Era of Whole-Genome Sequencing

REVIEW
published: 18 November 2016
doi: 10.3389/fcimb.2016.00141
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 November 2016 | Volume 6 | Article 141
Edited by:
Nikhil A. Thomas,
Dalhousie University, Canada
Reviewed by:
Fernando Navarro-Garcia,
CINVESTAV, Mexico
Jorge Blanco,
University of Santiago de Compostela,
Spain
*Correspondence:
Roy M. Robins-Browne
[e-mail]
Received: 31 August 2016
Accepted: 13 October 2016
Published: 18 November 2016
Citation:
Robins-Browne RM, Holt KE, Ingle DJ,
Hocking DM, Yang J and Tauschek M
(2016) Are Escherichia coli Pathotypes
Still Relevant in the Era of
Whole-Genome Sequencing?
Front. Cell. Infect. Microbiol. 6:141.
doi: 10.3389/fcimb.2016.00141Are Escherichia coli Pathotypes StillRelevant in the Era of Whole-GenomeSequencing?
Roy M. Robins-Browne 1, 2*, Kathryn E. Holt 3, 4, Danielle J. Ingle 1, 3, 4, Dianna M. Hocking 1,
Ji Yang 1 and Marija Tauschek 1
1 Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of
Melbourne, Parkville, VIC, Australia, 2 Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, VIC,
Australia, 3 Centre for Systems Genomics, The University of Melbourne, Parkville, VIC, Australia, 4 Department of
Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne,
Parkville, VIC, AustraliaThe empirical and pragmatic nature of diagnostic microbiology has given rise to severaldifferent schemes to subtype E.coli, including biotyping, serotyping, and pathotyping.These schemes have proved invaluable in identifying and tracking outbreaks, and forprognostication in individual cases of infection, but they are imprecise and potentiallymisleading due to the malleability and continuous evolution of E. coli. Whole genomesequencing can be used to accurately determine E. coli subtypes that are based onallelic variation or differences in gene content, such as serotyping and pathotyping. Wholegenome sequencing also provides information about single nucleotide polymorphisms inthe core genome of E. coli, which form the basis of sequence typing, and is more reliablethan other systems for tracking the evolution and spread of individual strains. A typingscheme for E. coli based on genome sequences that includes elements of both the coreand accessory genomes, should reduce typing anomalies and promote understandingof how different varieties of E. coli spread and cause disease. Such a scheme could alsodefine pathotypes more precisely than current methods.
Keywords: E. coli, diarrhoea, bacterial typing, pathotype, pathogenesis, sequence type, whole genome sequence
Escherichia coli is the most comprehensively studied bacterium on earth. Because it is relatively
easy to manipulate genetically, it has become a popular laboratory workhorse. Its natural habitat,
however, is the intestinal tract of humans and other mammals. For this reason it is used in public
health as an indicator of faecal contamination of water and other consumables.
Despite its ubiquity as a commensal, E. coli is also an important pathogen of humans and
domestic animals. It can become established and cause disease in tissues other than the intestinal
tract. These so-called extraintestinal pathogenic E. coli (ExPEC) are important causes of wound
infection, urinary tract infection, peritonitis, pneumonia, meningitis, and septicaemia. The ExPEC
group includes named subtypes, such as uropathogenic E. coli (UPEC), neonatal meningitis-
associated E. coli (NMEC), and sepsis-associated E. coli (SEPEC) (Pitout, 2012; Leimbach et al.,
2013).
Infections caused by ExPEC are usually opportunistic, i.e., they occur most often in hosts
who are compromised in some way, such as by having a dysfunctional urinary tract or systemic
immunocompromise due to neutropenia or extremes of age. Nevertheless, some ExPEC strains are
better equipped to cause extraintestinal infections than others due to factors that facilitate their

Robins-Browne et al. E. coli Pathotypes
ability to colonise tissues. These include type I fimbriae,
pyelonephritis-associated pili (PAP), and AfA/Dr adhesins in
the case of UPEC, or the K1 polysaccharide capsule, which
allows NMEC and SEPEC to evade complement-mediated killing
(Pitout, 2012; Leimbach et al., 2013).
