenicE. coli (ETEC), and
enteroinvasiveE. coli (EIEC), was also proposed as vaccine (Gohar et al. 2016 )
Balb/C mice were immunized subcutaneously, eliciting humoral immune resp
to each pathotype. In addition, the specifi c antibodies were protective when
were challenged intraperitoneally with the respective immunizing bacteria. A
such approach is based in a subcutaneous immunization, which is not commo
endemic areas, and only antibodies against EAEC 17-2 were generated.
W.P. Elias and F. Navarro-Garcia

47
3 Advances on EAEC in the Americas
3.1 Recent Epidemiological Information
A few studies about the etiology of diarrhea in the Americas have been publi
between 2013 and March 2016, including children and adults hospitalized wit
rhea or with diarrhea in the community (Table2.2 ). The studies that searched only for
viruses and/or parasitic enteropathogens were not included. When searched
ies, EAEC strains were found as the most prevalent diarrheagenicE. coli pathotype.
In Mexico, Patzi-Vargas and colleagues determined the prevalence of bac
enteropathogens in 831 children with acute diarrhea (Patzi-Vargas et al2015 ).
DiarrheagenicE. coli was the most common bacterial enteropathogen and the p
dominant pathotypes were diffusely adherent E. coli (35 %) and EAEC
EAEC was more frequent in children between 6 and 24 months old than in tho
younger than 6 months of age. In addition, all diarrheagenicE. coli strains were
searchedfor supplementaryvirulencegenes(SVG) mainly associatedwith
EAEC. Dispersin (aap ), dispersin-translocator (aatA), EAST-1 (astA ), plasmid-
encoded toxin (pet ), and cytolethal distending toxin (cdt ) were higher in diarrhea-
genicE. coli than non-diarrheagenicE. coli strains and 98 % of EAEC-infected
children harbored strains with SVG; 85 % carried theaap-aatAgene combination
and 33 % of these carriedastA. DiarrheagenicE. colicarrying SVG was a cause of
moderate to severe bacterial diarrhea in that population.
EAEC was found in 13 % of rural Panamanian children (Jimenez Guti
et al. 2014 ). In Brazil, coinfections of EAEC and norovirus were detected in 2
of fecal samples from hospitalized diarrheic children (Amaral et al. 201
another study from Brazil, EAEC was the most frequent pathotype found in ca
and controls (10 %) although without statistical differences between the tw
(Dias et al. 2016 ). A large study in Bolivia (3943 cases and 1026 controls) sh
that EAEC was the most prevalent pathotype found in 11.2 % of the c
(Gonzales et al. 2013 ).
3.2 Adhesins
Advances in the comprehension of EAEC binding mechanisms to the human
tinal tissue have been achieved by characterizing the structure, binding char
tics, and immunogenicity of AAF fi mbriae. These approaches are necessary i
to develop effi cient blocking strategies. Since AAF/II is the main adhesive str
of EAEC 042, much of the data is based on recent studies characterizing rece
for its pilin and its molecular structure.
Adhesion of EAEC 042 to extracellular matrix proteins (fi bronectin, lamin
and type IV collagen) was demonstrated in vitro. Also, purifi ed AafA bound fi
nectin in a dose-dependent manner (Farfan et al.2008 ). Since the major cellular
2 Enteroaggregative Escherichia coli (EAEC)

