International Journal of

Molecular Sciences

Review
Virulence Factors of Enteric Pathogenic Escherichia coli:
A Review
Babak Pakbin 1,2,3 , Wolfram M. Brück 1, * and John W. A. Rossen 2

1 Institute for Life Technologies, University of Applied Sciences Western Switzerland Valais-Wallis,
1950 Sion 2, Switzerland; [email protected]
2 Department of Medical Microbiology and Infection Prevention, University Medical Center Groningen,
University of Groningen, 9713 GZ Groningen, The Netherlands; [email protected]
3 Medical Microbiology Research Center, Qazvin University of Medical Sciences, Qazvin 15315-3419, Iran
* Correspondence: [email protected]

Abstract: Escherichia coli are remarkably versatile microorganisms and important members of the
normal intestinal microbiota of humans and animals. This harmless commensal organism can acquire
a mixture of comprehensive mobile genetic elements that contain genes encoding virulence factors,
becoming an emerging human pathogen capable of causing a broad spectrum of intestinal and
extraintestinal diseases. Nine definite enteric E. coli pathotypes have been well characterized, causing
diseases ranging from various gastrointestinal disorders to urinary tract infections. These pathotypes
employ many virulence factors and effectors subverting the functions of host cells to mediate their
virulence and pathogenesis. This review summarizes new developments in our understanding of
diverse virulence factors associated with encoding genes used by different pathotypes of enteric
pathogenic E. coli to cause intestinal and extraintestinal diseases in humans.






Keywords: enteric pathogenic Escherichia coli; E. coli pathotypes; virulence factor genes
Citation: Pakbin, B.; Brück, W.M.;
Rossen, J.W.A. Virulence Factors of
Enteric Pathogenic Escherichia coli: A
Review. Int. J. Mol. Sci. 2021, 22, 9922.
https://doi.org/10.3390/ 1. Introduction
ijms22189922 Escherichia coli, rod-shaped and gram-negative bacteria belonging to the Enterobacteri-
aceae family, were first isolated from infant stool and characterized by Theodor Escherich
Academic Editor: Tanneke Den in 1885 [1]. A few hours after birth, E. coli colonize and inhabit the gastrointestinal tract of
Blaauwen infants. Regarding several mutual benefits, humans and commensal E. coli strains coexist
without any adverse effects. However, commensal E. coli may cause disease in patients with
Received: 15 August 2021
breached gastrointestinal barriers or immunocompromised hosts [2]. Notably, certain E.
Accepted: 12 September 2021
coli mediate various diseases, including intestinal and extraintestinal disorders in humans
Published: 14 September 2021
and animals worldwide. Nine pathovars have been described for E. coli strains isolated
from humans causing diarrheagenic and extraintestinal diseases [3]. Of these, seven patho-
Publisher’s Note: MDPI stays neutral
types have been described as enteric pathogenic E. coli, including Enteropathogenic E. coli
with regard to jurisdictional claims in
(EPEC), Enterohaemorrhagic E. coli (EHEC), Enterotoxigenic E. coli (ETEC), Enteroinvasive
published maps and institutional affil-
E. coli (EIEC), Enteroaggregative E. coli (EAEC), Diffusely adherent E. coli (DAEC), and, a
iations.
new pathotype, Adherent-Invasive E. coli (AIEC), causing mostly diarrhea and intestinal
disorders. However, the EHEC pathotype has been implicated in extraintestinal diseases
such as Hemolytic Uremic Syndrome (HUS) [4]. Many of these pathotypes constitute public
health concerns as foodborne pathogens and caused several fatal outbreaks in developing
Copyright: © 2021 by the authors.
and developed countries [5,6]. Enteric E. coli pathotypes are implicated in many diseases
Licensee MDPI, Basel, Switzerland.
through distinctly different pathogenesis. Pathogenesis is the process by which pathogens
This article is an open access article
cause disease or disorder, often by expressing virulence-factor-encoding genes [7,8].
distributed under the terms and
Virulence factors are specific molecules, primarily proteins produced and released by
conditions of the Creative Commons
bacteria, fungi, protozoa, and viruses. These factors are encoded by specific genes located
Attribution (CC BY) license (https://
on the chromosome or mobile genetic elements (e.g., plasmids or transposons) in bacterial
creativecommons.org/licenses/by/
4.0/).
pathogens [9,10]. Each E. coli pathotype has its characteristic pathogenicity mechanisms and

Int. J. Mol. Sci. 2021, 22, 9922. https://doi.org/10.3390/ijms22189922 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 9922 2 of 18

a specific profile of virulence factors encoded by specific gene clusters. Genes associated
with pathogenicity may encode activities such as adhesion, invasion, attachment, iron
acquisition, motility, and toxin activity, among others. We can distinguish four main
virulence classes of E. coli pathotypes: colonization, fitness, toxins, and effectors; each
consists of several specific virulence factors with a definite function and activity (Table 1).
It is noteworthy that different enteric and extraintestinal pathotypes of E. coli isolates
share the same virulence factors and strategies [11]. Many virulence factors associated
with enteric E. coli pathotypes implicated in intestinal and extraintestinal disorders have
been identified in the last years [12]. The investigation of these virulence factors and the
associated encoding genes can provide explicit knowledge about the interaction between
these factors in enteric E. coli pathotypes and host proteins at the molecular level, indicating
how they lead to diseases and can implement preventive strategies against them [13]. This
review summarizes recent advances in our understanding of virulence factors and the
associated genes in enteric pathogenic E. coli.

Table 1. Virulence factor genes of enteric E. coli pathotypes: Colonization, fitness, toxins, and effectors.

