ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oc . 2003, p. 3332–3335 Vol. 47, No. 10
0066-4804/03/$08.00⫹0 DOI: 10.1128/AAC.47.10.3332–3335.2003
Copy igh © 2003, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
Role o Klebsiella pneumoniae OmpK35 Po in in
An imic obial Resis ance
An onio Dome´nech-Sa´nchez,
1,2
Luis Ma ínez-Ma ínez,
3,4
* San iago He na´ndez-Alle´s,
1,2
Ma ía del Ca men Conejo,
3
A
´l a o Pascual,
3,4
Juan M. Toma´s,
5
Sebas ia´n Albe í,
1,2,6
and Vicen e Ja ie Benedí
1,2
†
Labo a o y o Mic obiology, Depa men o Biology, Uni e si y o he Balea ic Island,
1
IMEDEA (CSIC-UIB),
2
and Resea ch Uni ,
Uni e si y Hospi al Son Du e a,
6
Palma de Mallo ca, Depa men o Mic obiology, School o Medicine, Uni e si y o Se ille,
3
and Uni e si y Hospi al V. Maca ena,
4
Se ille, and Depa men o Mic obiology,
Uni e si y o Ba celona,
5
Ba celona, Spain
Recei ed 25 Feb ua y 2003/Re u ned o modi ica ion 21 Ap il 2003/Accep ed 12 July 2003
OmpK35 om Klebsiella pneumoniae is he homologue o Esche ichia coli OmpF po in. Exp ession o
OmpK35 in K.pneumoniae s ain CSUB10R (lacking bo h OmpK35 and OmpK36) dec eased he MICs o
cephalospo ins and me openem >128- old and dec eased he MICs o imipenem, cip o loxacin, and chlo am-
phenicol >8- old. MIC educ ions by OmpK35 we e 4 imes (ce epime), 8 imes (ce o e an, ce o axime, and
ce pi ome), o 128 imes (ce azidime) highe han hose caused by OmpK36, bu he MICs we e simila o 1
dilu ion lowe o o he e alua ed agen s.
Klebsiella pneumoniae p oduces wo majo po ins (OmpK35
and OmpK36) and he quiescen po in OmpK37. De ails on
OmpK36 and OmpK37 ha e been p e iously epo ed (1, 6,
10). Mos clinical isola es o K.pneumoniae lacking ex ended-
spec um ␤-lac amase (ESBL) exp ess bo h OmpK35 and
OmpK36 po ins, while mos ESBL-exp essing K.pneumoniae
clinical isola es p oduce only OmpK36 (9). Un il now, he ew
clinical isola es lacking bo h OmpK35 and OmpK36 ha e been
ESBL-p oducing s ains (14).
Loss o OmpK36 is ela ed o ce oxi in esis ance and in-
c eased esis ance o oxyimino- and zwi e ionic cephalospo-
ins in s ains p oducing ESBL and o ca bapenem esis ance
in s ains p oducing plasmid-media ed AmpC- ype ␤-lac a-
mase (3, 4, 13, 15). Loss o OmpK36 also esul s in a mode a e
inc ease in luo oquinolone esis ance in s ains wi h al e ed
opoisome ases and/o ac i e e lux o quinolones.
P elimina y esul s (6) indica e ha OmpK35 allows e icien
pene a ion o ce oxi in, ce o axime, and ca bapenems, bu
he e has been some con o e sy on he ole o his po in in
cephalospo in pene a ion in K.pneumoniae (18). De ailed
s udies on he impo ance o OmpK35 in an imic obial esis-
ance a e lacking.
In o de o in es iga e he ole o OmpK35 in an imic obial
esis ance, we cloned he ompK35 gene. Fo his pu pose,
genomic DNA om K.pneumoniae s ain KT755 (19) was
diges ed wi h Sau3A. F agmen s we e liga ed o cosmid
pLA2917 (2) and used o ans o m Esche ichia coli DH5␣
(17). Recombinan s we e sc eened o ompK35 by PCR using
p ime s U681 (5⬘-CGG TTA CGG CCA GTG GGA ATA-3⬘)
and L1316 (5⬘-GAC GCA GAC CGA AAT CGA ACT-3⬘),
speci ic o en e obac e ial po ins and loca ed 215 and 850 bp
downs eam o he ompK36 s a codon, espec i ely (6). The
sizes o PCR-ampli ied p oduc s om ompF- ype genes a e
di e en om hose o o he po in genes (da a no shown).
One clone ca ying a plasmid, designa ed pSHA15, p oduced
an amplicon o he desi ed size. Ou e memb ane p o eins
(OMPs) we e isola ed as desc ibed p e iously (1, 13). Wes e n
blo analysis o OMPs was pe o med on Immobilon P il e s
(Millipo e, Bed o d, Mass.) using an i-OmpK35 an ibody (di-
lu ed 1:1,000) and alkaline phospha ase-labeled goa an i- ab-
bi immunoglobulin G (dilu ed 1:5,000) (13).
