ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,
0066-4804/98/$04.0010July 1998, p. 1850–1852 Vol. 42, No. 7
Copy igh © 1998, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
Ene gy-Dependen Accumula ion o Fluo oquinolones in
Quinolone-Resis an Klebsiella pneumoniae S ains
LUIS MARTI
´NEZ-MARTI
´NEZ,
1
* ISABEL GARCI
´A,
1
SOFI
´A BALLESTA,
1
VICENTE JAVIER BENEDI
´,
2
SANTIAGO HERNA
´NDEZ-ALLE
´S,
2
AND ALVARO PASCUAL
1
Depa men o Mic obiology, School o Medicine, Uni e si y o Se ille, Se ille,
1
and A ea o Mic obiology, Depa men
o En i onmen al Biology and IMEDEA (CSIC-UIB), Uni e si y o Balea ic Island, Palma de Mallo ca,
2
Spain
Recei ed 29 Oc obe 1997/Re u ned o modi ica ion 8 Janua y 1998/Accep ed 9 Ma ch 1998
The in acellula accumula ion o no loxacin and pe loxacin in Klebsiella pneumoniae was e alua ed. The
oles o lipopolysaccha ide, capsule, and ou e memb ane p o eins we e no impo an o he in abac e ial
accumula ion o luo oquinolones in isogenic s ains wi h known ou e memb ane al e a ions. In luo oquin-
olone- esis an clinical isola es also exp essing Gy A al e a ions, an ac i e e lux leading o dec eased accu-
mula ion o he d ugs enhanced hei esis ance o hese agen s.
Se e al mechanisms a e in ol ed in he esis ance o luo-
oquinolones (FQ) by g am-nega i e bac e ia. In Esche ichia
coli, poin mu a ions in he quinolone esis ance-de e mining
egion (QRDR) o gy A (21) a e he mos impo an cause o
esis ance. Mu a ions in gy B a e uncommon in clinical isola es
(22), while mu a ions in pa C (19) o (much less equen ly) in
pa E (4) con ibu e o inc eased le els o esis ance. The gy A
gene o Klebsiella pneumoniae has been cloned and sequenced
and is simila (abou 85% a he nucleo ide le el and abou
90% a he amino acid le el) o i s E. coli homolog (5). The
ole o gy A mu a ions in FQ- esis an K. pneumoniae has been
demons a ed, hese mu a ions being analogous o hose p e-
iously obse ed in E. coli (6, 7). Dec eased ac i i y o FQ
agains E. coli has also been ela ed o a educed in acellula
d ug accumula ion due o lipopolysaccha ide o po in al e -
a ions impai ing up ake o because o enhanced e lux (10, 11).
C oss- esis ance be ween FQ and be a-lac ams in K. pneu-
moniae mu an s has been ela ed o ou e memb ane p o ein
al e a ions (12, 17). Exp ession o he amA locus om K.
pneumoniae in E. coli con e s esis ance o no loxacin, de e -
mines he ac i e e lux o e acycline and chlo amphenicol,
and causes almos comple e loss o OmpF (8). I has been
epo ed ha he high hyd ophobici y o FQ is gene ally asso-
cia ed wi h dec eased accumula ion in bo h E. coli and Pseudo-
monas ae uginosa (14). The lipopolysaccha ide o he ou e
lea le o he ou e memb ane in some bac e ia blocks he
access o he d ug o he mic oo ganism. O he epo s indi-
ca e ha hyd ophylic compounds may c oss he ou e mem-
b ane h ough po ins (11).
The objec i es o his s udy we e o e alua e he accumula-
ion o wo FQ wi h di e en hyd ophobici ies (no loxacin
[NFX] and pe loxacin [PFX]) in K. pneumoniae by s udying
FQ-suscep ible isogenic s ains wi h known ou e memb ane
al e a ions and FQ- esis an clinical isola es.
(This wo k was p esen ed in pa a he 36 h In e science
Con e ence on An imic obial Agen s and Chemo he apy, New
O leans, La., 15 o 18 Sep embe 1996 [13].)
