Animal and human pathogenic Escherichia coli strains share common genetic

backgrounds
Olivier Clermont
a
, Maiwenn Olier
b
, Claire Hoede
a
, Laure Diancourt
c
, Sylvain Brisse
c
,
Monique Keroudean
b
, Je re my Glodt
a
, Bertrand Picard
d
, Eric Oswald
b,e
, Erick Denamur
a,
*
a
UMR722, INSERM and Universite Paris Diderot, Site Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France
b
INRA, UMR 1225, 31076 Toulouse, France
c
Genotyping of Pathogens and Public Health, Institut Pasteur, 75724 Paris, France
d
UMR722, INSERM and Universite Paris Nord, Site Xavier Bichat, 75018 Paris, France
e
Laboratoire de Bacteriologie-Hygie`ne, CHU de Toulouse, Institut Federatif de Biologie, 31059 Toulouse, France
1. Introduction
Escherichia coli is one of the most versatile bacterial species. It
alternates betweenits primary habitat, the gut of vertebrates, where
it lives as a commensal (Tenaillon et al., 2010), and its secondary
habitat, water and sediment (Savageau, 1983). It may also function
as anintra- and extraintestinal pathogeninhumans and many other
animal species (Kaper et al., 2004). This diversity of lifestyles is
achieved through a high degree of genome plasticity, with gene
losses and gains, through horizontal transfer (Rasko et al., 2008;
Touchon et al., 2009). This species has a core genome of less than
2000 genes, but more than 10,000 genes in total (Rasko et al., 2008;
Touchon et al., 2009). Thus, the diverse phenotypes observed result
principally from a large number of different gene combinations.
Despitethe highdegree of geneow, the populationstructureof this
species remains mostly clonal (Touchon et al., 2009), with the clear
delineationof at least sixprincipal phylogenetic groups (A, B1, B2, D,
E and F) (Jaureguy et al., 2008; Tenaillon et al., 2010) and the Shigella
strains, which belong to the E. coli species but cluster outside the
principal phylogenetic groups (Escobar-Paramo et al., 2003; Pupo
et al., 2000). It has been shown that genetic background plays a role
inthe acquisition, retentionandexpressionof foreignDNA(Escobar-
Paramo et al., 2004).
Besides the Shigella strains that are clearly restricted to human
host andhave inactivatednumerous genes during their evolutionary
history(Denamur et al., 2010), it has beensuggestedthat somegroup
B2 strains of the O81 serogroup may be specic for humans
(Clermont et al., 2008) and that some group B1 strains with the hly
gene may be specic for animals (Escobar-Paramo et al., 2006).
However, little is known about the relationships between phyloge-
netic groups and host specicity. In this context, the extent to
which bacterial strains from infected humans and animals are
Infection, Genetics and Evolution 11 (2011) 654662
A R T I C L E I N F O
Article history:
Received 12 October 2010
Received in revised form 4 February 2011
Accepted 7 February 2011
Available online 13 February 2011
Keywords:
Escherichia coli
Phylogeny
Host specicity
Pathogenic
A B S T R A C T
Escherichia coli is a versatile species encompassing both commensals of the digestive tracts of many
vertebrates, including humans, and pathogenic strains causing various intra- and extraintestinal
infections. Despite extensive gene ow between strains, the E. coli species has a globally clonal
population structure, consisting of distinct phylogenetic groups. Little is known about the relationships
between phylogenetic groups and host specicity. We therefore used multilocus sequence typing (MLST)
to investigate phylogenetic relationships and evaluated the virulence gene content of 35 E. coli strains
representative of the diverse diseases encountered in domestic animals. We compared these strains with
a panel of 101 human pathogenic and 98 non-human and human commensal strains representative of
the phylogenetic and pathovar diversity of this species. A global factorial analysis of correspondence
indicated that extraintestinal infections were caused mostly by phylogenetic group B2 strains, whereas
intraintestinal infections were caused mostly by phylogenetic group A/B1/E strains, with strains
responsible from extraintestinal or intraintestinal infections having specic virulence factors. It was not
possible to distinguish between strains of human and animal origin. A detailed phylogenetic analysis of
the MLST data showed that numerous pathogenic animal and human strains are very closely related, and
had a number of virulence genes in common. However, a set of specic adhesins was identied in animal
non-B2 group strains of all pathotypes. In conclusion, human and animal pathogenic strains share
common genetic backgrounds, but non-B2 strains of different origins seem to have different sets of
adhesins that could be involved in host specicity.
2011 Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: +33 1 57 27 75 34.
E-mail address: erick.denamur@inserm.fr (E. Denamur).
Contents lists available at ScienceDirect
Infection, Genetics and Evolution
j our nal homepage: www. el sevi er . com/ l ocat e/ meegi d
1567-1348/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.meegid.2011.02.005
phylogenetically related is unclear. Several studies based on
serotyping, multilocus enzyme electrophoresis, outer membrane
protein proles, pulsed-eld gel electrophoresis, ribotyping, ran-
domly amplied polymorphic DNA, phylogenetic group afliation
and virulence gene content have shown close relationships between
humanandanimal isolates (Achtmanet al., 1986; Cheriet al., 1991,
1994; Ewers et al., 2007; Girardeau et al., 2003, 2005; Johnson et al.,
2001, 2008; Mariani-Kurkdjian et al., 1993; Maynard et al., 2004;
Moulin-Schouleur et al., 2006; Pradel et al., 2001; Wu et al., 2008).
More recently, multilocus sequence typing (MLST) has been used to
study the phylogenetic relationships between strains in more detail.
These studies have focusedprincipallyonextraintestinal pathogenic
E. coli (ExPEC) [includingavianpathogenicE. coli (APEC) inparticular]
(Mora et al., 2009; Moulin-Schouleur et al., 2006, 2007) and
enterohemorrhagic E. coli (EHEC) (Feng et al., 2007; Newton et al.,
2009) strains. Large amounts of data have been amassed, but these
ndings are fragmented and difcult to compare, as different typing
approaches and non-redundant sets of strains were used in the
various studies. Consequently, there is currently no overview of the
global relationships between animal and human pathogenic strains
in the framework of the phylogeny of the E. coli species as a whole.
The aimof this work was to use MLST to study the phylogenetic
relationships and to assess the virulence gene content of 35 E. coli
strains representative of the diverse diseases encountered in
domestic animals, comparing these strains with a panel of 101
human pathogenic and 98 non human and human commensal
strains representative of the phylogenetic diversity of the species
and including well characterized archetypal strains.
