E. coli Pathotypes: Relevance in the Era of Whole-Genome Sequencing

This report, published in Frontiers in Cellular and Infection Microbiology, investigates the continuing relevance of Escherichia coli pathotypes in the age of whole-genome sequencing (WGS). The study explores the limitations of traditional subtyping schemes (biotyping, serotyping, pathotyping) and highlights the advantages of WGS in accurately identifying E. coli subtypes based on allelic variation and gene content. It delves into the various E. coli pathotypes associated with intestinal and extraintestinal diseases, including EPEC, EIEC, ETEC, EHEC, EAEC, DAEC, AIEC, and ExPEC subtypes like UPEC and NMEC, and their defining virulence factors. The report emphasizes the importance of understanding both core and accessory genomes for a comprehensive typing scheme that reduces anomalies and promotes a better understanding of E. coli spread and disease mechanisms. The authors also discuss the diagnostic markers, essential virulence determinants, and genetic characteristics of each pathotype, including the role of Shiga toxins, LEE pathogenicity island, and various adhesins. The paper concludes by suggesting that a typing scheme based on genome sequences could refine pathotype definitions and improve our understanding of E. coli pathogenesis.

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:
NikhilA. 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 coliPathotypes
StillRelevant in the Era of
Whole-Genome Sequencing?
Front. Cell. Infect. Microbiol. 6:141.
doi: 10.3389/fcimb.2016.00141
Are Escherichia coli Pathotypes Still
Relevant in the Era of Whole-Genome
Sequencing?
Roy M. Robins-Browne1, 2
*, Kathryn E. Holt3, 4, Danielle J. Ingle1, 3, 4
, Dianna M. Hocking1,
Ji Yang1 and Marija Tauschek1
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, RoyalChildren’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, Australia
The empiricaland pragmatic nature of diagnostic microbiology has given rise to sever
different schemes to subtype E.coli,including biotyping,serotyping,and pathotyping.
These schemes have proved invaluable in identifying and tracking outbreaks,and for
prognostication in individualcases ofinfection,but they are imprecise and potentially
misleading due to the malleability and continuous evolution ofE. coli.Whole genome
sequencing can be used to accurately determine E.coli subtypes thatare based on
allelic variation or differences in gene content, such as serotyping and pathotyping.
genome sequencing also provides information about single nucleotide polymorphism
the core genome of E. coli, which form the basis of sequence typing, and is more re
than other systems for tracking the evolution and spread of individualstrains. A typing
scheme for E. colibased on genome sequences that includes elements of both the cor
and accessory genomes, should reduce typing anomalies and promote understandin
of how different varieties of E. colispread and cause disease. Such a scheme could also
define 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 natura
however, is the intestinal tract of humans and other mammals. For this reason it is used
health as an indicator of faecal contamination of water and other consumables.
Despite its ubiquity as a commensal,E. coliis also an importantpathogen ofhumans and
domestic animals. It can become established and cause disease in tissues other than the
tract.These so-called extraintestinalpathogenic E.coli (ExPEC) are important causes of wound
infection, urinary tract infection, peritonitis, pneumonia, meningitis, and septicaemia. Th
group includes named subtypes,such as uropathogenic E.coli (UPEC), neonatalmeningitis-
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 mostoften in hosts
who are compromised in some way,such as by having a dysfunctional urinary tract or systemi
immunocompromise due to neutropenia or extremes of age. Nevertheless, some ExPEC
better equipped to cause extraintestinalinfections than others due to factors that facilitate their

Robins-Browne et al. E. coliPathotypes
ability to colonisetissues.Theseincludetype I fimbriae,
pyelonephritis-associated pili(PAP), and AfA/Dr adhesins in
the case ofUPEC, 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.colithat are associated with intestinal
disease are known collectively as intestinalpathogenic E.coli
(IPEC) or diarrheagenic E.coli (DEC)—although notall of
the subtypesin this group necessarily cause diarrhoea.The
individualpathotypes 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 resemblesEIEC in terms
of virulence attributes and pathogenicity,but is distinguishable
from other strains of E. coli by virtue of its biochemical activity
(Lan etal., 2004).Accordingly,shigellae can be regarded as
members of the EIEC pathotype.
