RESEARCH ARTICLE Open Access
The ST131 Esche ichia coli H22 subclone
om human in es inal mic obio a:
Compa ison o genomic and pheno ypic
ai s wi h hose o he globally success ul
H30 subclone
Ma ie-Hélène Nicolas-Chanoine
1,2,3*
, Ma ie Pe i jean
4,5
, Azucena Mo a
6
, Noémie Maye
1
, Jean-Philippe La igne
7,8
,
Oli ie Boule
9
, Vé onique Le lon-Guibou
1
, Jo ge Blanco
6
and Didie Hocque
4,5
Abs ac
Backg ound: In 2006, we ound heal hy subjec s ca ying ST131 Esche ichia coli in hei in es inal mic obio a
consis ing o wo popula ions: a subdominan popula ion o luo oquinolone- esis an E. coli belonging o subclone
H30 (H30-R o subclade C1), he cu en wo ldwide dominan ST131 subclone, and a dominan E. coli popula ion
composed o an ibio ic-suscep ible E. coli belonging o subclone H22 (clade B), he p ecu so o subclone H30. We
sequenced he whole genome o ecal H22 s ain S250, compa ed i o he genomes o ExPEC ST131 H30-Rx s ain
JJ1886 and commensal ST131 H41 s ain SE15, sough he H22-H30 genomic di e ences in ou ecal s ains and
assessed hei pheno ypic consequences.
Resul s: We de ec ed 173 genes ound in he Vi ulence Fac o Da abase, o which 148 we e sha ed by he h ee
ST131 genomes, whe eas some we e genome-speci ic, no ably hose allowing de e mina ion o i o ype (D o
S250 and C o JJ1886). We ound h ee sequences o he FimH si e in ol ed in adhesion: wo in S250 and SE15
close and iden ical, espec i ely, o ha p e iously epo ed o con e s ong in es inal adhesion, and one in JJ1886,
co esponding o ha commonly p esen in u opa hogenic E. coli. Among he genes in ol ed in suga me abolism,
one encoding a glucona e kinase lacked in S250 and JJ1886. Al hough his gene was also absen in bo h ou ecal
H22 and H30-R s ains, H22 s ains showed a highe capaci y o g ow in minimal medium wi h glucona e. Among
he genes in ol ed in glucona e me abolism, only he gh B gene di e ed be ween S250/H22 and JJ1886/H30-R
s ains, esul ing in di e en glucona e educ ases. O he genes in ol ed in bio ilm o ma ion, wo we e absen in
he h ee genomes and one, imB, in he JJ1886 genome. Ou ecal H30-R s ains lacking in ac imB displayed
delayed bio ilm o ma ion ela i e o ou ecal H22 s ains. The H22 s ains di e ed by subclade B ype and plasmid
con en , whe eas he H30-R s ains we e iden ical.
Conclusions: Pheno ypic analysis o ou ecal s ains based on obse ed genomic di e ences be ween S250 and
JJ1886 s ains sugges s he p esence o ai s ela ed o bac e ial commensalism in ou H22 s ains and ai s
commonly ound in u opa hogenic E. coli in ou H30-R s ains.
Keywo ds: E. coli ST131, H22 genome, Suga me abolism, Mannose-binding FimH egion, Bio ilm, Subclades B,
Plasmid eplicons
* Co espondence: [email p o ec ed]
1
Se ice de Mic obiologie, Hôpi al Beaujon, AP-HP, Clichy, F ance
2
Facul é de Médecine Pa is Dide o , Pa is, F ance
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Nicolas-Chanoine e al. BMC Mic obiology (2017) 17:71
DOI 10.1186/s12866-017-0984-8
Backg ound
Phylogene ic g oup B2, sequence ype (ST) 131 Esche i-
chia coli has been a wo ldwide dominan human ex a-
in es inal pa hogenic E. coli (ExPEC) since he beginning
o he 2000s, and is among hose esis an o luo oqui-
nolones and/o p oducing he ex ended-spec um β-
lac amase (ESBL) CTX-M-15 [1]. I s dominance was
shown o be d i en by he expansion o a subclone ha -
bo ing ype 1 imb iae-encoding imH allele 30 (sub-
clone H30), comp ised o mos ly s ains esis an o
luo oquinolones (H30-R) [2]. Wi hin his subclone, a
subg oup called H30-Rx, mos ly comp ising s ains e-
sis an o luo oquinolones and p oducing CTX-M-15,
has quickly eme ged and dissemina ed [2]. The e olu-
iona y his o y o clone ST131 e ealed ha be o e he
eme gence and dissemina ion o subclone H30 (also
called clade C), he ST131 popula ion consis ed o
mos ly wo subclones called H22 (clade B) and H41
(clade A), wi h subclone H22 comp ised o mos ly
luo oquinolone-suscep ible isola es [3–5]. I also e-
ealed ha subclone H22 was he p ecu so o subclone
H30 and ha sepa a e F- ype plasmids ha e shaped he
e olu ion o subclone H30 [2, 3, 5, 6]. The mos ecen
ST131 E. coli phylogene ic econs uc ion ca ied ou by
Ben Zakou e al. using 3779 non- ecombinan single
nucleo ide polymo phisms (SNP) ound in he high qual-
i y genomes o 172 clade B and C s ains, iden i ied i e
B subclades, o which he independen e olu iona y a-
jec o ies a e cha ac e ized by successi e inse ions and a
ecombina ion om ances al H22 (subclade B1) [7].
Subclade B2 is cha ac e ized by a Flag-2 locus inse ion,
subclade B3 by a Flag-2 locus inse ion and pa -C2 e-
combina ion, subclade B4 by Flag-2 locus and GI-PheV
inse ions, and subclade B5 by Flag-2 locus and Phi3 in-
se ions. O he inse ions, no ably ha o GI-PheV, e-
combina ion e en s, and mu a ions in gy A and pa C
occu ed wi hin s ains o subclade B5, esul ing in hei
e olu ion o clade C and subclades C1 (H30-R) and C2
(H30-Rx). Epidemiologically, he 172 isola es consis ed
o mos ly hose om No h Ame ica ob ained be ween
1948 and 2011, wi h mos collec ed be ween 2000 and
2010, i espec i e o he clade and subclade ypes. Since
he i s desc ip ion o he imH lineage in 2013 [3], he
imH ype has been ound in se e al epidemiological
s udies published on ExPEC o ecal ST131 isola es [8–17].
