U ina y T ac Physiological Condi ions P omo e Cip o loxacin
Resis ance in Low-Le el-Quinolone-Resis an Esche ichia coli
Guille mo Ma ín-Gu ié ez,
a,c
Je ónimo Rod íguez-Bel án,
a
José Manuel Rod íguez-Ma ínez,
b
Coloma Cos as,
a
Ja ie Azna ,
a,c
Ál a o Pascual,
b,c
Jesús Blázquez
a,d
Ins i u o de Biomedicina de Se illa (IBIS), Hospi al Uni e si a io Vi gen del Rocío, CSIC, Uni e sidad de Se illa, Se ille, Spain
a
; Depa amen o de Mic obiología, Uni e sidad
de Se illa, Se ille, Spain
b
; Unidad In e cen os de En e medades In ecciosas, Mic obiología y Medicina P e en i a, Hospi ales Uni e si a ios Vi gen Maca ena y Vi gen del
Rocío, Se ille, Spain
c
; Cen o Nacional de Bio ecnología (CNB), Consejo Supe io de In es igaciones Cien í icas (CSIC), Mad id, Spain
d
Esche ichia coli isola es ca ying ch omosomally encoded low-le el-quinolone- esis an (LLQR) de e minan s a e equen ly
ound in u ina y ac in ec ions (UTIs). LLQR mu a ions a e conside ed he i s s ep in he e olu iona y pa hway p oducing
high-le el luo oquinolone esis ance. The e o e, hei e olu ion and dissemina ion migh in luence he ou come o luo oquin-
olone ea men s o UTI. P e ious s udies suppo he no ion ha low u ine pH dec eases suscep ibili y o cip o loxacin (CIP) in
E. coli. Howe e , he e ec o he u ina y ac physiological pa ame e s on he ac i i y o cip o loxacin agains LLQR E. coli
s ains has ecei ed li le a en ion. We ha e s udied he ac i i y o cip o loxacin unde physiological u ina y ac condi ions
agains a se o well-cha ac e ized isogenic E. coli de i a i es ca ying he mos p e alen ch omosomal mu a ions (⌬ma R,
gy A-S83L, gy A-D87N, and pa C-S80R and some combina ions). The esul s p esen ed he e demons a e ha all he LLQR
s ains s udied became esis an o cip o loxacin (acco ding o CLSI guidelines) unde physiological condi ions whe eas he con-
ol s ain lacking LLQR mu a ions did no . Mo eo e , he su i al o some LLQR E. coli a ian s inc eased up o 100- old a e
challenge wi h a high concen a ion o cip o loxacin unde UTI condi ions compa ed o he esul s seen wi h Muelle -Hin on
b o h. These selec i e condi ions could explain he high p e alence o LLQR mu a ions in E. coli. Fu he mo e, ou da a s ongly
sugges ha ecommended me hods o MIC de e mina ion p oduce poo es ima ions o CIP ac i i y agains LLQR E. coli in
UTIs.
Cip o loxacin (CIP) is one o he agen s commonly u ilized o
ea men o u ina y ac in ec ions (UTIs) (1–3). I is used as
an app op ia e he apy in pa ien s wi h UTI no equi ing hospi-
aliza ion in a eas whe e he p e alence o esis ance is unde 10%.
In addi ion, i is conside ed an e ec i e ea men in he p e en-
ion o UTI in kidney ansplan ecipien s (4,5). Howe e , du ing
ecen yea s a clea inc ease in esis ance o luo oquinolones
(FQ) has been desc ibed wo ldwide (6,7).
The p edominan causa i e agen o communi y-acqui ed
UTI, Esche ichia coli, acqui es esis ance mainly h ough ch omo-
somal mu a ions in he genes encoding subuni s o he DNA gy-
ase (gy A and gy B genes) and opoisome ase IV (pa C and pa E
genes) o in egula o y genes a ec ing pe meabili y o e lux (8).
Some o hese mu a ions lead o gene a ion o low-le el-quinolo-
ne- esis an (LLQR) E. coli mu an s, wi h a CIP MIC ha is highe
han he epidemiological cu o alue bu s ill below he esis ance
b eakpoin o mos FQs. A high ( om 17% o 39%, depending on
he s udy) p e alence o LLQR E. coli in UTIs has been p e iously
desc ibed (8,9). Howe e , he causes o his p e alence a e un-
clea , as he high CIP le els ound in he bladde a e ea men
should be su icien o e adica e any suscep ible bac e ia.