2. Materials and methods
2.1. Bacterial strains
We studied 234 E. coli strains and one strain of Escherichia
fergusonii, the closest relative of E. coli (Lawrence et al., 1991) (Table
S1). Five groups of E. coli strains were represented: (i) a panel of 35
strains pathogenic in animals and representative of the various
diseases encountered in domestic animal species (from 8 birds and
27 mammals) comprising 15 ExPEC/APEC (the APEC strains
originating from the 8 birds) and 20 intraintestinal pathogenic E.
coli (InPEC) [8 enterotoxigenic E. coli (ETEC), 4 enteropathogenic E.
coli (EPEC), 5 Shiga toxin-producing E. coli (STEC)/EHEC and 3
unclassied InPEC] strains (Table 1), (ii) a panel of 93 pathogenic
human strains comprising 43 ExPEC [29, 8 and 6 involved in urinary
tract infection (UTI), newborn meningitis (NBM), septicemia and
miscellaneous infections, respectively] and50InPEC[7ETEC, 6EPEC,
8 EHEC, 10 enteroaggregative E. coli (EAEC), 1 enteroinvasive E. coli
(EIEC), 16diffusely adherent E. coli (DAEC) and 2 unclassiedInPEC],
(iii) 45 non human mammalian commensal strains, (iv) 53 human
commensal strains and (v) 8 human InPEC strains for which the
complete genome was available (Ogura et al., 2009; Rasko et al.,
2008); these strains were typed in silico in this study. The strains
fromgroups (ii), (iii) and (iv) inthis list originated mostly fromthree
published collections (Escobar-Paramo et al., 2004; Le Gall et al.,
2007; Ochman and Selander, 1984) and, withthe strains fromgroup
(v), may be considered representative of the phylogenetic and
pathovar diversity of E. coli. They encompass archetypal strains for
various diseases, and complete genome sequences are available for
some, in addition to group (v) strains.
2.2. Virulence factor (VF) screening
We tested for the presence of virulence factors involved in
extraintestinal (neuC, kpsE, sfa/foc, iroN, aer, iha, papC, papGI, papGII,
papGIII, hly, cnf1, hra, sat, ire, usp, chromosomal ompT, ibeA, malX,
irp2, fyuA and traT) (Diard et al., 2007; Johnson et al., 2006) and
intraintestinal [afaD, ipaH, stx1, stx2, eltB (LT), estA (ST), bfpA, eae,
aaiC and aatA] (Table S2) infections by PCR, as previously described
(Escobar-Paramo et al., 2004). The extraintestinal genes tested
correspond to the main classes of extraintestinal VFs, i.e. adhesin,
toxin, iron capture system, protectin. The intraintestinal genes
tested allow the classication in the main intestinal E. coli
pathovars, i.e. ETEC, EPEC, EHEC, EIEC, EAEC and DAEC (Kaper
et al., 2004). We also used PCR, as previously described (Bertin
et al., 1996; Boerlin et al., 2005; Dow et al., 2005; Franck et al.,
1998; Imberechts et al., 1994), to check for the presence of
adhesins classically associated with animal-specic pathogenic
strains of E. coli: K99 (fanA), K88 (faeG), F17 (f17A), F18 (fedA) and
Afr2 (afr2G), referred latter on as animal adhesins.
2.3. PCR O-typing
O-type was determined by an allele-specic PCR (Clermont
et al., 2007) using the primers given in Table S3. We assessed 28 O-
types with this assay. These 28 O-types were selected based on the
O-types already reported using the classical serological method for
the other strains of the collection.
2.4. MLST
MLSTwas performedwithpartial dinB, icdA, pabB, polB, putP, trpA,
trpB, anduidAsequences (Jaureguyet al., 2008). Allelesequences and
sequence types (STs) are available from Institut Pasteurs MLST
website, at www.pasteur.fr/mlst. Phylogenetic analysis was per-
formed with the concatenated sequences of the eight genes, by the
maximum likelihood (ML) method, as implemented in the PHYML
program (Guindon et al., 2005), as well as by the neighbor joining
(NJ) andmaximumparsimony (MP) methods using MEGA4(Tamura
et al., 2007), with E. fergusonii as the outgroup.
2.5. Factorial analysis of correspondence (FAC)
FAC was used to describe associations between the different
data sets. FAC uses a covariance matrix based on Chi squared
distances (Greenacre, 1992). This computation method determines
a plane dened by two principal axes of the analysis. The rst axis
(F1) accounts for most of the variance, and the second axis (F2),
orthogonal to F1, accounts for the largest part of the variance not
accounted for by F1. FAC was conducted with SPAD.N 4.5 software
(Cisia, Saint Mande , France), based on a two-way table. This table
had 234 rows, one for each E. coli strain, and 35 columns,
corresponding to 35 variables: human/animal origin, commensal,
ExPEC and InPEC characters, seven phylogenetic groups corre-
sponding to A, B1, B2, C, D, E, F and ungrouped (UG) strains
according to the MLST data (Escobar-Paramo et al., 2004; Jaureguy
et al., 2008), and 23 VFs (neuC, sfa/foc, iroN, papC, papGI, papGII,
papGIII, hlyC, cnf1, hra, fyuA, animal adhesins (AnAd), afaD, ipaH,
stx1, stx2, estA, eltB, bfpA, eae, aaiC and aatA). The data in this table
were attributed a binary code: 1 for present and 0 for absent.
The loading score for each variable on the plane of the variables
(factors F1 and F2, respectively) can be inferred from the
coordinates of the X and Y variables on the F1/F2 plane. Moreover,
the data corresponding to active variables were calculated directly
in the FAC, whereas the illustrative variables were only projected
onto the plane and not included in the computation.