Class Virulence Factor Activity/Function Pathotype

Colonization bfp adherence, EPEC
eae attaching and effacing of enterocyte, EPEC, EHEC
tir translocated intimin receptor, EPEC, EHEC
lifA initial attachment to the enterocytes, EPEC
csgA curli fimbriae, EPEC, EHEC
fimA type I fimbriae, EPEC, EHEC, DAEC
fimH type I fimbriae, ETEC, AIEC
bcsA cellulose structure, EPEC
eha biofilm formation, EHEC
saa biofilm formation, EHEC
sab biofilm formation, EHEC
toxB biofilm and adhesion establishment, EHEC
nleB biofilm formation, EHEC
nleE biofilm formation, EHEC
nleH biofilm formation, EHEC
bleG biofilm formation, EHEC
lfp long polar fimbriae, initial attachment, EHEC
CFA/I colonization factor, ETEC
CFA/II colonization factor, ETEC
CFA/IV colonization factor, ETEC
CS1-6 colonization (coli surface antigen), ETEC
etpA initial attachment, ETEC
aggR attachment and adherence, EAEC
aggA aggregative adhesion fimbria, EAEC
aafA aggregative adhesion fimbria, EAEC
agg3A aggregative adhesion fimbria, EAEC
agg4A aggregative adhesion fimbria, EAEC
agg5A aggregative adhesion fimbria, EAEC
aap dispersin, dispersion of EAEC, EAEC
afaA-E Afa/Dr adhesins, IL-8 secretion, DAEC
cytoskeleton rearrangement, destroying microvilli,
DraA-E DAEC
Afa/Dr adhesins, IL-8 secretion, expression of MICA
cytoskeleton rearrangement, destroying microvilli,
daaA-E DAEC
Afa/Dr adhesins, IL-8 secretion, expression of MICA
pop type I pili, adhesin, DAEC
Int. J. Mol. Sci. 2021, 22, 9922 3 of 18

Table 1. Cont.

Class Virulence Factor Activity/Function Pathotype

Fitness sdiA quorum sensing signaling, EPEC
iutA aerobactin synthesis, EIEC
iucB complex siderophore iron receptor, EIEC
yjaA polypeptide stress response protein, AIEC
fyuA ferric yersiniabactin uptake, AIEC
kpsMT II capsule synthesis, AIEC
shiga toxin, surface localization of nucleolin and
Toxins stx1 EHEC
cytotoxic effect,
shiga toxin, surface localization of nucleolin and
stx2 EHEC
cytotoxic effect,
ST I toxin, watery and secretory diarrhea, secretion of
estA ETEC
chemokines and cytokines,
ST II toxin, watery and secretory diarrhea, secretion of
estB ETEC
chemokines and cytokines,
LT I watery diarrhea, ETEC
LT II watery diarrhea, ETEC
eatA SPATE, ETEC, EIEC
astA enteroaggregative heat-stable toxin, secretory diarrhea, ETEC, EAEC, DAEC
ShET1 shigella enterotoxin 1, secretary intestinal activity, ETEC
ShET2 shigella enterotoxin 2, secretary intestinal activity, ETEC
SPATE, plasmid encoded toxin, inducing epithelial cell
pet EAEC, DAEC
extrusion, host cell entering,
SPATE, ShET1 expression, inducing epithelial cell
pic EIEC, EAEC
extrusion, mucolytic activity,
sigA SPATE, cytotoxin, accumulation of intestinal fluid, EIEC, EAEC, DAEC
SPATE, secreted autotransporter toxin, impairing tight
sat EAEC, DAEC
junction, mediating autophagy,
shigella extracellular enterotoxin, cytotoxin, IgA
sepA EAEC
protease like homologue,
hlyE alpha hemolysin toxin, EAEC
Effectors espA translocator structures of T3SS, E. coli common pili, EPEC, ETEC
espB translocator structures of T3SS, phagocytosis inhibition, EPEC
espC cleavage of T3SS translocator structures, EPEC
espD translocator structures of T3SS, EPEC
mitochondrial death, tight junction disruption, immune
espF EPEC, EHEC
evasion, host cell death,
espH phagocytosis inhibition, EPEC
espJ phagocytosis inhibition, biofilm formation, EPEC, EHEC
espP cleavage of T3SS translocator structures, EPEC, DAEC
espT host cell death, EHEC
disrupt mitochondrial membrane functionality, host cell
MAP EPEC
death,
inflammasome activation, tight junction disruption,
nleA EPEC
cytokines secretion inhibition,
etp Autotransporter protein, ETEC
tolC secretion of ST toxins, ETEC
nleF host cell death, inflammasome activation, EPEC, EHEC
cif cell cycle disruption, delays apoptosis, EPEC
Type III effector, cytoskeleton reorganization, cell death
ipaA EIEC
blockage,
Type III effector, adhesion, phagosome escape, cell
ipaB EIEC
turnover,
Type III effector, adhesion, actin polymerization,
ipaC EIEC
phagosome escape,
Type III effector, adhesion, phagosome escape, cell death
ipaD EIEC
blockage,
Int. J. Mol. Sci. 2021, 22, 9922 4 of 18

Table 1. Cont.

Class Virulence Factor Activity/Function Pathotype

ipaH dampen the inflammatory responses, invasion, EIEC, AIEC
inhibition of host cell trafficking membrane, inhibition
ipaJ EIEC
of inflammasomes,
ipgB1 cytoskeleton reorganization, ruffle formation, EIEC
ipgD cytoskeleton reorganization, EIEC
cytoskeleton reorganization, cell death blockage,
virA EIEC
autophagy inhibition,
virB virulence factor gene synthesis, EIEC
virF virulence factor gene expression, EIEC
virG Actin nucleation, EIEC
ospB dampen the inflammatory responses, EIEC
ospE cell detachment, EIEC
ospF dampen the inflammatory responses, EIEC
ospG dampen the inflammatory responses, EIEC
ospI dampen the inflammatory responses, EIEC
ospZ dampen the inflammatory responses, EIEC
icsB autophagy inhibition, ETEC
aaiA-Y type VI secretion system (T6SS), EAEC
ibeA invasion protein ibeA, AIEC

