ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,
0066-4804/99/$04.0010
Ap . 1999, p. 937–939 Vol. 43, No. 4
Copy igh © 1999, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
NOTES
De elopmen o Resis ance du ing An imic obial The apy Caused
by Inse ion Sequence In e up ion o Po in Genes
SANTIAGO HERNA
´NDEZ-ALLE
´S,
1
VICENTE J. BENEDI
´,
1
LUIS MARTI
´NEZ-MARTI
´NEZ,
2
A
´LVARO PASCUAL,
2
ALICIA AGUILAR,
3
JUAN M. TOMA
´S,
3
AND SEBASTIA
´N ALBERTI
´
1
*
A
´ ea de Mic obiologı´a, Depa amen o de Biologı´a, Uni e sidad de las Islas Balea es and IMEDEA (CSIC-UIB),
Palma de Mallo ca,
1
Depa amen o de Mic obiologı´a, Uni e sidad de Se illa, Se ille,
2
and
Depa amen o de Mic obiologı´a, Uni e sidad de Ba celona, Ba celona,
3
Spain
Recei ed 10 Augus 1998/Re u ned o modi ica ion 23 No embe 1998/Accep ed 22 Janua y 1999
We ha e demons a ed by using an in i o app oach ha in e up ion o he OmpK36 po in gene by inse -
ion sequences (ISs) is a common ype o mu a ion ha causes loss o po in exp ession and inc eased esis-
ance o ce oxi in in Klebsiella pneumoniae. This mechanism also ope a es in i o: o 13 po in-de icien ce oxi in-
esis an clinical isola es o K. pneumoniae, 4 p esen ed ISs in hei ompK36 gene.
Klebsiella pneumoniae is a majo nosocomial pa hogen, and
success ul chemo he apy is c i ical o he ea men o in ec-
ions caused by his mic oo ganism. An ibio ic- esis an s ains
eme ge wi h a iable equency, pa icula ly in he hospi al
en i onmen . K. pneumoniae esis ance o expanded-spec um
cephalospo ins caused by plasmid-bo ne ex ended-spec um
b-lac amases is a well-documen ed example and has inc eased
since he 1980s (11, 14, 17). Addi ionally, e ec s in po in ex-
p ession con ibu e o inc ease he esis ance le els p o ided
by he mechanism desc ibed abo e (12).
We ha e desc ibed po ins OmpK36 and OmpK35 (2, 7), he
K. pneumoniae homologues o Esche ichia coli po ins OmpC
and OmpF, espec i ely. Clinical isola es o his species exp ess
ei he OmpK36 o OmpK35 po ins o bo h (6). Recen ly, we
epo ed ha loss o po ins OmpK36 and OmpK35 in ex-
ended-spec um b-lac amase-p oducing clinical isola es o
K. pneumoniae caused inc eased esis ance o ce oxi in and
expanded-spec um cephalospo ins (3, 9). The ela ionship
be ween po in loss and an imic obial esis ance has also been
demons a ed in o he bac e ial species (1, 10). Howe e , he
mechanisms leading o po in de iciency and subsequen an i-
mic obial esis ance a e la gely unknown. The pu pose o he
p esen s udy was o iden i y he mechanisms causing po in
de iciency in clinical isola es o K. pneumoniae. Fo his pu -
pose, we i s used an in i o app oach. We selec ed ce oxi in-
esis an mu an s de i ed om s ain LB4(pSHA2) (9). S ain
LB4 is a po in-de icien , ce oxi in- esis an (MIC, 128 mg/ml)
clinical isola e ha e e ed o ce oxi in sensi i i y (MIC, 4
mg/ml) a e he plasmid pSHA2 ca ying he OmpK36 po in
gene was cloned. Ce oxi in- esis an mu an s we e ob ained by
pla ing LB4(pSHA2) on inc easing concen a ions o ce oxi in.