The pathotypes of E. coli that are associated with intestinal
disease are known collectively as intestinal pathogenic E. coli
(IPEC) or diarrheagenic E. coli (DEC)—although not all of
the subtypes in this group necessarily cause diarrhoea. The
individual pathotypes of DEC include enteropathogenic E. coli
(EPEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli
(ETEC), enterohemorrhagic E. coli (EHEC), enteroaggregative E.
coli (EAEC), diffusely-adherent E. coli (DAEC), and adherent-
invasive E. coli (AIEC) (Nataro and Kaper, 1998; Kaper et al.,
2004; Croxen et al., 2013). In addition, the entire genus Shigella
is a DEC pathotype, which closely resembles EIEC in terms
of virulence attributes and pathogenicity, but is distinguishable
from other strains of E. coli by virtue of its biochemical activity
(Lan et al., 2004). Accordingly, shigellae can be regarded as
members of the EIEC pathotype.
Each DEC pathotype represents a collection of strains that
possess similar virulence factors to each other and cause similar
diseases with similar pathology. Unlike ExPEC, where there are
no specific virulence determinants that exclusively define each
subtype, most DEC pathotypes are defined by the possession
of one or more pathotype-specific virulence markers, and
sometimes by the absence of others. Several of the defining
markers of DEC pathotypes are proven virulence determinants
of that pathotype, but for EAEC, DAEC, and AIEC the role of
these markers in virulence is not proven (Table 1).
DEC PATHOTYPES
In this section, we provide a brief overview of DEC pathotypes
with an emphasis on their defining characteristics and key
virulence determinants (where known). We also point out some
important gaps in the understanding of certain pathotypes.
For more detailed information on DEC in general, readers are
referred to reviews by Nataro and Kaper (1998), Kaper et al.
(2004), Clements et al. (2012), and Croxen et al. (2013). In the
bibliography, we have also included references to review articles
dealing with individual DEC pathotypes.
Enteropathogenic E. coli (EPEC)
EPEC was the first pathotype of DEC to be discovered, and is an
important cause of diarrhoea and premature death in children,
especially in developing countries (Robins-Browne, 1987). As
a group, EPEC is characterised by the presence of the locus
of enterocyte effacement (LEE) pathogenicity island (McDaniel
and Kaper, 1997; Robins-Browne and Hartland, 2002; Croxen
et al., 2013). This ∼40-kbp island encodes (i) an outer membrane
adhesive protein, known as intimin that is encoded by the
eae gene, (ii) a type 3 protein secretory system, (iii) several
type 3-secreted effectors, including the Tir protein which is the
translocated receptor for intimin (Kenny et al., 1997).
Expression of the LEE is associated with distinctive attaching-
effacing lesions in the intestinal epithelium which characterise
EPEC pathology (Moon et al., 1983; Tzipori et al., 1985). Almost
all genes within the LEE are required for the production of
these lesions, and studies in adult volunteers have demonstrated
that intimin and EspB, a key component of the type 3
secretion system, are essential virulence determinants of EPEC
(Donnenberg et al., 1993; Tacket et al., 2000). An accessory
virulence determinant, which EPEC also requires for virulence
in humans, is the bundle-forming pilus (BFP) (Girón et al., 1991;
Bieber et al., 1998). Some human isolates of EPEC naturally lack
BFP, but may cause disease (Trabulsi et al., 2002; Nguyen et al.,
2006). These strains, known as atypical EPEC are associated with
persistent diarrhoea in children (Nguyen et al., 2006). Atypical
EPEC are genetically diverse and appear to vary in virulence
(Tennant et al., 2009; Ingle et al., 2016a).