48Table 2.2Epidemiological studies on the etiology of acute diarrhea that included the detection of EAEC in American countries (2013–20
Country Study population Number of patientsDiarrheagenicE. coli pathotypes detected (%) References
Bolivia Hospitalized children with acute
diarrhea <5 years old
3943 cases and 1026
controls
Cases: EAEC (11.2 %), ETEC (6.6 %), EPEC (5.8 %)Gonzales et al.
(2013 )Controls: EAEC (7.4 %), ETEC (4.8 %), EPEC (4 %)
Brazil Adults and children (outbreaks,
serious cases of diarrhea, sporadic
diarrhea)
400 cases EAEC (0.7 %), aEPEC (3.2 %), DAEC (0.7 %), tEPEC
(0.2 %), STEC (0.2 %)
Assis et al.
(2014 )
Brazil Hospitalized children with acute
diarrhea <6 years old
63 cases with viral
agents of diarrhea
EAEC (15.9 %), aEPEC (6.3 %), tEPEC (3.2 %), EHEC
(1.6 %)
Amaral et al.
( 2015 )
Brazil Children < 5 years old 200 cases and 200
controls
Cases: EAEC (10 %), aEPEC (8 %), ETEC (0.5 %),
STEC (0.5 %)
Dias et al.
( 2016 )
Controls: EAEC (10 %), aEPEC (8.5 %), tEPEC (0.5 %),
ETEC (0.5 %), STEC (0.5 %)
Multicenter study
(data from Brazil)
Group 1: 0–11 months 129 diarrhea episode
stools and 2519
surveillance stools
Cases 0–11 months: EAEC (34.2 %), ETEC (21.1 %),
aEPEC (7.9 %), tEPEC (7.9 %), EIEC (5.3 %)
Platts-Mills et al.
(2015 )
Controls 0–11 months: EAEC (37.8 %), aEPEC
(13.3 %), EIEC (12.7 %), ETEC (5.9 %), tEPEC (5.8 %)
Group 2: 12–24 months
(community surveillance)
Cases 12–24 months: EAEC (34.2 %), ETEC (12.7 %),
EIEC (12.7 %), aEPEC (11.4 %), tEPEC (10.1 %)
Controls 12–24 months: EAEC (41.9 %), EIEC
(16.7 %), aEPEC (13.1 %), ETEC (9.9 %), tEPEC
(6.9 %)
Multicenter study
(data from Peru)
Group 1: 0–11 months 2047 diarrhea
episode stools and
3185 surveillance
stools
Cases 0–11 months: EAEC (15.1 %), ETEC (5.7 %),
aEPEC (4 %)
Platts-Mills et al.
(2015 )
Controls 0–11 months: EAEC (16.1 %), aEPEC (4.8 %),
ETEC (4.4 %)
Group 2: 12–24 months
(community surveillance)
Cases 12–24 months: EAEC (18.9 %), ETEC (7.6 %),
aEPEC (7.2 %)
Controls 12–24 months: EAEC (20.2 %), aEPEC
(7.3 %), ETEC (7.1 %)
W.P. Elias and F. Navarro-Garcia

49
Colombia Children <5 years old 466 cases and 349
controls
Cases: EAEC (0.3 %), ETEC (4.9 %), EPEC (1.4 %),
EIEC (0.3 %), mixed DEC (0.6 %)
Gomez- Duarte
et al. (2013 )
Controls: EAEC (0.9 %), ETEC (2.3 %), EPEC (0.3 %)
Mexico Hospitalized children with acute
diarrhea <5 years old
831 cases EAEC (24 %), DAEC (35 %), aEPEC (16 %), ETEC
(9 %), tEPEC (4 %), STEC (0.4 %), EIEC (0.4 %), mixed
DEC (10 %)
Patzi-Vargas
et al. (2015 )
Nicaragua Children <5 years old
(households)
337 cases and 106
controls
Cases: EAEC (3.6 %), EPEC (11.3 %), ETEC (7.7 %),
EHEC (3 %)
Becker-Dreps
et al. (2014 )
Controls: EAEC (4.7 %), EPEC (14.2 %), ETEC (6.6 %),
EHEC (0.9 %)
Panama Children <5 years old (rural
residents)
87 cases EAEC (12.6 %) Jimenez
Gutierrez et al.
(2014 )
Uruguay Children <5 years old (high
socioeconomic level, households)
59 cases EAEC (6.8 %), aEPEC (10.2 %), tEPEC (5.1 %), STEC
(5.1 %)
Varela et al.
( 2015 )
EAEC enteroaggregativeE. coli, aEPEC atypical enteropathogenicE. coli, tEPEC typical enteropathogenicE. coli, ETEC enterotoxigenicE. coli, STEC Shiga
toxin-producingE. coli, EHEC enterohemorrhagicE. coli, EIEC enteroinvasiveE. coli, DAEC diffusely adherentE. coli, DEC diarrheagenicE. coli
2 Enteroaggregative Escherichia coli (EAEC)