2. Enteropathogenic Escherichia coli (EPEC)

EPEC is the principal cause of diarrheal diseases and outbreaks in infants characterized
for the first time in the UK in 1945, with a high morbidity and mortality rate in children
under six months of age. The EPEC rate in developed countries has decreased in recent
decades. However, it is one of the major public health concerns for infants and adults in
developing countries [14]. Some pathogenesis mechanisms of EPEC, such as the attaching
and effacing (A/E) virulence factors, are shared with pathogens such as rabbit-EPEC,
Citrobacter rodentium in mice, EHEC, and Escherichia albertii [15]. Foodstuffs, particularly
milk and ground beef, are a vehicle of EPEC transmission to humans and animals and lead
to intestinal infections [16–18].
Histopathologically, EPEC belong to the A/E pathogens. The main virulence-factor-
encoded genes of EPEC are located on a 35.62 kb chromosomal pathogenicity island (PAI)
within 41 open reading frames (ORFs) and a pathogenic plasmid-termed locus of enterocyte
effacement (LEE) and EPEC adherence factor (EAF), respectively [19]. LEE gene expression
is modulated by the LEE-encoded regulator gene, located on the first part of the LEE.
Another PAI of EPEC is termed the EspC island, encoding a serine protease autotransporter
enterotoxin gene with the same name, and this is the only toxin released by EPEC, which
can lead to epithelial cell necrosis [14]. Regarding the EAF plasmid presence/absence,
EPEC is classified into typical (t-EPEC) with complete virulence genes and atypical EPEC
without EAF-harboring operon for bundle type IV pili (bfp) [20]. The most prominent
virulence factors of EPEC in both typical and atypical pathogens are located on the LEE.
This PAI is a type-three secretion system (T3SS) comprising the EspA, EspB, and EspD
(EPEC secreted protein) translocator proteins, effector proteins (Map, EspF, EspG, EspH,
EspZ), the proteins involved in intimate adherence, and regulatory elements [21]. Gut
microbiome surface molecules, oxygen levels, and hormones in the environment mediate
the signaling of LEE-activating transcription. A/E is initiated with the reaction between
the outer membrane intimin and the translocated intimin receptor encoded by eae and tir
genes, respectively [22]. Before T3SS expression following a stable attachment by intimin-
Tir interaction and effectors injection, EPEC sticks to the surface of the host cells (initial
attachment) using pili-like structures [23]. Type-IV pili (bfp) in a-EPEC and the lymphocyte
inhibitory factor (lifA/efa1) in t-EPEC mediate the initial attachment to the enterocytes
located in the small bowel considering the first step of A/E following the formation
of localized microcolonies. T3SS is a complex injectisome machine in molecular nano-
Int. J. Mol. Sci. 2021, 22, 9922 5 of 18

scale including more than 20 main proteins and consisting of three principal extracellular
appendages: the needle, filament, and translocation pore; structurally, it is composed of a
syringe with a central channel embedded into the bacterial membrane by ring structures
and a supramolecular structure on the top of the needle connected to the host cell [24].
EspA, espB, and espD proteins construct different translocator structures of T3SS, and these
proteins are cleaved by espP and espC [25]. After the translocation of Tir, a strong attachment
is formed while the translocated protein interacts with intimin. The first result of the
Tir–intimin interaction is the induction of a cytoskeletal rearrangement and the pedestal
formation underneath the pathogen attachment side in the host cell [26]. The translocated
Tir protein is phosphorylated by tyrosine kinase in the host cell. The phosphorylation
of Tir recruits the NcK protein leading to the activation of the neural Wiskott–Aldrich
syndrome protein (N-WASP), the stimulation of the actin-related protein (ARP) 2/3, and the
mediation of actin polymerization and rearrangement, which results in pedestal formation.
This cytoskeletal rearrangement contributes to immune modulation and diarrhea. A thick
biofilm formation also has a crucial role in EPEC pathogenesis [27]. The activation of
biofilm formation is mediated by Quorum-sensing encoded by the suppressor of the
division inhibitor (SdiA) gene. However, for biofilm formation, bacteria demand structures
to support these characteristic curli fimbriae, type-I fimbriae, and the cellulose encoded in
EPEC by csgA, fimA, and bcsA genes, respectively [28].
The next step in EPEC pathogenesis is the translocation of LEE and Nle effectors and
proteins into the host cell through the T3SS. Some of these effectors efface the host cell with
multiple functionalities. In addition to attachment and actin pedestal formation, Tir inhibits
the NF-kB signaling as the immune evasion mechanism in EPEC pathogenesis [29]. The
mitochondrial-associated protein (MAP) shares the Trp-xxx-Glu motif conferring GTPase
GEF activity. GEF induces the Cdc42 factor and contributes to filopodia formation at the
bacterial attachment site. MAP also disrupts the mitochondrial membrane functionality,
contributing to the host cell’s death. Another prominent effector is NleA (espI), having
many roles, as proceeding with the NLRP3 inflammasome activation, tight junction dis-
ruption, and inhibition of the secretion of host cell cytokines [30]. EspF is a multi-function
effector inducing mitochondrial death, disrupting the tight junction integrity, and impli-
cated in phagocytosis inhibition, contributing to the immune evasion of EPEC during the
pathogenesis. EspB, H, and J genes also expressed proteins to inhibit phagocytosis [31].
Another multi-functional non-LEE effector is nleF, which activates the inflammasome by
binding to caspase-4, dampening the cellular immune response. EspF, espT, MAP, nleF, and
the cycle-inhibiting factor (Cif) induce host cell death by blocking the progression of the
cell cycle. Despite the identification of the functions of these effectors, multi and single
effects of many Nle and esp proteins secreted by EPEC and other A/E pathogens have not
been characterized yet (Table 1) [32].

3. Enterohaemorrhagic Escherichia coli (EHEC)

EHEC cause diarrhea, hemorrhagic colitis (HC) with bloody diarrhea, and hemolytic
uremic syndrome (HUS) in humans and are implicated in several foodborne outbreaks in
developed countries. The main reservoir of this pathogen is the intestinal tract of cattle.
EHEC was first isolated from a patient with bloody diarrhea and gastrointestinal disorder
in 1982 and led to a worldwide pandemic [33]. EHEC as foodborne A/E pathogens are
mainly transmitted to humans through contaminated food and water [34]. Outbreaks
of EHEC serotypes usually have occurred via the fecal-oral route by person-to-person
transmission, animal contact, and consumption of under-thermally processed foods as well
as undercooked meat products, unpasteurized apple juice, raw milk, or cross-contaminated
raw vegetables such as lettuce and bean sprouts. Raw flour has also been recently identified
as a source of EHEC outbreaks [8,35–37].
The most important serotype playing a role in EHEC outbreaks is O157: H7, which
is still considered a serious health concern in Japan, Europe, and North America. EHEC
serotype O104: H4 was first isolated from a gastrointestinal infection and HUS outbreak
Int. J. Mol. Sci. 2021, 22, 9922 6 of 18