I has been s a ed ha small inc eases in MIC alues p oduced
by LLQR mu a ions ha e a p o ound impac on he mu an , p e-
en ing concen a ion o FQs and aising he likelihood ha
s ains wi h high-le el esis ance will appea (10). The e o e,
LLQR mu an s a e a guably he i s s ep on he e olu iona y
pa hway o p oducing high-le el quinolone esis ance, and hus
hei dissemina ion and e olu ion migh in luence he ou come o
FQ ea men o UTI.
One o he de e minan s o his ou come is he en i onmen
ound in he u ina y ac , which is cha ac e ized mainly by he
p esence o u ine and anae obic condi ions. U ine is a luc ua ing
and complex luid composed o o e 95% wa e , plus sodium,
ammonia, phospha e, sul a e, u ea, c ea inine, p o eins, and
p oduc s p ocessed by he kidney and li e (11). The a iabili y in
he physicochemical p ope ies o u ine is bes illus a ed by he
wide pH ange o human u ine, which no mally a ies om 5 o 8
(12). Addi ionally, he bladde en i onmen is mainly anae obic,
wi h a concen a ion o dissol ed oxygen (DO) in u ine o abou
4.2 ppm (13); he concen a ion is also a iable and mainly e lec s
he enal me abolic s a e. Mo eo e , in pa ien s wi h u ina y in-
ec ions, he u ine DO concen a ion is signi ican ly educed as a
esul o oxygen consump ion by he in ec ing mic obes (13).
Howe e , he ela i e con ibu ions o hese pa ame e s o he
possible ou comes o CIP ea men agains LLQR s ains ha e
ecei ed li le a en ion. CIP is an ampholy e consis ing o an
acidic g oup and a basic g oup ha can exis in ou di e en
pH-dependen p o ona ion o ms (14). The zwi e ionic o m is
necessa y o bac e ial pe meabili y, while he neu al o m p o-
ides good in es inal abso p ion (15). The e o e, he wide ange o
Recei ed 16 Ma ch 2016 Re u ned o modi ica ion 24 Ap il 2016
Accep ed 27 Ap il 2016
Accep ed manusc ip pos ed online 2 May 2016
Ci a ion Ma ín-Gu ié ez G, Rod íguez-Bel án J, Rod íguez-Ma ínez JM, Cos as C,
Azna J, Pascual A, Blázquez J. 2016. U ina y ac physiological condi ions
p omo e cip o loxacin esis ance in low-le el-quinolone- esis an Esche ichia coli.
An imic ob Agen s Chemo he 60:4252–4258. doi:10.1128/AAC.00602-16.
Add ess co espondence o Jesús Blázquez, [email p o ec ed].
G.M.-G. and J.R.-B. con ibu ed equally o his a icle.
Copy igh © 2016, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
c ossma k
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pH ound in human u ine can a ec he ioniza ion s a us o CIP
and can hus modi y i s an imic obial ac i i y (16–19). Rega ding
oxygen le els, i has been epo ed ha g ow h unde condi ions
o anae obiosis a ec s bac e ial suscep ibili y o CIP (20). The e-
o e, bo h pa ame e s could, in p inciple, in luence he esis ance
p o iles o LLQR UTI s ains, hus a ec ing he p ognosis o ea -
men .
Pha macokine ic and pha macodynamic (PK/PD) s udies in-
dica e ha e y high (up o 990 ␮g/ml) concen a ions o CIP can
be eached in u ine du ing ea men (21), sugges ing ha , despi e
apossible e ec o pH and o he u ine pa ame e s on CIP ac i i y,
such ea men should be su icien o kill any suscep ible bac e ia.
Howe e , o da e, no s udies on he e ec o hese pa ame e s on
he ac i i y o CIP agains s ains con aining LLQR mu a ions
ha e been pe o med.
Consequen ly, we aimed o e alua e he impac o he abo e-
men ioned pa ame e s (namely, g ow h in u ine, pH, and anae -
obiosis) on he an imic obial ac i i y o CIP agains a se o well-
cha ac e ized isogenic LLQR s ains ca ying he mos equen
ch omosomal mu a ions (9,22) and agains a se ies o E. coli clin-
ical isola es wi h known LLQR mu a ions. MICs o CIP in bo h
Muelle -Hin on b o h (MH) and u ine unde a ious pH and
oxygen supply condi ions we e s udied. In addi ion, he su i al
a es o hese s ains in he p esence o he maximum concen a-
ion o CIP eached in he bladde we e also analyzed.
MATERIALS AND METHODS
S ains and g ow h condi ions. Wild- ype E. coli ATCC 25922 and nine
di e en isogenic s ains ca ying combina ions o he mos p e alen
ch omosomal mu a ions (⌬ma R,gy A-S83L, gy A-D87N, and pa C-
S80R) we e used. Isogenic s ains we e hose cons uc ed p e iously by
Machuca e al. (23). Mo eo e , six u opa hogenic E. coli (UPEC) s ains
wi h well-cha ac e ized LLQR mu a ions ha had been isola ed om pa-
ien s in he Uni e si y Hospi al Ma ques de Valdecilla and Uni e si y
Hospi al Vi gen Maca ena du ing 2009 we e s udied (Table 1).
