We desc ibe he epidemiology, clinical ea u es, and
molecula cha ac e iza ion o en e ohemo hagic Esche-
ichia coli (EHEC) in ec ions caused by he singula hyb id
pa ho ype O80:H2, and we examine he in luence o
an ibio ics on Shiga oxin p oduc ion. In F ance, du -
ing 2005–2014, a o al o 54 pa ien s we e in ec ed wi h
EHECO80:H2;91%hadhemoly icu emicsynd ome.Two
pa ien s had in asi e in ec ions, and 2 died. All s ains
ca ied s x2 ( a ian s s x2a, 2c, o 2d); he a e in imin
gene (eae-ξ);anda leas 4genescha ac e is ico pS88,
a plasmid associa ed wi h ex ain es inal i ulence. Simi-
la s ains we e ound in Spain. All isola es belonged o
he same clonal g oup. A subinhibi o y concen a ions,
azi h omycindec easedShiga oxinp oduc ionsigni ican -
ly,cip o loxacininc easedi subs an ially,andce iaxone
had no majo e ec . An ibio ic combina ions ha included
azi h omycinalsowe e es ed.EHECO80:H2,whichcan
induce hemoly ic u emic synd ome complica ed by bac-
e emia, is eme ging in F ance. Howe e , azi h omycin
migh e ec i ely comba hese in ec ions.
En e ohemo hagic Esche ichia coli (EHEC) a e a sub-
se o Shiga oxin–p oducing E. coli (STEC) ha cause
dia hea and hemo hagic coli is; illness can p og ess o
hemoly ic u emic synd ome (HUS) in 5%–10% o cases
(1,2). HUS is he mos equen e iology o pedia ic acu e
enal ailu e, and i s le hali y is 3%–5% wo ldwide (1,3)
and 1% in F ance (4). Long- e m enal inju ies occu in
20%–30% o HUS pa ien s (1,3,5).
EHEC se o ype O157:H7 accoun s o ≈60% o HUS
cases wo ldwide (6,7). O he well-known se og oups as-
socia ed wi h HUS include O26, O111, O145, O55, O103,
O121, and O91. EHEC O80:H2 s ains a e a ely epo ed in
he li e a u e bu ha e been de ec ed in F ance. In 2013, an
EHEC O80:H2 s ain was esponsible o a se e e case o
HUS wi h elapse associa ed wi h bac e emia (8). We iden i-
ied se e al gene ic ai s in his isola e, such as a a e a ian
o he in imin gene (eae-ξ) and gene ic de e minan s ela ed
o he pS88 plasmid associa ed wi h ex ain es inal- i ulence
pa hogenic E. coli (ExPEC) (9). This plasmid, mainly ound
in a ian pa hogenic E. coli and E. coli s ains ha cause
neona al meningi is, may pa ly explain he bac e emia ob-
se ed, which has been epo ed in pa ien s wi h HUS.
The occu ence o bac e emia du ing EHEC in ec ions
wa an s an ibio ic ea men o hose in ec ions. How-
e e , an ibio ics usually a e no ecommended o EHEC
in ec ion because o he isk o wo sening HUS, no ably
by induc ion o syn hesis o sec e ion o Shiga oxin (S x)
(1,10–12). The e o e, bac e emia du ing EHEC in ec ion
ep esen s a new he apeu ic challenge. Howe e , a 2011
ou b eak in Ge many linked o EHEC O104:H4 (13) un-
de sco ed he po en ial bene i o ce ain an ibio ics when
HUS occu s (14,15). Thus, he use o an ibio ics du ing
EHEC in ec ions emains a sou ce o deba e (2,16).
Ou s udy aimed o de e mine he incidence a e o
HUS cases associa ed wi h he singula EHEC O80:H2,
desc ibe hei clinical ea u es, and examine he molecula
cha ac e is ics o he s ains. In addi ion, we assessed he
e ec s o di e en an ibio ics on S x p oduc ion in ep e-
sen a i e s ains.