by the consumption of sprouts in Germany in 2011. From there, it was spread out around
the world. However, the genome sequencing of this serotype revealed that it belongs to
both EHEC and enteroaggregative E. coli (EAEC) pathotypes, introducing a new emerged
pathotype of E. coli termed enteroaggregative hemorrhagic E. coli (EAHEC) [34,38,39].
The shiga-like toxin (SLT), also termed verotoxin and encoded by stx genes, is the main
virulence factor in EHEC serotypes which belongs to the shiga-toxin producing E. coli group
and is responsible for pathological manifestations leading to specific disease symptoms
caused during EHEC infections, such as HUS and renal failure [8,40]. SLT includes two
subgroups, stx1 and stx2, and different subtypes. Stx2a, stx2c, and stx2d positive EHEC
isolates are strongly associated with HC and HUS compared to other stx-subgroups and
subtypes [41,42]. The shiga toxin is an AB5 toxin. Subunit A is cleaved into two fragments,
A1 and A2 , by reducing a disulfide bond, releasing the A1 fragment into the cytoplasm. It
exerts the cytotoxic effect by enzymatically deadenylating position 4324 of 28s rRNA of the
60s ribosome, leading to the inhibition of protein synthesis and cell death [43]. In addition,
to triggering ribotoxic stress, the A1 fragment of stx induces cytokine production and
activates the apoptotic cell pathways. Protein translation inhibition and immune response
modulation are mainly responsible for the pathogenesis of shiga toxin’s subunit A. The A
subunit fragments need non-covalent binding with homopentameric B subunits to enter
the target cell and induce the cytotoxic effect. B subunits of shiga toxins specifically bind
to the carbohydrate moiety of glycosphingolipid globotriaosylceramide (Gb3 or CD77), a
specific receptor molecule found on the surface of kidney epithelial and intestinal Paneth
cells in humans. After receptor binding and Gb3 -stx complex formation, the attached
toxins form a cluster, membrane invagination, and endocytic pit formation at the plasma
membrane of the cell [44]. These invaginations separate from the plasma membrane to form
intracellular toxin carriers. Intracellular trafficking of the endocytic carriers is performed
by moving from the early endosome to the Golgi and the endoplasmic reticulum before stx
escapes from the Gb3 -stx complex to exert the cytotoxic effect. Stx toxins are transported
to the non-target (Gb3 -negative) cells by neutrophil transmigration, micropinocytosis,
and Gb3 -independent transcytosis. Inside Gb3 -negative cells, stx toxins only induce an
inflammatory response and do not prevent protein synthesis [45]. There is a lack of a
definite secretion system to release stx toxins by EHEC serotypes. Shiga toxins are released
through phage-mediated lysis while specific triggers, including SOS-inducing agents such
as antibiotic therapy or DNA damages, depress the transcription of lytic phase genes. This
contributes to the lysis of the STEC bacterial cell and the release of shiga toxins into the
extracellular milieu [46].
EHEC serotypes are a subgroup of STEC strains containing adhesin and attachment
virulence factors encoded by the LEE pathogenicity island. Adhesin proteins are involved in
the colonization and biofilm formation of EHEC on the abiotic and biological surfaces [47].
The first step of EHEC adherence to intestinal epithelial cells is initial attachment. Recent
studies showed that the initial contact and attachment between the pathogen and the host
cell are mediated through the interaction between the pathogen’s long polar fimbriae (lfp
gene) and extracellular membrane proteins in the host cell, including fibronectin, collagen
IV, and laminin [48]. They closely intimate the attachment, and the A/E effect occurs via
the interactions of intimin (encoded by eae gene) and the receptor proteins in the host cell
membrane, consisting of Tir (injected through T3SS), nuclein, and β1-integrins. The A/E
pathogenic mechanism in EHEC is different from that in EPEC. Pedestal formation and
actin rearrangement are mediated through an Nck-independent mechanism. Cell actin
cytoskeleton rearrangement is induced while the Tir–intimin complex links to the EspF
protein by a homologue protein of insulin receptor substrate [49]. N-WASP and ARP2/3 are
activated for actin assembly via interaction with the induced EspF. Curli (encoded by csg
genes) are recognized as adherence and colonization factors. Colonization, microcolonies,
and biofilm formation are mediated by E. coli common pilus (ECP) and Hemorrhagic
E. coli common pilus (HCP) via interaction with the surface of the host cell [49]. Some
proteins, such as type 1, F9, and E. coli laminin-binding fimbriae are involved in the
Int. J. Mol. Sci. 2021, 22, 9922 7 of 18

adhesion to the host cell. Autotransporters including Eha, Saa, and Sab proteins released
by EHEC contribute to biofilm formation and adhesion [50]. Moreover, a plasmidal gene
termed ToxB, located on the pO157 plasmid, may be involved in the adhesion in serotype
O157: H7 and other EHEC strains. It is worthwhile to note that specific environmental
conditions such as pH, temperature, and nutrient limitations induce the transcription and
expression of adhesin genes through some signaling systems, such as the secretion of
quorum-sensing molecules [51]. EHEC serotypes secrete twice as many effectors into the
host cell via T3SS than EPEC. Host inflammatory responses induced by A/E, colonization,
and biofilm formation also contribute to the symptoms of GI disorders. NleF, as a non-LEE
effector, is considered a prominent virulence factor because of the counteraction to the
host inflammatory response and inhibition of cell death. Several non-LEE-effectors such as
NleB, NleE, BleG, NleH, and EspJ have been recently shown to mediate EHEC survival and
biofilm formation (Table 1) [52–54].

