ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,
0066-4804/97/$04.0010Feb. 1997, p. 274–277 Vol. 41, No. 2
Copy igh q1997, Ame ican Socie y o Mic obiology
Up ake and In acellula Ac i i y o T o a loxacin in Human
Phagocy es and Tissue-Cul u ed Epi helial Cells
ALVARO PASCUAL,* ISABEL GARCI
´A, SOFI
´A BALLESTA, AND EVELIO J. PEREA
Depa men o Mic obiology, School o Medicine, Uni e si y o Se ille, Se ille 41080, Spain
Recei ed 28 May 1996/Re u ned o modi ica ion 9 Sep embe 1996/Accep ed 12 No embe 1996
The pene a ion o o a loxacin in o human polymo phonuclea leukocy es (PMNs), human pe i oneal
mac ophages, and issue-cul u ed epi helial cells (McCoy cells) was e alua ed. The cellula concen a ion o
ex acellula concen a ion (C/E) a ios o o a loxacin we e g ea e han 9 o ex acellula concen a ions
anging om 0.5 o 25 mg/ml. The up ake o o a loxacin by PMNs was apid, e e sible, nonsa u able, no
ene gy dependen , and signi ican ly inc eased a pH 6. The C/E a ios o o a loxacin we e no a ec ed by cell
iabili y bu we e signi ican ly inc eased a 4&C. Inges ion o opsonized zymosan, bu no opsonized S aphylo-
coccus au eus, signi ican ly inc eased he amoun o PMN-associa ed o a loxacin. This agen a concen a-
ions o 0.5 and 1 mg/ml induced a g ea e educ ion in he su i al o in acellula S. au eus in PMNs han
cip o loxacin and o loxacin. I was concluded ha o a loxacin eaches concen a ions wi hin phagocy ic and
nonphagocy ic cells se e al imes highe han he ex acellula ones, while i emains ac i e in PMNs.
Mos luo oquinolones a e able o concen a e in phagocy ic
and nonphagocy ic cells, eaching concen a ions se e al imes
highe han he ex acellula ones (9). Mo eo e , hese agen s
ha e been shown o emain ac i e in acellula ly agains di -
e en acul a i e in acellula pa hogens.
Mos s udies on he in acellula pha macology o an imi-
c obial agen s ha e used human phagocy es, and only a ew
s udies ha e e alua ed he pene a ion in o o he ypes o cells.
Ne e heless, he pa hogenic mechanism o se e al in ec ions
in ol es he su i al o bac e ia in nonphagocy ic cells, as has
been desc ibed o Chlamydia achoma is and en e oin asi e
Esche ichia coli in epi helial cells.
An impo an limi a ion o mos comme cially a ailable
luo oquinolones is hei limi ed ac i i y agains g am-posi i e
pa hogens. In ecen yea s, howe e , new quinolones ha o e
good ac i i y agains hese bac e ia ha e been desc ibed.
Among hem, spa loxacin has been shown o pene a e apidly
in o phagocy ic cells, emaining ac i e agains S aphylococcus
au eus (4).
T o a loxacin (CP-99,219) is a new azabicyclo naph hy i-
done agen wi h a b oad spec um o an ibac e ial ac i i y
which includes g am-posi i e bac e ia such as S ep ococcus
pneumoniae and S. au eus (6). Mo eo e , his agen showed
high in i o ac i i y agains Chlamydia achoma is (2).
The pu pose o his s udy was o e alua e he up ake o
o a loxacin by human polymo phonuclea leukocy es (PMNs),
human pe i oneal mac ophages (PM s), and issue-cul u ed
epi helial cells. The mechanism in ol ed in he pene a ion o
his agen in o human PMNs and i s in acellula ac i i y com-
pa ed wi h hose o cip o loxacin and o loxacin we e also e al-
ua ed.
MATERIALS AND METHODS
Isola ion o phagocy es. PMNs we e eco e ed om he hepa inized enous
blood o heal hy dono s and we e pu i ied by p e iously desc ibed me hods (10).