Howe e , hese ha e mos ly conce ned luo oquinolone-
esis an and /o ESBL-p oducing isola es. In 2006, we
e ie ed non-ESBL-p oducing ST131 E. coli isola es,
suscep ible o esis an o luo oquinolones, om he in es-
inal mic obio a o 7% o heal hy subjec s li ing in he Pa is
a ea [18]. The luo oquinolone- esis an isola es accoun ed
o a subdominan E. coli popula ion in ou independen
heal hy subjec s, whe eas hose suscep ible o luo oquino-
lones accoun ed o he dominan E. coli popula ion in
h ee o he independen subjec s. The la e inding
s ongly sugges s ha ST131 E. coli is pa o he no mal
in es inal mic obio a o humans. In he p esen s udy, we
aimed o de e mine whe he hese isola es belong o
di e en Hsubclones, based on hei luo oquinolone-
suscep ibili y pa e n. The dominan luo oquinolone-
suscep ible ST131 E. coli popula ions belonged o subclone
H22. Assembled and anno a ed genomes o H22 s ains
we e no a ailable when we pe o med he p esen s udy.
Thus, we i s sequenced he whole genome o one o ou
commensal H22 s ains (S250) and compa ed i o he
genome o wo e e ence ST131 s ains: he ExPEC H30-Rx
s ain JJ1886 and he commensal H41 s ain SE15. This
genome compa ison ocused on i ulence ac o (VF)-en-
coding genes and hose encoding p ocesses o s uc u es
(suga me abolism, bio ilm o ma ion, and he FimH
mannose-binding si e) known o be in ol ed in he adap a-
ion o he bac e ia o di e en en i onmen s including
human in es ine. The ou luo oquinolone- esis an ecal
ST131 isola es we e H30-R s ains. Thus, we compa ed
hem o he h ee ecal H22 s ains conside ing he
genomic di e ences iden i ied be ween H22 s ain S250
and H30-Rx s ain JJ1886, and analyzed he pheno ypic
impac o some o hese di e ences.
Me hods
Bac e ial s ains
The se en ecal ST131 isola es (s ains 02, 39, 183, 187,
196, 208, and S250) ob ained om he in es inal mic o-
bio a o se en heal hy subjec s li ing in he Pa is a ea in
2006 we e included in he s udy. They display se o ype
O25:H4, do no p oduce ESBL, and a e ei he suscep-
ible (196, 208 and S250) o esis an (02, 39, 183 and
187) o luo oquinolones [18]. The luo oquinolone-
esis an s ains we e e ie ed by pla ing he eces o
heal hy subjec s on nalidixic acid-con aining pla es. The
luo oquinolone-suscep ible s ains accoun ed o he
dominan E. coli popula ion o h ee subjec s. We p e i-
ously assessed he i ulence po en ial o s ain S250 in
he Caeno habdi is elegans and zeb a ish models and
analyzed i s genome by op ical mapping. This s ain had
a le el o i ulence simila o ha o he mul id ug
esis an ST131 isola es, wi h which i sha ed 86% gen-
ome simila i y [19]. We also included he E. coli K-12
MG1655 e e ence s ain in he s udy as a con ol in he
expe imen s assessing he use o glucona e as he sole
sou ce o ca bon.
imH ype
imH yping was based on he in e nal 489-nucleo ide
(n ) sequence o he imH gene as p e iously
desc ibed [20].
Nicolas-Chanoine e al. BMC Mic obiology (2017) 17:71 Page 2 o 12
An ibio ic suscep ibili y
An ibio ic suscep ibili y was de e mined by he aga
di usion me hod and in e p e ed ollowing he 2015
EUCAST ecommenda ions (www.eucas .o g). The ol-
lowing an ibio ics we e es ed: amoxicillin, amoxicillin +
cla ulanic acid, cip o loxacin, gen amicin, amikacin,
co imoxazole and os omycin.
Molecula analysis o esis ance mechanisms
The genes encoding esis ance o amoxicillin (TEM and
SHV enzymes) we e iden i ied by PCR and sequencing
me hods as p e iously desc ibed [21]. The qn genes en-
coding plasmid-media ed esis ance o luo oquinolones
we e es ed and he quinolone esis ance de e mining
egion (QRDR) o he genes gy A,gy B,pa C and pa E
ampli ied and sequenced using me hods p e iously
desc ibed [21–23]. The QRDRs o ou s ains we e
compa ed wi h hose o he luo oquinolone-suscep ible
e e ence s ain E. coli K-12 MG1655 [GenBank:
CP014225.1] and he allelic gy A and pa C p o iles we e
compa ed wi h hose p e iously desc ibed o ST131
isola es [3].
Sequencing and analysis o he whole genome o H22
s ain S250
The comple e genomic sequence was de e mined o
H22 s ain S250. To al DNA was ex ac ed using he
Qiagen Blood & Cell Cul u e DNA Mini Ki (Qiagen,
Cou aboeu , F ance). Lib a ies we e cons uc ed using
Nex e a echnology and sequenced on an Illumina
HiSeq-2000 using a 2 × 100 nucleo ides (n ) pai ed-end
s a egy. All eads we e p ocessed o emo e low
quali y o a e ac ual nucleo ides, using sequen ially
sickle (www.gi hub.com/najoshi/sickle), AlienT imme
[24] and qDuplica e ( p.pas eu . /pub/genso /p o-
jec s/ q ools). Read pai s we e assembled using clc_as-
semble om he CLC Genomics Wo kbench analysis
package (www.clcbio.com/p oduc s/clc-genomics-wo k-
bench). All con igs o ≥500 n we e eo de ed and eo -
ien ed, using he genomic sequence o s ain E. coli K-12
MG1655 as a e e ence, wi h Mau e Con ig Mo e [25].
