ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2007, p. 2236–2239 Vol. 51, No. 6
0066-4804/07/$08.00⫹0 doi:10.1128/AAC.01444-06
Copy igh © 2007, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
Mu an P e en ion Concen a ions o Fluo oquinolones o
En e obac e iaceae Exp essing he Plasmid-Ca ied
Quinolone Resis ance De e minan qn A1
䌤
J. M. Rod ı´guez-Ma ı´nez,
1
* C. Velasco,
1
I. Ga cı´a,
1
M. E. Cano,
3
L. Ma ı´nez-Ma ı´nez,
3
and A. Pascual
1,2
Depa men o Mic obiology, Uni e si y o Se ille, Se ille,
1
Se ice o Mic obiology, Uni e si y Hospi al Vi gen Maca ena, Se ille,
2
and Se ice o Mic obiology, Uni e si y Hospi al Ma que´s de Valdecilla, San ande ,
3
Spain
Recei ed 17 No embe 2006/Re u ned o modi ica ion 18 Decembe 2006/Accep ed 19 Ma ch 2007
The in luence o qn A1 on he de elopmen o quinolone esis ance in En e obac e iaceae was e alua ed by
using he mu an p e en ion concen a ion pa ame e . The exp ession o qn A1 conside ably inc eased he
mu an p e en ion concen a ion compa ed o s ains wi hou his gene. In he p esence o qn A1, mu a ions
in gy A and pa C genes we e easily selec ed o p oduce high le els o quinolone esis ance.
Fluo oquinolone esis ance in En e obac e iaceae is usually
he esul o mu a ions in ch omosomal genes o ype II opo-
isome ases o in genes coding o o egula ing e lux pumps
and/o po ins (6). Recen epo s indica e ha quinolone e-
sis ance may also be plasmid media ed by he qn A,qn S,qn B,
o aac(6⬘)-Ib-c genes (12, 16). Al hough qn A (and, p esum-
ably, ela ed) genes con e low-le el esis ance on hei own,
when ound in he p esence o o he mechanisms, hey a e able
o help selec o mu an s wi h inc eased le els o luo oquin-
olone esis ance (6, 11, 16).
A majo goal o an imic obial he apy is o achie e su icien
d ug exposu e in ela ion o MIC o op imal e icacy a he si e
o in ec ion. Ac i i y can be assessed by using he mu an
p e en ion concen a ion (MPC). This is he d ug concen a-
ion ha p e en s he g ow h o he leas -suscep ible single-
s ep mu an p esen in a la ge bac e ial popula ion, aking in o
accoun esis an mu an subpopula ions p esen p io o an-
imic obial ea men (1, 5). The mu an selec ion window, he
an ibio ic concen a ion ound be ween MIC and MPC, is
whe e single-s ep mu an s can be en iched (5).
The aim o he p esen s udy was o de e mine he in luence
o qn A1 on he de elopmen o quinolone esis ance in En-
e obac e iaceae using he MPC pa ame e . Fo his pu pose,
14 s ains we e e alua ed. These included: (i) ou Klebsiella
pneumoniae clinical s ains con aining qn A1 wi h di e en le -
els o luo oquinolone suscep ibili y (Tables 1 and 2) (11, 17)
and (ii) h ee g oups o isogenic s ains ha bo h exp ess and
do no exp ess qn A1 (Table 1). The p ime s used o clone
qn A1 in pACYC184 (New England Biolabs, Inc., Ba celona,
Spain) we e 5⬘-CGGCAGTTAAAATTGGGGCT-3⬘and 5⬘-G
ACCAGACTGCATAAGCAACAC-3⬘.
qn A1 exp ession as he only quinolone esis ance mecha-
nism did no b ing abou luo oquinolone esis ance; howe e ,
he associa ion o qn A1 wi h o he mechanisms, such as po in
loss and Se 83Phe subs i u ion in he Gy A p o ein o he K.
pneumoniae C2 pMG252 s ain, did p oduce a pheno ype o
in e media e luo oquinolone suscep ibili y, which suppo ed
he addi i e na u e o he mechanism (Table 2) (10). This
could imply an impo an ool in he acquisi ion o high quin-
olone esis ance le els in ela ion o plasmid-media ed quino-
lone esis ance (14, 17, 21).