PMN p epa a ions we e .97% pu e. PM s we e isola ed om pe i oneal e lu-
en s o pa ien s unde going con inuous ambula o y pe i oneal dialysis (CAPD)
and who we e being ollowed up by he Neph ology Depa men o he Uni e -
si y Hospi al o Se ille, as desc ibed p e iously (13). Dono s we e clinically
unin ec ed a he ime o he s udy. Cell p epa a ions om he CAPD dono
always con ained .75% PM s and ,15% PMNs. Final cell suspensions we e
adjus ed o 2 310
6
PM s/ml o 2 310
6
PMNs/ml in Hanks balanced sal
solu ion (HBSS) con aining 0.1% gela in. Bo h popula ions o cells we e $95%
iable by ypan blue exclusion c i e ia.
Tissue cul u e cells. McCoy cells (Flow Labo a o ies, I ine, Uni ed King-
dom) we e g own in minimal essen ial medium (Flow) supplemen ed wi h 1 mM
HEPES (N-2-hyd oxye hylpipe azine-N9-2-e hanesul onic acid; Flow) and con-
aining 10% e al cal se um (Flow) wi hou an ibio ics. Fo each expe imen , he
cells we e de ached om he issue cul u e bo les wi h ypsin-EDTA (Flow),
washed once wi h minimal essen ial medium con aining e al cal se um (10%),
and suspended in HBSS a a concen a ion o 5 310
6
cells pe ml.
T o a loxacin up ake by cells. Up ake o adiolabeled o a loxacin by phago-
cy ic and epi helial cells was de e mined by a eloci y g adien cen i uga ion
echnique desc ibed by Klempne and S y (7). [
14
C] o a loxacin (21.53 mCi/
mg) and o a loxacin we e kindly supplied by P ize Cen al Resea ch (G o on,
Conn.). In hese expe imen s, phagocy es o issue cells we e incuba ed in HBSS
con aining di e en concen a ions o o a loxacin (0.5, 1, 2, 5, 10, and 25
mg/ml). A e di e en incuba ion pe iods a 378C, he cells we e sepa a ed om
he ex acellula solu ion by cen i uga ion h ough a wa e -impe meable silicone
oil ba ie in a mic ocen i uge ube. A 10-ml aliquo o he ex acellula medium
and he en i e cell pelle , ob ained by cu ing o he po ion o he mic ocen i-
uge ube con aining he pelle , we e placed in 3 ml o scin illa ion luid (Ready
Mic o; Beckman Ins umen s, Inc., Fulle on, Cali .), and he adioac i i y was
coun ed in a liquid scin illa ion coun e (model LS 1801; Beckman). A e de-
e mina ion o he cell olume wi h adiolabeled polye hylene glycol and wa e
(New England Nuclea ), he a e o accumula ion o he an imic obial agen in
PMNs, PM s, o issue cells was calcula ed and exp essed as cellula concen-
a ion o ex acellula concen a ion (C/E) a io (13).
Cha ac e iza ion o o a loxacin up ake. Fu he s udies o elucida e he
mechanism o o a loxacin up ake by PMNs we e pe o med as desc ibed p e-
iously (10). The impo ance o cell iabili y was s udied by using PMNs killed by
exposu e o 10% o malin o 30 min. These cells we e hen washed and sus-
pended in esh medium. Mo eo e , he in luence o en i onmen al empe a u e,
pH, and me abolic inhibi o s was e alua ed. The in luence o empe a u e was
examined by compa ing an imic obial up ake a 4 and 378C. The pH p o iles o
o a loxacin up ake in media p eadjus ed o di e en ex e nal pHs (pH 6, 7, and
8) by he addi ion o 10 N HCl o 10 N NaOH we e measu ed. An inhibi o o
glycolysis (sodium luo ide, 1.5 310
23
M; Sigma Chemical Co., S . Louis, Mo.),
an inhibi o o mi ochond ial oxida i e me abolism (sodium cyanide; 1.5 310
23
M; Sigma), a blocke o he p o on g adien (ca bonyl cyanide m-chlo ophenyl-
hyd azone; 1.5 310
25
M; Sigma), and an uncouple o oxida i e phospho yla-
ion (2,4-dini ophenol; 1 310
24
M; Sigma) we e used as me abolic inhibi o s.