Ma e ials and Me hods
Clinical Da a
Fo his s udy, we conside ed all E. coli O80:H2 isola es
ecei ed du ing Janua y 2005–Oc obe 2014 by he Cen-
e Na ional de Ré é ence Associé Esche ichia coli (Pa is,
F ance). We hen collec ed demog aphic and clinical da a
om pa ien s’ medical eco ds (e.g., age, sex, loca ion);
p esence o dia hea (wi h o wi hou blood); possible
En e ohemo hagic Esche ichia coli
Hyb id Pa ho ype O80:H2 as a
New The apeu ic Challenge
Nu can Soysal, Pa icia Ma iani-Ku kdjian, Yasmine Smail, Sand ine Liguo i, Malika Gouali,
Es elle Loukiadis, Pa ick Fach, Ma hias B uyand, Jo ge Blanco, Philippe Bide , S éphane Bonaco si
1604 Eme gingIn ec iousDiseases•www.cdc.go /eid•Vol.22,No.9,Sep embe 2016
RESEARCH
Au ho a ilia ions:Cen eHospi alo-Uni e si ai eRobe -Deb é
(AP-HP),Pa is,F ance(N.Soysal,P.Ma iani-Ku kdjian,
Y.Smail,S.Liguo i,P.Bide ,S.Bonaco si);Ins i u Na ionaldela
San ée delaReche cheMédicale,Pa is(N.Soysal,
P.Ma iani-Ku kdjian,P.Bide ,S.Bonaco si);Uni e si éPa is
Dide o ,Pa is(N.Soysal,P.Ma iani-Ku kdjian,P.Bide ,
S. Bonaco si); Ins i u Pas eu , Pa is (M. Gouali); Labo a oi e
Na ionaldeRé e encepou lesEsche ichia coli, Ma cy l’E oile,
F ance(E.Loukiadis);ANSES,Maison-Al o ,F ance(P.Fach);
Ins i u de Veille Sani ai e, Sain Mau ice, F ance (M. B uyand);
Uni e sidade de San iago de Compos ela, Lugo, Spain (J. Blanco)
DOI:h p://dx.doi.o g/10.3201/eid2209.160304
En e ohemo hagic E. coliO80:H2
sou ce o in ec ion; p esence o neu ologic o o he compli-
ca ions (including panc ea i is, hepa i is, myoca di is, and
bac e emia); whe he he pa ien had HUS; and ou come a
ime o ollow-up (e.g., elapse, esidual enal inju ies [in-
cluding p o einu ia and enal ailu e], a e ial hype ension,
o dea h). HUS was de ined as anemia (hemoglobin <10
g/dL), h ombopenia (pla ele s <150,000/mm3), and enal
ailu e (c ea inine abo e e e ence o age, weigh , and sex,
o >0.2 p o ein/c ea inine a io).
Bac e ia S ains
We eco e ed isola es 35344 and 35431 om s ool and
blood cul u es, espec i ely, o a HUS pa ien who was he
subjec o a ecen case epo (8). These s ains belonged
o sequence ype 301 and ha bo ed 4 in es inal i ulence
genes (s x2c, s x2d, hlyA, and eae-ξ) and mos o he ex-
ain es inal i ulence genes ca ied by plasmid pS88 (8).
The Labo a oi e Na ional de Ré e ence pou les Esche ich-
ia coli (Ma cy l’E oile, F ance) and e e ence labo a o ies
om Spain, I aly, and Ge many we e associa ed wi h his
s udy and p o ided us wi h hei EHEC O80 s ains when
a ailable. The e e ence s ain EDL933 (O157:H7, s x1a,
s x2a, and eae-γ) se ed as he con ol in he s udy o S x
p oduc ion (17). We s o ed all EHEC s ains a –80°C in
5% glyce ol.
Se o yping
The Cen e Na ional de Ré é ence des Esche ichia coli,
Shigella, e Salmonella a he Ins i u e Pas eu (Pa is,
F ance) ini ially de e mined he O80 se og oup by using a
me hod based on he analysis o he O an igen genes clus-
e b es ic ion agmen s leng h polymo phism (18); his
esul was ecen ly con i med by O80-speci ic PCR wi h
p ime s a ge ing O80 polyme ase gene wzy (GenBank ac-
cession no. AB812032). This new PCR was included in ou
p e iously desc ibed O-se og oup mul iplex PCR (19). We
assessed speci ici y o he new mul iplex PCR on empla e
DNA ex ac ed om 130 O e e ence s ains as p e iously
desc ibed (20). P ime s used o he EHEC O-se og ouping
mul iplex PCR a e p o ided (online Technical Appendix
Tables 1, 2, h p://wwwnc.cdc.go /EID/a icle/22/9/16-
0304-Techapp1.xlsx). We de e mined he H se og oup by
using PCR a ge ing he liC genes (21).
Molecula Cha ac e iza ion
Among EHEC O80 s ains, we sc eened se e al gene ic de-
e minan s by mul iplex PCR as p e iously desc ibed (22),
including in es inal i ulence genes (s x1, s x2, eae, and
hlyA) and ex ain es inal i ulence genes associa ed wi h
plasmid pS88 (si A, ei B, cia, iss, iucC, i oN, hlyF, e sC,
c aA, and ompTp) (9,23). We also de e mined he a ian s
o s x2 (s x2a, s x2b, s x2c, and s x2d) and he a ian o
eae by using PCR-based me hods (24,25).