MPC assays we e pe o med on all 14 s ains, as p e iously
desc ibed (9), pe mi ing colony g ow h o up o 96 h. Mos
s ains equi ed be ween 48 and 96 h o incuba ion be o e
esis an colonies we e obse able, excep in he N5 and 1960
s ains o moxi loxacin and in he C2 and C2/pMG252 s ains
o cip o loxacin and moxi loxacin (Table 2). The MPC alues
o each luo oquinolone o he di e en s ains exp essing he
qn A1 gene as he only known quinolone esis ance mechanism
(Esche ichia coli DH10B⫹qn A1;E. coli J53 ansconjugan s
con aining qn A1) eached le els (2 o 4 ␮g/ml) app oaching
he peak se um concen a ions a ained du ing he apy (Table
2) (5, 7, 20). MPC alues o he non-qn A1-p oducing isogenic
con ols anged om 0.015 o 0.125 ␮g/ml (Table 2). These
alues we e much highe in s ains con aining addi ional esis-
ance mechanisms. The MPCs o clinical qn A1-con aining K.
pneumoniae s ains anged om 4 o 64 ␮g/ml o he a ious
luo oquinolones. MPC alues o K. pneumoniae C2 and K.
pneumoniae C2 pMG252 s ains we e 4 o 8 ␮g/ml and 32 o
128 ␮g/ml, espec i ely, showing, once again, he e ec o he
qn A1 gene on he MPC pa ame e . A po en ial consequence
o a subop imal he apy selec ing o esis ance can be exem-
pli ied by conside ing he case o mu an s selec ed one dilu ion
s ep below he MPC whose MIC is equal o o g ea e han i s
MPC alue (Table 2).
Plasmid-media ed quinolone esis ance enables mu an bac-
e ia wi h low le els o esis ance o su i e long enough o
hem o g ow and eme ge du ing luo oquinolone ea men .
In e es ingly, he MICs o some mu an s om ansconjugan s
con aining qn A1 and de i ing om E. coli J53 and K. pneu-
moniae C2/pMG252 we e as high as 4 ␮g/ml and 32 o 128
␮g/ml, espec i ely (Table 2). P ime s used o de ec mu a ions
in gy A,pa C, and qn A1 ha e been desc ibed p e iously (8,
19). Mu a ions in he quinolone esis ance-de e mining e-
* Co esponding au ho . Mailing add ess: Depa men o Mic obi-
ology, Uni e si y o Se ille, Sanchez Pizjuan s/n, Se ille 41009, Spain.
Phone: 34 954 55 28 63. Fax: 34 954 37 74 13. E-mail: jm od iguez
@us.es.
䌤
Published ahead o p in on 2 Ap il 2007.
2236
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gions o gy A o pa C, which a e ela ed o he luo oquinolone
esis ance in En e obac e iaceae, we e ound in qn A1-exp ess-
ing mu an s (Se 83Phe and Asp87Ty in Gy A, and Se 80Ile
and Val87Asp in Pa C). No mu a ions we e ound ei he in he
qn A1 coding sequences o in hei p omo e sequences (da a
no shown) (18). Addi ional mu a ions in qn A1-con aining
s ains ela ing o quinolone esis ance and de ec ed in he
ype II opoisome ase genes a e a e lec ion o he abili y o
his mechanism o selec o mu an s wi h addi ional ch omo-
somal quinolone esis ance mechanisms. Such mechanisms,
because o hei addi i e na u e, lead o he apeu ic ailu e
(10). In i o selec ion o luo oquinolone- esis an E. coli ex-
p essing he qn A1 gene was epo ed ecen ly in he case o a
pa ien wi h a u ina y ac in ec ion being ea ed wi h no -
loxacin o an in ec ion due o a cip o loxacin-suscep ible iso-
la e (13). These da a indica e he ole o qn A1 in he selec ion
and gene a ion o quinolone- esis an mu an s.