4. Enterotoxigenic Escherichia coli (ETEC)

The E. coli pathotype ETEC releasing enterotoxins in the human small intestine is the
principal cause of travelers’ and children diarrhea in developing countries. The highest
infection and mortality rates of ETEC are found in children under the age of two. Ac-
cording to the World Health Organization’s reports, ETEC annually causes more than
157,000 human diarrheal cases that lead to death [55]. The main symptoms caused by ETEC
infection are mild to severe diarrhea and abdominal pain. Other symptoms, such as vom-
iting, nausea, fever, and headache have rarely been observed [56]. ETEC are also animal
pathogens causing acute diarrheal diseases in cattle, poultry, and piglets [57]. Generally,
the two main virulence factors in ETEC that lead to diarrhea in humans are colonization
factors and enterotoxins [58].
ETEC first adhere to the small intestine epithelial cells via surface structures and
then secrete the enterotoxins. Colonization plays a fundamental role in the initial step of
the pathogenesis mechanism of ETEC. ETEC engage with small bowel epithelial cells by
various plasmid-encoded colonization factor (CFs) genes encoding fimbrial, non-fimbrial
and fibrillar structures [59]. Among many CFs, CFA/I, CFA/II, and CFA/IV are the main
factors to mediate the colonization of ETEC and have been highly detected in ETEC isolated
from clinical samples. Other proteinaceous fimbrial and non-fimbrial structures include
coli surface antigens (CS) such as CS1-CS6, type 1 fimbriae (fimH), E. coli common pili
(ecpA) [60], autotransporter proteins such as the Etp group of proteins, and EatA (a member
of SAPTE family). Flagella promote the initial attachment, adhesin, and colonization of
ETEC to the host intestinal epithelial cells [61]. The EtpA secreted by ETEC is a glycoprotein
containing N-acetylgalactosamine (NAG). NAG also presents as the sugar moiety on A
blood group glycans. Therefore, ETEC contributes to a more severe diarrheal disease
among humans with Type A blood [62].
The most prominent and effective virulence factor of ETEC is the secretion of entero-
toxins. Two types of enterotoxins, heat-stable toxins (STs) and heat-labile toxins (LTs), are
secreted by ETEC and activate cyclic nucleotide production, contributing to intestinal net
water, salt, and fluid loss, causing secretory diarrhea in humans and animals. LTs, STs, or
a combination of both toxins might be expressed by ETEC strains [63]. Genes encoding
enterotoxins are located on both plasmids and the chromosome (prophages), which can
be transferred among the different E. coli pathotypes. A 72 amino-acid (aa) protein (as the
toxin precursor) is synthesized, of which a 19–18 aa region is released as the active form
of the heat-stable toxin. These toxins are divided into STI and STII encoded by estA and
estB genes, respectively located on plasmids. Considering the host species, STI is classified
into STp (18 aa) and STh (19 aa) subtypes, mainly found in porcine and human ETEC
strains, respectively. However, both subtypes have been detected in ETEC isolated from
humans [64]. The structure of these toxins mimics the biologic function of mammalian
peptides, including guanylin and uroguanylin, binds to the guanylate cyclase C receptor,
and increases the intracellular cyclic guanosine monophosphate (cGMP) via the hydrolysis
Int. J. Mol. Sci. 2021, 22, 9922 8 of 18

of guanosine triphosphate. The increase in cGMP concentration activates the protein kinase
that phosphorylates the cystic fibrosis transmembrane regulatory (CFTR) channel. The
activated CFTR channel inhibits ion exchange and sodium reabsorption, contributing to
the release of water and salt into the intestinal lumen and thus net fluid loss, leading to
watery and secretory diarrhea [65,66]. The putative export channel for the secretion of ST
is an outer membrane protein encoded by the tolC gene. The EtpA adhesin factor gene,
located on a plasmid, expresses a protein to construct a molecular bridge structure between
bacteria and the host cell. Without the expression of tolC and EtpA genes, the delivery of
ST to the target cells is not performed. These genes have also been considered the main
virulence factors of ETEC [67]. Another mechanism of ST triggering diarrhea is the tight
junction modulation of intestinal epithelial cells, leading to an increase in permeability by
suppressing the expression of proteins and disturbing the integrity of the tight junction.
In addition, ST toxins mediating the innate immune responses led to the secretion of
chemokines and cytokines such as IL-6 and IL-8 [63].
Another toxin secreted by ETEC after colonization of the small intestine is a heat-
labile toxin. Two types of LT have been identified, LT type I and II. LT (type I) is also
categorized into LTh and LTp subtypes, detected in strains isolated from humans and pigs.
However, LT II is mainly associated with strains isolated from animals and occasionally
from humans [67]. LT is initially synthesized as a holotoxin in the periplasm and then
released via two secretion systems: a type-II secretion system (T2SS) and outer membrane
vesicles (OMV). ETEC mainly secretes LT through the OMV system. ST and LT toxins
are composed of a single A subunit with enzymatic activity and a pentameric B subunit
binding to the GM1 ganglioside receptor on the host cell surface [68]. The structure and
pathogenesis mechanism of LT is identical to that (about 80%) of the cholera toxin secreted
by Vibrio cells. After delivery and translocation into the host cell, the A subunit induces
the ADP-ribosylation factor and inhibits GSα GTPase activity, leading to the activation of
adenylate cyclase and the increase of intracellular cyclic adenosine monophosphate (cAMP)
concentrations. Higher levels of cAMP contribute to the activation of the CFTR ion channel,
followed by the secretion of water and electrolytes, leading to watery diarrhea [69]. By
interacting with the carbohydrate-binding sites, LT also binds to blood groups A and B
determinants. LT also facilitates the ETEC pathogenesis by improving the initial adherence,
intestinal colonization, and increased virulence factors expression [64]. There are other
virulence factors detected in ETEC strains isolated from humans with diarrhea. Secretion
of an enteroaggregative heat-stable toxin (EAST1) by ETEC, encoded by the ast gene, with
a similar function to the ST toxin, leads to increased intracellular levels of cGMP. The
SPATE EatA, located on a large virulence plasmid, also increases the virulence of ETEC by
facilitating the LT delivery (Table 1) [70].

5. Enteroinvasive Escherichia coli (EIEC)

Severe mucosal and bloody diarrhea with abdominal cramps and fever are typical
clinical features of bacillary dysentery or shigellosis caused by different species of Shigella
and EIEC as invasive foodborne pathogens [13,71]. Initially known as Bacillus dysenteriae,
Shigella was previously characterized in 1897 by Kiyoshi Shiga in Japan after a severe
epidemic causing more than 91,000 cases, with 20% mortality [72]. Fifty years later, EIEC
was discovered and characterized with the same genetic, pathogenic, and biochemical
properties as were observed for Shigella. Thus, they may be categorized in a single pathovar.
However, a considerable distinction is still maintained regarding the clinical significance
of Shigella species [1,73]. EIEC and Shigella are unable to ferment lactose, are non-motile,
and lysine decarboxylase negative. These properties are considered to differentiate Shigella
species and EIEC from other bacteria [74]. EIEC differs from other pathovars of E. coli
because it is an obligate intracellular pathogen with neither adherence nor flagella fac-
tors [71]. Shigella comprise four species, including S. sonnei, S. flexneri, S. boydii and S.
dysenteriae, which are responsible for varying degrees of diarrhea and acute intestinal and
Int. J. Mol. Sci. 2021, 22, 9922 9 of 18