To in es iga e he gene ic di e si y o he O80:H2
s ains s udied, we used he Di e siLab geno yping me hod
(bioMé ieux, Ma cy-l’É oile, F ance), which is based on
PCR ampli ica ion o epea sequences o DNA ( ep-PCR)
as p e iously desc ibed (26). We hen compa ed hese
geno ypes wi h ep esen a i e s ains o se og oups (O157,
O104, O121, and O111) p e iously yped and eco ded in
ou Di e siLab da abase (S. Bonaco si, unpub. da a).
E ec o Va ious An ibio ics on S x P oduc ion
We p epa ed inocula o assess S x p oduc ion in he p es-
ence o absence o an ibio ics as p e iously desc ibed (27).
We ob ained log-phase g ow h o s ains in b ain–hea in-
usion b o h by using o e nigh incuba ion a 37°C and hen
dilu ed he esul wi h Lu ia-Be ani b o h o an inoculum
o 106 CFU/mL. We added 3 an imic obial agen s (azi h o-
mycin, cip o loxacin, and ce iaxone) a inal MICs o 0.5
and 0.25 o a single assay. We also es ed combina ions
o an ibio ics a a MIC o 0.5. Fo each s ain, we also pe -
o med an an ibio ic- ee assay. We collec ed he bac e ial
cul u es a e 18 h o incuba ion a 37°C and cen i uged
a 4°C a 2,000 pm o 10 min. We il e ed supe na an s
h ough a 0.22-µm po e-size il e (Millipo e, Bed o d,
MA, USA) and s o ed a –20°C un il needed.
We quan i ied S x1 and S x2 by using a chemilumi-
nescen immunoassay o EHEC oxins (Liaison, DiaSo in,
Spain). We exp essed he esul s in ela i e ligh uni s, and
con e ed each esul o a concen a ion o S x (ng/mL) by
using a s anda d cu e ob ained by se ial dilu ion o a high-
ly posi i e sample and he manu ac u e -p o ided posi i e
con ol (70 ng/mL S x concen a ion). We pe o med each
measu e 3 imes.
S a is ical Analysis
We calcula ed means, medians, and SDs in Excel (Mic oso
Co p., Redmond, WA, USA). S uden pai ed - es was used
o compa e means o S x concen a ions; p alues <0.05
we e conside ed s a is ically signi ican . Quan i a i e a i-
ables a e p esen ed as median and ange o qua ile ange.
Resul s
Du ing Janua y 2005–Oc obe 2014, he Cen e Na ional
de Ré é ence Associé Esche ichia coli collec ed 57 s ains
o EHEC O80:H2 in F ance. These s ains we e isola ed,
mos ly om s ool specimens, om 54 pa ien s; 2 and 3
isola es each we e eco e ed om 2 pa ien s. Clinical da a
we e a ailable o all bu 1 pa ien .
The spa io empo al dis ibu ion o he O80:H2 in ec-
ions clea ly indica es an inc eased numbe o in ec ions
du ing he pas 5 yea s (Figu e 1). In 2014, he EHEC O80
se og oup was he second-leading cause o pedia ic HUS
in F ance (4). Mos o he cases occu ed du ing summe
and he beginning o au umn. Geog aphic dis ibu ion o
Eme gingIn ec iousDiseases•www.cdc.go /eid•Vol.22,No.9,Sep embe 2016 1605
RESEARCH
O80:H2 EHEC in ec ions in F ance e ealed high 10-yea
cumula i e incidences (>1/100,000) in F anche-Com é
(2.83/100,000 child en) and in Rhône-Alpes (1.19/100,000
child en), con as ing wi h he dis ibu ion o O157 in ec-
ions, which a e a ely de ec ed in hese a eas (Figu e 2).