Mu an s wi h inc eased esis ance o ce oxi in (MIC, .48
mg/ml), de e mined acco ding o he Na ional Commi ee o
Clinical Labo a o y S anda ds ecommenda ions, we e selec -
ed and u he cha ac e ized. The ou e memb ane p o eins
(OMPs) om hese mu an s we e isola ed om bac e ial en-
elopes as sodium lau yl sa cosina e-insoluble ma e ial (5) and
analyzed by sodium dodecyl sul a e-polyac ylamide gel elec-
opho esis. We ound ha exp ession o po in OmpK36 was
comple ely abolished in hose mu an s. In o de o s udy pos-
sible mu a ions a ec ing he ompK36 gene, we isola ed he
pSHA2 plasmid om he ce oxi in- esis an mu an s. Plasmids
we e analyzed by Sou he n blo ing (4) wi h an ompK36 p obe
ob ained by PCR ampli ica ion o plasmid pSHA2 wi h p ime s
U228 (59-GGTAAAAAAAACCGGATGCG-39) and L1730
(59-CGTGCTTAGAACTGGTAAACC-39), which anneal o
sequences 427 bp ups eam and 1,097 bp downs eam o he
ompK36 s a codon (accession no. Z33506), espec i ely.
These analyses e ealed ha di e en e en s occu ed in he
ompK36 gene con ained in pSHA2: poin mu a ions o small
dele ions, dele ions o he en i e gene, and inse ions. These
mu an plasmids de i ed om pSHA2 we e designa ed pSHA
ollowed by an a bi a y numbe (Table 1). As shown in Table
1, he mos equen ypes o changes de ec ed in he mu an
* Co esponding au ho . Mailing add ess: UIB-Mic obiologı´a, Ca -
e e a de Valldemosa Km. 7.5, 07071-Palma de Mallo ca, Spain. Phone:
34-971-173335. Fax: 34-971-173184. E-mail: [email p o ec ed].
TABLE 1. Analysis o andomly selec ed mu an plasmids
con e ing inc eased esis ance o ce oxi in (MIC,
.48 mg/ml) de i ed om s ain LB4(pSHA2)
Plasmid Type o mu a ion IS posi ion
a
pSHA119 Poin mu a ion o small dele ion
pSHA124 Poin mu a ion o small dele ion
pSHA125 Poin mu a ion o small dele ion
pSHA128 Inse ion o IS187
pSHA123 Inse ion o IS903 8
pSHA103 Inse ion o IS524
pSHA100 Inse ion o IS26 14
pSHA146 Inse ion o IS195
pSHA149 Inse ion o IS57
pSHA151 Inse ion o IS529
pSHA101 Inse ion o IS26 77
pSHA154 Inse ion o IS903 28
pSHA157 Dele ion
pSHA104 Dele ion
a
Values indica e he posi ions (in base pai s) o he ISs wi h espec o he
ompK36 s a codon.
937
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plasmids we e inse ions. Among 14 andomly selec ed mu-
an s, 9 (64.3%) p esen ed an inse ion. De ailed cha ac e iza-
ion o he na u e and posi ion o he inse ed DNA was ca -
ied ou by sequencing he mu an plasmids. DNA sequencing
was pe o med wi h p ime s U681 (59-CGGTTACGGCCAG
TGGGAATA-39) and L1316 (59-GACGCAGACCGAAATC
GAACT-39), which ep esen DNA sequences 230 and 345 bp
downs eam o he ompK36 s a codon, and p ime s U228 and
L1730. We de ec ed ou di e en inse ion sequences (ISs;
IS26,IS5,IS903, and IS1) loca ed in di e en si es o he
ompK36 gene. Fu he mo e, wi h p obes speci ic o he iden-
i ied ISs (ob ained by elu ion o an IS in e nal es ic ion
agmen om he co esponding mu an plasmid), we e i ied
by Sou he n blo analysis ha he ISs we e p esen in many
copies in he ch omosome o he o iginal s ain LB4 (da a no
shown).