An en i onmen al isola e, K. pneumoniae C3 (18), and he
de i ed mu an s KT717, KT793, and KT5003 (1–3, 9) we e
used. K. pneumoniae LB2 and LB4 (12) and K. pneumoniae
CSUB10S and CSUB10R (kindly p o ided by J. Lin˜a es [Ba -
celona, Spain]) a e clinical isola es; LB and CSUB isola es a e
epidemiologically un ela ed, as de e mined by pulse- ield gel
elec opho esis (da a no shown). The ou e memb ane cha -
ac e is ics o all s ains a e shown in Table 1. MICs o NFX
(Sigma, Mad id, Spain) and PFX (Rhoˆne-Poulenc, An ony,
F ance) we e de e mined by mic odilu ion, acco ding o Na-
ional Commi ee o Clinical Labo a o y S anda ds NCCLS
guidelines (16). To assess FQ accumula ion in K. pneumoniae
C3, bac e ial cells we e incuba ed wi h 2, 5, 10, o 50 mg o each
FQ pe ml a 37°C o di e en pe iods o ime. Fo he o he
s ains a concen a ion o 10 mg/ml was used. The cells we e
sepa a ed om he ex acellula solu ion by cen i uga ion
h ough a silicon oil ba ie (1.029 g/cm
3
). The en i e cell pelle
was placed in 2 ml o 0.1 M glycine-HCl bu e (pH 3.0),
o exed, and cen i uged o 5 min a 12,800 3g. The amoun
o FQ in he supe na an was de e mined by spec o luo om-
e y. Bac e ially associa ed FQ was exp essed as nanog ams o
FQ pe millig am [d y weigh ] o cells. The e ec s o he
ene gy inhibi o s ca bonyl cyanide m-chlo ophenylhyd azone
(CCCP) (0.1 mM; Sigma) and 2,4-dini ophenol (Sigma) we e
also e alua ed. Da a a e exp essed as means 6s anda d de i-
a ions. Di e ences among g oups we e compa ed by analysis
o a iance, which was used o assess s a is ical signi icance a
P alues o #0.05.
To analyze al e a ions in he Gy A subuni o DNA gy ase
and he Pa C subuni o opoisome ase IV, he QRDR o gy A
and he analogous egion o pa C we e sequenced wi h p e i-
ously desc ibed p ime s (6). MICs o PFX we e iden ical o K.
pneumoniae C3 and he de i ed mu an s. A wo-dilu ion s ep
inc ease in he MIC o NFX o mu an KT5003 was obse ed
in compa ison wi h he pa en al s ain K. pneumoniae C3.
MICs o bo h quinolones o K. pneumoniae LB4 and
CSUB10R we e highe han o K. pneumoniae LB2 and
CSUB10S, espec i ely (Table 1). The in acellula accumula-
ion o NFX and PFX in K. pneumoniae C3 was apid (Fig. 1)
and no sa u able a ex acellula concen a ions anging om
2 o50mg/ml. Nonsigni ican di e ences in he accumula ion
o FQ we e obse ed in isogenic s ains KT717, KT793, and
KT5003. These esul s indica e ha he oles o cell wall com-
ponen s in he in abac e ial accumula ion o FQ wi h di e en
g ades o hyd ophobici y a e small o nonexis en . The high
suscep ibili ies o he nonclinical s ains, K. pneumoniae C3
and he mu an s, o FQ a e p obably ela ed o he absence o
speci ic mechanisms o esis ance o FQ. The accumula ions o
* Co esponding au ho . Mailing add ess: Depa men o Mic obi-
ology, School o Medicine, Apdo 914, 41080 Se ille, Spain. Phone:
34-5-4557448. Fax: 34-5-4377413. E-mail: [email p o ec ed].
1850
on Augus 1, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om on Augus 1, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om on Augus 1, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om
bo h FQ we e signi ican ly lowe in he clinical K. pneumoniae
isola es LB4 and CSUB10R. P eincuba ion o s ains in he
p esence o CCCP did no modi y he accumula ion o NFX in
K. pneumoniae C3, bu i signi ican ly inc eased he accumula-
ion o NFX in bo h K. pneumoniae LB4 and CSUB10R (Table
1). Simila esul s we e ob ained wi h 2,4-dini ophenol. This
sugges s ha , a leas in pa , he educed accumula ion ob-
se ed in bo h clinical isola es could be caused by an ene gy-
dependen mechanism ha pumps FQ ou o he bac e ial cell.
Deguchi e al. ha e also desc ibed an ac i e e lux sys em o
NFX in FQ- esis an K. pneumoniae (7).