Among he 53 pa ien s o whom clinical da a we e
a ailable, 48 (91%) had HUS; 27 (51%) we e male. Me-
dian age o hese 48 pa ien s was 1.2 yea s ( ange 0.2–
39 yea s, in e qua ile ange [IQR] 0.7–1.6 yea s). Only
1 adul HUS pa ien (a 39-yea -old) was epo ed. The 5
(9%) non-HUS pa ien s we e la gely olde (1, 2, 6, 21, and
40 yea s old). Among HUS pa ien s, e e was p esen in
45%; median leukocy e coun was 13,000 cells/mm3 (da a
we e no a ailable o 14 pa ien s), and 56% had leuko-
cy osis (>11,500 leukocy es/mm3) (online Technical Ap-
pendix Table 3). Dia heal illness was epo ed o 83%
o HUS pa ien s (bloody dia hea o 30%); median ime
om onse o dia hea o diagnosis o HUS was 6 days
(da a a ailable o 37 pa ien s). Dia heal illness in amily
membe s was eco ded in only 2 HUS cases. One pa ien
had a elapse complica ed by bac e emia (8), 1 pa ien died
a e myoca dial complica ion wi h panc ea ic abscess
om which an O80:H2 EHEC s ain was isola ed, and 1
pa ien died om sep ic shock wi h in es inal nec osis and
pe i oni is. Eigh (17%) cases o neu ologic complica ion
(17%) we e epo ed. All he neu ologic complica ions
we e seizu es (documen ed in 6 cases by imaging) wi h
ischemic s okes. Among he 8 pa ien s wi h neu ologic
complica ions, 2 died, and 1 has di icul y concen a ing
(19 mon hs a e HUS); o 1 pa ien , he neu ologic s a e
could no be assessed, and o 4 o he s, he neu ologic ou -
come was a o able.
O he 48 HUS pa ien s, 27% needed acu e dialysis
suppo (median du a ion 10 days, ange 3–21 days, IQR
7.4–13.5 days), and 28% had long- e m enal sequelae,
including p o einu ia o all cases, hype ension in 3 cas-
es, and ch onic enal ailu e in 1 case (median ollow-
up du a ion 8 mon hs, ange 1–108 mon hs, IQR 2–41
mon hs). Finally, only 21% o medical eco ds men ioned
he possible sou ce o in ec ion, and no hypo hesis could
be o mula ed.
Gene ic cha ac e iza ion showed ha all O80:H2
s ains o human o igin collec ed in F ance ca ied he s x2
genes and no s x1 genes (online Technical Appendix Ta-
ble 3). The s x2 sub ype could no be de e mined o he 2
s ains (isola ed in 2006 and in 2010) because hey had los
hei s x2 gene despi e p ese a ion a –80°C. Among he
emaining s ains, 69% had a combina ion o s x2 a ian s,
s x2c/2d (62%) and s x2a/2d (7%); 31% ha bo ed unique
a ian s, s x2a (22%) and s x2d (9%). All s ains had he in-
imin encoding gene eae and i s a ian eae-ξ, and 87% ca -
ied he en e ohemolysin ehxA gene. All 57 s ains sha ed
>4 cha ac e is ic genes o he pS88 plasmid, si A, cia, hlyF,
and ompTp; 98% had he iss and i oN genes; 96% had he
c aA gene; and 61% had he iucC and e sC genes (online
Technical Appendix Table 3).
An imic obial d ug suscep ibili y es ing e ealed ha
mos s ains we e mul id ug esis an ; a es o esis ance
we e 91% o amoxicillin, 89% o nalidixic acid, 82% o
co imoxazole, and 71% o kanamycin (online Technical
Appendix Table 3). O e all, 52% o he s ains we e esis-
an o all 4 an ibio ics.
To examine whe he an animal could be he po en ial
sou ce o EHEC O80:H2, we solici ed he Labo a oi e Na-
ional de Ré e ence pou les Esche ichia coli. Only 1 s ain
om an animal sou ce (LNR-511-4, isola ed om aw cow
milk cheese) was a ailable. This s ain ca ied eae-ξ, ehxA,
and s x2a genes and 7 genes associa ed wi h he pS88 plas-
mid ela ed o ExPEC (si A, cia, iss, i oN, hlyF, c aA, and
ompTp). This s ain also was esis an o amoxicillin, kana-
mycin, and nalidixic acid.
To in es iga e he dis ibu ion in Eu ope o his eme g-
ing EHEC se og oup, se e al na ional e e ence labo a o ies
in Eu ope we e associa ed wi h his s udy and we e asked
o send us hei a ailable EHEC O80 s ains. We ob ained 5
s ains om Spain, whe eas I aly and Ge many had no such
s ain in hei collec ions. Among he 5 s ains om Spain,
3 we e o human o igin (IH42632/03a, IH33264/ 07a, and
IH102878/12a) and 2 we e o animal o igin (VTB-262
om a cow and FV4476 om a pig [ he pig-o igin s ain
was ac ually isola ed in Slo akia]) (28). All 5 s ains sha ed
eae-ξ, bu s x2 (s x2a) was ound in only 2 s ains (bo h
o human o igin), and none had ehxA. Only he 3 human
s ains ha bo ed mos o he in es iga ed genes associa ed
wi h pS88. The con as be ween animal s ains and human
s ains om he na ional e e ence labo a o y in Spain was
also e iden in hei an ibio ic- esis ance p o iles; only he
human s ains we e mul id ug esis an (online Technical
Appendix Table 3).