MH (Conda P onadisa, Spain) was used as a con ol medium. Fo
g ow h a e de e mina ions, o e nigh cul u es o he s ains we e dilu ed
1:100 in o MH and cul u ed wi h ae a ion a 37°C du ing2ha 180 pm
o ob ain bac e ia in he exponen ial-g ow h phase. G ow h assays we e
conduc ed in clea la -bo om 96-well Mic oWell pla es (Nunc, Roskilde,
Denma k), con aining 8 eplica es o 100 ␮l pe sample. The pla es we e
incuba ed a 37°C wi h shaking on an au oma ed mic opla e eade (In-
ini e M200; Tecan, Männedo , Swi ze land), and he abso bance a 595
nm o each well was measu ed e e y 30 min. The du a ion o each assay
was 24 h.
U ine ob ained om 3 heal hy olun ee s who had no ecei ed an i-
bio ic ea men du ing he p e ious 6 mon hs was pooled, s e ilized by
il a ion h ough 0.22-mm-po e-size il e s (polye he sul one [PES]
memb ane) (VWR, Uni ed Kingdom), di ided in o aliquo s, and s o ed
a ⫺20°C. The u ine was adjus ed o pH alues o 5.0, 6.0, and 7.0 wi h
HCl o NaOH (bo h om Sigma-Ald ich), and he samples we e again
subjec ed o s e ile il a ion be o e use. U ine-aga pla es we e made by
mixing 4:1 pH-adjus ed pooled human u ine wi h 6% aga (Conda P o-
nadisa, Spain). Final pH alues we e de e mined o u ine-aga be o e
pla ing and we e eadjus ed when necessa y.
Measu emen o pH o u ine om pa ien s wi h communi y-ac-
qui ed E. coli UTI. F om Ma ch 2015 o June 2015, ⱖ18-yea -old pa ien s
wi h UTI symp oms ecei ing ca e a he P ima y Ca e Uni s o Vi gen del
Rocío Uni e si y Hospi al we e included in his s udy. Mids eam ca ch
u ine was collec ed in s e ile p ese a i e ubes (SRO-1-25B; So ia Mel-
guizo S.A., Mad id, Spain) and was anspo ed on ice. U ine samples
we e p ocessed wi hin 4 o 8 h a e collec ion. Fo each sample, 10 ␮lo
u ine was quan i a i ely cul u ed on o B illiance UTI Cla i y aga pla es
(Oxoid, Basings oke, Uni ed Kingdom). All pla es we e ae obically incu-
ba ed o 18 o 24 h a 37°C, and he esul s we e exp essed as he numbe
o CFU pe millili e . U ine pH was measu ed in each collec ed specimen
wi h a pH me e .
Cul u es in which E. coli le els we e abo e h esholds o ⱖ10
5
CFU/ml
o women and ⱖ10
3
CFU/ml o men we e de ined as posi i e. In o al,
pH alues o u ine om 136 pa ien s (wi h p o en UTI caused by E. coli)
we e included.
Suscep ibili y es ing. MICs o CIP we e de e mined in iplica e o
each bac e ial s ain using he b o h mic odilu ion me hod, acco ding o
he Clinical and Labo a o y S anda ds Ins i u e (CLSI) e e ence me hods
(24). O e nigh cul u es we e p epa ed and adjus ed o a densi y o 0.5
McFa land. To ep oduce u ine physiological condi ions, he MICs o
CIP we e measu ed in MH and u ine a pH alues o 5, 6, and 7. To
de e mine i he e ec was due only o he dec ease in pH, MICs we e also
measu ed in MH a pH 5 and pH 6, in addi ion o he s anda d pH o MH
b o h (7.3 ⫾0.1). Mic opla es we e incuba ed unde ae obic and anae -
obic condi ions.
MICs o CIP we e also de e mined by a g adien MIC s ip me hodol-
ogy (Lio ilchem s l, I aly) acco ding o CLSI c i e ia. Bac e ial suspen-
sions we e p epa ed om o e nigh cul u es and adjus ed o a densi y o
0.5 McFa land. G adien MIC s ip expe imen s we e pe o med on MH
aga (Di co, So ia Melguizo, Spain) and u ine-aga wi h pH alues o 5, 6,
and 7. Pla es we e incuba ed unde ae obic condi ions o 24 h (48 h o
u ine-aga es s) a 37°C.