Enterohemorrhagic E. coli (EHEC)
EHEC first came to attention as the cause of two outbreaks
of haemorrhagic colitis (HC) in the USA during 1982 (Riley
et al., 1983). The defining virulence determinant of EHEC
is the phage-encoded Shiga toxin (also known as Verotoxin),
of which there are several varieties (O’Loughlin and Robins-
Browne, 2001; Melton-Celsa et al., 2012). Although volunteer
studies with EHEC are prohibited for ethical reasons, vast
quantities of epidemiological data leave no doubt that Shiga toxin
is responsible for the life-threatening manifestations of EHEC
infections, namely, HC and the haemolytic uraemic syndrome
(HUS). Evidence supporting a role for Shiga toxin in these
conditions include the observation that infections with other
bacteria which produce Shiga toxin, such as Shigella dysenteriae
serotype 1 and occasional strains of EAEC, may also cause HC
and HUS (Rohde et al., 2011; Walker et al., 2012).
Not all strains of Shiga toxin-producing E. coli (STEC or
VTEC) cause HC or HUS, and the term “EHEC” is generally
reserved for those that do. Thus, although all EHEC are STEC,
not all STEC are EHEC. The properties that distinguish EHEC
from those STEC that do not cause HC or HUS are accessory
virulence factors which allow the bacteria to adhere to the
intestinal epithelium, such as the LEE pathogenicity island in so-
called “typical EHEC” or a number of other adhesins that are
present in LEE-positive and/or LEE-negative strains (reviewed in
McWilliams and Torres, 2014).
Typical EHEC strains of serotype O157:H7 also generally
carry a virulence-associated plasmid, known as pO157, which
encodes a number of putative virulence determinants (Burland
et al., 1998). Related plasmids occur in EHEC of other
serogroups, including O26, O103, O111, and O145 (Ogura
et al., 2009). One of the virulence-associated factors encoded
by these plasmids is a serum-sensitive haemolysin, known
as EHEC haemolysin or enterohaemolysin. Many EHEC
isolates produce this protein, including some that carry
plasmids only distantly related to pO157 (Beutin et al., 1989).
Accordingly, the production of enterohaemolysin can be used as
a diagnostic marker of EHEC (Feldsine et al., 2016). Interestingly,
enterohaemolysin is also produced by some LEE-positive, Shiga
toxin-negative strains of E. coli obtained from cattle and sheep
(Cookson et al., 2007). This observation provides evidence of
the evolutionary relationship between atypical EPEC and EHEC,
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 2 November 2016 | Volume 6 | Article 141

Robins-Browne et al. E. coli Pathotypes
TABLE 1 | Virulence-associated markers of diarrheagenic E. coli from humans.
Pathotype Defining marker Essential virulence
determinant(s)
Location of essential
virulence determinant(s)
Major diagnostic
target(s) for PCR
Other diagnostic
target(s)
EPEC LEE PAI LEE PAI Pathogenicity island eae bfpAa
EIEC/Shigella pINV pINV Plasmid ipaH Other ipa genes
ETEC ST or LT ST and/or LT plus colonisation
factors
Plasmid; transposon elt, est
EHEC Shiga toxin Shiga toxin 1 and/or 2 Prophages stx1, stx2 eaea, ehxAa
EAEC pAA; aggregative adhesion Not known Plasmid (probably); possibly
others
aggR, aatA, aaiC
DAEC Afa/Dr adhesins Not known Not known Afa/Dr adhesinsb
AIEC Adherent-invasive phenotype Not known Not known none none
aaiC, gene for a secreted protein of enteroaggregative E. coli; aatA, gene for a transporter protein of enteroaggregative E. coli; Afa, afimbrial adhesin; aggR, gene for a transcriptional
regulator; AIEC, adherent-invasive E. coli; bfpA, gene for a structural protein of bundle-forming pili; DAEC, diffusely-adherent E. coli; EAEC, enteroaggregative E. coli; EIEC, enteroinvasive
E. coli; elt, gene for heat-labile enterotoxin; EPEC, enteropathogenic E. coli; est, gene for heat-stable enterotoxin; ETEC, enterotoxigenic E. coli; ipaH, gene for a type 3-secreted effector
protein of enteroinvasive E. coli and Shigella; LEE PAI, locus of enterocyte effacement pathogenicity island; LT, heat-labile enterotoxin; pAA, virulence plasmid of enteroaggregative E.
coli; pINV, virulence plasmid of enteroinvasive E. coli and Shigella; ST, heat-stable enterotoxin.
a Not present in all strains.
b These are under review following concerns about specificity.
which is also evident from the high degree of relatedness between
atypical EPEC strains of serotype O55:H7 and EHEC O157:H7
(Feng et al., 1998).