To analyze he gene ic di e si y and gene ic ela ed-
ness wi h o he EHEC se og oups, all he O80:H2 s ains
1606 Eme gingIn ec iousDiseases•www.cdc.go /eid•Vol.22,No.9,Sep embe 2016
Figu e 1.Numbe o en e ohemo hagicEsche ichia coliO80:H2
s ains de ec ed annually, F ance, Janua y 2005–Oc obe 2014.
En e ohemo hagic E. coliO80:H2
we e analyzed by using ep-PCR and compa ed wi h some
o he se og oups (O157, O104, O111, and O121) (Figu e
3). All EHEC O80:H2 s ains sha ed 95% simila i y excep
s ain IH102878/12a om Spain (90% simila i y). These
da a sugges ha almos all EHEC O80 s ains belong o a
unique clonal g oup, ega dless o hei geog aphic o igin
and sou ce (human o animal). EHEC O80:H2 s ains we e
gene ically dis an o ep esen a i e s ains o se og oups
O157, O104, O111, and O121.
To s udy he e ec o an ibio ics on S x p oduc ion in
O80:H2 EHEC s ains, we selec ed 4 ep esen a i e s ains
(33115, 35344, 35431, and 36047) based on hei geno yp-
ic and clinical cha ac e is ics (online Technical Appendix
Table 3). S ain EDL933 (O157:H7) se ed concomi an ly
as he con ol. We examined suscep ibili y o he 3 an ibio -
ics (Table). Fi s , we es ima ed he basal p oduc ion o S x
in di e en s ains a e 18 h o g ow h wi hou an ibio -
ics (Figu e 4). The basal p oduc ion among he di e en
O80:H2 s ains we e compa able bu signi ican ly lowe
compa ed wi h ha o s ain EDL933, which p oduces
≈100- old mo e S x han ce ain O80:H2 s ains (35344
and 36047). We could no sepa a ely es ima e he espec-
i e a es o S x1 and S x2.
Fo each s ain, we examined he in luence o he 3
an ibio ics a concen a ions below he MICs exp essed
as ela i e sec e ion o S x compa ed wi h basal sec e ion
(wi hou an ibio ics) a e 18 hou s’ incuba ion pe iod (Fig-
u e 5, panels A–E). O e all, azi h omycin was esponsible
o >5- old dec eases o S x p oduc ion in all O80:H2
s ains a a MIC o 0.5. As expec ed, he same e ec was
obse ed wi h he EDL933 s ain. A all concen a ions
es ed, cip o loxacin signi ican ly induced a majo inc ease
o S x sec e ion o all s ains excep s ain 35344. The in-
c ease was pa icula ly ma ked o O80:H2 s ains com-
pa ed wi h O157 s ains; a 100- old inc ease was obse ed
o 33115 and 35431 (Figu e 5, panels B and C), compa ed
wi h a 6- old inc ease o EDL933 (Figu e 5, panel E).
Ce iaxone, in con as wi h o he an ibio ics, did no sig-
ni ican ly al e S x p oduc ion excep o 1 O80:H2 s ain
(Figu e 5, panel C).
Finally, we wan ed o de e mine whe he he bene icial
e ec o azi h omycin on S x p oduc ion would pe sis in
he p esence o 2 o he an ibio ics, a combina ion which
migh be used in cases o bac e emia. The combina ions
o an ibio ics we e es ed on 2 O80:H2 s ains (Figu e 6).
Azi h omycin pai ed wi h cip o loxacin signi ican ly e-
duces S x p oduc ion compa ed wi h cip o loxacin alone.
Howe e , his p oduc ion was highe han ha obse ed
wi h azi h omycin alone, and o bo h s ains, S x le els
we e highe o azi h omycin/cip o loxacin compa ed wi h
no an ibio ic. These da a indica e ha he mac olide migh
only pa ially inhibi he noxious e ec o cip o loxacin.