3. Results and discussion
3.1. Multidimensional analysis
We assessed the global relationships between the phylogenetic
groups, the VF content and origin (human versus animal and
O. Clermont et al. / Infection, Genetics and Evolution 11 (2011) 654662 655
commensal versus pathogenic) of the strains, by carrying out a FAC
with phylogenetic groups and VFs as active variables and the
animal/human, commensal, ExPEC and InPEC variables as illustra-
tive variables. Strains were assigned to phylogenetic groups on the
basis of MLST data. Seven groups were considered: the six
previously recognized groups (A, B1, B2, D, E and F) and the C
group plus ungrouped strains (see below for the denition of the C
group and ungrouped strains). On the F1/F2 plane, which
accounted for 29.37% of the total variance, the variables papGI,
papGIII, cnf1, sfa/sfoc, hlyC, iroN, hra, papC, neuC, kpsE, fyuA, B2 group
and ExPEC were projected onto the positive values of the F1 axis,
whereas the variables stx2, E group, eae, stx1, bfpA, eltB, estA, B1
group, InPEC, AnAd, ipaH, aatA and Agroup were projected onto the
negative values of this axis. The variables F group, papGII, D group,
aer, afaDand aaiC were projected onto the negative values of the F2
axis. The variables human, animal and commmensal were grouped
around the origin of the axes and it was therefore not possible to
differentiate between them in this FAC (Fig. 1). When projecting
the strains on the plane, most of the ExPEC strains had positive F1
coordinates (Fig. 2A), whereas the InPEC strains had negative
coordinates on this axis (Fig. 2B). Moreover, it was not possible to
distinguish between ExPEC and InPEC strains of human and animal
origin in this FAC (Fig. 2).
This global analysis suggests that extraintestinal infections are
caused principally by B2 strains, which have many extraintestinal
VFs, whereas intraintestinal infections are caused mostly by A/B1/E
strains exhibiting intestinal VFs. It was not possible to distinguish
between strains of human and animal origin.
Table 1
Main characteristics of the 35 pathogenic animal E. coli strains studied.
Table 1. Main characteristics of the 35 pathogenic animal E. coli strains studied 1
Strain ID Host
Country
origin
Patho-
genicity
Phylo-
genetic
group
and
sub-
group
ST
a
O-type
n
e
u
C

b
k
p
s
E
s
f
a
/
f
o
c
i
r
o
N
a
e
r
p
a
p
C
p
a
p
G

I
p
a
p
G

I
I
p
a
p
G
I
I
I
h
l
y
C
c
n
f
1
h
r
a
f
y
u
A "Animal"
adhesin a
f
a
D
i
p
a
H
s
t
x
1
s
t
x
2
e
l
t
B
(
L
T
)
e
s
t
A
(
S
T
)
b
f
p
A
e
a
e
a
a
i
C
a
a
t
A
25KH9 Bos taurus Belgium ETEC A I 2 101 - - - + - + - - - - - + - F17a - - - - - - - - - -
S1191 Sus scrofa USA STEC A I 2 139 - - - - - - - - - - - - - F18 - - - + - + - - - -
510 Bos taurus Belgium ETEC A I 160 101 - - - - - - - - - - - + - K99 - - - - - + - - - -
431 Sus scrofa USA ETEC A I 160 101 - - - - - - - - - - - + - K99 - - - - - + - - - -
262KH89 Bos taurus Belgium Diarrhea A I 230 26 - - - - - - - - - + - - - K88 - - - - - - - - + -
255/1-1 Bos taurus France EHEC A I 140 Unknown - - - - - - - - - - - - - - + - + - - - - - - -
126A Bos taurus Belgium ETEC UG 228 8 - - - - - - - - - - - + - K99 - - - - - + - - - -
86-1390 Sus scrofa Canada EPEC B1 245 45b - - - - - - - - - - - - + - - - - - - - - + - -
193 Bos taurus USA EHEC B1 246 26 - - - - + - - - - - - + + - - - - - - - - + - -
C/15333 Bos taurus
Northern
Ireland
EPEC B1 232 26 - - - - - - - - - + - - + - - - - - - - - + - -
RDEC-1
Oryctolagus
cuniculus
USA EPEC B1 493 15 - - - - + - - - - - - - + - - - - - - - - + - -
5131 Sus scrofa Canada ExPEC B1 248 115 - - + + + + - - + - - + - - - - - - - - - - - -
987 Sus scrofa USA ETEC B1 249 9 - - - - - - - - - - - - - K99 - - - - - + - - - -
31A Bos taurus France Diarrhea B1 NC 153 - - - - + + - - - - - + + F17c/GAF - - - - - - - - - -
211 Bos taurus Belgium ETEC B1 247 Rough + + - - - - - - - - - + - - - - - - - - - - - -
S5 Ovis aries UK ExPEC B1 266 15 + + - - - - - - - + - - + F17b - - - - - - - - - -
E22
Oryctolagus
cuniculus
France EPEC B1 135 103 - - - - - - - - - - - - - AFR2 - - - - - - - + - -
111KH86 Bos taurus Belgium Diarrhea B1 227 Unknown - - - - - - - - - - - + - F111 - - - - - - - - - -
789 Gallus gallus Israel APEC C 148 78 - - + + + - - - - - - - + - - - - - - - - - - -
BEN 0265 Gallus gallus France APEC C 147 78 - - - + + - - - - - - - + - - - - - - - - - - -
1404 Bos taurus France ExPEC C 159 78 - - - - - - - - - + - - + F17b - - - - - - - - - -
239KH89 Bos taurus Belgium ExPEC C 229 Unknown - - - - + + - - + + + + + - - - - - - - - - - -
DEC7a Sus scrofa USA ETEC C 157 157 - - - - - - - - - + - - + K88 - - - - + + - - - -
248/1-2 Bos taurus France EHEC C 270 Unknown - - - + - + - - - - - + + - + - + - - - - - - -
G7 Sus scrofa UK ETEC C NC 8 - - - - - - - - - + - - + K88 - - - - + - - - - -
DEC4a Bos taurus Argentina EHEC E 284 157 - - - - - - - - - - - - - - - - + - - - - + - -
BEN 1189 Gallus gallus Belgium APEC D 145 78 - - - + + - - - - - - - - - - - - - - - - - - -
BM2-1 Bos taurus France ExPEC B2 II 150 2 - - + + - - - - - - - + + - - - - - - - - - - -
M623 Sus scrofa Spain ExPEC B2 IV 223 2 + + - + - + - - + + + + + - - - - - - - - - - -
28C Sus scrofa Spain ExPEC B2 VII 141 75 - - + + - + - - + + + + + - - - - - - - - - - -
APECO1 Gallus gallus USA APEC B2 IX 418 1 + + - + + + - + - - - - + - - - - - - - - - - -
BEN 0139
Meleagridis
gallopavo
France APEC B2 IX 146 2 + + - + + - - - - - - - + - - - - - - - - - - -
BEN 2908 Gallus gallus France APEC B2 IX 146 2 + + + + + - - - - - - + + F111 - - - - - - - - - -
BEN 0374 Gallus gallus Spain APEC B2 IX 146 18 + + + + + - - - - - - - + - - - - - - - - - - -
BEN 0079 Gallus gallus France APEC B2 IX 146 18 + + + + + - - - - + + + + - - - - - - - - - - -
a
ST numbers correspond to the IP scheme (www.pasteur.fr/mlst). NC: non-coded because more than one gene over the 8 studied are not ampliable.
b
Colors correspond to the different PAIs and plasmid determined as in (Bingen-Bidois et al., 2002): orange, PAI II
J96
; green, PAI III
536
; violet, HPI; grey, plasmid origin.