extra-intestinal diseases in humans [73,75,76]. EIEC strains primarily elicit watery diarrhea
and cause invasive inflammatory colitis [77].
EIEC/Shigella infection commences with the penetration of the pathogen into the
epithelial cells in the colon, passing through the microfold cells and reaching the underlying
submucosa by a transcytosis mechanism [78,79]. The disruption and damage of tight
junctions caused by inflammation also give EIEC access to the underlying submucosa [80].
EIEC is taken up by macrophages, where it lyses the endocytic vacuole and escapes
from the phagosome. Following the release from dead macrophages, EIEC invades the
basolateral sides of the colonocytes, survives and multiplicates intracellularly, moves
directionally through the cytoplasm, and extends into the adjacent epithelial cell [13]. The
virulence of this pathogen is primarily due to 220-kb plasmid-encoding virulence factors
including the T3SS complex, chaperones, transcriptional regulators, translocators, and
more than 25 effector proteins [4]. A T3SS needle, strongly required for the invasion,
apoptosis of macrophages, and cell survival, is encoded on the mxi-spa locus of the
virulence plasmid [11]. Before the invasion, the adhesion of EIEC to the epithelial cells
is mediated via the binding of proteins encoded by the IpaBCD complex and IpaB genes
to the α1 β5 integrin and hyaluronan CD44 receptors, respectively. Membrane ruffling
and epithelial cell cytoskeleton reorganization are mediated by IpaA, IpaC, IpgB1, IpgD,
and virA to provide pathogen uptake [81]. IpaH7.8, IpaB, IpaD, and IpaC have also been
implicated in phagosome escape [82]. VirG nucleates actin by inducing the acquisition of
N-WASP, contributing to ARP2/3 formation and mediated unipolar actin tail formation on
the surface of the bacteria [83]. IpaC also activates SRC-family protein kinases to recruit the
ARP2/3 complex at the site of the bacterial contact and induces actin polymerization to
form a ruffle for bacterial entry. IpgB1 as mimicry of RhoG also promotes ruffle formation
by the activation of RAC1. Actin microfilaments and their polymerization provide the
propulsive forces needed for the directed movement of the pathogen throughout the host
cell cytoplasm [80]. EIEC provides host cell integrity and survival in the early stages of
infection by inhibiting intestinal epithelial cell detachment and turnover utilizing protein
effectors OspE and IpaB, respectively. Some effectors, including VirA, IpaA, and IpgD,
destabilize microtubules and actin and mediate the blockage of death in host cells to
promote colonization and maintain its replicative niche [4,82].
Once localized in the epithelial cell cytoplasm, EIEC suppresses the host immune
response and counteracts the immune defense system by using protein effectors to persist
and survive inside the colonocytes [84]. OspF and OspG inhibit the activation and gene
transcription of NF-кB. IpaH9.8 and OspB also suppress the expression of inflammatory
cytokine levels such as interleukin-8. Other effectors, including OspZ, OspI, and IpaH4.5 ,
also help EIEC downregulate and dampen the inflammatory responses [82]. Moreover,
effectors IcsB and VirA promote host-cell-autophagy-mediated degradation in EIEC by
sequestering the VirG and destabilizing microtubules, respectively [84]. Common clinical
symptoms of watery diarrhea in EIEC and Shigella infections have been attributed to the
additional virulence factors, including Shigella enterotoxins 1 and 2 encoded by ShET1 and
ShET2 genes, respectively. ShET1 is located on the chromosome and a pathogenicity island,
whereas ShET2 has been found in a virulence plasmid. Both enterotoxins contribute to
secretary intestinal activity. ShET2 induces inflammation in the host intestinal epithelial
cells [85]. Moreover, other toxins encoded by virulence factor genes such as Pic, SepA,
SigA, and Sat are expressed and secreted in EIEC and Shigella species. Pic, encoded by
chromosome locus, regulates ShET1 expression [80]. SepA and Pic toxin genes belong to the
serine protease autotransporters of the Enterobacteriaceae (SPATEs) family toxin. SPATEs
are protease-based toxins released by autotransporter pathways and secreted usually by
pathogenic E. coli and Shigella species [86]. The SigA-encoded cytotoxin contributes to
intestinal fluid accumulation in the EIEC or Shigella-infected hosts [86]. Recently, other
virulence factors have also been detected and identified prevalently in EIEC and Shigella
isolates, including iutA, iucB, EatA, VirF, VirB, IpaJ, and OspC3 implicated for aerobactin
synthesis, complex siderophore iron receptor, SPATE toxin, the regulation of virulence
Int. J. Mol. Sci. 2021, 22, 9922 10 of 18

factor gene expression, the control of virulence factor gene synthesis, the inhibition of
the host cell trafficking membrane, and the inhibition of inflammasomes, respectively
(Table 1) [72,79].