OMP p o iles o E.coli DH5␣ca ying pSHA15 exhibi ed a
band wi h he same mobili y as ha o OmpK35 exp essed by
K.pneumoniae KT755 (da a no shown). The exp ession o
OmpK35 was down egula ed in a high-osmola i y cul u e me-
dium, as occu s wi h he OmpF-like po ins (9), and OmpK35
eac ed wi h an i-OmpK35 in immunoblo expe imen s (da a
no shown). The OmpK35 p o ein exp essed by he E.coli
clone was ex ac ed by po in ex ac ion me hods based on he
ypsin esis ance o po ins and hei s ong nonco alen asso-
cia ion wi h he pep idoglycan, and i also e ained i s hea
modi iabili y.
The ompK35 gene o K.pneumoniae KT755 was sequenced
(EMBL da abase accession no. AJ011501). The amino acid
sequence o OmpK35 was aligned wi h he sequences o o he
en e obac e ial po ins (5, 7) (Fig. 1) on he basis o he con-
se a ion o he ␤-s ands and some key esidues ha a e well
conse ed in po ins: Lys16, A g38, Glu58, A g75, Asp106,
Glu110, and A g126. OmpK35 is an OmpF homologue and
p esen s a ypical 16 ␤-s and s uc u e, wi h eigh sho
pe iplasmic u ns and eigh ex acellula loops o a iable
leng hs. OmpK35 loop 3, which de ines he size o he ans-
memb ane po e in o he po ins, ex ends inside he ba el and
is he mos conse ed loop and con ains only one mo e esidue
han OmpF and OmpC om E.coli and OmpK36 om K.
pneumoniae.
Fo suscep ibili y es ing expe imen s, he ompK35 gene was
cloned in pWSK30 (20) and endowed wi h a kanamycin esis-
* Co esponding au ho . Mailing add ess: Depa men o Mic obi-
ology, School o Medicine, Uni e si y o Se ille, Apdo. 914, 41080
Se ille, Spain. Phone: 34-955008287. Fax: 34-954377413. E-mail:
[email p o ec ed].
† Deceased.
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ance casse e om pCS12 (8) o gi e pSHA16K. The
pSHA16K plasmid was cloned in o he po in-de icien K.pneu-
moniae CSUB10R clinical isola e and in o he clonally ela ed
(3) isola e K.pneumoniae CSUB10S (exp essing OmpK36).
The exp ession o OmpK35 does no in e e e wi h ␤-lac a-
mase ac i i y in CSUB10R, as de e mined spec opho ome i-
cally wi h c ude supe na an s om sonica ed cells as he en-
zyme sou ce (15) (da a no shown).
MICs o ce oxi in (Sigma, Mad id, Spain), ce o e an (Zen-
eca, Mad id, Spain), ce azidime (Glaxo, Ba celona, Spain),
ce o axime (Sigma), ce epime (B is ol-Mye s Squibb, Mad id,
Spain), ce pi ome (Hoechs Ma ion-Roussel, Romain ille,
F ance), imipenem (Me ck Sha p & Dohme, Mad id, Spain),
me openem (Zeneca), cip o loxacin (Sigma), clina loxacin
(Pa ke-Da is, Ann A bo , Mich.), amikacin (Sigma), gen ami-
cin (Sigma), e acycline (Sigma), and chlo amphenicol (Sig-
ma) o s ains CSUB10S and CSUB10R and OmpK35-ex-
p essing ansconjugan s de i ed om hese wo s ains (Table
1) 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) guide-
lines (16). An imic obial agen MICs o K.pneumoniae
CSUB10R con aining plasmids pKSK ( ec o ) and pSHA25K
(OmpK36) we e also de e mined o compa ison.
Exp ession o OmpK35 in K.pneumoniae CSUB10R e-
duced (Fig. 2) he MICs o all agen s es ed wo o mo e imes.
The highes educ ions (ⱖ128- old) we e obse ed o cepha-
mycins, oxyimino-cephalospo ins, zwi e ionic cephalospo ins,
and me openem. Signi ican educ ions (ⱖ8- old) we e also
no ed o imipenem, cip o loxacin, and chlo amphenicol. The
lowes educ ions we e ob ained o hose agen s o which K.
FIG. 1. Compa ison by alignmen o he deduced OmpK35 sequence om K.pneumoniae wi h he sequences o OmpF and OmpC om E.coli
and OmpK36 om K.pneumoniae a ailable in GenBank, EMBL, and DDBJ. Seconda y s uc u e mo i s a e desc ibed on he basis o he c ys al
s uc u e o OmpK36. The numbe ing is based on he ma u e OmpK36. Conse ed amino acids (shaded and boxed) and gaps in oduced o
maximize alignmen (hyphens) a e indica ed.