Su i al a es. O e nigh cul u es o he di e en s ains we e g own
a 37°C wi hou he an imic obial agen in bo h MH and u ine a di e en
pH alues. Then, bac e ia a a densi y o ⬃5⫻10
5
CFU/ml om hese
cul u es we e inocula ed in o esh MH o u ine con aining CIP and we e
incuba ed a 37°C o 6 h in anae obiosis. The quinolone concen a ion
used o de e mina ion o su i al a es was 1,000 ␮g/ml, sligh ly abo e
he maximum concen a ion o CIP eached in u ine wi hin he i s 6 h
a e adminis a ion o an o al dose o 500 mg CIP (21), which is he
ecommended dose o adul s (4). An ibio ic- ee b o h was e alua ed in
pa allel as a con ol. Viable coun s we e de e mined by pla ing se ial di-
lu ions on o MH aga pla es incuba ed a 37°C o up o 24 h. The su i al
a e was calcula ed by no malizing he numbe o su i ing bac e ia o he
es ima ed ini ial popula ion.
TABLE 1 Geno ypes o isogenic and UPEC s ains
a
S ain
Geno ype
gy A1 gy A2 pa C ma R
Isogenic
ATCC 25922
EC01 ⌬
EC02 S83L
EC03 S83L ⌬
EC04 S83L S80R
EC05 S83L S80R ⌬
EC06 S83L D87N
EC07 S83L D87N ⌬
EC08 S83L D87N S80R
EC09 S83L D87N S80R ⌬
UPEC
Val09 S83L
MAC02 S83L
MAC10 S83L
MAC11 S83L S80R
MAC21a S83L S80I
MAC33 D87Y
a
UPEC, u opa hogenic Esche ichia coli s ains; ⌬, p ecise dele ion.
LLQR E. coli Resis ance in UTI
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RESULTS
Va iabili y o pH in u ine om UTI pa ien s. We i s conduc ed
a p ospec i e s udy o measu e he u ine pH om pa ien s wi h
communi y-acqui ed E. coli UTI. The esul s p esen ed in Fig. 1
demons a e a wide a iabili y in pH alues, anging om 5 o 8,
sugges ing ha physiological u ina y pH is highly a iable among
pa ien s. We ound ha he as majo i y (90%) o pH alues we e
below neu ali y, wi h mos samples being ma kedly acidic (63%
wi h pH ⫽ⱕ6). This esul , which ag ees wi h a p e ious epo
(25), indica es ha low u ine pH is he ule a he han he excep-
ion in UTI pa ien s.
E ec o u ine pH on he g ow h o E. coli ATCC 25922. We
hen s udied he abili y o he E. coli ATCC 25922 s ain o g ow in
u ine a physiological pH alues. G ow h cu es in MH and u ine
we e ca ied ou using an au oma ed spec opho ome e unde
con olled condi ions o empe a u e and shaking. Figu e 2 shows
ha E. coli ATCC 25922 was able o p ospe in u ine, al hough i s
g ow h was somewha lowe han ha in MH, p obably due o he
low concen a ion o nu ien s. Acidi ica ion o u ine, including
pH 5, led o only small dec eases in g ow h, demons a ing ha E.
coli is well adap ed o ex eme pH alues. Simila ly, all LLQR de-
i a i es we e able o g ow in u ine a di e en pH alues (da a no
shown). I should be no ed he e ha we we e unable o pe o m
g ow h cu e de e mina ions in basic u ine (pH 8) because, unde
ou condi ions, u ine componen s p ecipi a e a pH alues highe
han 7. This, oge he wi h he ac ha basic u ine is uncommon
among UTI pa ien s (⬍5%) (Fig. 1), led us o exclude his condi-
ion om u he expe imen s.
E ec o pH and anae obiosis on he ac i i y o CIP agains
LLQR E. coli s ains g owing in u ine. To de e mine i CIP ac i -
i y is a ec ed by u ine acidi ica ion, we pe o med p elimina y
MIC de e mina ions using o a combina ion o he g adien MIC
s ip me hodology and pH-adjus ed u ine aga pla es (see Ma e-
ials and Me hods). As expec ed, pH alues had a la ge in luence
on he CIP MICs de ec ed by g adien MIC s ips o all LLQR
s ains. Figu e 3 shows an illus a i e example wi h ATCC 25922
and i s gy A-S83L–pa C-S80R de i a i e EC05.