Since esis ance o ce oxi in in labo a o y-de i ed mu an s
was dependen on mu a ions o he OmpK36 po in gene, p e-
dominan ly inse ion o ISs, we in es iga ed whe he his ype
o mechanism also occu ed in i o. Fo his pu pose, we am-
pli ied he en i e ompK36 gene om wo pai s o s ains iso-
la ed om wo pa ien s be o e and a e an imic obial he apy:
s ains LB1 and LB4 (9) and s ains CMD1 and CMD2 (19).
LB4 and CMD2 a e po in-de icien ce oxi in- esis an clinical
isola es de i ed om he po in-su icien , ce oxi in-sensi i e
s ains LB1 and CMD1, espec i ely. Addi ionally, we cha ac-
e ized he OmpK36 po in gene in 11 addi ional po in-de i-
cien K. pneumoniae clinical isola es: s ains HUSR2/94, C1,
LB66, LB68, LB73, CSUB2, CSUB8R, CSUB9R, CSUB10R,
CSUB11, and CSUB12.
Analysis o he amplicons wi h he ompK36 gene p obe de-
ec ed inse ions, seen as inc eases in he size o ompK36,in
some o hese clinical isola es (Fig. 1A). In addi ion, we ob-
se ed in s ain HUSR2/94 a dele ion on he OmpK36 po in
gene (Fig. 1A, lane 2). Fu he cha ac e iza ion o he abo e-
desc ibed inse ions was ob ained by sequencing he PCR am-
plicons. We iden i ied he p esence o IS102 in s ain CMD2,
and we iden i ied inse ions wi h a high deg ee o sequence
iden i y o IS5in he amplicons om s ains LB73, CSUB2,
and CSUB11. These ISs we e loca ed in di e en posi ions
wi hin he ompK36 coding egion (Fig. 1B). These esul s dem-
ons a e ha po in loss due o inse ions o ISs wi hin he po in
genes is a mechanism ha ope a es in i o in pa ien s unde -
going an ibio ic ea men .
I has been sugges ed ha a low le el o andom ansposi-
ion migh help cells o adap o en i onmen al changes and o
inc ease he su i al a e (18), and i is also well known ha ISs
can in e up o al e gene exp ession. The e a e many exam-
ples in he li e a u e whe e ISs ha e been in ol ed in esis ance
o ce ain an ibio ics by c ea ing no el p omo e s o an imi-
c obial esis ance genes (8, 15). Howe e , o ou knowledge,
his is he i s epo whe e an imic obial esis ance is caused
by in e up ion o po in genes by ISs. Mechanisms o po in loss
and inc eased esis ance o imipenem we e also s udied in
Pseudomonas ae uginosa by Yoneyama and Nakae by using an
in i o app oach (20): only dele ions in he Op D2 po in gene
we e obse ed. Howe e , we ha e shown ha , bo h in i o and
in i o, ISs cause po in de iciency in K. pneumoniae. ISs a e
widesp ead in all bac e ial species s udied, and, al hough hei
p esence has no been co ela ed wi h an imic obial esis ance,
hey ha e also been ound in E. coli po ins (13, 16). Thus,
in e up ion o po in genes by ISs is he e o e no an exclusi e
phenomenon o K. pneumoniae. Fu he s udies will be e-
qui ed o de e mine he impo ance o his mechanism in o he
bac e ial species.
This wo k was suppo ed by g an s om he Comisio´n In e minis-
e ial de Ciencia y Tecnologı´a (CICYT). S.H.A. and S.A. we e sup-
po ed by a p edoc o al ellowship and a pos doc o al con ac om
CICYT, espec i ely.
We hank J. A. M. an de Klunde (Uni e si y Hospi al Leiden),
G. Jacoby (Lahey Hi chcock Clinic), and J. Lin˜a es and C. A danuy
(Hospi al de Bell i ge) o clinical isola es, he Se icio de Secuencia-
cio´n del Cen o de In es igaciones Biolo´gicas o DNA sequencing,
and J. Casadesu´s (Uni e sidad de Se illa) o help ul discussions
h oughou he wo k.
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