The eo de ed con igs we e analyzed. The genome o
H22 s ain S250 was compa ed o ha o wo e e ence
ST131 s ains, JJ1886 (H30-Rx) [GenBank: CP006784.1]
and SE15 (H41) [GenBank: AP009378.1], ocusing on
VF-encoding genes and genes in ol ed in suga me abol-
ism, bio ilm o ma ion, and me hyla ion. We down-
loaded VFs (www.mgc.ac.cn/VFs/) a ailable om he
Vi ulence Fac o Da abase (VFDB) and sea ched he
h ee genomes o hei p esence using P odigal 2.6.1
[26] and clus e ed hem a 90% iden i y using CD-hi
4.6 [27]. We ex ac ed he sequences o he genes in-
ol ed in bio ilm o ma ion in s ain E. coli K12
BW25113 [28] om i s genome [GenBank: CP009273.1]
and clus e ed hem a 90% iden i y wi h CD-hi . We
assessed he pe cen age iden i y be ween he genes om
E. coli K12 BW25113 and he h ee s udied genomes.
Using he NBCI basic local alignmen sea ch ool
(BLAST), we blas ed genes om E. coli K12 MG1655
agains he S250, JJ1886, and SE15 genomes o e i y he
possible absence o any genes in ol ed in bio ilm o ma-
ion. We hen sea ched he genomes o ST131 s ains
S250, JJ1886, and SE15 o genes in ol ed in suga me-
abolism as de ined by Mal by e al. [29] in commensal
s ains E. coli HS [GenBank: CP000802.1] and E. coli
Nissle 1917 [GenBank: CP007799.1] using BLAST.
Mo eo e , we also sea ched o all he genes in ol ed in
glucona e me abolism (main pa hway: gn R,gn T,gn U,
gn P,gn K; seconda y pa hway: idnT,idnDOTR; gene
idnK (gn V) which plays a ole in he wo pa hways; he
En ne -Doudo o pa hway: edd and eda; addi ional
genes in ol ed in o he pa hways: kdgT,kdgK,gnd, k A,
gh B,kduD,dkgA and dkgB) [30], as well as he 100 base
pai s (bps) ups eam o he s a codon o each, in he
genome o s ains S250 and JJ1886. The nucleo ide se-
quence o he 100 bp-ups eam egions and he se-
quences o he deduced p o eins o each gene o he
s ains S250 and JJ1886 we e compa ed. We also
sea ched o genes encoding me hyl ans e ases in
ST131 s ain EC958 [GenBank: HG941718.1] [31] in he
genome o ST131 s ains S250, JJ1886, and SE15 using
BLAST.
Subclade B and clade C yping
Acco ding o he wo k o Ben Zakou e al. [7], we de-
e mined he subclade B ype o H22 s ain S250 and he
clade C ype o s ain JJ1886 by di ec blas ing o he
genes o he Flag-2 locus (Flag-2 locus om s ain E. coli
042 [EMBL: CR 753847]), Phi3 ( om s ain EC958), and
GI-PheV ( om s ain JJ1886) agains he genome o
s ains S250 and JJI886. We hen de e mined he ype o
subclade B displayed by s ain S250 and he wo o he
ecal H22 s ains by PCR using p ime s speci ic o he
Flag-2 locus, Phi3, and GI-PheV (Addi ional ile 1: Table
S1) and ou H30-R ecal s ains as a posi i e con ol.
Plasmid con en
The plasmid con en o H22 s ain S250 was de e mined
using he PlasmidFinde sys em [32] and he FII, FIA,
and FIB o mula o he de ec ed IncF plasmid by PCR-
based eplicon yping (h p://pubmls .o g/plasmid/). The
la e me hod was also applied o he wo emaining
ecal H22 s ains and he ou ecal H30-R s ains.
ExPEC s a us and i o ype
Acco ding o he s udy o Johnson and Adam, ExPEC
s a us is de ined by he p esence o ≥2 VF genes among
he ollowing genes: pap,s a/ ocDE,a a/d aBC,iu A,
Nicolas-Chanoine e al. BMC Mic obiology (2017) 17:71 Page 3 o 12
and kpsMT II [33]. As he la e gene is no included in
he VFDB, we sea ched o i by blas ing he kpsMT II
genes [GenBank: X53819] agains he S250, JJ1886, and
SE15 genomes. Based on he s udy o Blanco e al., he
majo i o ypes o E.coli ST131 a e de ined using ou
genes as ollows: i o ype A = a aFM955459
+
i oN
−
ibeA
−
sa
+/−
, i o ype B = a aFM955459
−
i oN
+
ibeA
−
sa
+/−
,
i o ype C = a aFM955459
−
i oN
−
ibeA
−
sa
+
, and i o-
ype D = a aFM955459
+/−
i oN
+/−
ibeA
+
sa
+/−
[34]. As
a aFM955459 is no included in he VFDB, we sea ched
o i by blas ing he a aFM955459 ope on [EMBL:
FM955459] agains he S250, JJ1886, and SE15 genomes
[35]. Then, classic mul iplex PCR was used o sea ch o
genes encoding ExPEC-associa ed VFs (Addi ional ile 2:
Table S2) [34] in he se en ecal s ains o con i m he
esul s p o ided by he di ec genome analysis o s ain
S250 and o de e mine he VF p o ile, i o ype, and
ExPEC s a us o he six emaining ecal s ains.