The ac i i y o CIP agains all LLQR isogenic de i a i es was
also de e mined in u ine unde di e en condi ions (pH and an-
ae obiosis) by he mic odilu ion me hod. Table 2 con i ms ha
pH had a la ge in luence on MICs, wi h 4- old o 16- old MIC
inc eases in he ATCC 25922 s ain and i s isogenic LLQR de i -
a i es when he pH was changed om neu al o 6. Impo an ly,
wi h he excep ion o he ATCC 25922 and EC01 (⌬ma R) s ains,
all suscep ible s ains became esis an (MIC o ⬎4␮g/ml) ac-
co ding o CLSI classi ica ion. The MIC inc eases we e e en
highe when he u ine pH was se o 5. Unde hose condi ions,
CIP ac i i y dec eased 32- old o 256- old in compa ison o he
ac i i y seen a pH 7. A pH 5, all s ains excep ATCC 25922 we e
esis an o CIP, as de ined by CLSI guidelines. Taken oge he ,
hese esul s indica e ha he p esence o any o he s udied LLQR
mu a ions was enough o con e clinical le els o esis ance a
acidic bu physiological pH. Rema kably, he ATCC 25922 s ain,
which lacks any o hese esis ance de e minan s, emained sus-
cep ible unde hese condi ions.
Rega ding he e ec o anae obiosis, Table 2 (nO
2
columns)
shows ha g ow h unde anae obic condi ions inc eased he CIP
MICs 2- old o 8- old a pH 7 compa ed o he alues ob ained
FIG 1 Dis ibu ion o u ine pH alues in 136 samples om pa ien s wi h UTI
caused by E. coli.
FIG 2 G ow h cu es o E. coli ATCC 25922 g owing in MH and u ine a
di e en pH alues. E o ba s ep esen s anda d e o s o he means o e-
sul s om a leas ou eplica es. MH, Muelle -Hin on; UR, u ine; OD, op ical
densi y.
FIG 3 CIP suscep ibili y es ing o wo ep esen a i e s ains (ATCC 25922
and EC05), using he g adien MIC s ip me hodology. I was obse ed ha pH
has a g ea in luence on CIP ac i i y, wi h a signi ican inc ease in MICs when
pH dec eases. MICs (shown in mic og ams pe millili e ) a e indica ed a he
igh bo om o each condi ion. UR 7, u ine-aga pH 7; UR 6, u ine-aga pH
6; UR 5, u ine-aga pH 5.
Ma ín-Gu ié ez e al.
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wi h oxygen, con e ing all s ains, excep ATCC 25922 and EC01,
o in e media e o esis an . The inc eases due o he absence o
oxygen a pH 6 we e minimal ( om 2- old o 4- old), and he e
we e almos no changes a pH 5.
In summa y, hese esul s demons a e ha he pH o u ine
had a g ea e ec on he ac i i y o CIP agains he E. coli ATCC
25922 LLQR isogenic de i a i es, wi h a pH o 5 capable o con-
e ing all LLQR s ains o in e media e o esis an o CIP ac-
co ding o CLSI b eakpoin s. Fu he mo e, g ow h in u ine a
neu al pH unde anae obic condi ions p oduced an inc ease in
MIC alues su icien o classi ying eigh s ains as esis an which
would ha e been conside ed suscep ible by adi ional MIC de e -
mina ions in MH.
Table 3 shows ha , simila ly o he esul s ob ained wi h u ine,
acidi ica ion o MH had a g ea e ec on CIP ac i i y, wi h in-
c eases o 8- old o 16- old and 32- old o 64- old a pH 6 and 5,
espec i ely. A pH 6, all s ains, excep ATCC 25922 and EC01,
became esis an o in e media e, as de ined by he CLSI clinical
b eakpoin s. On he o he hand, g ow h in he absence o oxygen
had a smalle e ec in MH han in u ine, wi h CIP ac i i y in-
c eases o 2- old o 4- old a pH 7 and almos no e ec a pH 6 and
5. The e o e, g ow h in u ine exace ba ed he e ec o pH and
anae obiosis on he CIP ac i i y wi h espec o g ow h in MH.
MIC de e mina ions unde physiological condi ions. Cu -
en me hods employed o de e mine MICs in clinical mic obiol-
ogy labo a o ies a e based on g ow h in MH b o h o on MH aga
pla es in which he pH is close o neu ali y (s anda d pH is 7.3 ⫾
0.1). The e o e, acco ding o he esul s desc ibed abo e, when
CIP eaches he bladde , he en i onmen al condi ions could al e
i s an imic obial ac i i y. Table 4 shows he old change in MICs
when he s ains a e g own in u ine a di e en pH and oxygen
le els in compa ison o hose ob ained using classical MIC de e -
mina ion me hods (MH b o h, pH 7.3, and ae obiosis). These
esul s indica e ha anae obic g ow h in u ine inc eased he CIP
MIC a pH 7 (2- old o 8- old), pH 6 (16- old o 64- old), and pH
5 (128- old o 512- old).