O. Clermont et al. / Infection, Genetics and Evolution 11 (2011) 654662 656
3.2. Fine-scale phylogenetic analysis
We investigated the relationships between human and animal
pathogenic strains in more details, at the clonal level and within
the species as a whole, by carrying out MLST analysis on the 234
strains. Three main MLST schemes are currently available for E. coli,
i.e. the scheme used in this work (Institut Pasteur, IP, scheme)
(Jaureguy et al., 2008), the Whittam scheme (http://www.shiga-
tox.net/ecmlst/cgi-bin/dbquery) (Reid et al., 2000) and the Acht-
man scheme (http://mlst.ucc.i.e./mlst/dbs/Ecoli) (Wirth et al.,
2006), each using a different combination of genes. The MLST
data are usually studied in two ways, with the alleles at different
loci providing an allelic prole, which denes the sequence type
(ST). No weighting is given to take into account the number of
nucleotide differences between the alleles. Alternatively, nucleo-
tide sequences may be used for phylogenetic reconstructions
(Tenaillon et al., 2010). The results obtained with these three
schemes are highly correlated (Gordon et al., 2008), suggesting
that the clonal structure of the species is robust.
We chose to use the set of genes described in a previous study
(Jaureguy et al., 2008), as we have previously used this MLST
scheme to characterize a unique set of strains representative of the
phylogenetic and lifestyle diversity of the species. Large amounts
of data are available for these strains. The phylogenetic tree
reconstructed from the concatenated sequences using the ML
method (Fig. 3) showed the major phylogenetic groups previously
described (Tenaillon et al., 2010): A, B1, B2, D, E and F. An
additional group, closely related to the B1 group but identied as A
by PCR triplex phylogrouping (Clermont et al., 2000), was also
identied and called C (Escobar-Paramo et al., 2004; Moissenet
et al., 2010). Only six strains (ECOR42, ECOR31, 126A, DEC9a, 101-1
and DAEC5) were not included in these groups and were classied
as ungrouped, indicating the robustness of the phylogenetic
classication. The F and B2 groups are the most basal, having
emerged rst, whereas the A and B1/C groups diverged more
recently. Furthermore, a clear genetic structure was identied
within the B2 phylogenetic group, with at least nine subgroups (I
IX) in addition to the EPEC 1 cluster commonly represented by the
O127:H6 strain E2348/69 (Le Gall et al., 2007; Reid et al., 2000).
Two subgroups (I and II) were identied within the A phylogenetic
group, the subgroup II corresponding to the strains with an A
0
genotype with the PCR triplex typing method, i.e. the absence of
amplication of any of the genetic markers (chuA, yjaA and
TSPE4.C2) (Clermont et al., 2000; Gordon et al., 2008). No
phylogenetically robust subgroup was observed within the B1
and D phylogenetic groups, only some strains were grouped with
high bootstrap values. The phylogenetic groups and subgroups
delineated above were retrieved when the phylogenetic tree from
the concatenated sequences was reconstructed using NJ (Fig. S1)
and MP (Fig. S2) methods. Furthermore, a minimum spanning tree
analysis based on allelic prole data gave similar results for the
shallow phylogenetic grouping (data not shown). The ST numbers
of the strains using the IP scheme is given in Table 1 and Table S1,
as well as, when available, the correspondence with the ST using
the Achtman scheme (Table S1).
With this phylogenetic approach, we clearly identied animal
and human strains belonging to the same phylogenetic subgroups
or clonal complexes (closely related STs), as presented below.
Bothanimal andhumanExPECstrains were foundinall but three
of the subgroups of the B2 phylogenetic group(I, Vand VIII) (Table 1
and Table S1, Fig. 3). The most highly represented subgroup was
subgroup IX, which corresponds to ST95 of the Achtman scheme
(Wirth et al., 2006), ST29 of the Whittam scheme (Newton et al.,
2009), and the B2-1 group of a previous study (Moulin-Schouleur
et al., 2007). This subgroup encompasses the APEC strains of
serogroup O1, O2 and O18 (Johnson et al., 2007; Mora et al., 2009;
Moulin-Schouleur et al., 2007) and the archetypal human strains
UTI89(Chenet al., 2006), a straincausing urinarytract infection, and
RS218, an isolate from a neonate with meningitis (Xie et al., 2006),
bothof serotype O18:K1:H7. Non-humanmammalianExPECstrains
were also found in subgroup II (O2-type strain, archetypal UTI
human strain CFT073 of O6-type), subgroup IV (O2-type strain,
archetypal UTI human strain IAI74 of O2-type) and subgroup VII
(O75-type strain, archetypal human UTI strain IH11128 of O75-
type). Furthermore, strains of serotype O6:H31, which have been
reported in dog urinary tract infections (Cheri et al., 1991; Johnson
et al., 2001), belongtosubgroupIII (archetypal humanUTI strain536
of O6-type) and O4:H5 strains, also isolated in urinary tract
Fig. 1. Factorial analysis of correspondence (FAC) of the 234 E. coli strains. Projections of the 35 variables human/animal origin, commensal, ExPEC and InPEC characters, 7
phylogenetic groups (A, B1, B2, C, D, E and F) and ungrouped strains (UG) and 23 VFs (see Section 2) onto the F1/F2 plane calculated in the FAC (Greenacre, 1992). AnAd:
Animal adhesin.
O. Clermont et al. / Infection, Genetics and Evolution 11 (2011) 654662 657
infections indogandcat (Johnsonet al., 2001), belongtosubgroupVI
(archetypal human UTI strain J96 of O4-type) (Le Gall et al., 2007)
(data not shown). As expected, no pathogenic animal strain
belonged to subgroup VIII, as this clone has been described as
commensal inhumans (Clermont et al., 2008). Similarlyonlyhuman
EPECstrains testingpositivefor bfpAbyPCRwerepresent intheEPEC
1 cluster.