Each DEC pathotype represents a collection ofstrains 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,mostDEC pathotypes are defined by the possession
of one or more pathotype-specificvirulencemarkers,and
sometimesby the absence ofothers.Severalof 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 emphasison their defining characteristicsand key
virulence determinants (where known). We also point out some
importantgapsin the understanding ofcertain pathotypes.
For more detailed information on DEC in general,readers are
referred to reviews by Nataro and Kaper (1998),Kaper etal.
(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 ofthe 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 asintimin thatis 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 intestinalepithelium which characterise
EPEC pathology (Moon et al., 1983; Tzipori et al., 1985). Almo
all genes within the LEE are required for the production of
these lesions, and studies in adult volunteers have demonstra
that intimin and EspB,a key componentof the type 3
secretion system,are essentialvirulence determinants of EPEC
(Donnenberg etal., 1993;Tacketet al., 2000).An accessory
virulence determinant,which EPEC also requires for virulence
in humans, is the bundle-forming pilus (BFP) (Girón et al., 199
Bieber et al., 1998). Some human isolates of EPEC naturally la
BFP,but may cause disease (Trabulsi et al.,2002;Nguyen et al.,
2006). These strains, known as atypical EPEC are associated
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 oftwo outbreaks
of haemorrhagic colitis (HC) in the USA during 1982 (Riley
et al., 1983).The defining virulencedeterminantof EHEC
is the phage-encoded Shiga toxin (also known as Verotoxin),
of which there are severalvarieties (O’Loughlin and Robins-
Browne,2001;Melton-Celsa etal.,2012).Although volunteer
studieswith EHEC are prohibited forethicalreasons,vast
quantities of epidemiological data leave no doubt that Shiga t
is responsible for the life-threatening manifestations ofEHEC
infections,namely,HC and the haemolytic uraemic syndrome
(HUS). Evidence supporting a role forShiga toxin in these
conditions include the observation thatinfections with other
bacteria which produce Shiga toxin, such as Shigella dysente
serotype 1 and occasional strains of EAEC,may also cause HC
and HUS (Rohde et al., 2011; Walker et al., 2012).
Not all strains ofShiga 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 allSTEC are EHEC.The properties that distinguish EHEC
from those STEC that do not cause HC or HUS are accessory
virulence factorswhich allow the bacteria to adhere to the
intestinal epithelium, such as the LEE pathogenicity island in
called “typicalEHEC” or a number of other adhesins that are
present in LEE-positive and/or LEE-negative strains (reviewed
McWilliams and Torres, 2014).
TypicalEHEC strainsof serotype O157:H7 also generally
carry a virulence-associated plasmid,known as pO157,which
encodes a number of putative virulence determinants (Burlan
et al., 1998).Relatedplasmidsoccur in EHEC of other
serogroups,including O26,O103,O111,and O145 (Ogura
et al.,2009).One ofthe virulence-associated factors encoded
by theseplasmidsis a serum-sensitivehaemolysin,known
as EHEC haemolysinor enterohaemolysin.Many EHEC
isolatesproducethis protein,includingsome that carry
plasmids only distantly related to pO157 (Beutin et al.,1989).
Accordingly, the production of enterohaemolysin can be used
a diagnostic marker of EHEC (Feldsine et al., 2016). Interestin
enterohaemolysin is also produced by some LEE-positive, Shi
toxin-negative strains of E.coli obtained from cattle and sheep
(Cookson etal.,2007).This observation provides evidence of
the evolutionary relationship between atypical EPEC and EHE
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Robins-Browne et al. E. coliPathotypes
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 phenotypeNot 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, afimbrialadhesin; 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,
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-sec
protein of enteroinvasive E. coliand Shigella; LEE PAI, locus of enterocyte effacement pathogenicity island; LT, heat-labile enterotoxin; pAA, virulence plasmid of enteroaggreg
coli; pINV, virulence plasmid of enteroinvasive E. coliand Shigella; ST, heat-stable enterotoxin.
aNot present in allstrains.
bThese are under review following concerns about specificity.
which is also evident from the high degree of relatedness between
atypicalEPEC 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 ofeach ofthese 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 smallintestine.In order to deliver
thesetoxinsto the smallintestinalepithelium,ETEC need
to attach to epithelialcells,which they achieve by means of
specific colonisation factors (Qadriet 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 virulencedeterminantshas been demonstrated
experimentally (Qadriet al., 2005;Madhavan and Sakellaris,
2015).