On he o he hand, qn A1 g ea ly eased he mu an selec ion
(Fig. 1). Fo example, in he p esence o qn A1 (E. coli
DH10B⫹qn A1) and a 0.25 ␮g/ml, mo e han 10
3
esis an
colonies g ew om an ini ial popula ion o 10
10
bac e ia, and
colonies could s ill be eco e ed a cip o loxacin concen a-
ions o 1 o 2 ␮g/ml (Fig. 1A). Exp ession o qn A1 om a
na u al plasmid in E. coli J53 p oduced o e 10
5
esis an
colonies g own a 1 ␮g/ml o J53 pMG252, and colonies could
s ill be eco e ed a moxi loxacin concen a ions o 2 o 4
␮g/ml (Fig. 1B). In he case o isogenic s ains o K. pneu-
moniae C2, he exp ession o qn A1 om a na u al plasmid
TABLE 1. Quinolone suscep ibili y cha ac e is ics o s ains included in his s udy
S ain Desc ip ion CIP
suscep ibili y
a
Mechanisms associa ed wi h quinolone
esis ance Sou ce o
e e ence
qn A1
b
Gy A Pa C Po ins
d
UAB1 K. pneumoniae clinical s ain I ⫹w
c
w ⫹11
N5 K. pneumoniae clinical s ain S ⫹w w ⫹17
1960 K. pneumoniae clinical s ain S ⫹Se 83Phe w ⫹17
1132 K. pneumoniae clinical s ain R ⫹w w ⫹17
DH10B E. coli e e ence lab s ain S – w w ⫹
DH10B/pACYC184 E. coli DH10B con aining pACYC184 ec o S – w w ⫹This s udy
DH10B⫹qn A1 E. coli DH10B con aining qn A1 cloned in
pACYC184 ec o
S⫹w w ⫹This s udy
E. coli J53 E. coli e e ence lab s ain S – w w ⫹
J53/pMG252 E. coli J53 ansconjugan de i ed om UAB1 S ⫹w w ⫹11
J53/pN5 E. coli J53 ansconjugan de i ed om N5 S ⫹w w ⫹17
J53/p1960 E. coli J53 ansconjugan de i ed om 1960 S ⫹w w ⫹17
J53/p1132
⫺
E. coli J53 ansconjugan de i ed om 1132 S – w w ⫹17
C2 K. pneumoniae clinical s ain S – Se 83Phe w – 11
C2/pMG252 C2 ansconjugan de i ed om UAB1 I ⫹Se 83Phe w – This s udy
a
S, suscep ible, I, in e media e suscep ibili y, R, esis ance acco ding o CLSI guidelines (4).
b
⫹, P esence and exp ession o qn A1;⫺, no exp ession.
c
w , wild ype.
d
⫹o ⫺indica es he p esence o absence o po ins, espec i ely.
TABLE 2. Fluo oquinolone MPCs and MICs o clinical and isogenic s ains used in his s udy and de i ed esis an mu an s
a
S ain MIC MPC MPC/MIC (MSW) MIC o mu an s
b
MPC ime (h)
window
c
CIP LFX MXF CIP LFX MXF CIP LFX MXF CIP LFX MXF CIP LFX MXF
UAB1 2 2 4 16 16 32 8 8 8 16–64 16–32 32–64 72–96 96 72
N5 0.25 0.5 0.5 4 4 16 16 8 32 8 8 4–8 48 48 24–48
1960 1 2 2 64 64 32 64 32 16 32–64 64 16 72 72 24–48
1132 4 4 8 32 32 32 8 8 4 16 16 16 48 48 72
E. coli DH10B 0.002 0.004 0.002 0.015 0.015 0.06 7.5 3.8 30 0.008 0.008 0.06 72 72 96
E. coli DH10B/pACYC184 0.002 0.004 0.002 0.015 0.015 0.06 7.5 3.8 30 0.008 0.008 0.06 72 72 72
E. coli DH10B⫹qn A 0.125 0.125 0.125 2 2 2 16 16 16 1 1 0.5 72–96 96 96
E. coli J53 0.004 0.008 0.015 0.06 0.06 0.125 15 7.5 16.7 0.03 0.03 0.5 48 48 72
J53/pMG252 0.25 0.25 0.5 4 4 4 16 16 8 4 2 2–4 96 96 72
J53/pN5 0.125 0.125 0.125 4 4 4 32 32 32 2 4 2 72 96 72
J53/p1960 0.125 0.125 0.125 4 4 4 32 32 32 1 2 4 72 96 72
J53/p1132
⫺
0.004 0.008 0.015 0.06 0.06 0.25 15 7.5 16.7 0.03 0.06 0.25 72 48 72
C2 0.25 0.25 0.125 8 8 4 32 32 32 4–8 8 2–8 24–48 48 24–48
C2/pMG252 2 2 1 128 128 32 64 64 32 64–128 64 32–64 24–48 72 24–48
a
MICs (in ␮g/ml) we e de e mined by mic odilu ion o cip o loxacin (CIP), le o loxacin (LFX), o moxi loxacin (MXF). MPC alues (in ␮g/ml) we e de e mined
o cip o loxacin, le o loxacin, o moxi loxacin on Muelle -Hin on pla es as he lowes an ibio ic concen a ion (in he ange o concen a ion s eps analyzed) a which
esis an colonies do no o m. The MSW is he mu an selec ion window, he an ibio ic concen a ion ound be ween he MIC and he MPC.