FimH s uc u e
Acco ding o he s udy o Soku enko e al., he amino
acid a ia ions obse ed wi hin he sequence o he
adhesin, FimH, o ype 1 imb iae, esul in di e en
le els o binding o mono-mannose (M
1
) s uc u es,
whe eas hey ha e no impac on he no mal high le el o
binding o i-mannose (M
3
) s uc u es [36]. By measu -
ing he a io o M
1
/M
3
binding in di e en expe imen al
models, hey de ined low M
1
-binding (M
1
/M
3
< 0.1) and
high M
1
-binding (M
1
/M
3
> 0.90) FimH pheno ypes and
showed ha hey a e ela ed o speci ic FimH sequences
[37]. The e o e, he deduced p o ein sequence o FimH
o H22 s ain S250 was compa ed wi h ha o H30-Rx
ST131 s ain JJ1886 [GenBank: AGY86963.1], H41
ST131 s ain SE15 [GenBank: BAI57801.1], phylog oup
AE. coli s ain MG1655 [GenBank: AMC97175.1], and
wo o he phylog oup B2 s ains: ST73 s ain CFT073
[GenBank: AAN83822.1] and ST131 uk_P46212 [Gen-
Bank: ALT52319.1]. The deduced p o ein sequence o
FimH was hen cha ac e ized o he six emaining ecal
s ains by using p ime s o which he sequences ha e
been p e iously published [20].
Ampli ica ion and sequencing o he idnK (gn V) and gh B
genes o he se en ecal s ains and g ow h wi h
glucona e as he sole ca bon sou ce
The idnk (gn V) gene, encoding a he mosensi i e D-
glucona e kinase, and he gh B gene, encoding a gluco-
na e educ ase, we e ampli ied wi h p ime s indica ed in
Addi ional ile 1: Table S1. Addi ional p ime s we e used
o sequence he gh B gene (Addi ional ile 1: Table S1).
We e alua ed he abili y o he se en s ains o g ow in
he p esence o glucona e as he sole ca bon sou ce, as
p e iously desc ibed [38]. Expe imen s we e conduc ed
h ee imes independen ly and all incuba ions we e
pe o med o e nigh a 37 °C wi h shaking (150 pm).
B ie ly, bac e ia we e i s cul u ed in Muelle Hin on
b o h be o e washing wice in minimal media M63
[15 mM (NH
4
)
2
SO
4
; 100 mM KH
2
PO
4
; 0.002 mM
FeSO
4
(7H
2
O)]. We inocula ed 5 ml o M63 supple-
men ed wi h 0.2% glucose (Sigma-Ald ich, F ance) wi h
10 μl o he washed bac e ia. One ml o his cul u e was
washed wice in M63. We ans e ed 10 μl o his
washed cul u e in o 5 ml o M63 wi h 0.2% glucona e
(Sigma-Ald ich, Sain -Quen in Falla ie , F ance). The
cul u es we e hen adjus ed o 0.002 a OD
600
in 30 ml
esh M63 wi h 0.2% glucona e and incuba ed a 37 °C
wi h shaking (150 pm). We es ima ed he bac e ial
g ow h a e 48 h by measu ing he OD
600
[39]. Tukey’s
es was used o in e g oup compa isons and R so wa e
o s a is ical analyses. P alues <0.01 we e conside ed o
be s a is ically signi ican .
Ampli ica ion and sequencing o he imB gene and
kine ics o bio ilm o ma ion in he se en ecal s ains
The imB gene was ampli ied and sequenced wi h he
p ime s indica ed in Addi ional ile 1: Table S1. The kin-
e ics o ea ly bio ilm o ma ion was assessed using he
BioFilm Ring Tes ® (BioFilm Con ol, Sain Beauzi e,
F ance), as desc ibed [40]. B ie ly, s anda dized bac e ial
cul u es we e incuba ed a 37 °C in a 96-well mic o i e
pla e in he p esence o magne ic beads. A a ious ime
poin s, he pla es we e placed on o a magne ic es block
and pu in he eade . The images o each well be o e
and a e magne ic a ac ion we e analyzed using
BioFilm Con ol so wa e ha gi es a BioFilm Index
(BFI). The BFI was con e ed in o he p opo ion o
immobilized beads ela i e o a e e ence condi ion (%
RBI) using he o mula: % RBI = √[(1-(BFI
assay
-BFI
min
)/
(BFI
con ol
-BFI
min
)] ×100, whe e BFI
assay
is he BFI o he
es ed s ain, BFI
con ol
is he BFI o he con ol, co e-
sponding o he maximum BFI, and BFI
min
is he
minimal obse ed BFI when all he beads a e blocked.
The mo e RBI app oaches a alue o 1, he mo e he bio-
ilm is ully o med (beads a e immobilized). Th ee
expe imen s we e pe o med in duplica ed pe s ain
and pe incuba ion ime. The kine ics o bio ilm o ma-
ion we e compa ed using a wo-way ANOVA ollowed
by Dunne ’s mul iple compa isons es .
Resul s
imH ype, an ibio ic suscep ibili y, molecula analysis o
esis ance mechanisms, and allelic p o iles o he gy A
and pa C genes
The h ee luo oquinolone-suscep ible s ains (S250, 208
and 196) we e o he imH22 ype. All bu one we e
suscep ible o all o he an ibio ics es ed (Table 1). H22
s ain 196 was esis an o bo h amoxicillin and co i-
moxazole. The ou luo oquinolone- esis an s ains
Nicolas-Chanoine e al. BMC Mic obiology (2017) 17:71 Page 4 o 12
(187, 183, 39 and 02), which we e all esis an o amoxi-
cillin bu suscep ible o he o he an ibio ics es ed, we e
o he imH30 ype (H30-R) (Table 1). QRDR nucleo ide
sequence analysis showed ha he h ee H22 s ains dis-
played gy A1a and pa C1 alleles, whe eas he ou H30-
R s ains displayed he gy A1AB allele, encoding amino
acid subs i u ions S83 L and D87N, and he pa C1aAB
allele, encoding amino acid subs i u ions S80I and E84V
(Table 1). We ound he subs i u ion I529L in Pa E in
he se en ecal s ains (Table 1). None o hese s ains
ha bo ed plasmid-media ed qn genes. H22 196 and he
ou H30-R s ains ha we e esis an o amoxicillin ha -
bo ed a TEM-1-encoding gene (Table 1).
Genomic and pheno ypic cha ac e iza ion
We assembled he whole genome sequence o he ecal
H22 s ain S250 in o 50 con igs and analyzed and com-
pa ed i wi h hose o he ExPEC H30-Rx s ain JJ1886
and he commensal H41 s ain SE15.