Enteroinvasive E. coli (EIEC)
EIEC are closely related to Shigella,especially in termsof
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
allow shigellae/EIEC to penetrate epithelialcells,move within
these cells and invade neighbouring cells (Marteyn et al., 2012).
Both shigellae and EIEC carry severalother putative virulence
determinants including adhesins and secreted toxins, but pIN
which appears to be restricted to these bacteria, is the key to
virulence (Marteyn et al., 2012; Croxen et al., 2013).
EIEC and shigellae exemplify the changes thatE. coli can
make to adjust to a pathogenic lifestyle (Day et al., 2001). Th
by acquiring pINV,and other genetic elements thatallow the
bacteria to adopt an intracellular lifestyle, the capacity of E. c
to live inside cells is continuously enhanced by the deletion o
inactivation of genes that are inimical to this lifestyle (Day et
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 ofwhich restrict
intracellulargrowth (Day etal., 2001;Prunieret al., 2007).
Moreover,since flagella are not required for colonisation of the
large intestine or for motility within cells, all shigellae and ma
strains of EIEC are non-motile. The capacity of E. coli to adapt
new environments in this way provides fascinating insights in
the extraordinary versatility of this species as a pathogen.
Enteroaggregative E. coli (EAEC)
This relatively recently discovered E.colipathotype is mainly
associated with paediatric diarrhoea in developing countries,
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” patte
of adherenceto tissueculturecellsin vitro (Nataro etal.,
1987; Figure 1). This phenotype is attributable to one of sev
differenthydrophobic aggregative fimbriae,known as AAF/I,
AAF/II, AAF/III, and AAF/IV, encoded by pAA orsimilar
plasmids.Other putative virulence factors of EAEC include (i)
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Robins-Browne et al. E. coliPathotypes
FIGURE 1 | Light micrographs showing the distinctive patterns of
adherence of enteroaggregative E. coli (left) and diffusely-adherent E.
coli (right) to cultured epithelial cells (adapted from Nataro et al., 1987).
These patterns were responsible for the names of these pathotypes and were
originally used to identify them in vitro.
a pAA-encoded cytotoxin (Pet),(ii) a pAA-encoded heat-stable
enterotoxin,known as enteroaggregative stable toxin (EAST-1)
that is related to STa of ETEC,but not restricted to EAEC,and
(iii) ShET1,a putative enterotoxin that is also found in Shigella
flexneri (Okeke and Nataro, 2001; Croxen et al., 2013). Although
the pathogenicity of EAEC is evident from foodborne outbreaks
in several countries and infection studies of volunteers (Nataro
et al.,1995;Harrington et al.,2006),the contribution of these
and other putative virulence-associated determinants of EAEC is
not known (Harrington et al., 2006; Croxen et al., 2013). As with
atypical EPEC, EAEC are genetically diverse with the likelihood
that some types are more virulent than others (Boisen et al., 2012;
Zhang et al., 2016).
In 2011, a Shiga toxin-producing derivative of an EAEC strain
of serotype O104:H4,shotto prominence by causing a major
foodborne outbreak ofdiarrhoea and HUS in Germany,with
serious outcomes for human health and the internationalfood
trade (Buchholz et al., 2011; Rohde et al., 2011).
Few studies of diarrhoea today use the aggregative adherence
phenotype to identify EAEC.Instead most investigators target
the pAA-borne genes, aatA and aggR (that encode a transporter
of a virulence protein and a virulence regulator, respectively), or
the chromosomally-encoded aaiC gene which is also associated
with virulence (Table 1;Panchalingam etal.,2012).Although
PCR-based identification ofEAEC is convenient,the presence
or absence of these genes does not necessarily concur with the
aggregative phenotype,nor is it known whether this phenotype
or the presence of aatA,aggR and/or aaiC is the more reliable
predictor ofvirulence (Weintraub,2007;Croxen etal.,2013).
This issue is not trivial,because until it is resolved we will lack
a clear definition of what really constitutes EAEC.