Non B2 ExPEC animal strains of the O78-type belong to
phylogenetic groups D (one APEC strain) and C (2 APEC and 2
bovine ExPEC strains) (Table 1). Group D is diverse, and no close
relationship was identied between the animal ExPEC strain
(BEN1189) and human strains, although numerous human ExPEC
strains belong to this group (Bingen et al., 1998; Picard et al., 1999)
(Fig. 3). Conversely, the group C is clonal with short branch lengths
(Fig. 3). Thus, the numerous APEC strains of the O78-type assigned
to phylogroup A by the triplex PCR method (Ewers et al., 2007;
Johnson et al., 2008) probably belong to this clone. One of the group
C strains tested, the bovine ExPEC strain 1404, which carries the Vir
plasmid, has been shown to be closely related to avian and human
ExPEC strains, as it belongs to esterase electrophoretic type 2
Fig. 2. Factorial analysis of correspondence (FAC) of the 234 E. coli strains. Projections of (A) the ExPEC strains (black symbols) of animal (triangle) and human (square) origin,
the non-ExPEC strains (white symbols) and (B) the InPEC strains of animal (triangle) and human (square) origin and the non-InPEC strains (white symbols) onto the F1/F2
plane calculated in the FAC (Greenacre, 1992).
O. Clermont et al. / Infection, Genetics and Evolution 11 (2011) 654662 658
(Cheri et al., 1994). The group C strains in our collection also
include one human ExPEC strain, ECOR72, and animal and human
InPEC strains. A case of neonatal meningitis due to a group C strain
has recently been reported (Moissenet et al., 2010). This strain was
shown to be virulent in a mouse model of septicemia (Picard et al.,
1999). One ExPEC strain from pig (5131) and another from sheep
(S5), belong to the B1 group (Table 1) and appear closely related to
human InPEC strains (Fig. 3).
Fig. 3. Phylogenetic tree of the 234 E. coli strains studied, reconstructed fromthe partial sequences of 8 housekeeping genes (www.pasteur.fr/mlst) by PHYML (Guindon et al.,
2005) and rooted on E. fergusonii. Bootstrap values are indicated at the corresponding nodes only when they exceed 70%. Pathogenic strains are indicated in bold and boxed in
grey. The name of the strain is followed by H (for human origin) or A (for animal origin). The phylogenetic groups and subgroups are indicated on the right part of the gure.
O. Clermont et al. / Infection, Genetics and Evolution 11 (2011) 654662 659
Animal InPEC strains were found to belong to phylogenetic
groups A, B1, C and E. Group E, which includes human and animal
EHEC O157:H7 strains, is clonal, whereas groups A and B1 are more
diverse, with longer branch lengths (Fig. 3). All animal InPEC (ETEC
and EHEC) strains of phylogenetic group A belong to subgroup I,
which also contains human InPEC (DAEC and EAEC with the
archetypal EAEC strain JM221) and ExPEC strains, as well as the
human commensal laboratory derived strain K-12 (Fig. 3). Within
the B1 group, bovine O26-type EHEC (193) and EPEC (C/15333),
together with O45b-type (86-1390) and O15-type EPEC (RDEC-1)
strains, are closely related to human O26-type and O111-type
EHEC strains. The swine ETEC strain 987 appears closely related to
the human EPEC strain E110019, as are the bovine InPEC 111KH86
and the human ETEC DEC13a strains (Fig. 3). The rabbit EPEC strain
E22 (O103-type) is very closely related, with a high bootstrap
value, to the human O103-type EHEC strain 12009, as previously
reported (Mariani-Kurkdjian et al., 1993) and to the human O111/
O128-EPEC 2 strains B171 (Rasko et al., 2008), DEC12a and DEC11a
(Czeczulin et al., 1999; Reid et al., 2000) (Fig. 3). Animal ETEC and
EHEC strains were found in group C, together with human InPEC
and animal and human ExPEC strains, as stated above (Table 1).
In sum, MLST analysis corroborates the FAC analysis by showing
that animal and human pathogenic strains are in most of the cases
closely phylogenetically related and that the B2 phylogenetic
group encompasses mainly ExPEC strains.
3.3. Fine-scale VF pattern analysis
Animal ExPEC strains have a variable pattern of extraintestinal
VFs, whether considered individually or in the pathogenicity island
(PAI) context, even within a clonal lineage (Table 1). Different
patterns were also observed between human and animal strains of
the same lineage (for example, within B2 subgroups II, VII and IX;
Table 1 and Table S1). Furthermore, no particular pattern of VFs
was found to be specic to a particular type of extraintestinal
disease (e.g. UTI, NBM, septicemia or avian colibacillosis), as
previously reported (Bauchard et al., 2010; Ewers et al., 2004;
Johnson et al., 2007; Kariyawasamet al., 2007; Mokady et al., 2005;
Moulin-Schouleur et al., 2006). This is in agreement with the recent
ndings based on comparative genomics showing that multiple
genetic paths of gain and loss of genes can lead to convergent
phenotypes (Mokady et al., 2005; Touchon et al., 2009). The
intraintestinal VFs studied were pathotype-specic and therefore
did not distinguish between human and animal strains. In a very
small number of strains in our collection, we did not nd the
expected pathotype-dening VFs (ST, LT, Bfp). However, in these
cases, the VFs were plasmid-encoded and the plasmid was
probably lost during the many subcultures of the strains (Table 1).
By contrast, adhesins usually described in pathogenic animal
strains (see Section 2), were found in 17 animal strains, of which 15
are pathogenic, but only in four human strains: the F111 adhesin in
one DAEC strain (DAEC221), one EAEC strain (11074) and one
ExPEC strain (ECOR71), and the F17c/GAF adhesin in one ExPEC
strain (IAI64) (Table 1 and Table S1). The expression of the F17c/
GAF adhesin, whichis specic for terminal N-acetyl-D-glucosamine
and blood group M antigen, by human strains (particularly those
isolated from cases of urinary tract infection) is well documented,
although the reported incidence is low (Rhen et al., 1986). By
contrast, to our knowledge, the presence of genes encoding F111
adhesins has not previously been reported in any E. coli strains
pathogenic in humans. As suggested by the FAC (Fig. 1), these
animal adhesins were found almost exclusively in non-B2
strains, as only three of the 21 strains concerned belonged to
phylogenetic group B2 (animal ExPEC strain BEN2908, animal
commensal strain ASP47b and human ExPEC strain IAI64) (Table 1
and Table S1). In sum, a set of specic adhesins was preferentially
associated to the animal pathogenic strains of non-B2 phylogenetic
groups.