PMNs in HBSS wi h and wi hou me abolic inhibi o s we e incuba ed o 30 min
a 378C, o a loxacin ( inal concen a ion, 2 mg/ml) was hen added, and he
up ake was measu ed, as desc ibed abo e.
In a se ies o expe imen s, o a loxacin (ex acellula concen a ion, 2 mg/ml)
up ake by human PMNs was measu ed a e he s imula ion o cells wi h 200 nM
pho bol my is a e ace a e (PMA; Sigma) and a e he phagocy osis o ei he
opsonized zymosan (0.9 mg/li e ; Sigma) o S. au eus ATCC 25923, opsonized in
5% pooled human se um (15 min a 378C) a a 10/1 a io o bac e ia o PMNs.
* 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, Se ille
41080, Spain. Phone: 34.5.4557448. Fax: 34.5.4377413.
274
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PMA o opsonized pa icles we e added o PMN suspensions a he same ime
ha he an imic obial agen was added, and he up ake was measu ed as de-
sc ibed abo e.
The e lux o e e sibili y o he binding o cell-associa ed o a loxacin was
also s udied. PMNs, PM s, o McCoy cells we e incuba ed o 10 min a 378C
wi h o a loxacin (ex acellula concen a ion, 2 mg/ml), collec ed by cen i u-
ga ion, and apidly suspended in quinolone- ee medium. Cell-associa ed o a-
loxacin was quan i ied a a ious ime in e als (5, 10, and 20 min) a e he
emo al o he ex acellula an imic obial agen . All assays we e pe o med in
duplica e wi h PMNs om i e di e en dono s.
Pa i ion coe icien o quinolones. The pa i ion coe icien s o quinolones
we e de e mined by he modi ied me hod o Nikaido (8). Solu ions (10 mg/ml) o
quinolones we e made in 0.1 M phospha e bu e (pH 7.2). A e being shaken
wi h an equal olume o n-oc anol a 258C o 48 h and cen i uged a 1,870 3g
o phase sepa a ion, he concen a ions o quinolones in he aqueous phase
we e de e mined by a spec opho ome ic assay measu ing he A
260
o o a-
loxacin and he A
286
o o loxacin (Hoechs AG, Ba celona, Spain). The pa i-
ion coe icien s we e exp essed as he a io o he amoun o he compound in
he n-oc anol o ha in he aqueous phase.
O ganisms and suscep ibili y es ing. S. au eus ATCC 25923 was used o he
killing assays. Suscep ibili y s udies we e de e mined by dilu ion assay. The MICs
and minimum bac e icidal (MBCs) o cip o loxacin (Baye AG, Le e kusen,
Ge many), o loxacin (Hoechs AG), concen a ions and o a loxacin o his
s ain we e 0.25, 0.25, and 0.03 mg/ml, espec i ely.
In acellula ac i i ies o an imic obial agen s. To e alua e he in acellula
ac i i ies o an imic obial agen s, a p e iously desc ibed me hod was used (13).
B ie ly, 0.1 ml o opsonized bac e ial suspension (5 310
7
CFU/ml) and 0.1 ml o
PMNs (5 310
6
PMNs pe ml) we e combined in a se ies o polyp opylene
bio ials (Beckman), and he ials we e incuba ed in a shake (250 pm) o 60
min a 378C. A e incuba ion, he mix u es we e washed h ee imes wi h 2.5 ml
o ice-cold phospha e-bu e ed saline by using di e en ial cen i uga ion (160 3
g; 5 min a 48C) o emo e he ex acellula bac e ia. The cells we e hen
suspended in 0.2 ml o RPMI medium (Sigma). A his ime, di e en concen-
a ions o he di e en luo oquinolones we e added, and he ials we e incu-
ba ed in a shake (50 pm) a 378C. The ials we e emo ed a ime ze o and a e
3 h o incuba ion (con ol and samples wi h an imic obial agen s). Cells we e
lysed in dis illed wa e , and samples we e dilu ed and pou pla ed in aga .