b
MIC o CIP (mic odilu ion) o cip o loxacin- o moxi loxacin- esis an colonies we e eco e ed on Muelle -Hin on pla es one s ep below he MPC alue.
c
Ea lies ime (in hou s) a which esis an colonies we e isible one s ep below he MPC.
VOL. 51, 2007 NOTES 2237
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p oduced mo e han 10
3
esis an colonies g own a 16 ␮g/ml,
and colonies we e s ill isualized a cip o loxacin concen a-
ions o 64 o 128 ␮g/ml (Fig. 1C), le els highe han peak
se um concen a ions a ained du ing d ug he apy. In cases o
s ains con aining qn A1, doses exceeding MPC in mono-
he apy would no be ad isable wi h app o ed dosing p oce-
du es and e alua ions o d ug oxici y (2, 3). The inc eased
equency o quinolone- esis an mu an s in s ains which ex-
FIG. 1. MPC assays. MIC and MPC alues a e indica ed on igu es by a ows. CIP, cip o loxacin; MXF, moxi loxacin.
2238 NOTES ANTIMICROB.AGENTS CHEMOTHER.
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p ess qn A1 a di e en concen a ions below he MPC p o-
ides suppo o he ole o low-le el esis ance media ed by
he mechanism in selec ing o quinolone esis ance (11, 15),
pa icula ly i we ake in o accoun he MICs obse ed in
mu an s selec ed below he MPC and include some mu an s
ob ained om s ains whose only quinolone esis ance mech-
anism was qn A1 (Table 2).
Bac e ia su i ing a MPC we e coun ed a e 96 h, as de-
sc ibed elsewhe e (9). The MIC and MPC o hese o ganisms
(isola ed a e y low equencies [da a no shown]) we e simila
o hose o he o iginal s ains. Al hough some bac e ia su -
i ed MPC o a p olonged pe iod, no quinolone- esis an
mu an s we e selec ed, showing ha he MPC pa ame e was
wo king as speci ied. I is possible ha su i al is ela ed o
mu a ions in he hip genes (high pe sis ence), being enhanced
in he p esence o ␤-lac ams o luo oquinolones, and p oduc-
ing a pe sis en pheno ype a high an imic obial concen a ions
(22).
Mechanisms, such as qn o he ecen ly desc ibed aac(6⬘)-
Ib-c gene, which a e implica ed in low-le el luo oquinolone
esis ance o plasmid-media ed de e minan s, may play a sig-
ni ican ole in he gene a ion o esis an mu an s and he a-
peu ic ailu e. This may be because, i s , many clinical s ains
epo ed as con aining genes ha code o plasmid-media ed
luo oquinolone esis ance a e suscep ible and, second, he
p esence o such genes in conjuga i e plasmid o o he mobile
elemen s may acili a e apid dissemina ion in En e obac e ia-
ceae and o he bac e ia o clinical signi icance. Animal models
will be necessa y in u u e o pe o m in i o alida ion o
hese in i o da a ob ained by using he MPC pa ame e .
This s udy was suppo ed by he Di eccio´n Gene al de In es igacio´n
del Minis e io de Ciencia y Tecnologı´a o Spain (p ojec SAF2005-
04704), he Minis e io de Sanidad y Consumo, Ins i u o de Salud
Ca los III (p ojec PI060580), and he Spanish Ne wo k o he Re-
sea ch in In ec ious Diseases (REIPI RD06/0008) o Spain. This s udy
was pa ially suppo ed by a g an om Baye .
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