Su i al a es a he maximum physiological CIP concen a-
ion. The esul s p esen ed abo e sugges ha CIP ea men o
LLQR s ains could be ine icien unde UTI physiological condi-
ions. To es his possibili y, o e nigh cul u es o he di e en
s ains we e g own a 37°C wi hou he an imic obial agen in
bo h MH and u ine a di e en pH alues. Then, bac e ia a a
densi y o abou 5 ⫻10
5
CFU/ml we e inocula ed in o esh MH
o u ine con aining CIP (1,000 ␮g/ml) and g own a 37°C du ing
6 h. The a ionale o his expe imen al design was o mimic he
bes -case scena io, using he addi ion o he highes CIP concen-
a ion e e epo ed wi hin he i s 6 h a e an o al dose o 500
TABLE 5 Cip o loxacin MICs agains clinical s ains in MH b o h and
u ine a di e en pH alues
a
S ain
MIC (␮g/ml)
MH b o h U ine pH 7 U ine pH 6 U ine pH 5
O
2
nO
2
O
2
nO
2
O
2
nO
2
O
2
nO
2
Val09 1 218 8 16 256 256
MAC02 0.125 0.25 0.25 0.5 1 1 32 16
MAC10 0.25 0.5 0.125 0.5 1 43216
MAC11 0.5 20.25 2246464
MAC21a 0.5 20.25 2246464
MAC33 0.125 0.5 0.25 0.25 0.5 1 88
a
In e media e and esis an MIC alues acco ding o he CLSI guideline a e shown in
bold. O
2
, ae obic incuba ion; nO
2
, anae obic incuba ion.
TABLE 2 Cip o loxacin MICs o E. coli s ains in u ine a di e en pH
alues
a
S ain
MIC (␮g/ml)
UR pH 7 UR pH 6 UR pH 5
O
2
nO
2
O
2
nO
2
O
2
nO
2
ATCC 25922 0.015 0.03 0.06 0.25 1 1
EC01 0.03 0.06 0.25 0.5 42
EC02 0.25 1 443232
EC03 0.5 28166432
EC04 0.5 28166464
EC05 1 4 16 64 256 256
EC06 1 1 443232
EC07 0.5 28166464
EC08 4 8 32 64 512 512
EC09 8 16 64 256 512 512
a
In e media e and esis an MIC alues acco ding o he CLSI guideline a e shown in
bold. O
2
, ae obic incuba ion; nO
2
, anae obic incuba ion.
TABLE 3 MICs o cip o loxacin o isogenic E. coli s ains in MH b o h
a di e en pH alues
a
S ain
MIC (␮g/ml) in MH b o h a pH:
765
O
2
nO
2
O
2
nO
2
O
2
nO
2
ATCC 25922 0.004 0.008 0.03 0.06 0.125 0.125
EC01 0.008 0.06 0.125 0.125 0.5 0.25
EC02 0.125 0.25 1 1 44
EC03 0.25 1 2488
EC04 0.5 1 441616
EC05 1 4886432
EC06 0.125 0.25 1 1 44
EC07 0.25 1 441616
EC08 2 4 16 16 64 128
EC09 4 16 32 64 256 128
a
In e media e and esis an MIC alues acco ding o he CLSI guideline a e shown in
bold. O
2
, ae obic incuba ion; nO
2
, anae obic incuba ion.
TABLE 4 Mean old changes in MICs o cip o loxacin in MH b o h and
u ine a di e en alues o pH and anae obiosis
a
S ain
MIC (␮g/ml) in MH b o h and u ine a pH:
765
O
2
nO
2
O
2
nO
2
O
2
nO
2
ATCC 25922 4 8 32 64 128 256
EC01 1 8 8 64 32 256
EC02 4 8 16 32 128 256
EC03 2 8 16 64 32 128
EC04 2 4 16 32 64 128
EC05 1 4 16 64 64 256
EC06 4 8 16 32 128 256
EC07 2 8 16 64 64 256
EC08 2 4 16 32 128 256
EC09 1 4 16 64 32 128
a
O
2
, ae obic incuba ion; nO
2
, anae obic incuba ion.
LLQR E. coli Resis ance in UTI
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mg, al hough he concen a ion ha CIP eaches in he bladde is
ypically lowe (21). No e ha we decided o use such a high con-
cen a ion because we an icipa ed ha he median CIP concen a-
ion du ing he i s hou s o ea men (268 ␮g/ml) (21) would be
in a ange nea o below he MIC o se e al LLQR s ains in u ine
unde condi ions o low pH (see Table 2); hence, he e ec s o CIP
ea men agains hese s ains would mos likely be innocuous.