The e ec o azi h omycin in combina ion wi h ce iax-
one depended on he s ain es ed. Fo s ain 35344, he as-
socia ion sligh ly inc eased S x p oduc ion compa ed wi h
ce iaxone, whe eas o s ain 33115, he opposi e was
Eme gingIn ec iousDiseases•www.cdc.go /eid•Vol.22,No.9,Sep embe 2016 1607
Figu e 2. Regional 10-yea cumula i e incidence a es o hemoly ic u emic synd ome cases caused by en e ohemo hagic Esche ichia
colise o ypesO157:H7andO80:H2,F ance,Janua y2005–Oc obe 2014.A)Se o ypeO157:H7.B)Se o ypeO80:H2.Whi e,<0.5
cases/100,000child en;ligh g ayshading,0.5–0.7cases/100,000child en;mediumg ayshading,0.8–0.9cases/100,000child en;da k
g ayshading,1–2cases/100,000child en;black,>2cases/100,000child en.
RESEARCH
1608 Eme gingIn ec iousDiseases•www.cdc.go /eid•Vol.22,No.9,Sep embe 2016
Figu e 3. Dend og am
ob ained a e Di e siLab
geno yping analysis (based on
PCRampli ica iono epea
sequenceso DNA)o 56
en e ohemo hagic Esche ichia
coli(EHEC)O80s ains om
humans in F ance compa ed
wi h o he isola es de ec ed
in F ance, Ge many, and
Spain, Janua y 2005–Oc obe
2014. O he isola es include 1
animal-o igin s ain om F ance
(LNR511-4,bo ine,2012);
5 animal- and human-o igin
isola es om Spain (FV4476,
po cine; VTB-262, bo ine;
IH43632/03a,IH33264/07a,
andIH102878/12a,human);
and 6 compa ison s ains om
o he se og oups(EDL933and
32031, O157; 32527 and 32573,
O104, isola ed du ing a 2011
ou b eak in Ge many; 36061,
O121;and34593,O111).
ID,iden i ica ion.
En e ohemo hagic E. coliO80:H2
obse ed. Howe e , in his expe imen , he obse ed S x
a e was lowe in ce iaxone and azi h omycin/ce iaxone
assays compa ed wi h basal sec e ion.
Discussion
In his s udy, we desc ibed he eme gence in F ance o a
new i ulen EHEC se o ype, O80:H2, ha ha bo s sin-
gula gene ic cha ac e is ics o a hyb id STEC/ExPEC
pa ho ype. Se e al impo an conclusions can be d awn.
Fi s , he EHEC O80:H2 s ain appea s o be a leas as
i ulen as he EHEC O157:H7 s ains p esen in F ance.
Indeed, he a e o HUS-associa ed complica ions, such as
enal inju ies (28%) and dea h (4%), o he O80:H2 s ain
we e compa able wi h ha o O157:H7 (1,4). Mo eo e ,
EHEC O80:H2 isola es ha e he pa icula p ope ies o in-
duce in asi e in ec ions; >2 o he 53 pa ien s a ec ed had
bac e emia o deep abscess. Ex ain es inal in ec ions e y
a ely a e associa ed wi h EHEC; o ou knowledge, only 6
cases o bac e emia ha e been p e iously desc ibed in he
con ex o HUS (8). None o hese 6 epo s ex ensi ely
sea ched o ex ain es inal i ulence ac o s. Whe he he
ex ain es inal i ulence o EHEC O80:H2 is ela ed o he
p esence o gene ic ai s cha ac e is ic o an ex ain es inal
i ulence–associa ed plasmid emains o be de e mined.
Iden i ica ion o he salmochelin-encoding genes (i oN) in
98% o he EHEC O80:H2 om F ance is o pa icula in-
e es . This gene is clea ly in ol ed in he pa hophysiology
o E. coli bac e emia and meningi is (9,29).
Because he su eillance sys em o STEC is olun a y
in F ance and because s x-speci ic PCR is pe o med only
in cases in ol ing dia hea wi h HUS suspicion, accu a e
da a a e no a ailable on he a es o dia hea wi hou HUS
o o bloods eam in ec ions caused by his se o ype and
o he s. Only cases o dia hea occu ing among HUS pa-
ien con ac s a e sys ema ically in es iga ed. The e o e,
many cases o O80:H2- ela ed dia hea a e p obably un-
diagnosed, and we canno d aw any conclusion conce ning
he isk o HUS in cases o dia hea associa ed wi h STEC
O80:H2. Mo eo e , non-HUS cases o bac e emia caused
by O80:H2 s ains will escape de ec ion.