Subclade B and C ype
We we e unable o ind he genes composing he Flag-2
locus in he genome o H22 s ain S250 using BLAST.
We only ound a agmen o app oxima ely 1700 bp
which was e y simila o he end o he i s gene, I hA,
and ano he o 769 bp simila o he end o he las gene,
la U, o he Flag-2 locus (da a no shown). We ound
nei he Phi3 no GI-PheV in he genome o H22s ain
S250, whe eas we ound hem, as well as he Flag-2
locus, in he genome o JJ11886. The use o speci ic
p ime s allowed us o con i m he absence o hese gen-
e ic elemen s in H22 s ain 250 and o de ec he Flag-2
locus in H22 s ains 196 and 208, as well as GI-PheV in
H22 s ain 196. These h ee gene ic elemen s we e amp-
li ied om ou ou H-30R s ains (Table 1).
VF-encoding genes
Using a gene iden i y le el o ≥90%, a o al o 173 genes
among he 2520 Esche ichia sp. VF-encoding genes o
he VFDB was iden i ied in he genomes o he h ee
ST131 s ains s udied. H22 s ain S250, H30-Rx s ain
JJ1886, and H41 s ain SE15 had 160, 159, and 152 VF-
encoding genes, espec i ely. The h ee ST131 genomes
sha ed 148 i ulence genes (Fig. 1). Nine genes we e
ound speci ically in H22 s ain S250: he i e i oBCDEN
genes ha encode p o eins ela ed o a ca echola e sid-
e opho e, he h ee pixCDH genes encoding Pix pilus
adhesion, and he ibeA gene in ol ed in in asion o
b ain endo helium (Fig. 1). Ten genes we e speci ically
ound in H30-RX s ain JJ1886: he ou iucABCD and
iu A genes encoding p o eins in ol ed in he binding
and anspo o i on, he pap1 and papX genes
encoding pap ope on egula o y p o eins, he sa gene
encoding a oxin, he lu gene encoding au o anspo e
p o ein Ag43a, and he iha gene encoding he adhesion-
side opho e ecep o . Th ee genes, including he imB
gene, we e p esen in s ains H22 S250 and H41 SE15,
bu no H30-RX s ain JJ1886 (Fig. 1). The ECP_2810
gene, encoding a Val-Gly Repea s- ela ed p o ein, was
p esen in H41 s ain SE15 and H30-RX s ain JJ1886.
Vi o ype and ExPEC s a us
The VF genes iden i ied in he VFDB would sugges
i o ype D (ibeA
+
,i oN
+
, and sa
−
) o s ain S250 and
i o ype C (sa
+
,ibeA
−
, and i oN
−
) o s ain JJ1886.
These i o ypes we e con i med by he absence o he
Table 1 Cha ac e iza ion o he se en ecal s ains o E. coli ST131
S ain/ FimH
ype
Suscep ibili y β-lac amase Allele ype
a
(amino acid subs i u ion)
Flag-2 Phi3 GI-PheV Subclade IncF
plasmid
eplicon
AMX AMC CIP GEN AMK SXT FOS gy A pa C pa E
S250/H22 S S S S S S S −1a 1 NA
(I529L)
−−− B1 F89:A-:B62
208/H22 S S S S S S S −1a 1 NA
(I529L)
+−− B2 −
196/H22 R S S S S R S TEM-1 1a 1 NA
(I529L)
+−+ B4 F24:A-:B6
187/H30 R S R S S S S TEM-1 1AB
(S83 L/D87N)
1aAB
(S80I/E84V)
NA
(I529L)
+ + + C1 F1:A2:B20
183/H30 R S R S S S S TEM-1 1AB
(S83 L/D87N)
1aAB
(S80I/E84V)
NA
(I529L)
+ + + C1 F1:A2:B20
39/H30 R S R S S S S TEM-1 1AB
(S83 L/D87N)
1aAB
(S80I/E84V)
NA
(I529L)
+ + + C1 F1:A2:B20
02/H30 R S R S S S S TEM-1 1AB
(S83 L/D87N)
1aAB
(S80I/E84V)
NA
(I529L)
+ + + C1 F1:A2:B20
a
acco ding o e e ence [3], AMX amoxicillin, AMC amoxicillin + cla ulanic acid, CIP cip o loxacin, GEN gen amicin, AMK amikacin, SXT co imoxazole, FOS
os omycin, R esis an , Ssuscep ible, −: absence, +: p esence, NA no a ailable
Nicolas-Chanoine e al. BMC Mic obiology (2017) 17:71 Page 5 o 12

a aFM955459 ope on in he genome o s ains S250 and
JJ1886, shown using BLAST. We we e unable o de e -
mine a i o ype o s ain SE15, as none o he ou
genes we e de ec ed in he genome o his s ain. Com-
bining VFDB-based and BLAST analysis o he kpsMT II
gene, we ound ha s ains S250 and SE15 did no dis-
play an ExPEC s a us, as hey ha bo ed only one (kpsMT
II) o he genes used o de ine his s a us. Mul iplex PCR
classically used o sea ch o genes encoding ExPEC-
associa ed VF (Addi ional ile 2: Table S2) o he se en
ecal s ains showed ha he wo emaining ecal H22
s ains displayed i o ype D, as s ain S250, and he ou
ecal H30-R s ains i o ype C, as s ain JJ1186 (Table 2).
I also showed ha only i o ype C H30-R s ain 39 dis-
played an ExPEC s a us ela ed o he p esence o he
iu A and kpsMT II genes. We ound 11 o he ampli ied
VF genes ( imH, ma B, pe , chuA, yuA, i p2, si A, aT,
malX, usp and ompT) in all bu one (s ain H22 208)
s ain, i e (F10 papA,iha,sa ,iucD and iu A) in only
H30-R s ains, and ou (cd ,i oN,iss and ibeA) in only
H22 s ains. The numbe o ampli ied VF-encoding
genes a ied om 15 o 17 in H30-R s ains and 13 o
16 in H22 s ains.