Figu e 4 shows ha he su i al a es o all s ains, excep ATCC
25922 and EC01, whose su i al a es all below he de ec ion limi
o ou assay, inc eased subs an ially (10- old o 100- old) in u ine
a pH 5 compa ed wi h hose seen wi h MH. A highe pH alues,
no s a is ically signi ican di e ences in su i al we e ound, ex-
cep o EC02.
E ec o u ine, pH, and anae obiosis on CIP ac i i y agains
LLQR clinical isola es. To disca d he possibili y ha he ob-
se ed e ec s we e speci ic o he E. coli ATCC 25922 s ain and i s
de i a i es, we sco ed he MICs o CIP agains six well-cha ac e -
ized LLQR E. coli clinical s ains. As o he ATCC 25922 s ain, he
MIC showed a ma ked inc ease when de e mina ions we e pe -
o med in u ine a di e en pHs and unde anae obic condi ions.
In his case, h ee o six s ains became in e media e, as de ined by
he CLSI b eakpoin s, when MIC de e mina ions we e pe o med
in u ine a pH 7 unde condi ions o anae obiosis. Fu he in-
c eases we e displayed in u ine wi h acidic pHs; all clinical s ains
demons a ed esis ance a pH 5 (Table 5).
DISCUSSION
The e ec i eness o CIP o he ea men o UTI does no depend
on pa hogen suscep ibili y alone. Physicochemical and pha ma-
cological p ope ies o an ibio ics a e impo an ac o s ela ed o
success ul he apy. Because CIP is an ampholy e (14), he pH o
u ine a ec s i s ioniza ion s a us, which is c i ical o i s ac i i y
and cellula accumula ion in bo h bac e ia and bladde cells. Re-
sul s o p e ious s udies suppo he no ion ha low u ine pH
dec eases suscep ibili y o CIP in a numbe o bac e ial pa hogens
(16–19,25).
Ou esul s con i m ha g ow h in u ine a neu al pH sligh ly
inc eases esis ance o CIP in E. coli (26). This inc ease is u he
enhanced in an anae obic en i onmen , such as ha ound in he
bladde . Impo an ly, g ow h in acidic u ine ende s all LLQR
s ains esis an o CIP acco ding o CLSI b eakpoin s. These e-
sul s sugges ha adi ional suscep ibili y es ing in MH b o h
may esul in poo es ima ions o CIP ac i i y in UTI caused by
LLQR s ains.
I has been sugges ed ha he high concen a ions o FQ a -
ained in he u ina y ac a e enough o e adica e bac e ial pa ho-
gens, e en hose which, ollowing CLSI guidelines, would ha e
been conside ed esis an (27). This assump ion is made based on
he empi ical success o ea men s wi h CIP agains esis an
clones and he p edic i e alues o PK/PD modeling o FQ e ec-
i eness, which, gi en he high alues o maximum concen a ion
o d ug in se um (C
max
) and a ea unde he concen a ion- ime
cu e (AUC) eached in he u ina y ac , p edic he clinical e-
sponse o s ains wi h d ug MIC alues as high as ⬃20 ␮g/ml
(28). Acco ding o ou esul s, he applica ion o his c i e ion o
MIC da a o LLQR s ains sugges s ha g ow h in human u ine,
unde physiological condi ions o pH and oxygen concen a ion,
could cause ea men ailu e in mos cases. Simila beha io is
expec ed o o he luo oquinolones; o ins ance, he ac i i y o
le o loxacin, moxi loxacin, and o loxacin is also a ec ed by
g ow h in u ine, pH, and anae obiosis (29,30).
To u he alida e his p edic ion, we de eloped a su i al
assay in which bac e ia we e exposed o an ex emely high con-
cen a ion o CIP (app oxima ely 4- old he median concen a-
ion in u ine) wi hin he i s 6h(
21) in u ine adjus ed o di e en
pH alues and in anae obiosis. Ou esul s show ha , especially
unde acidic condi ions, su i al is enhanced 10- old o 100- old
in mos s ains compa ed o he a e seen wi h MH a neu al pH.
Fu he mo e, mos LLQR s ains showed a ela i ely small (1 o 2
log) dec ease in iabili y, despi e he high CIP concen a ion ha
was used. In con as , he s ain lacking any FQ esis ance de e -
minan (ATCC 25922) was unable o cope wi h he simula ed CIP
ea men , highligh ing he impo ance o LLQR mu a ions in
ea men ailu e. Once again, hese esul s aise issues ega ding
FIG 4 Su i al a es o E. coli isogenic s ains ea ed wi h 1,000 ␮g/ml o CIP a e 6 h unde anae obic condi ions. Su i al a es we e calcula ed by di iding he
numbe o CFU pe millili e a e CIP incuba ion by he alue ob ained a ime ze o. All esul s we e ob ained by a leas h ee independen expe imen s. E o
ba s ep esen s anda d de ia ions. As e isks deno e s a is ically signi ican di e ences (P⬍0.05 [S uden =s es ]) be ween condi ions in compa ison o he
espec i e su i al a e in Muelle -Hin on o e e y s ain.