We we e no able o iden i y he po en ial sou ce o his
eme ging EHEC pa ho ype. All epo ed cases ha e been
spo adic, and he possibili y o oodbo ne in ec ion emains
specula i e, e en hough O80:H2 s ains we e isola ed om
ew animals. The highes incidence o O80:H2 in ec ion was
obse ed in egions o F ance whe e EHEC O157 in ec ions
a e no p edominan , sugges ing a possible a ypical ou e
o sou ce o in ec ion. Al hough Spain does no sha e bo -
de s wi h he high-incidence egions o F ance, i was he
only coun y whe e a signi ican numbe o EHEC O80:H2
s ains we e ound. Two O80:H2 EHEC s ains om Spain
(IH102878/12a and IH33264/07a) we e e y simila o
F ench s ains belonging o he same clonal g oup wi h a sim-
ila i ulence geno ype, sugges ing a di ec lineage be ween
he isola es in Spain and F ance. As is usually obse ed o
o he se o ypes, mos o he in ec ions wi h hese s ains oc-
cu ed du ing summe and he beginning o au umn (4,7).
The molecula cha ac e iza ion o he EHEC O80:H2
s ains was o pa icula in e es . The p esence in all
s ains om Spain and F ance o he e y a e eae-ξ gene
combined wi h esul s o he gene ic di e si y s udy us-
ing ep-PCR s ongly indica e ha hese s ains, wha -
e e hei o igin, ha e a common ances o combining he
O80:H2 se o ype and an eae-ξ gene con aining LEE. On
his unique gene ic backg ound, se e al gene ic e en s oc-
cu ed: he acquisi ion o >1 p ophage encoding S x, he
p esence o a plasmid simila o ha ound in ExPEC, and
he p esence o a plasmid encoding en e ohemolysin. The
ch onology o hese e en s emains specula i e. Howe e ,
acquisi ion o S x2-encoding genes seems o emain pa -
icula ly ac i e because 3 di e en a ian s in >4 di e -
en modes (in combina ion o alone) we e obse ed. The
di e si y o he ex ain es inal i ulence plasmidic genes
Eme gingIn ec iousDiseases•www.cdc.go /eid•Vol.22,No.9,Sep embe 2016 1609
Table. An ibio ic suscep ibili y o en e ohemo hagic Esche ichia coli O80:H2andO157:H7isola es used in an an ibio ics assay,
F ance, Janua y 2005–Oc obe 2014
Isola e (sou ce)
Se o ype
MIC,µg/mL
Azi h omycin
Cip o loxacin
Ce iaxone
35344 (s ool)
O80:H2
1
0.5
0.25
35431 (blood)
O80:H2
16
0.5
0.06
33115 (panc eas)
O80:H2
16
0.25
0.06
36047 (s ool)
O80:H2
16
0.06
0.06
EDL933(g oundbee )
O157:H7
32
0.12
0.12
Figu e 4. Mean concen a ions (loga i hmic scale) o Shiga
oxin p oduced in he absence o an ibio ics by selec ed s ains
o en e ohemo hagic Esche ichia colise o ypesO80,F ance,
Janua y2005–Oc obe 2014.O157 e e ences ain(EDL933)
was used as con ol. E o ba s indica e SDs.
RESEARCH
combina ion sugges s he in e play o ce ain plasmidic
de e minan s and in es inal pa hogenic ai s. The mini-
mal combina ion o plasmidic genes common o all s ains
was he associa ion o ompTp and hlyF, which migh ep-
esen a bene icial in luence on he in es inal pa hogenic
i ulence o E. coli O80:H2. Ch omosomal ompT and
hlyF a e in ol ed in he sec e ion o ou e memb ane
esicles, which migh se e as anspo e s o oxins and
hus migh boos he i ulence o S x-p oducing E. coli
(30,31). Whe he ompTp has a simila ole emains o be
u he in es iga ed.
Finally, clinical ea u es and molecula cha ac e -
iza ion indica ing he po en ial in asi e pa hogenici y o
EHEC O80:H2 aise he ques ion o which an ibio ic should
be used in such in ec ions. Se e al clinical s udies ha e
sugges ed a dele e ious e ec o an ibio ics du ing EHEC
in ec ion, leading o ecommenda ions o no use such
ea men (2). The majo explana ion o such an ad e se
e ec is he induc ion o S x sec e ion h ough he SOS
esponse, which is s imula ed by an ibio ics such as luo-
oquinolones in i o and in expe imen al models (32,33).
Se e al s udies demons a ed ha he e ec o an ibio ics
on HUS depends on hei class (27,33–37). Cip o loxacin
aises he p oduc ion and elease o S x in i o and is asso-
cia ed wi h a highe mo ali y a e in pigs (33). O he s ud-
ies ha e shown ha some an ibio ics such as azi h omycin
migh be associa ed wi h a dec ease o he p oduc ion and
elease o S x in i o (37) and wi h a o able ou comes in
in i o s udies in pigle s and mice (33,38). Finally, du ing
a 2011 ou b eak o EHEC O104 in ec ion in Ge many, a
pa ien ea ed by cip o loxacin plus imipenem unexpec -
edly had a be e p ognosis han all o he s (14). This esul
sugges s ha esponse o an ibio ics migh also di e de-
pending o he s ain in ol ed (34).