3.4. Concluding remarks
Our data for a representative set of animal pathogenic strains
analyzed within the context of the overall phylogeny of the E. coli
species clearly show that human and animal pathogenic strains
share common genetic backgrounds, i.e. are very closely related by
MLST analysis. Human and animal strains causing the same disease
in different hosts share a common pool of virulence genes, but a set
of adhesins specic to animal non-B2 strains was identied.
It has been suggested that, on many occasions, strains derived
from a common recent ancestor have become specialized for a
particular host through subtle genetic changes (Ron, 2006). Our data
indicate that the gain (or loss) of few genes, as genes coding for
adhesins, could participate to the host specicity. But more subtle
changes as single nucleotide polymorphisms (SNPs) in coding or
regulatory regions, in multiple combinations, could also be involved
in the host specicity. The recent identication of numerous SNPs in
O157:H7 EHEC strains specic for humans or cattle, including a SNP
in the translocated intimin receptor protein (Clawson et al., 2009),
and of specic gene expression patterns in B2 phylogenetic group
APEC and human urinary tract infection strains in the chicken and
UTI mouse models (Zhao et al., 2009) is consistent with this
hypothesis. Complete genome sequences for many animal and
humanpathogenic strains, generatedbynext-generation sequenc-
ing technologies (MacLean et al., 2009), will facilitate identication
of the subtle genetic elements involved in host specialization.
Ethical statement
There is no need for ethical statement. The work is based on
pathogenic animal strains and previously published collection of
strains. There is no animal model experiment.
Acknowledgments
We thank J. Fairbrother, J. Mainil and M. Moulin-Schouleur for
providing us with E. coli strains. ED was supported in part by the
Fondation pour la Recherche Me dicale.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.meegid.2011.02.005.
References
Achtman, M., Heuzenroeder, M., Kusecek, B., et al., 1986. Clonal analysis of
Escherichia coli O2:K1 isolated from diseased humans and animals. Infect.
Immun. 51, 268276.
Bauchard, P., Germon, P., Bre e, A., Oswald, E., Hacker, J., Dobrindt, U., 2010.
Pathogenomic comparison of human extraintestinal and avian pathogenic
Escherichia coli search for factors involved in host specicity or zoonotic
potential. Microb. Pathog. 49, 105115.
Bertin, Y., Martin, C., Oswald, E., Girardeau, J.P., 1996. Rapid and specic detection of
F17-related pilin and adhesin genes in diarrheic and septicemic Escherichia coli
strains by multiplex PCR. J. Clin. Microbiol. 34, 29212928.
Bingen, E., Picard, B., Brahimi, N., Mathy, S., Desjardins, P., Elion, J., Denamur, E.,
1998. Phylogenetic analysis of Escherichia coli strains causing neonatal menin-
gitis suggests horizontal gene transfer from a predominant pool of highly
virulent B2 group strains. J. Infect. Dis. 177, 642650.
Bingen-Bidois, M., Clermont, O., Bonacorsi, S., Terki, M., Brahimi, N., Loukil, C.,
Barraud, D., Bingen, E., 2002. Phylogenetic analysis and prevalence of urosepsis
strains of Escherichia coli bearing pathogenicity island-like domains. Infect.
Immun. 70, 32163226.
Boerlin, P., Travis, R., Gyles, C.L., et al., 2005. Antimicrobial resistance and virulence
genes of Escherichia coli isolates fromswine in Ontario. Appl. Environ. Microbiol.
71, 67536761.
O. Clermont et al. / Infection, Genetics and Evolution 11 (2011) 654662 660
Chen, S.L., Hung, C.S., Xu, J., et al., 2006. Identication of genes subject to positive
selection in uropathogenic strains of Escherichia coli: a comparative genomics
approach. Proc. Natl. Acad. Sci. U.S.A. 103, 59775982.
Cheri, A., Contrepois, M., Picard, B., Goullet, P., Orskov, I., Orskov, F., De Rycke, J.,
1991. Clonal relationships among Escherichia coli serogroup 06 isolates from
human and animal infections. FEMS Microbiol. Lett. 64, 225230.
Cheri, A., Contrepois, M., Picard, B., Goullet, P., Orskov, I., Orskov, F., 1994. Clonal
relationships among Escherichia coli serogroup O78 isolates from human and
animal infections. J. Clin. Microbiol. 32, 11971202.
Clawson, M.L., Keen, J.E., Smith, T.P., Durso, L.M., McDaneld, T.G., Mandrell, R.E.,
Davis, M.A., Bono, J.L., 2009. Phylogenetic classication of Escherichia coli
O157:H7 strains of human and bovine origin using a novel set of nucleotide
polymorphisms. Genome Biol. 10, R56.
Clermont, O., Bonacorsi, S., Bingen, E., 2000. Rapid and simple determination of the
Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66, 45554558.
Clermont, O., Johnson, J.R., Menard, M., Denamur, E., 2007. Determination of
Escherichia coli Otypes by allele-specic polymerase chain reaction: application
to the O types involved in human septicemia. Diagn. Microbiol. Infect. Dis. 57,
129136.
Clermont, O., Lescat, M., OBrien, C.L., Gordon, D.M., Tenaillon, O., Denamur, E., 2008.
Evidence for a human-specic Escherichia coli clone. Environ. Microbiol. 10,
10001006.
Czeczulin, J.R., Whittam, T.S., Henderson, I.R., Navarro-Garcia, F., Nataro, J.P., 1999.
Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia
coli. Infect. Immun. 67, 26922699.
Denamur, E., Picard, B., Tenaillon, O., 2010. Population genetics of pathogenic
Escherichia coli. In: Robinson DA, F.D., Feil, E.J. (Eds.), Bacterial Population
Genetics in Infectious Disease. Wiley-Blackwell, Hoboken, pp. 269286.
Diard, M., Baeriswyl, S., Clermont, O., Gouriou, S., Picard, B., Taddei, F., Denamur, E.,
Matic, I., 2007. Caenorhabditis elegans as a simple model to study phenotypic
and genetic virulence determinants of extraintestinal pathogenic Escherichia
coli. Microbes Infect. 9, 214223.