6. Enteroaggregative Escherichia coli (EAEC)

EAEC has been considered an emerging foodborne pathogen primarily associated
with persistent and acute childhood diarrhea and growth retardation in developed and
developing countries [13]. Moreover, EAEC is the second leading cause of acute and
persistent travelers’ diarrhea after ETEC in developed and developing nations, and one of
the major causes of enteric infections in patients with HIV/AIDS [4,52]. Other symptoms
of EAEC infection include vomiting, nausea, borborygmi, anorexia, and tenesmus [87,88].
As mentioned before, a hybrid strain of EAEC/STEC (serotype O104: H4) caused a large
outbreak in 2011 in Germany, resulting in more than 4300 diarrhea cases and 50 deaths [89].
Several studies indicated that EAEC has the potential for efficient gut inflammation and
enteric colonization, which might intensify the effects of other pathogenic bacterial vir-
ulence strategies [7]. EAEC often causes watery diarrhea, occasionally accompanied by
blood or mucus. EAEC colonizes the mucosa of small and large bowels, contributing to
mild to severe inflammation in the colon [90]. Traditionally, EAEC has been characterized
by a “stacked-brick” pattern adherence to Hep-2 cells. Due to the heterogenicity of EAEC,
the pathogenic mechanisms of this pathogen are very complicated. EAEC pathogenesis
involves three steps: adherence to the intestinal epithelium via adherence aggregative
fimbriae, biofilm formation, and secretion of toxins, mucosal inflammation, and cytotoxic
damages [32,88,91].
The virulence factors of EAEC are encoded on a family of virulence plasmids, the plas-
mid of aggregative adherence called pAA, and pathogenicity islands distributed through-
out the chromosome [92]. AggR is the main virulence factor regulator of EAEC. It is a
member of a bacterial transcriptional regulator family, AraC, and is located on the pAA
plasmid to control the expression of plasmid-borne and chromosomal virulence factor-
encoded genes [93]. Epidemiological studies showed a significant association between the
presence of the AggR gene, diarrheal diseases, and the concentrations of fecal cytokines
in patients with diarrhea infected with EAEC strains [94–96]. Therefore, EAEC strains are
divided into typical, carrying AggR, and AggR-regulated virulence factors and atypical
EAEC, lacking the AggR regulon. The first stage of EAEC pathogenesis is the attachment
and adherence to the intestinal mucosal cells [97]. The aggregative adherence of EAEC
is associated with the aggregative adhesion fimbria (AAF), which is encoded by AAF
genes [97]. Moreover, the initial attachment of EAEC to the epithelial cells is facilitated
by AAFs. Five variants of AAFs have been identified, including AAF/I–V encoded by
aggA, aafA, agg3A, agg4A, and agg5A, respectively. All of them are located on pAA. AAF
genes have exclusively been detected in EAEC pathotypes [90]. The adherence medi-
ated by AAFs and flagellin also induces cytokine responses [98]. The isoelectric point of
AAFs is relatively high (pI: 8.9–9.4) compared with other adhesins. Throughout the gut
(pH: 6–7.4), the AAFs bear a high positive charge [97]. Regarding the negative charge of
lipopolysaccharide on the surface of gram-negative bacteria, an extension of AAFs away
from the cell surface is mediated with the help of the secretion of a protein called dispersin.
Dispersin is encoded by the aap gene located on pAA and can mask the negative charge
conferred by lipopolysaccharides [99]. This protein counteracts excessive aggregation and
causes the dispersion of EAEC across the mucosal epithelial cells. Other, less critical factors
associated with EAEC adherence are alternative fimbrial structures, including type-IV
pili [100]. Biofilm formation is another pathogenic mechanism of EAEC, and it is entirely
distinct from that mediated by non-pathogenic E. coli. A type-VI secretion system (T6SS)
encoded by aaiA-Y genes, located on a pathogenicity island identified on the chromosome
and activated by AggR, mediates biofilm formation in EAEC. However, aaiA and aaiC
genes have more commonly been found in typical EAEC strains worldwide [90,101].
Int. J. Mol. Sci. 2021, 22, 9922 11 of 18

The final stage of EAEC pathogenesis is toxin secretion, causing several effects, in-
cluding enlarged crypt opening, microvillus vesiculation, and epithelial cell extrusion [88].
These putative toxins include a plasmid-encoded toxin, a protein involved in intestinal
colonization, a Shigella extracellular enterotoxin, a secreted autotransporter toxin, and an
enteroaggregative heat-stable toxin, which are encoded by the pet, pic, sigA, sepA, sat, and
astA genes, respectively [90]. The sigA and pic genes are located on pathogenicity islands,
and the other toxin genes are localized on pAA [102]. A thick mucus layer surrounds
the EAEC biofilms, and the bacteria penetrate it through the secretion of the SPATE pic
toxin with mucolytic activity [90]. Shigella extracellular enterotoxins (ShET1) are cytotoxin,
enterotoxin, and IgA protease like homologues inducing intestinal cyclic GMP (cGMP)
and cAMP-mediated secretion. This toxin has also been found in EAEC/STEC hybrid
strains [88,103]. Pet, a 108 kDa protein belonging to the SPATE family, mediates the actin-
binding protein fodrin, induces exfoliation, disrupts the actin skeleton, is trafficked through
the endoplasmic reticulum, and induces the entry into the host cell. Pet also functions
as a cytotoxin and heat-labile enterotoxin [104]. Sat, a SPATE toxin with cytotoxic and
enterotoxic activities, impairs the tight junction and mediates autophagy in the epithelial
cells. The enteroaggregative heat-stable toxin has mechanistic and physical similarities to
the enterotoxin STa secreted by ETEC (Table 1) [105].

7. Diffusely Adherent Escherichia coli (DAEC)

DAEC is identified as a heterogenous pathotype of E. coli characterized by a diffuse
pattern on Hep-2 and HeLa cells [88]. DAEC is associated with diarrhea in children
between the ages of 1.5 and 5 years, urinary tract infection (UTI) in adults, pregnancy
complications, and part of the intestinal commensal microflora in adults and children [106].
Watery diarrhea caused by DAEC can become more persistent in younger children. Adults
are asymptomatic carriers of DAEC strains contributing to some chronic inflammatory
intestinal diseases, including Crohn’s, coeliac, and inflammatory bowel diseases [107]. It
has been shown that DAEC strains belong to the phylogenetic group B2, and it is worth
noting that this phylogenetic group is predominant among the human commensal E. coli
strains [32,88,108]. Despite the isolation of this strain from stools and duodenal cultures,
none of the adult patients developed diarrhea [88]. The pathogenesis of DAEC starts
with the attachment of the pathogen to specific host cells. The attachment of DAEC
strains onto the urinary tract and enteric epithelial cells allows them to resist clearance
by micronutrition and peristalsis, respectively [109]. The attachment also allows DAEC
to interact with the host cell, release and deliver toxins, and trigger signaling events in
host cells. DAEC attaches and adhere to the host cells using non-classical patterns and is
mediated by Afa/Dr adhesins [110].
An important step in DAEC infection is the specific colonization of the small bowel
mediated by the Afa/Dr family of adhesins, including the two main classes of adhesins
presented on the surface of the pathogen: fimbrial adhesins (including linear polymers) and
afimbrial adhesins (homotrimers or single proteins) [111–113]. Afa/Dr adhesins include
Dr, DrII, AfaE/I–III, AfaE-V, NFA-I, and F1845 adhesins. Afa, Dr, and F1845 adhesins
are encoded by afaA-E, DraA-E, and daaA-E genes, respectively [107,114]. F1845 and Dr
adhesins bind to the decay-accelerating factor (DAF), a molecule expressed and found
on the apical surface of urinary and intestinal epithelial cells, inducing cytoskeleton rear-
rangement and destroying microvilli [11,32,88]. The increased levels of DAF expression
caused by the binding of Afa/Dr adhesins in patients with Crohn’s disease (CD) may
contribute to the inflammation [110]. Some Afa/Dr adhesins can also bind to the epithe-
lial cell surface receptor human carcinoembryonic antigen-related cell adhesion molecule
(CEACAM), resulting in CDC42 activation, CEACAM aggregation at the site of bacterial
adhesion, brush border microvilli effacement, and the mediation of a process leading to
the microfilament-independent, microtubule- and lipid-raft-dependent internalization of
the bacterial pathogen. These processes are thought to play a major role in the infection of
intestinal epithelial cells by DAEC strains [115]. Moreover, Dr adhesins bind to type-IV
Int. J. Mol. Sci. 2021, 22, 9922 12 of 18