Deduced p o ein FimH
As indica ed in Fig. 2, he deduced p o ein sequence o
adhesin FimH o ep esen a i es o H30-Rx ST131 E. coli
(s ains JJ1886 and uk_P46212), H41 ST131 E. coli
(s ain SE15), H22 ST131 E. coli (s ain S250), UPEC
(ST73 s ain CFT073), and E. coli K12 (s ain MG1655)
displayed amino acid di e ences a he posi ions p e i-
ously shown o cha ac e ize di e en M
1
/M
3
a io phe-
no ypes, con ibu ing o di e en le els o coloniza ion
o di e en niches [37]. Thus, we iden i ied he sequence
-A27, N70 and S78 o N78 and S70, V163, and R166-
co esponding o he lowe M
1
/M
3
a io (0.08-0.09) in
Table 2 Vi ulence ac o -encoding genes, i o ype, and ExPEC s a us o human ecal ST131 Esche ichia coli H22 and H30 subclones
S ain imH Vi ulence ac o -encoding gene Vi o ype ExPEC s a us
S250 H22 imH, ma B, pe , i oN, chuA, yuA, i p2, si A, kpsMII-K5, iss, aT, ibeA, malX, usp, ompT D-
208 H22 imH, ma B, cd B, pe , chuA, yuA, i p2, si A, kpsMII-K5, ibeA, malX, usp, ompT D-
196 H22 imH, ma B, cd B, pe , i oN, chuA, yuA, i p2, si A, kpsMII-K5, iss, aT, ibeA, malX, usp, ompT D-
187 H30 imH, F10 papA, iha, ma B, sa , pe , iucD, iu A, chuA, yuA, i p2, si A, aT, malX, usp, ompT C-
183 H30 imH, F10 papA, iha, ma B, sa , pe , iucD, iu A, chuA, yuA, i p2, si A, aT, malX, usp, ompT C-
39 H30 imH, F10 papA, iha, ma B, sa , pe , iucD, iu A, chuA, yuA, i p2, si A, kpsMII-K5, aT, malX, usp, ompT C+
02 H30 imH, F10 papA, ma B, sa , pe , iucD, iu A, chuA, yuA, i p2, si A, aT, malX, usp, ompT C-
Fig. 1 Lis o genes ound in he genomes o H22 s ain S250, H30-Rx s ain JJ1186 and, H41 s ain SE15. Each s ain is ep esen ed by a ci cle and
each gene commonly ound in he genome o wo s ains o in he genome o he h ee s ains is indica ed in he co esponding in e sec ing
egions, whe eas genes speci ic o each s ain a e indica ed in he sec ion o he ci cle no sha ed wi h ano he ci cle. The 148 genes p esen in
he h ee s ains a e lis ed a he bo om o he igu e
Nicolas-Chanoine e al. BMC Mic obiology (2017) 17:71 Page 6 o 12
he s udy o Soku enko e al. in s ain SE15. We ound
he subs i u ions A27V and V163A, ha a e each ela ed
o a en- old inc ease in he M
1
/M
3
a io in he Soku -
enko e al., s udy in s ain MG1655 and s ain CFT073,
espec i ely. We also ound he subs i u ion R166H
ela ed o a M
1
/M
3
a io o 0.33 in he same s udy in
H30-Rx s ains JJ1886 and uk_P46212. H22 s ain S250
displayed a sequence no published in he Soku enko e
al. s udy as esidue N was p esen a bo h posi ions 70
and 78. Howe e , we ound none o he subs i u ions
known o induce an inc ease in he M
1
/M
3
a io in he
FimH sequence o s ain S250. We ound he same ea-
u e in he wo o he ecal H22 s ains, whe eas he
FimH sequence o ou ou H30-R s ains was iden i-
cal o ha o H30-Rx s ains JJ1886 and uk_ P46212
(da a no shown).
Genes in ol ed in suga me abolism and g ow h wi h
glucona e as he sole ca bon sou ce
All genes in ol ed in suga me abolism in commensal E.
coli s ains HS and Nissle we e de ec ed in he genome
o H41 s ain SE15, whe eas one, he D-glucona e
kinase-encoding idnK (gn V) gene, was no de ec ed in
ei he s ain H30-Rx JJ1886 o H22 S250 (Table 3). I s
absence in ou six emaining s ains was e ealed by he
PCR-sequencing assay. The e o e, we assessed he abili y
o ou s ains o g ow wi h glucona e as he sole ca bon
sou ce in he absence o he idnK (gn V) gene and he
p esence o he gn K gene, which bo h ans o m D-glu-
cona e o 6P-glucona e. A e 48 h o g ow h, he bio-
mass o ou H30-R s ains was signi ican ly lowe han
ha o ou H22 s ains (Tukey’s es p< 0.01) (Fig. 3). This
unexpec ed di e ence led us o compa e he genomes o
s ains H22 S250 and H30-Rx JJ1886 ocusing on all he
genes, o he han he idnk (g nV) gene, in ol ed in gluco-
na e me abolism, as well as he 100 bp ups eam o he s a
codon o he ope ons o genes. All we e p esen in he gen-
ome o s ains S250 and JJ1886. The 100 bp ups eam o
he s a codon and he sequence o he deduced p o eins
showed 100% iden i y (da a no shown) be ween hese wo
s ains, excep o he coding egion o he gh B gene,
which showed a T968C subs i u ion, esul ing in amino
acid subs i u ion V323A in s ain JJ1886. This subs i u ion
was con i med in ou ou ecal H30-R s ains, whe eas
V323 was iden i ied in he wo emaining H22 ecal s ains.