Dow, M.A., Toth, I., Alexa, P., Davies, M., Malik, A., Oswald, E., Nagy, B., 2005.
Predominance of afr2 and ral mbrial genes related to those encoding the K88
and CS31A mbrial adhesins in enteropathogenic Escherichia coli isolates from
rabbits with postweaning diarrhea in Central Europe. J. Clin. Microbiol. 43,
13661371.
Escobar-Paramo, P., Giudicelli, C., Parsot, C., Denamur, E., 2003. The evolutionary
history of Shigella and enteroinvasive Escherichia coli revised. J. Mol. Evol. 57,
140148.
Escobar-Paramo, P., Clermont, O., Blanc-Potard, A.B., Bui, H., Le Bouguenec, C.,
Denamur, E., 2004. A specic genetic background is required for acquisition
and expression of virulence factors in Escherichia coli. Mol. Biol. Evol. 21, 1085
1094.
Escobar-Paramo, P., Le Menach, A., Le Gall, T., Amorin, C., Gouriou, S., Picard, B.,
Skurnik, D., Denamur, E., 2006. Identication of forces shaping the commensal
Escherichia coli genetic structure by comparing animal and human isolates.
Environ. Microbiol. 8, 19751984.
Ewers, C., Janssen, T., Kiessling, S., Philipp, H.C., Wieler, L.H., 2004. Molecular
epidemiology of avian pathogenic Escherichia coli (APEC) isolated from coli-
septicemia in poultry. Vet. Microbiol. 104, 91101.
Ewers, C., Li, G., Wilking, H., et al., 2007. Avian pathogenic, uropathogenic, newborn
meningitis-causing Escherichia coli: how closely related are they? Int. J. Med.
Microbiol. 297, 163176.
Feng, P.C., Monday, S.R., Lacher, D.W., et al., 2007. Genetic diversity among clonal
lineages within Escherichia coli O157:H7 stepwise evolutionary model. Emerg.
Infect. Dis. 13, 17011706.
Franck, S.M., Bosworth, B.T., Moon, H.W., 1998. Multiplex PCR for enterotoxigenic,
attaching and effacing, and Shiga toxin-producing Escherichia coli strains from
calves. J. Clin. Microbiol. 36, 17951797.
Girardeau, J.P., Lalioui, L., Said, A.M., De Champs, C., Le Bouguenec, C., 2003.
Extended virulence genotype of pathogenic Escherichia coli isolates carrying
the afa-8 operon: evidence of similarities between isolates from humans and
animals with extraintestinal infections. J. Clin. Microbiol. 41, 218226.
Girardeau, J.P., Dalmasso, A., Bertin, Y., Ducrot, C., Bord, S., Livrelli, V., Vernozy-
Rozand, C., Martin, C., 2005. Association of virulence genotype with phyloge-
netic background in comparison to different seropathotypes of Shiga toxin-
producing Escherichia coli isolates. J. Clin. Microbiol. 43, 60986107.
Gordon, D.M., Clermont, O., Tolley, H., Denamur, E., 2008. Assigning Escherichia coli
strains to phylogenetic groups: multi-locus sequence typing versus the PCR
triplex method. Environ. Microbiol. 10, 24842496.
Greenacre, M., 1992. Correspondence analysis in medical research. Stat. Methods
Med. Res. 1, 97117.
Guindon, S., Lethiec, F., Duroux, P., Gascuel, O., 2005. PHYML online a web server
for fast maximum likelihood-based phylogenetic inference. Nucleic Acids Res.
33, W557559.
Imberechts, H., Van Pelt, N., De Greve, H., Lintermans, P., 1994. Sequences related to
the major subunit gene fedA of F107 mbriae in porcine Escherichia coli strains
that express adhesive mbriae. FEMS Microbiol. Lett. 119, 309314.
Jaureguy, F., Landreau, L., Passet, V., et al., 2008. Phylogenetic and genomic diversity
of human bacteremic Escherichia coli strains. BMC Genomics 9, 560.
Johnson, J.R., Delavari, P., Stell, A.L., Whittam, T.S., Carlino, U., Russo, T.A., 2001.
Molecular comparison of extraintestinal Escherichia coli isolates of the same
electrophoretic lineages from humans and domestic animals. J. Infect. Dis. 183,
154159.
Johnson, J.R., Clermont, O., Menard, M., Kuskowski, M.A., Picard, B., Denamur, E.,
2006. Experimental mouse lethality of Escherichia coli isolates, in relation to
accessory traits, phylogenetic group, and ecological source. J. Infect. Dis. 194,
11411150.
Johnson, T.J., Kariyawasam, S., Wannemuehler, Y., et al., 2007. The genome
sequence of avian pathogenic Escherichia coli strain O1:K1:H7 shares strong
similarities with human extraintestinal pathogenic E. coli genomes. J. Bacteriol.
189, 32283236.
Johnson, T.J., Wannemuehler, Y., Johnson, S.J., Stell, A.L., Doetkott, C., Johnson, J.R.,
Kim, K.S., Spanjaard, L., Nolan, L.K., 2008. Comparison of extraintestinal patho-
genic Escherichia coli strains from human and avian sources reveals a mixed
subset representing potential zoonotic pathogens. Appl. Environ. Microbiol. 74,
70437050.
Kaper, J.B., Nataro, J.P., Mobley, H.L., 2004. Pathogenic Escherichia coli. Nat. Rev.
Microbiol. 2, 123140.
Kariyawasam, S., Scaccianoce, J.A., Nolan, L.K., 2007. Common and specic genomic
sequences of avian and human extraintestinal pathogenic Escherichia coli as
determined by genomic subtractive hybridization. BMC Microbiol. 7, 81.
Lawrence, J.G., Ochman, H., Hartl, D.L., 1991. Molecular and evolutionary relation-
ships among enteric bacteria. J. Gen. Microbiol. 137, 19111921.
Le Gall, T., Clermont, O., Gouriou, S., Picard, B., Nassif, X., Denamur, E., Tenaillon, O.,
2007. Extraintestinal virulence is a coincidental by-product of commensalism
in B2 phylogenetic group Escherichia coli strains. Mol. Biol. Evol. 24, 23732384.
MacLean, D., Jones, J.D., Studholme, D.J., 2009. Application of next-generation
sequencing technologies to microbial genetics. Nat. Rev. Microbiol. 7, 287296.