Colonies we e coun ed a e 24 h o incuba ion a 378C. The da a we e exp essed
as he pe cen ages o s aphylococci su i ing compa ed wi h he le els in he
con ols (wi hou an imic obial agen s) a 3 h. In addi ion o de e mining bac-
e ial su i al, mo phologic s udies we e also ou inely pe o med a ime ze o
and a e 3ho incuba ion o e alua e he disposi ion o he bac e ia (cell
associa ed o ex acellula ). Samples (50 ml) we e emo ed om he bio ials and
we e deposi ed on glass slides. A e being s ained wi h W igh s ain, he samples
we e examined by ligh mic oscopy. All assays we e pe o med in duplica e wi h
PMNs om i e di e en dono s.
S a is ical analysis o da a. The da a we e exp essed as he means 6s anda d
de ia ions. Di e ences among g oups we e compa ed by analysis o a iance,
used o assess s a is ical signi icance a P#0.05.
RESULTS
Up ake o o a loxacin by PMNs, PM s, and McCoy cells.
Figu e 1 shows he kine ics o up ake o o a loxacin by hu-
man PMNs. T o a loxacin up ake by hese cells was apid and
high. Wi h ex acellula concen a ions o 2 mg/ml, he C/E
a ios we e g ea e han 10 a e 1 min o incuba ion. The e ec
o he ex acellula concen a ions o o a loxacin on up ake
by PMNs is p esen ed in Fig. 2. Cell-associa ed o a loxacin
was no sa u able a ex acellula concen a ions anging om
0.5 o 25 mg/ml. The up ake o o a loxacin by human PM s
and issue-cul u ed epi helial cells was simila o ha ob ained
wi h PMNs. A ex acellula concen a ions o 2 mg/ml (30 min;
378C), he C/E a ios we e 10.6 62.0 o PMNs, 10.2 62.0 o
PM s, and 9.6 61.9 o McCoy cells.
The e ec s o pH, en i onmen al empe a u e, cell iabili y,
and me abolic inhibi o s on he up ake o o a loxacin by
human PMNs a e indica ed in Table 1. T o a loxacin up ake
by PMNs was signi ican ly highe a pH 6 and when iable
PMNs a 48C we e used. None o he me abolic inhibi o s used
a ec ed he in acellula pene a ion o his quinolone.
The s imula ion o PMNs by a memb ane ac i a o (PMA)
and he phagocy osis o opsonized S. au eus cells did no a ec
he in acellula pene a ion o o a loxacin (C/E a ios, 9.0 6
1.7 and 8.2 61.6, espec i ely). The phagocy osis o opsonized
zymosan, howe e , signi ican ly inc eased he up ake o o a-
loxacin by PMNs (C/E a io, 13.9 61.7; P,0.05).
To e alua e whe he o a loxacin ha had been aken up by
human PMNs, PM s, and issue-cul u ed epi helial cells was
igh ly bound o cellula componen s, we e alua ed he kine ics
o e lux (Fig. 3). The e e sibili y o binding o o a loxacin
was apid, wi h 80, 65, and 72% o he cell-associa ed d ug
being los by 5 min in PMNs, PM s, and McCoy cells, espec-
i ely. The kine ics o he e lux o o a loxacin om PMNs a
378C was simila o ha obse ed in PMNs a 48C o dead
PMNs a 378C (da a no shown).