Diffusely-Adherent E. coli (DAEC)
As with EAEC, DAEC were originallyidentified bytheir
distinctive pattern of adherence to tissue culture cells (Scaletsky
et al., 1984;Nataro etal., 1985,1987;Figure 1).The first
determinantof diffuseadherenceto be identified wasan
autotransporterprotein,known asAIDA-I, for the Adhesin
Involved in DiffuseAdherence(Benz and Schmidt,1992).
E. coli strainsthatexpressAIDA-1, however,generally carry
other virulence determinants,such as STb,making them ETEC
(Dubreuil, 2010), or the LEE pathogenicity island, making the
EPEC (Servin,2005,2014;Table 1).Accordingly,few ofthese
strains are considered DAEC, despite their phenotype.
AIDA-I-negativeDAEC strains typciallyexpressAfa/Dr
adhesinsand cause urinary tractinfections,placing them in
the UPEC subgroup ofExPEC.Although E.coli thatexpress
afimbrialadhesins (Afa) and Dr fimbriae have been associated
with diarrhoea in children, the specificity of the probes and PC
primers that were used to detect and identify these bacteria
questionable,in that they may also react with EAEC and some
other types of E.coli (Servin,2014).This,and the fact that two
prototypical DAEC strains failed to cause diarrhoea in volunte
who ingested up to 1010 colony-forming units,casts doubt on
the role ofDAEC in diarrhoea,notwithstanding considerable
evidence of the deleterious effects of these bacteria on intest
epithelial cells in vitro (reviewed in Servin, 2014).
Adherent-Invasive E. coli (AIEC)
AIEC are unusual amongst DEC pathotypes in that they are no
associated with diarrhoea. Instead they are thought to contrib
to the developmentof Crohn’sdisease,which is a chronic
inflammatory bowel disease.The aetiology of Crohn’s disease is
uncertain,but is likely to involve both host and environmental
factors (Alhagamhmad et al., 2016). AIEC strains are discernib
from other varieties of E.coli,including commensals,by virtue
of their ability to adhere to and invade epithelialcells and to
replicate within macrophages (Martinez-Medina etal.,2009).
Analysis ofwhole genome sequences ofseveralAIEC isolates,
however,has shown that the AIEC phenotype may not be due
to one or more specific virulence determinants (O’Brien et al.
2016), suggesting that the distinctive phenotype of these bac
may resultfrom metabolic processes thatenhances growth in
tissues affected by Crohn’s disease.Thus,although AIEC are
recovered more commonly from patients with Crohn’s diseas
than from healthy people,it is unclear whether these bacteria
contribute to the pathogenesis of Crohn’s disease or are mere
adapted to or enriched in intestinal tissue affected by this dis
E. COLI GENOMICS
The first complete genome sequence of an E.coli strain (E.coli
K-12) was published in 1997 (Blattner et al.,1997).Since then
many thousands of E. coli isolates from a wide range of sourc
have also been sequenced, although most of these genomes
not been fully assembled into a finished and complete genom
sequence.Nevertheless,from the available data we can glean
thatthe size ofthe E.coli genome (which includes plasmids
and prophage) ranges from approximately 4.6 million base pa
(Mbp) to around 5.9 Mbp—a difference of more than 1.3 Mbp.
Each individual E.coli strain carries between 4200 and 5500
genes.As more E.colistrains are sequenced the core genome
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Robins-Browne et al. E. coliPathotypes
(i.e.,the backbone ofchromosomalgenes thatare presentin
every E.coli strain)shrinks.The size of the core genome
currently stands at fewer than 1500 genes and willto continue
to diminish, albeit slowly, as more strains are sequenced. Genes
thatare notpartof the core are referred to as the accessory
genome. These include all of the genes that encode bacteriophage
elements,virulence determinantsand acquired resistance to
antimicrobials.The E. coli pangenome—the totalnumberof
unique genes that have been identified in E. coli—comprises more
than 22,000 and willcontinue to increase as more strains are
sequenced.
All of the genesfor E. coli virulence determinantswere
mostlikely acquired by horizontalgene transferfrom other
bacteria via plasmids, bacteriophages, pathogenicity islands, and
transposons (Leimbach et al., 2013; Table 1). Thus, every E. coli
strain comprises a mosaic ofcore and accessory genes,with
almost allof the latter,including the virulence determinants of
DEC, being transmissible between strains. For these reasons, it is
inevitable that new pathotypes of DEC will continue to emerge,
either through novel assemblies of E. coli virulence determinants,
as in the case ofEHEC (Feng etal.,1998) and Shiga toxin-
producing EAEC (Rohde et al., 2011), or through the acquisition
of virulence genes from other bacterial species.