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pneumoniae CSUB10R was al eady suscep ible: clina loxacin,
e acycline, amikacin, and gen amicin. Exp ession o OmpK36
in CSUB10R also dec eased he MICs o all an imic obial
agen s es ed, excep clina loxacin, o alues simila o he
MICs o he ela ed clinical isola e CSUB10S. MIC educ-
ions caused by OmpK35 exp ession we e 4 imes (ce epime),
8 imes (ce o e an, ce o axime, and ce pi ome), o 128 imes
(ce azidime) highe han hose caused by OmpK36 exp es-
sion. These esul s, howe e , do no necessa ily mean ha
OmpK35 should be conside ed speci ic o hese agen s, as
exp ession o OmpK36 also signi ican ly educed hei MICs.
MIC educ ions caused by OmpK35 we e he same (me o-
penem, amikacin, gen amicin, and e acycline) o 1 dilu ion
s ep lowe (ce oxi in, imipenem, cip o loxacin, clina loxacin,
and chlo amphenicol) han hose caused by OmpK36.
Exp ession o OmpK35 in K.pneumoniae CSUB10S, leading
o he simul aneous exp ession o he wo majo po ins o K.
pneumoniae (Fig. 2), esul ed in he MICs o he e alua ed
agen s being he same o one dilu ion s ep highe han hose
agains he ans o man exp essing only OmpK35.
Exp ession o bo h OmpK35 and o a lesse ex en OmpK36
dec eases he MICs o cip o loxacin o K.pneumoniae
CSUB10R (which con ains a Se 83Phe change in he A subuni
o DNA gy ase and exp esses ac i e e lux o luo oquinolones
[12]), indica ing ha bo h po ins allow pene a ion o his d ug.
Po in exp ession was minimally ele an o he ac i i y o
clina loxacin, a luo oquinolone much mo e ac i e agains
CSUB10R han cip o loxacin. OmpK35 and OmpK36 exp es-
sion also dec eased he MICs o e acycline and chlo amphen-
icol. These da a suppo he gene al ole o bo h po ins as
hyd ophilic po es. MICs o aminoglycosides did no signi i-
can ly change a e po in exp ession, p esumably because o
he pene a ion o hese agen s by po in-independen pa h-
ways.
Mos ESBL-p oducing K.pneumoniae s ains lack OmpK35
(11). Loss o his po in may be one o he ac o s con ibu ing
o an imic obial esis ance in ESBL-p oducing K.pneumoniae
and may a o he selec ion o addi ional mechanisms o e-
sis ance, including loss o OmpK36 and/o ac i e e lux (14).
OmpK35 is no no mally exp essed in high-osmola i y me-
dia, which may esul in ep ession o i s exp ession in K.
pneumoniae in i o. This may be o he apeu ic impo ance
because o he limi ed en ance o ce ain an imic obial agen s
in K.pneumoniae. New s udies on po in exp ession in K.pneu-
moniae g own in i o a e needed.
This wo k was suppo ed in pa by g an s om he Fondo de
In es igaciones Sani a ias o he Minis e io de Sanidad y Consumo o
Spain o L.M.-M. and V.J.B and Plan Nacional de I⫹D (Minis e io de
Ciencia y Tecnología) o J.M T. A.D.-S. was suppo ed by a p edoc-
o al ellowship om he Minis e io de Educacio´n y Cul u a o he
Spanish go e nmen .
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TABLE 1. MICs o an imic obial agen s agains K. pneumoniae s ains wi h di e en pa e ns o po in exp ession
S ain Po in(s) MIC (␮g/ml)
a
FOX CTT CAZ CTX FEP PIR IPM MEM CIP CLX AMK GEN TET CHL
CSUB10S OmpK36 2 0.06 256 8 2 4 0.125 0.03 0.5 0.06 1 4 2 16
CSUB10R None 128 32 ⬎512 512 512 512 1 4 4 0.125 1 8 4 64
CSUB10R/pSHA16K OmpK35 1 0.03 2 0.5 0.125 0.5 0.125 0.03 0.25 0.06 0.5 4 1 8
CSUB10R/pSHA25K OmpK36 2 0.125 256 4 0.5 4 0.25 0.03 0.5 0.125 0.5 4 1 16
CSUB10S/pSHA16K OmpK35 and OmpK36 1 0.03 4 1 0.125 0.5 0.125 0.03 0.5 0.06 1 4 1 8
CSUB10R/pKSK None 128 16 ⬎512 256 128 256 1 4 2 0.125 1 8 2 64
a
Abb e ia ions: FOX, ce oxi in; CTT, ce o e an; CAZ, ce azidime; CTX, ce o axime; FEP, ce epime; PIR, ce pi ome; IPM, imipenem; MEM, me openem; CIP;
cip o loxacin; CLX, clina loxacin; AMK, amikacin; GEN, gen amicin; TET, e acycline; CHL, chlo amphenicol.
3334 NOTES ANTIMICROB.AGENTS CHEMOTHER.
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