The ST131 Escherichia coli H22 subclone from human intestinal microbiota: Comparison of genomic and phenotypic traits with those of the globally successful H30 subclone

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 Full lis o au ho in o ma ion is a ailable a he end o he a icle © The Au ho (s). 2017 Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. 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