E. COLI SUBTYPES
Apart from pathotype,individualstrainsof E. coli can be
subtyped using a variety ofcriteria thatmay vary between
individualstrains.These includesequencetype, serotype,
pulsotype, phage type, and biotype.
Sequence Type
The conserved natureof the E. coli core genomeallows
determination ofthe genetic distance between strainsbased
on nucleotidepolymorphismsin shared genes.For more
than a decade multi-locus sequence typing (MLST),in which
sequence types (STs) are defined on the basis of combinations
of allelicvariation in 6–11 so-called “housekeepinggenes”
(Maiden etal., 1998),hasbeen the gold standard forDNA
sequence-based typing ofbacterialpathogens.ThreeMLST
schemeshavebeen proposed forE. coli, each based on a
differentset of 7–8 genes(Reid et al., 2000;Wirth et al.,
2006;Jaureguy etal.,2008),of which the 7-locus scheme of
Mark Achtman appears to be the moststable and congruent
with whole genome phylogenies (Chaudhuriand Henderson,
2012;Clermontet al., 2015).The principleof MLST has
recently been extended to core gene MLST (cgMLST) (Maiden
et al., 2013),and a new E. coli schemeincorporating
more than 2500genesis now available(alongsidethe 7-
locus scheme ofMark Achtman)in the Enterobase database
hostedat the Warwick Medical School (http://enterobase.
warwick.ac.uk).Sequence typing hasproved usefulin many
settings,e.g.,in tracing thespread ofparticularstrainsin
differentregions,such as E.coliST131,a multidrug resistant
UPEC clone (Nicolas-Chanoineet al., 2014;Petty et al.,
2014).
Serotype
Serotypingbased on antigenicvariation in thesurfaceO-
(polysaccharide)and H- (flagella)antigensof E. coli was
previouslyused for the preliminaryidentification ofDEC
pathotypes. Indeed, much of the early evidence linking EPEC
the cause of outbreaks of diarrhoea was based on the antigen
relatedness of strains obtained from patients in different loca
(Robins-Browne,1987).ETEC, EIEC, EHEC, and EAEC also
belong to a limited number of serotypes,but serotyping is no
longer used for the preliminary identification of these categor
having been replaced by directtesting forthe presenceof
virulence-associated genes (Table 1). Moreover, E. coli serot
are not immutable,and can change due to mutation or phage-
mediated transduction (Mavris et al., 1997; Kido and Kobayas
2000).The superiority ofsequence typing over serotyping is
illustrated by the ST131 UPEC pandemic strain,in which most
isolates are serotype O25b:H4,butsome are serotype O16:H5
(Nicolas-Chanoine etal.,2014).Importantly,E. coliserotypes
can be reliably predicted from whole genome sequences (Ing
et al., 2016b).Indeed,in-silico serotyping offersa number
of advantages over traditionalserotyping,including the non-
reliance on typing sera that may vary in quality,and the ability
to type strains that do not express the O- or H-antigens in vit
or that autoagglutinate (Ingle et al.,2016b).For these reasons,
in-silico serotyping is likely to replace traditionalserotyping in
future.
Nevertheless, many food microbiology laboratories current
use serotyping for the preliminary identification of EHEC, mos
notably E.coli O157:H7 and the so-called “big six” serogroups
(O26,O45,O103,O111,O121,and O145)of EHEC strains
(Brooks et al., 2005).
Interestingly,even the identification ofserotype,together
with the demonstration ofa suite ofshared virulence genes,
may notprovide sufficiently refined information to identify a
particular subclone or clade ofEHEC (Manning etal.,2008).