Bio ilm geno ype and pheno ype
The exp ession o a wide panel o genes can in luence
bio ilm o ma ion [28]. We speci ically sea ched o
hese genes in he genome o s ains JJ1886, S250, and
SE15 (Addi ional ile 3: Table S3). Two genes o he bio-
ilm gene panel, liC and liD, we e missing om he
h ee es ed genomes. We aligned he liC and liD genes
om E. coli s ain MG1655 on o he h ee genomes
(JJ1886, S250 and SE15) o e i y hei absence and
ound dele ions wi hin liC and he absence o liD in
he h ee s ains. An in ac e sion o he imB gene was
p esen in s ains H22 S250 and H41 SE15, bu i was
dis up ed in H30-Rx JJ1886 due o he inse ion o IS3-
like, as p e iously desc ibed in a ious H30 s ains [34,
41]. The di e ence in he s uc u e o he imB gene in
s ains H30-Rx JJ1886 and H22 250 led us o sequence
he imB gene o he emaining ecal s ains. We ound
ha he imB nucleo ide sequence in he ou H30-R
s ains was iden ical o ha o H30-Rx s ain JJ1886,
Fig. 2 Alignmen o FimH sequence om a ious Esche ichia coli
s ains. Phylog oup B2 E. coli: ST73 s ain CFT073, H30 ST131 JJ1886,
H30 ST131 uk_P46212, H22 ST131 S250 and H41 ST131 SE15;
phylog oup A: MG1655. “1”indica es he s a o he FimH p o ein.
Amino acid posi ions in ol ed in binding o mono-mannose and
i-mannose s uc u es a e indica ed in g ey
Table 3 Dis ibu ion o he genes in ol ed in suga me abolism
in commensal Esche ichia coli s ains HS and Nissle [29] in he
genomes o ep esen a i e H30, H22, and H41 subclones o
Esche ichia coli ST131
Commensal
s ain/gene
Ca abolized
suga
H30
JJ1886
H22
S250
H41
SE15
E. coli HS
a aA A abinose + + +
a aB A abinose + + +
a aD A abinose + + +
ucK Fucose + + +
galK Galac ose + + +
gn K Glucona e + + +
idnK Glucona e −−+
lacZ Lac ose + + +
manA Mannose + + +
nagE N-ace ylglucosamine + + +
nanA N-ace ylneu amina e + + +
nanT1 N-ace ylneu amina e + + +
bsK Ribose + + +
uxaC Glucu ona e + + +
E. coli Nissle 1917
agaA N-ace ylgalac osamine + + +
agaE N-ace ylgalac osamine + + +
agaF N-ace ylgalac osamine + + +
agaW N-ace ylgalac osamine + + +
Nicolas-Chanoine e al. BMC Mic obiology (2017) 17:71 Page 7 o 12
whe eas ha o he wo emaining H22 s ains was iden-
ical o ha o s ain S250. Niba e al. p e iously
epo ed ha he dele ion o liC and/o liD induces a
subs an ial dec ease in bio ilm o ma ion, whe eas dele-
ion o imB esul s in in i s nea comple e absence
(Addi ional ile 3: Table S3). We hus es ed whe he his
imB polymo phism a ec s bio ilm o ma ion. Kine ic
measu emen s o ea ly bio ilm o ma ion showed ha
he h ee H22 s ains o med bio ilms signi ican ly ea l-
ie (p< 0.0001) han he ou H30-R s ains (Fig. 4). O
no e, H30-R s ain 39, which was he single ecal s ain
wi h an ExPEC s a us, showed signi ican ly (p< 0.0001)
slowe bio ilm p oduc ion (incomple e bio ilm o ma ion
a e 24 h) han he o he H30-R s ains.
Genes encoding me hyl ans e ases
We ound all bu one o he en me hyl ans e ases, p e i-
ously iden i ied in he genome o H30 ST131s ain EC958
[31], in he genome o H30-Rx s ain JJ1886 (Table 4). We
ound six in he genome o H41 s ain SE15 and only
h ee in he genome o H22 s ain S250 (Table 4).
Plasmid con en
We de ec ed an IncFII plasmid in con ig 4 and an
IncFIB elemen in con ig 41 o he S250 genome using
PlasmidFinde . Using NCBI BLAST and he plasmid
MSLT yping sys em, we iden i ied he eplicon alleles
Fig. 3 G ow h o human ecal subclone H30 and subclone H22 s ains o ST131 Esche ichia coli in minimal medium plus 0.2% glucona e. S ain
g ow h was assessed by measu ing he OD a 600 nm a e 48 h o incuba ion. Ba s ep esen he s anda d de ia ion ob ained om h ee
independen expe imen s
Fig. 4 Ea ly bio ilm o ma ion by human ecal subclone H30 and
subclone H22 s ains o ST131 Esche ichia coli. BioFilm Con ol®
image analysis so wa e was used and esul s a e exp essed as he
p opo ion o immobilized beads ela i e o e e ence condi ions
(% RBI) acco ding o incuba ion ime. Ba s ep esen he s anda d
de ia ion ob ained om h ee independen expe imen s pe o med
in duplica e. The bio ilm is mo e ully o med (beads a e
immobilized) as RBI app oaches a alue o 1. Signi ican di e ences
be ween H22 and H30 using Dunne ’s es a e indica ed by *
(p< 0.05), ** (p< 0.01) and *** (p< 0.001)
Table 4 Dis ibu ion o he 10 me hyl ans e ases p e iously
desc ibed in H30 s ain EC958, [31] in he genomes o
ep esen a i e H30, H22, and H41 subclones o Esche ichia coli ST131
Me hyl ans e ase (H30 EC958) H30 JJ1886 H22 S250 H41 SE15
M.EcoMIV + −−
M.EcoMV + −−
M.EcoMDcm + + +
EcoMIII + −+
M.EcoMVI + + +
M.EcoMDam + + +
M.EcoMVII −−−
M1.EcoMI + −+
M2.EcoMI + −+
M.EcoMII + −−
To al numbe 9 3 6
+: p esence,-: absence
Nicolas-Chanoine e al. BMC Mic obiology (2017) 17:71 Page 8 o 12
F89 and B62 in he whole genome o s ain S250. We
con i med his esul o s ain S250 by PCR-sequencing,
and also ob ained he ollowing esul s o he six
emaining ecal s ains: F1:A2:B20 o he ou H30-R
s ains, no IncF plasmid o H22 s ain 208, and F24:B6
o H22 s ain 196 (Table 1).