50
receptor of fi bronectin is integrin α5β1, Izquierdo and colleagues evaluated t
participation of this receptor in the fi bronectin-mediated adherence of
strain 042 to intestinal cells (Izquierdo et al.2014a ). They identifi ed the complex
AafA/fi bronectin/integrin α5β1 and showed that EAEC strain 042 has the abil
to bind directly and indirectly to integrin α5β1; the indirect binding is mediate
AAF/II and fi bronectin. Subsequent studies confi rmed the binding to laminin
showed the involvement of the major subunit of AAF/II fi mbriae (AafA) in the
binding to cytokeratin 8, indicating a role of CK8 as a potential receptor for EA
(Izquierdo et al.2014b ).
New atomic resolution insights on the structure of AAFI and AAF/II w
achieved by X-ray crystallography and nuclear magnetic resonance stru
(Berry et al.2014 ). The major pilin subunits (AggA and AafA) assemble into line
polymers by donor strand complementation, where a single minor subunit (Ag
and AAfB) is inserted at the tip of the polymer by accepting the donor strand
the terminal major subunit. The minor subunits are conserved while the majo
units display large structural differences. In spite of that, both AAF recofi bronectin as receptor. All together, these data reinforce the role of
cytokeratin 8, and laminin as receptors for AAF.
3.3 Hybrid EAEC/STEC Strain
Rapid genome sequencing and public availability of these data from the EAEC
STEC outbreak strain allowed the identifi cation of an O-antigen-specifi c bact
riophage tail spike protein encoded in the genome (Scholl et al.2012 ). These
authors synthesized this gene and fused it to the tail fi ber gene of an R-type
cin, a phage tail-like bacteriocin, and expressed the novel bacteriocin such th
the tail fi ber fusion was incorporated into the bacteriocin structure. The resu
particles have bactericidal activity specifi cally againstE. coli strains that produce
the O104 lipopolysaccharideantigen,includingthe outbreakstrain. This
O-antigen tail spike-R- type pyocin strategy provides a platform to respond ra
idly to emerging pathogens upon the availability of the pathogen’s gen
sequence (Scholl et al.2012 ).
Recently, Carbonari et al. (2014 ) reported the fi rst isolation of an EAEC O104:
strain associated with an acute diarrhea case in Argentina (Carbonari et al.2014 ).
The identifi ed E. coli strain was susceptible to all antimicrobials tested and
the aggR , aaiC , pAA plasmid,lpfO113 , rfbO104 ,fl iCH4, and terD genes. Although
serotype EAEC O104:H4 rarely spreads and sporadic cases have been r
global concern increased after the large-scale outbreak in Europe in 2011. Th
ing of EAEC O104:H4 reinforces the need for improved methodologies f
detection of allE. coli pathotypes (Carbonari et al. 2014 ).
W.P. Elias and F. Navarro-Garcia

51
More recently, Ross et al. (2015 ) studied the role of long polar fi mbriae (lpf )lpf1
and lpf2 operons encoded in E. coli O104:H4 (Ross et al. 2015 ). IsogeniclpfA1 and
lpfA2major fi mbrial subunit mutants were assessed their ability to adhere to
tinal epithelial cells. The Δ lpfA1showed decreased adherence in both cell systems
while the Δ lpfA2 only showed a decrease in adherence to polarized Caco-2 c
Additionally, it was found that the Δ lpfA1 was unable to form a stable biofi lm
in an in vivo murine model of intestinal colonization, the Δ lpfA1 had a reduc
ity to colonize the cecum and large intestine. Further, in competitive a
presence of the wild-type O104:H4 facilitates increased adherence of the Δ lp
levels exceeding that of the wild type in the in vitro and in vivo models. Thus,
data demonstrated that Lpf1 is one of the factors responsible for O104:H4 in
adhesion and colonization (Ross et al.2015 ).
4 Conclusions
In conclusion, much progress has been made in recent years towards unders
ing the pathogenesis and epidemiology of EAEC. It has been consistently sho
that EAEC is a globally important pathogen, affecting both children and adult
unlike other diarrheagenicE. coli pathotypes that are only prevalent in developing
countries or agents of sporadic outbreaks. The role of EAEC as an agent of ur
tract infections was also demonstrated recently, indicating its relevance
pathogen of extraintestinal infections, another peculiar characteristic of EAEC
The ability of a strain of EAEC O104:H4 in acquiring the Stx phage generate
highly virulent pathogen that was responsible for the largest outbreak of diar
and HUS reported to date. This hybrid EAEC/STEC was detected in strains of
other serotypes but related to small HUS outbreaks or cases of bloody diarrhe
Another advance in the knowledge of the pathogenesis of EAEC was the close
relationship between growth and cognitive impairment in malnourished child
and EAEC as the agent of repeated cases of diarrhea or even in asymptomati
colonization. New challenges concerning EAEC research reside in the defi niti
of which set of virulence genes defi ne the virulent strains within the
heterogeneity of this pathotype; the choice of immunogenic antigens with pa
protection coverage for these heterogeneous strains; and the comprehension
mechanisms and bacterial factors involved in the intestinal infl ammation pro
induced by EAEC.
AcknowledgmentsThe authors were supported by a Conacyt grant (221130) to FNG and by
FAPESP and CNPq grants to WPE. We also thank Paul S. Ugalde for the artistic work in Fig.2.2,
Lucia Chavez-Dueñas for organizing the reference database and Fernando H. Martins for h
with collecting epidemiological data in American countries.
2 Enteroaggregative Escherichia coli (EAEC)

52
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2 Enteroaggregative Escherichia coli (EAEC)
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