In such instances,further subtyping may be required to track
outbreaks.Traditionally,this has included phage typing (which
is based on the susceptibility ofisolates to infection with one
or more specific virulentbacteriophages)or typing based on
restriction fragment length polymorphism (pulsotyping),which
permits the discernment of outbreak strains from background
“noise”(Benderet al., 1997).The valueof pulsotypingis
exemplified byPulseNet,a surveillancenetworkof public
health laboratories thatuse DNA fingerprinting for the early
identification ofcommon sourcesof foodborne outbreaksof
disease (Swaminathan et al., 2001). More recently, public hea
laboratories have been shifting to analysis of whole genome s
nucleotide polymorphisms (SNPs) to trace outbreaks of E.coli
and other foodborne pathogens.This approach firstcaptured
the attention ofthe internationalpublichealth community
during the high-profile 2011 outbreak ofdiarrhoea and HUS
in Germany caused by Shiga toxin producing EAEC (Buchholz
et al., 2011; Rohde et al., 2011), and is now being used for ro
analysis in many laboratories, e.g., to investigate E. coli O157
outbreaks by Public Health England (Cowley et al.,2016),and
the GenomeTrakr project established by the US Food and Dru
Administration (Allard et al., 2016).
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Robins-Browne et al. E. coliPathotypes
Biotype
Biotyping was once relied upon to group and separate individual
strains ofE. coli,particularly in the period before serotyping
became established for this purpose. Currently, biotyping is still
used to distinguish shigellae from othervarietiesof E. coli.
Although at present there is no comprehensive scheme to predict
E. coli biotype from whole genome sequences,this may be
possible in future should biotyping still be required.
Biochemicalprofiles also play a centralrole in the isolation
and preliminary identification ofE. colistrains in general,on
media such as McConkey and eosin methylene blue agar,and
of EHEC on sorbitol MaConkey (SMAC) agar and CHROMagar
STEC medium (de Boer et al., 2015).
Pathotype
As mentioned above,the subdivision ofDEC into pathotypes
has may uses.However,some isolates do not comply with the
standard pathotyping scheme (Table 2).Such strainsinclude
isolates of EPEC that carry genes for the heat-labile enterotoxin
of ETEC (Dutta et al., 2015); and strains of ETEC and EAEC that
secrete Shiga toxin (Zhang et al.,2007;Buchholz et al.,2011).
Even Shigella dysenteriae type 1,which carries the Shiga toxin
gene on its chromosome is atypical, as far as the Shigella biotype
is concerned,since no other strain in this “genus” produces
this toxin.In addition,Shigella boydii serotype 13 is unusual in
thatit carries the LEE pathogenicity island ofEPEC (Walters
et al., 2012), although this particular clone is evidently incorrectly
classified,being more closely related to E.albertiithan to E.
coli (Hyma etal., 2005).E. albertiiis a disctinctEscherichia
species that is characterised in part by its carriage ofthe LEE
pathogenicity island (Huys et al., 2003).
Hybrid strains of E.coli pathotypes are not surprising given
the mobility of most of the genes that encode virulence in DEC.
Whatis perhaps more surprising is thathybrids don’toccur
more often. In this regard, DEC strains that infect humans seem
somewhat limited in the combinations of virulence determinants
TABLE 2 | Examples of clinically significant diarrheagenic E. coli strains
that do not comply with established pathotypes.
Strain Comments References
Shiga-toxin producing
EAEC
Some investigators have
deemed these to a new
pathotype named STEAEC
Clements et al., 2012
Shiga-toxin producing
ETEC
Most of these strains are
associated with pig edema
disease
Zhang et al., 2007
LT-producing EPEC We have found this
uncommon hybrid in our
studies of paediatric diarrhoea
(unpublished)
Dutta et al., 2015
Shigella B13 carrying the
LEE pathogenicity island
This clone is more closely
related to E. albertiithan to
E. coli
Hyma et al., 2005;
Walters et al., 2012
EAEC, enteroaggregative E. coli; EPEC, enteropathogenic E. coli; ETEC, enterotoxigenic
E. coli; LEE, locus of enterocyte effacement; LT, heat-labile enterotoxin; STEAEC, Shiga-
toxin producing enteroaggregative E. coli.
that occurtogether,otherthan thosethat are already well
characterised (Table 1). Thus, whereas EPEC, EHEC, ETEC, a
shigellae have allemerged on severaldifferent occasions (Pupo
et al., 2000; Sahl et al., 2011; von Mentzer et al., 2014; Ingle
2016a),hybrids of these are uncommon (Nyholm et al.,2015).