Ou analysis o S x p oduc ion in he p esence o an-
ibio ics has clea ly indica ed ha cip o loxacin should
no be used in cases o EHEC O80 in ec ion. In con as ,
azi h omycin p o ided a bene icial in i o e ec and migh
be use ul in cases o EHEC O80–associa ed dia hea. These
esul s a e consis en wi h he sys ema ic e iew by Agge
e al. (16), who concluded ha a p o ein syn hesis inhibi o
can be conside ed du ing EHEC in ec ions when speci ic
c i e ia a e me . To ou knowledge, only a ew s udies ha e
1610 Eme gingIn ec iousDiseases•www.cdc.go /eid•Vol.22,No.9,Sep embe 2016
Figu e 5. Rela i e p oduc ion a e o
Shiga oxin p oduced in 5 s ains o
en e ohemo hagic Esche ichia coli
(4O80s ainsand1O157s ain)
a subinhibi o y concen a ions o
azi h omycin,cip o loxacin,and
ce iaxone, compa ed wi h basal
p oduc ion a e (no an ibio ics), F ance,
Janua y 2005–Oc obe 2014. A) Isola e
35344. B) Isola e 33115. C) Isola e
35431. D) Isola e 36047. E) Isola e
EDL933.E o ba s indica e SDs.
Figu e 6. Rela i e p oduc ion a e o Shiga oxin p oduced in
2 s ains o en e ohemo hagic Esche ichia coliO80(isola es
35344 and 33115) a subinhibi o y concen a ions o azi h omycin,
cip o loxacin,ce iaxone(aloneandincombina ion),compa ed
o basal p oduc ion a e (no an ibio ics), F ance, Janua y 2005–
Oc obe 2014.AZM,azi h omycin;AZM/CIF,azi h omycin/
cip o loxacin;AZM/CRO,azi h omycin/ce iaxone;CIF,
cip o loxacin;CRO,ce iaxone.E o ba sindica eSDs.
En e ohemo hagic E. coliO80:H2
es ed he e ec o an ibio ics in combina ion (14,39). Ou
combina ion assay esul s would sugges ha azi h omycin
plus ce iaxone migh be a easonable choice in cases o
sys emic in ec ion.
These indings should be ega ded as p elimina y and
equi e con i ma ion. Howe e , despi e hese p omising in
i o esul s and because ou assays we e pe o med wi h-
ou measu ing cy o oxici y, we canno ye ad oca e he use
o hese an ibio ics o ea men o pa ien s in ec ed wi h
EHEC O80:H2. Howe e , a planned na ional clinical ial
in F ance (NCT02336516) o es he e icacy o azi h o-
mycin in child en wi h pos dia heal HUS migh soon p o-
ide some answe s.
In conclusion, a clonal g oup o EHEC O80:H2
s ains o unknown o igin and wi h he abili y o induce
in asi e in ec ions and le hali y is eme ging in F ance
and ep esen s a new he apeu ic challenge. The in e play
be ween in es inal and ex ain es inal i ulence ac o s in
his new hyb id STEC/ExPEC pa ho ype emains o be
elucida ed. Azi h omycin migh be a possible op ion o
p e en in asi e in ec ions caused by EHEC O80:H2,
whe eas azi h omycin/ce iaxone migh be use ul in
ea ing such in ec ions.
Acknowledgmen s
We hank he F ench Socie y o Pedia ics o i s suppo o
his s udy.
Wo k in he Lab o o io de e e encia de Esche ichia coli was
inanced by g an no. CN2012/303 om Conselle ía de Cul u a,
Educación e O denación Uni e si a ia (Xun a de Galicia) and he
Eu opean Regional De elopmen Fund.
D . Soysal is a pedia ician wo king in Assis ance Publique–
Hôpi aux de Pa is (AP-HP). He esea ch domain is pedia ic
in ec ions, pa icula ly pa hogenici y and he apeu ic
managemen o E. coli in es inal in ec ions.
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Add ess o co espondence: S éphane Bonaco si, Hôpi al Robe -Deb é, 48
Boule a d Sé u ie , 75019 Pa is, F ance; email: [email p o ec ed]
1612 Eme gingIn ec iousDiseases•www.cdc.go /eid•Vol.22,No.9,Sep embe 2016