Discussion
CTX-M-15 p oducing, luo oquinolone- esis an ST131
E. coli, which has been shown o be a wo ldwide human
ExPEC since he beginning o he 2000s, has also been
shown o colonize he human diges i e ac [1]. In
2006, we ound heal hy subjec s wi h a subdominan in-
es inal popula ion o luo oquinolone- esis an ST131 E.
coli, as well as hose wi h a dominan in es inal popula-
ion o ST131 E. coli suscep ible o luo oquinolones
[18]. The la e inding sugges s ha ST131 E. coli may
be a human in es inal commensal. He e, we i s showed
ha ou h ee an ibio ic-suscep ible ecal ST131 E. coli
s ains displayed imH22, whe eas he ou esis an o
luo oquinolones displayed imH30 (H30-R). We hus
cen e ed ou s udy on he compa ison be ween hese
wo g oups o s ains ocusing on p ocesses and s uc-
u es in ol ed in en i onmen al adap a ion, conside ing
ha subclone H22 is he p ecu so o subclone H30,
which eme ged a he end o 1990s [3, 7]. We i s se-
quenced he whole genome o one o ou ecal H22
s ains, s ain S250, as he e we e no assembled and an-
no a ed genomes o H22 s ains when we s a ed ou
wo k. In con as , he e we e sequenced whole genomes
o ep esen a i e mul id ug esis an H30 ExPECs, such
as H30-RX s ain JJ1886 and a commensal ST131 E. coli,
s ain SE15, belonging o ano he ST131 subclone, cha -
ac e ized by he allele imH41. Compa ison o he ge-
nomes o H22 s ain S250, H30-Rx s ain JJ1886, and
H41 s ain SE15 showed high simila i y in e ms o he
numbe and ypes o VF genes om he VFDB. This
may explain he simila le el o i ulence ha we ound
p e iously be ween H22 s ain S250 and an H30-Rx
s ain in he C. elegans model [19, 42]. Ne e heless,
s ains S250 and JJ1886 displayed wo di e en i o ypes:
D o s ain S250 and C o s ain JJ1886. Mo eo e ,
s ain S250 did no display he VF genes equi ed o
ExPEC s a us, whe eas s ain JJ1886 did [43]. The e was
also high simila i y be ween he genes in ol ed in bio-
ilm o ma ion in he h ee genomes [28], excep o he
imB gene, which was dis up ed in he H30-Rx s ain
JJ1886 and in ac in bo h H22 s ain S250 and H41
s ain SE15. As he in ac imB gene was also absen
om ou ecal H30-R s ains and p esen in ou
emaining ecal H22 s ains, we analyzed he kine ics o
ea ly bio ilm o ma ion in ou se en ecal s ains. Bio ilm
o ma ion was signi ican ly delayed in he H30-R s ains.
This is he i s s udy o compa e he o ma ion o
bio ilms by H22 and non-ESBL-p oducing H30-R s ains
[4, 42, 44–46]. Fu he s udies a e equi ed o e i y he
in ol emen o he imB gene in he wo di e en bio-
ilm pheno ypes. Compa ison o he genes o he h ee
genomes in ol ed in he me abolism o a ious suga s
showed he absence o one o he wo genes encoding
glucona e kinases in s ains S250 and JJ1886. Al hough
his gene was absen in bo h ou ecal H22 and H30-R
s ains, we ound ha H22 s ains had a highe capaci y
o g ow in medium wi h glucona e as he sole ca bon
sou ce. Thus, glucona e consump ion may con e an
ad an age o H22 s ains o esiding in he human in-
es ine, as glucona e is a componen o in es inal mucus
[47]. Indeed, i has been shown ha E. coli labo a o y
mu an s wi h impai ed g ow h on glucona e a e less able
o colonize he la ge in es ine o mice [38]. Compa ison
o all genes, and hei p omo e egions, in ol ed in he
di e en pa hways o glucona e me abolism showed one
di e ence be ween s ains S250 and JJ1886, namely a
mu a ion leading o an amino acid subs i u ion in he
2-ke o-D-glucona e educ ase Gh B in s ain JJ1886. Al-
hough his gene ic di e ence was ound be ween he
ecal H22 and H30-R s ains, u he s udies a e equi ed
o cla i y he ole o his mu a ion o he di e ence in
he g ow h o he wo s ains when glucona e is he sole
ca bon sou ce. Ano he di e ence we iden i ied be ween
s ain S250 and s ain JJ1886 conce ned he sequence o
he ype 1 imb iae FimH adhesin, shown by Soku enko
e al. o be in ol ed in a ia ions o he M
1
/M
3
a io
associa ed wi h issue opism and he shi o bac e ial
adap a ion om a commensal o pa hological habi a
[36, 37]. The FimH sequence o s ain JJ1886, which was
also p esen in ou H30-R ecal s ains, co esponded o
ha iden i ied by Soku enko e al. in s ains wi h a M
1
/
M
3
a io = 0.33. This sequence and M
1
/M
3
a io a e
commonly obse ed in u opa hogenic s ains [36, 37].
The FimH sequence iden i ied in s ain S250 and he
o he ecal H22 s ains was no ound in he s ains
s udied by Soku enko e al. and was only ound a a low
equency in hose s udied by Chen e al. [48]. This se-
quence showed none o he amino acid subs i u ions in-
ol ed in he inc ease o he M
1
/M
3
a io. Thus i is
likely o be associa ed wi h he lowes M
1
/M
3
a io
shown by Soku enko e al., which was associa ed wi h
s ong adhesion o in es inal cells [36, 37]. Al oge he ,
al hough he H22 and H30-R s ains in es iga ed in his
wo k we e all isola ed om he diges i e ac o heal hy
subjec s, only H22 s ains displayed p ope ies p e i-
ously shown o be associa ed wi h human in es inal
commensalism, including compe i ion o nu ien s in
he in es ine (glucona e use) and a high in es inal adhe-
si eness [49].
We sough o con i m he gene ic di e ences iden i-
ied he e be ween he lineages H22 and H30 wi h he
Nicolas-Chanoine e al. BMC Mic obiology (2017) 17:71 Page 9 o 12