By contrast, some EPEC strains from animals carry colonisatio
fimbriae that closely resemble those from ETEC (Adams et al.
1997), and ETEC from swine may express Shiga toxin as well
STa and/or STb (Zhang et al., 2007; DebRoy et al., 2010).
A particular limitation ofpathotyping concerns its limited
capacityto accommodatenew strainsthat do not comply
with known categories.These include Shiga toxin producing
strains ofEAEC, which some authors have assigned to a new
pathotype,designated Shiga-toxin producing enteroaggregative
E. coli (Clementset al., 2012).This is not unreasonable
considering that EHEC, which is a well-accepted pathotype its
appears to have emerged relatively recently from EPEC (Feng
et al., 1998), but the nomenclature is unwieldy and inflexible.
example,it may be more accurate to use the term “Shiga toxin
producing atypicalEPEC” for EHEC given the origin of these
strains,and Shiga toxin producing ETEC for the bacteria that
cause oedema disease in pigs.
Anotherproblem with thecurrentdefinitionsof DEC
pathotypes is that some strains are defined in part by negativ
criteria.For example,EPEC is defined ashaving theLEE
pathogenicity island, but lacking Shiga toxin (otherwise it wou
be EHEC),and atypicalEPEC is defined as lacking both Shiga
toxin and bundle-forming pili (Kaper, 1996; Trabulsi et al., 20
We believe that characterising pathogens on the basis of thei
lack of one or more virulence determinants may group severa
typesof distantly related orunrelated bacteria together,and
cause some distinct pathogenic categories with uncharacteris
virulence determinants to be overlooked.
CONCLUSION
The abilityto divide E. coli into subtypesis essentialto
understand the epidemiology and pathogenesisof particular
clones.The use of sequence typing,biotyping,serotyping,and
pathotyping to group similar bacteria together while separatin
them from others is helpful in many circumstances, such as w
tracing outbreaks, but can be misleading when serotypes cha
or classification systems struggle to accommodate novel stra
The subdivisionof DEC into pathotypesis critical
for understandinghow thesebacteriacausedisease.The
identification ofpathotypesis also invaluableclinically(to
determiningprognosisand guide clinical management)
and epidemiologically to detectoutbreaksand estimatethe
contribution ofdifferenttypes ofDEC to the overallburden
of disease,as wellas for the controlof these diseases by public
health interventionsand immunisation (Levine etal., 1983;
Sjöling et al., 2015).
Whole genomesequencingof E. coli strainshas vastly
enhanced our understanding of the evolution and pathobiolog
of this highly adaptableand versatilespecies.A major
advantage ofwhole genome sequencing is thatmostsubtypes
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Robins-Browne et al. E. coliPathotypes
and otherpropertiescan be predicted with ahigh degree
of accuracyfrom sequencedata.Combinedwith clinical,
pathologicaland epidemiologicalmetadata,whole genome
sequencing willalso permit elucidation of which strains within
a subtype are more virulence than others.For these reasons,
we expectthatsome ofthe typing schemesin currentuse
will eventuallybe replaced bya system thatis based on
a combination ofgeneswithin thecore genome(probably
cgMLST) and the accessory genome, comprising major virulence
determinants and associated pathogenic potential. In this regard,
the coordinated sharing ofwhole genome sequence data via
GenomeTrakr,coupled with standardised extraction ofE. coli
typing information including sequence type, serotype, pathotype
and antimicrobial resistance from genome data using tools such
as Enterobase is likely to become the new gold standard for E.
coli analysis.Thus,although whole genome sequencing will not
replace pathotyping in the short-term,it should,together with
clinical,field,and experimentaldata,be used to enhance our
understanding of what constitutes a pathotype, while allowing
more pathotypes to be identified by permitting the identificat
of particularcombinationsof genesthatare associated with
specific clinicalsyndromes and pathology.This is particularly
important for loosely defined pathotypes, such as EAEC, DAEC
AIEC, and atypical EPEC.
AUTHOR CONTRIBUTIONS
All of the authorscontributed tothe preparation ofthe
manuscript, and to the ideas and concepts contained in it.
ACKNOWLEDGMENTS
Research in the authors’ laboratories is funded by the Austral
NHMRC.
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Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 9 November 2016 | Volume 6 | Article 141