Di e en au ho s ha e ound disc epan amino acids a po-
si ion 83 o he Gy A p o ein in FQ-suscep ible K. pneumoniae:
Th (accession no. X16817 [5] and s ain C3 [ his wo k]) and
Se (ATCC 13833 [6]). In ei he case, a change in his posi ion
was obse ed in FQ- esis an s ains LB2-LB4 (Ty ) and
CSUB10S-CSUB10R (Phe). Bo h pai s o s ains also ha e
changes a posi ion 112 (Val o Ile). These changes should
con ibu e o he obse ed esis ance o FQ, as simila changes
ha e been obse ed in o he g am-nega i e o ganisms (6, 20,
21). No changes in he QRDRs o he pa C genes o he ou
clinical isola es s udied we e obse ed, in compa ison wi h he
sequence o K. pneumoniae C3. New s udies a e in p og ess o
elucida e he oles o po in de iciency, e lux, and opoisome -
ase modi ica ion in he esis ance o K. pneumoniae o FQ.
Deguchi e al. ha e sugges ed ha he sys em esponsible o
ac i e e lux o NFX may con ibu e o inc eased esis ance o
be a-lac ams, chlo amphenicol, and e acycline. The ela i e
impo ance o he e lux sys ems o K. pneumoniae LB4 and
CSUB10R o an imic obial agen s o he han FQ is no known.
Bo h s ains p oduce b oad- and ex ended-spec um be a-lac-
amases. Loss o po ins may explain he inc eased esis ance o
be a-lac ams in compa ison wi h hei espec i e pa en al
s ains LB2 and CSUB10S (12), bu his does no discoun he
impo ance o he e lux mechanism as a con ibu o o be a-
lac am esis ance in K. pneumoniae, as has p e iously been
shown o P. ae uginosa (15). As soon as he gene ics o he
e lux mechanisms in hese wo s ains a e de e mined, hei
impo ance as ac o s con ibu ing o esis ance o an imic o-
bial agen s o he han FQ can be de ined.
This wo k was suppo ed by g an s om Comisio´n In e minis e ial
de Ciencia y Tecnologı´a (CICYT) o he Spanish go e nmen (PB96-
0197). S.H.-A. was suppo ed by a p edoc o al ellowship om CICYT
(FP94-41497233).
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FIG. 1. Time-dependen accumula ion o NFX (p) and PFX (Œ)inK. pneu-
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TABLE 1. Suscep ibili ies o and accumula ion o NFX in ou isogenic and ou clinical K. pneumoniae s ains
S ain P esence in s ain o
a
:NFX
MIC
(mg/ml)
Accumula ion o NFX
(ng/mg [d y weigh ])
b
O-Ag K-Ag OmpK34 OmpK35 OmpK36 Con ol CCCP
Isogenic s ains
C3 O1 K66 1110.015 366 665 346 646
KT717 O1 21110.03 330 667 ND
KT793 221110.03 299 668 ND
KT5003 221220.06 314 654 ND
Clinical s ains
LB2 ND ND 1122 286 624 385 665
LB4 ND ND 1224 116 621 263 638
CSUB10S ND ND 1124 313 66ND
CSUB10R ND ND 12216 66 620 76 69
a
OmpK34, OmpK35, and OmpK36 a e he K. pneumoniae homologs o E. coli OmpA and po ins OmpF and OmpC, espec i ely. O-Ag, O an igen; K-Ag, K an igen;
ND, no de e mined.
b
Da a a e means 6s anda d de ia ions o i e expe imen s.
VOL. 42, 1998 NOTES 1851
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1852 NOTES ANTIMICROB.AGENTS CHEMOTHER.
on Augus 1, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om
ERRATUM
Ene gy-Dependen Accumula ion o Fluo oquinolones in
Quinolone-Resis an Klebsiella pneumoniae S ains
LUIS MARTI
´NEZ-MARTI
´NEZ, ISABEL GARCI
´A, SOFI
´A BALLESTA, VICENTE JAVIER BENEDI
´,
SANTIAGO HERNA
´NDEZ-ALLE
´S, AND ALVARO PASCUAL
Depa men o Mic obiology, School o Medicine, Uni e si y o Se ille, Se ille, and A ea o Mic obiology, Depa men o
En i onmen al Biology and IMEDEA (CSIC-UIB), Uni e si y o Balea ic Island, Palma de Mallo ca, Spain
Volume 42, no. 7, p. 1850–1852, 1998. Page 1851, Table 1: “76 69” o accumula ion o NFX a e CCCP ea men o s ain
CSUB10R should ead “161 618.”
2772