collagen, necessary for DAEC-mediated urinary tract infections [88,115]. The interaction
between Afa/Dr adhesins, flagella, and DAF receptors mediates IL-8 secretion and pro-
motes the transmigration of polymorphonuclear neutrophils (PMNs). This causes DAF
upregulation on the epithelial cell surface, providing more adhesion receptors [107,112].
The interaction between PMNs and Afa/Dr adhesins accelerates PMN-associated apoptosis
and decreases the rate of phagocytosis mediated by PMNs. Across the epithelial cells,
DAEC strains also induce the expression of MICA, which is potentially responsible for
mediating inflammatory bowel disease (IBD) [112,116].
Although the pathogenesis of DAEC strains is mainly mediated through Afa/Dr
adhesins, some SPATE toxins are secreted by this pathotype [112]. Afa/Dr DAEC strains
release two main classes of SPATE toxins: class I, with cytotoxic activity against epithelial
cells, including a secreted autotransporter toxin, a plasmid-encoded toxin, an extracellular
serine protease, and sigA toxins, encoded by sat, pet, EspP, and sigA genes, respectively;
class II, which are not cytotoxic and consist of a protein involved in intestinal colonization
encoded by the pic gene [12,88,115]. In addition, other virulence factors, including type-I
pili, pilus adhesin and enteroaggregative heat-stable toxins encoded by pap, fim, and astA
genes, respectively, have been recently detected in DAEC strains (Table 1) [117].

8. Adherent Invasive Escherichia coli (AIEC)

AIEC is one of the most important causative agents of idiopathic inflammatory disor-
ders, including IBD and CD, and primarily affect the human small bowel [118]. However,
this pathotype may also be present as part of the normal microbiota of the human gut
in healthy individuals and does not cause any disease [119]. No single distinct agent is
defined as the leading cause of idiopathic IBD [120,121]. In addition to AIEC, other enteric
pathogens have been reported to be involved in IBD and CD, including Campylobacter
species, Mycobacterium paratuberculosis, and Cytomegalovirus [122,123]. The AIEC patho-
type is an enteric pathogen able to adhere to and invade the intestinal epithelial cell layer
and multiply within macrophages and epithelial cells. No particular virulence factors
found in other E. coli pathotypes have been determined in AIEC strains [124,125]. AIEC
pathogenesis consists of three stages: adhesion, invasion, and multiplication within the
epithelial host cells. Initially, AIEC requires the adhesion to the epithelial host cells in
the ileum, using the CAECAM6 expressed on these cells. Notably, the expression levels
of CAECAM6 in CD patients is increased due to the stimulation of TNF-α production,
causing intestinal inflammation by the colonization of AIEC [126]. Several virulence factors,
such as outer membrane vesicles, outer membrane proteins, and long polar fimbriae, are
expressed and utilized by AIEC strain after the adhesion to the epithelial host cells to
mediate invasion, infect, and replicate within the macrophages [124,127].
Several virulence factors and associated encoding genes have been identified to play a
role in the pathogenic interaction with the intestinal mucosa. Higher levels of expression
of genes encoding virulence factors involved in adhesion, invasion, and survival encoding
were detected in AIEC isolated from patients with CD, IBD, and ulcerative colitis [128–130].
Virulence factors present in AIEC isolated from patients with intestinal disorders include
the adhesive subunit of type-1 fimbriae, the polypeptide stress response protein, the
invasion protein ibeA, the invasion plasmid antigen, and virulence factors involved in
ferric yersiniabactin uptake and capsule synthesis encoded by fimH, yjaA, ibeA, ipaH, fyuA,
and kpsMT II genes, respectively [114,131,132]. AIEC strains have been considered a new
therapeutic target for treating patients with IBD and CD [123]. However, several aspects of
its pathogenesis are unknown and more genomic investigations, especially to determine
the virulence factor-encoding genes, are required (Table 1) [118,127,129].

9. Conclusions
Enteric E. coli pathotypes display a high diversity of pathogenicity mechanisms and
virulence factors profiles. They remain a significant concern in public health and food safety.
The evolution of enteric E. coli pathotypes has contributed to the emergence of distinct E. coli
Int. J. Mol. Sci. 2021, 22, 9922 13 of 18

pathotypes capable of toxin secretion, aggregative colonization, multiplying in the gastroin-

testinal tract, and damaging different environments through the adaptation of key genetic
elements, resulting in the formation of new pathotypes [133]. The emergence of new E. coli
pathotypes such as the hybrid EAEC/STEC (E. coli serotype O104: H4) strain that caused
fatal outbreaks throughout Europe shows the importance of having a surveillance system
in place. As food, water, animals, and people are potential vectors of enteric pathogenic E.
coli transmission and contamination [5], such surveillance systems should be embedded
in one-health approaches/networks. A wide range of functions including protein synthe-
sis, specific gene transcription, the secretion of diverse micro/macro-molecules and ions,
cytoskeleton rearrangement, apoptosis, autophagy, mitochondrial activities, cell division,
and signal transduction in epithelial intestinal and extraintestinal host cells are affected by
enteric E. coli pathotypes. This involves a broad spectrum of virulence factors encoded by
specific gene clusters on the chromosome or mobile genetics elements. Therefore, for an
optimal surveillance, new technologies such as high-throughput sequencing are required
to gather precise information about the virulence profiles of E. coli pathotypes [134,135]. In
addition, a comprehensive investigation of the role of E. coli pathotype virulence factors in
pathogenesis will facilitate the development of novel therapeutics, the improvement of the
design of effective vaccines, and the prevention of a further spreading of these diverse and
widespread pathogens.

Author Contributions: Conceptualization, B.P., W.M.B. and J.W.A.R.; writing—original draft prepa-
ration, B.P.; writing—review and editing, W.M.B., B.P. and J.W.A.R. All authors have read and agreed
to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: We confirm that all data supporting the findings of this study are
available within the article.
Conflicts of Interest: The authors declare no conflict of interest.

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