Mariani-Kurkdjian, P., Denamur, E., Milon, A., et al., 1993. Identication of a clone of
Escherichia coli O103:H2 as a potential agent of hemolytic-uremic syndrome in
France. J. Clin. Microbiol. 31, 296301.
Maynard, C., Bekal, S., Sanschagrin, F., Levesque, R.C., Brousseau, R., Masson, L.,
Lariviere, S., Harel, J., 2004. Heterogeneity among virulence and antimicrobial
resistance gene proles of extraintestinal Escherichia coli isolates of animal and
human origin. J. Clin. Microbiol. 42, 54445452.
Moissenet, D., Salauze, B., Clermont, O., Bingen, E., Arlet, G., Denamur, E., Merens, A.,
Mitanchez, D., Vu-Thien, H., 2010. Meningitis caused by Escherichia coli pro-
ducing TEM-52 extended-spectrum beta-lactamase within an extensive out-
break in a neonatal ward: epidemiological investigation and characterization of
the strain. J. Clin. Microbiol. 48, 24592463.
Mokady, D., Gophna, U., Ron, E.Z., 2005. Extensive gene diversity in septicemic
Escherichia coli strains. J. Clin. Microbiol. 43, 6673.
Mora, A., Lopez, C., Dabhi, G., et al., 2009. Extraintestinal pathogenic Escherichia coli
O1:K1:H7/NMfromhuman and avian origin: detection of clonal groups B2 ST95
and D ST59 with different host distribution. BMC Microbiol. 9, 132.
Moulin-Schouleur, M., Schouler, C., Tailliez, P., et al., 2006. Common virulence
factors and genetic relationships between O18:K1:H7 Escherichia coli isolates of
human and avian origin. J. Clin. Microbiol. 44, 34843492.
Moulin-Schouleur, M., Reperant, M., Laurent, S., Bree, A., Mignon-Grasteau, S.,
Germon, P., Rasschaert, D., Schouler, C., 2007. Extraintestinal pathogenic
Escherichia coli strains of avian and human origin: link between phylogenetic
relationships and common virulence patterns. J. Clin. Microbiol. 45, 3366
3376.
Newton, H.J., Sloan, J., Bulach, D.M., et al., 2009. Shiga toxin-producing Escherichia
coli strains negative for locus of enterocyte effacement. Emerg. Infect. Dis. 15,
372380.
Ochman, H., Selander, R.K., 1984. Standard reference strains of Escherichia coli from
natural populations. J. Bacteriol. 157, 690693.
Ogura, Y., Ooka, T., Iguchi, A., et al., 2009. Comparative genomics reveal the
mechanismof the parallel evolution of O157 and non-O157 enterohemorrhagic
Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 106, 1793917944.
Picard, B., Garcia, J.S., Gouriou, S., Duriez, P., Brahimi, N., Bingen, E., Elion, J.,
Denamur, E., 1999. The link between phylogeny and virulence in Escherichia
coli extraintestinal infection. Infect. Immun. 67, 546553.
Pradel, N., Boukhors, K., Bertin, Y., Forestier, C., Martin, C., Livrelli, V., 2001.
Heterogeneity of Shiga toxin-producing Escherichia coli strains isolated from
hemolytic-uremic syndrome patients, cattle, and food samples in central
France. Appl. Environ. Microbiol. 67, 24602468.
Pupo, G.M., Lan, R., Reeves, P.R., 2000. Multiple independent origins of Shigella
clones of Escherichia coli and convergent evolution of many of their character-
istics. Proc. Natl. Acad. Sci. U.S.A. 97, 1056710572.
Rasko, D.A., Rosovitz, M.J., Myers, G.S., et al., 2008. The pangenome structure of
Escherichia coli: comparative genomic analysis of E. coli commensal and patho-
genic isolates. J. Bacteriol. 190, 68816893.
Reid, S.D., Herbelin, C.J., Bumbaugh, A.C., Selander, R.K., Whittam, T.S., 2000. Parallel
evolution of virulence in pathogenic Escherichia coli. Nature 406, 6467.
Rhen, M., Klemm, P., Korhonen, T.K., 1986. Identication of two newhemagglutinins
of Escherichia coli, N-acetyl-D-glucosamine-specic mbriae and a blood group
M-specic agglutinin, by cloning the corresponding genes in Escherichia coli K-
12. J. Bacteriol. 168, 12341242.
Ron, E.Z., 2006. Host specicity of septicemic Escherichia coli: human and avian
pathogens. Curr. Opin. Microbiol. 9, 2832.
Savageau, M.A., 1983. Escherichia coli habitats, cell types, and moleculat mechan-
isms of gene control. Am. Nat. 122, 732744.
Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: Molecular Evolutionary
Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 15961599.
Tenaillon, O., Skurnik, D., Picard, B., Denamur, E., 2010. The population genetics of
commensal Escherichia coli. Nat. Rev. Microbiol. 8, 207217.
O. Clermont et al. / Infection, Genetics and Evolution 11 (2011) 654662 661
Touchon, M., Hoede, C., Tenaillon, O., et al., 2009. Organised genome dynamics in
the Escherichia coli species results in highly diverse adaptive paths. PLoS Genet.
5, e1000344.
Wirth, T., Falush, D., Lan, R., et al., 2006. Sex and virulence in Escherichia coli: an
evolutionary perspective. Mol. Microbiol. 60, 11361151.
Wu, G., Carter, B., Mafura, M., Liebana, E., Woodward, M.J., Anjum, M.F., 2008.
Genetic diversity among Escherichia coli O157:H7 isolates and identication of
genes linked to human infections. Infect. Immun. 76, 845856.
Xie, Y., Kolisnychenko, V., Paul-Satyaseela, M., Elliott, S., Parthasarathy, G., Yao, Y.,
Plunkett 3rd, G., Blattner, F.R., Kim, K.S., 2006. Identication and characteriza-
tion of Escherichia coli RS218-derived islands in the pathogenesis of E. coli
meningitis. J. Infect. Dis. 194, 358364.
Zhao, L., Gao, S., Huan, H., Xu, X., Zhu, X., Yang, W., Gao, Q., Liu, X., 2009. Comparison
of virulence factors and expression of specic genes between uropathogenic
Escherichia coli and avian pathogenic E. coli in a murine urinary tract infection
model and a chicken challenge model. Microbiology 155, 16341644.
O. Clermont et al. / Infection, Genetics and Evolution 11 (2011) 654662 662