Enterotoxigenic E. coli (ETEC)
ETEC is a leading cause of diarrhoea in children in developing
countries and in travellers to these countries (Qadri et al., 2005;
Lanata et al., 2013). ETEC is also an important cause of diarrhoea
in domestic animals, notably calves and piglets, where ETEC-
induced diarrhoea is of considerable economic importance (Nagy
and Fekete, 1999; Fairbrother et al., 2005).
As the name suggests, the ETEC pathotype is defined by the
capacity of the bacteria to produce one or more enterotoxins. In
ETEC the specific enterotoxins are the heat-labile and heat-stable
enterotoxins (LT and ST) and their various subtypes (Qadri et al.,
2005). The two major subtypes of ST are STa (also known as STI)
and Stb (STII), of which only STa is important in humans (Qadri
et al., 2005; Taxt et al., 2010). Most ETEC strains isolated from
humans with diarrhoea produce STa, often together with LT. The
role of each of these toxins in disease has been established in
volunteer studies (Levine et al., 1977, 1979).
Both ST and LT exert their maximum impact on water and
electrolyte transport in the small intestine. In order to deliver
these toxins to the small intestinal epithelium, ETEC need
to attach to epithelial cells, which they achieve by means of
specific colonisation factors (Qadri et al., 2005; Madhavan and
Sakellaris, 2015). These factors are highly variable structurally
and antigenically, and also differ between isolates from humans
and animals. In several instances, the role of colonisation factors
as accessory virulence determinants has been demonstrated
experimentally (Qadri et al., 2005; Madhavan and Sakellaris,
2015).
Enteroinvasive E. coli (EIEC)
EIEC are closely related to Shigella, especially in terms of
the disease they cause, i.e., bacillary dysentery, and their key
virulence determinant: a plasmid known as pINV. This plasmid
encodes a type 3 secretion system and a number of effectors that
allow shigellae/EIEC to penetrate epithelial cells, move within
these cells and invade neighbouring cells (Marteyn et al., 2012).
Both shigellae and EIEC carry several other putative virulence
determinants including adhesins and secreted toxins, but pINV,
which appears to be restricted to these bacteria, is the key to their
virulence (Marteyn et al., 2012; Croxen et al., 2013).
EIEC and shigellae exemplify the changes that E. coli can
make to adjust to a pathogenic lifestyle (Day et al., 2001). Thus,
by acquiring pINV, and other genetic elements that allow the
bacteria to adopt an intracellular lifestyle, the capacity of E. coli
to live inside cells is continuously enhanced by the deletion or
inactivation of genes that are inimical to this lifestyle (Day et al.,
2001; Feng et al., 2011; Prosseda et al., 2012). Examples of such
genes include some that encode anti-virulence factors, such as
nadA, nadB, and ompT, and those for metabolic pathways such
as lysine decarboxylation, the end products of which restrict
intracellular growth (Day et al., 2001; Prunier et al., 2007).
Moreover, since flagella are not required for colonisation of the
large intestine or for motility within cells, all shigellae and many
strains of EIEC are non-motile. The capacity of E. coli to adapt to
new environments in this way provides fascinating insights into
the extraordinary versatility of this species as a pathogen.
Enteroaggregative E. coli (EAEC)
This relatively recently discovered E. coli pathotype is mainly
associated with paediatric diarrhoea in developing countries, but
has also been linked to diarrhoea in adults, including travellers
(Okeke and Nataro, 2001; Harrington et al., 2006). EAEC was
originally identified by its characteristic “stacked-brick” pattern
of adherence to tissue culture cells in vitro (Nataro et al.,
1987; Figure 1). This phenotype is attributable to one of several
different hydrophobic aggregative fimbriae, known as AAF/I,
AAF/II, AAF/III, and AAF/IV, encoded by pAA or similar
plasmids. Other putative virulence factors of EAEC include (i)
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