Ma ín-Gu ié ez e al.
4256 aac.asm.o g July 2016 Volume 60 Numbe 7An imic obial Agen s and Chemo he apy
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he clinical success o CIP agains LLQR s ains unde physiolog-
ical condi ions and should be in e p e ed as cau iona y.
Despi e he gene al belie ha mul iple mu a ions a e equi ed
o gene a e clinically impo an esis ance in E. coli (31), he e-
sul s p esen ed he e show ha UTIs could p oduce ideal condi-
ions o he gene a ion and selec ion o single LLQR mu an s.
Fu he mo e, he high su i al a es ha we ound upon CIP ex-
posu e could be a de e mining ac o in he de elopmen o mul-
is ep esis ance mu an s. FQs ha e been demons a ed o ac as
mu agens and o ha e a s imula o y e ec on ecombina ion e-
quency (32,33), accele a ing bac e ial e olu ion and an ibio ic
esis ance. I is concei able, he e o e, ha in u ine a low pH,
p e alen mu a ions con e ing LLQR such as Gy A S83L could
p o ide he basal le el o esis ance needed o wi hs and he i s
hou s o ea men . This may p o ide a window o he eme gence
o u he esis ance ueled by he mu agenic e ec o CIP. In
ag eemen , i has been epo ed ha pa ien s p e iously ea ed
wi h FQ a e p one o u ina y ac in ec ion caused by CIP- esis-
an E. coli (34). In summa y, u ina y ac condi ions may ep e-
sen an en i onmen in which LLQR mu an s would be selec ed
unde condi ions o ea men wi h CIP (and possibly o he FQs).
This could explain, a leas pa ially, he high p e alence o hese
mu a ions and aises conce ns abou he app op ia eness o CIP
ea men s when LLQR s ains a e de ec ed.
Conce ning he clinical and physiological cha ac e is ics o pa-
ien s wi h UTI caused by E. coli, he e a e se e al ac o s ha
should be aken in o conside a ion when CIP is employed. I has
been epo ed ha he composi ion o he die a ec s he acid-
base balance in he body. A highe p o ein in ake signi ican ly
inc eases enal acid exc e ion by inc easing ammonium ou pu ,
hus acidi ying he u ine (35,36). Mo eo e , he e ec o u ine pH
on CIP ac i i y may become ele an in pa ien s wi h ce ain un-
de lying diseases. Thus, i is known ha pa ien s wi h hype en-
sion a e associa ed wi h a lowe u ina y ci a e le el and a highe
le el o acid exc e ion, esul ing in lowe u ine pH alues (37).
This e ec can also be ound in pa ien s wi h ype 2 diabe es o
me abolic acidosis (38,39). The e a e some pa ien cha ac e is ics
ha a e ela ed o lowe pH alues in u ine, such as olde age o
highe body weigh (38,40,41). Some medica ions, such as loop
diu e ics o mine aloco icoids, can also acidi y u ine (42,43).
The e o e, ea men wi h CIP o UTI in pa ien s wi h ac o s
ela ed o u ine acidi ica ion could esul in he apeu ic ailu e
and inc eased selec i e p essu e, p omo ing he de elopmen o
bac e ial esis ance. Thus, u ina y pH alues could ha e p ac ical
in e es in he managemen o hese pa ien s, whe e he physician
should selec he cou se bes sui ed o he indi idual pa ien : ei he
basi ying he u ine by alkaliniza ion (e.g., by he use o po assium
ci a e o sodium bica bona e) (44,45) wi h he aim o inc easing
he ac i i y o CIP o elec ing o use o he an ibio ics no a ec ed
by low pH alues.
ACKNOWLEDGMENT
We a e g a e ul o Michael McConnell o use ul scien i ic commen s and
English e ision o he manusc ip .
FUNDING INFORMATION
This wo k was suppo ed by he Subdi ección Gene al de Redes y Cen os
de In es igación Coope a i a, Minis e io de Economía y Compe i i idad,
Ins i u o de Salud Ca los III, Spanish Ne wo k o Resea ch in In ec ious
Diseases (g an RD12/0015/0012; REIPI RD12/0015/0012), co inanced by
Eu opean De elopmen Regional Fund ⬙A way o achie e Eu ope⬙ERDF,
and Fondo de In es igación Sani a ia (g an PI13/00063).
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