Pa i ion coe icien o quinolones. The pa i ion coe icien s
o o a loxacin and o loxacin we e 0.646 and 0.168, espec-
i ely. This means ha o a loxacin is app oxima ely ou
imes mo e hyd ophobic han o loxacin.
In acellula ac i i y o o a loxacin. The in acellula ac-
i i y o o a loxacin agains S. au eus compa ed wi h hose o
cip o loxacin and o loxacin was e alua ed by a 3-h assay (Fig.
4). A ex acellula concen a ions o 0.5 and 1 mg/ml, he
in acellula ac i i y o o a loxacin was signi ican ly g ea e
han hose o cip o loxacin and o loxacin.
FIG. 1. Kine ics o o a loxacin up ake by human PMNs (n55). The
ex acellula concen a ion was 2 mg/ml. E o ba s indica e s anda d de ia ions.
FIG. 2. T o a loxacin up ake by human PMNs a di e en ex acellula con-
cen a ions (n55). Incuba ions we e ca ied ou o 20 min. E o ba s indica e
s anda d de ia ions.
VOL. 41, 1997 UPTAKE OF TROVAFLOXACIN BY PHAGOCYTES 275
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DISCUSSION
The up ake o adiolabeled o a loxacin by phagocy ic cells
(PMNs and PM s) and nonphagocy ic cells has been e alu-
a ed. A he apeu ic ex acellula concen a ions, o a loxacin
eached concen a ions in PMNs 10 o mo e imes highe han
he ex acellula concen a ions. These alues we e sligh ly
highe han hose obse ed wi h o he luo oquinolones such
as o loxacin, lome loxacin, spa loxacin, and BAY-Y-3118 (4,
5, 11, 12). This could be ela ed o he ac ha o a loxacin is
mo e hyd ophobic han hose quinolones and heo e ically
could pass mo e easily h ough he cell memb ane.
The up ake o o a loxacin by PM s was simila o ha
obse ed o PMNs. This inding could be in e es ing since
in aleukocy ic su i al o bac e ia has been pos ula ed as an
impo an cause o pe sis en pe i oni is in pa ien s unde going
CAPD (1) and li le in o ma ion on he pene a ion o an imi-
c obial agen s in human PM s is a ailable. A highe ex acel-
lula concen a ions (10 mg/ml) and unde di e en expe imen-
al condi ions, Edels ein e al. (3) epo ed C/E a ios o
o a loxacin o g ea e han 20 o guinea pig al eola mac-
ophages.
T o a loxacin also eached high in acellula concen a ions
(C/E a ios, $9) in issue-cul u ed epi helial cells. These alues
a e highe han hose obse ed o o loxacin and spa loxacin
(4, 11). Since o a loxacin yielded high in insic ac i i y
agains C. achoma is and since hese mic oo ganisms mul iply
wi hin epi helial cells, hese esul s could ein o ce he po en-
ial use o his agen agains chlamydial in ec ions.
The up ake o o a loxacin by human PMNs was apid,
nonsa u able (ex acellula concen a ions anged om 0.5 o
25 mg/ml), e e sible (as was he case o PM s and McCoy
cells), and no ene gy dependen . I was signi ican ly inc eased
a acidic pH, as was epo ed elsewhe e o o he quinolones
(4, 5), bu o a lesse ex en . This phenomenon could be ela ed
o he ac ha such quinolones display a ee ca boxyl g oup
ha becomes p o ona ed when he pH changes om alkaline
o acidic. I has been obse ed ha he ionized o ms o weak
o ganic acids di use h ough biological memb anes much
mo e slowly han hei un-ionized o ms (14). The up ake o
o a loxacin by human PMNs was signi ican ly inc eased a
48C and was no a ec ed by cell iabili y. We ha e p e iously
obse ed ha he up ake o some quinolones such as ema-
loxacin and lome loxacin signi ican ly dec eased a 48C and
was a ec ed by cell iabili y (12), while he up ake o o he
quinolones such as spa loxacin was no a ec ed by hese pa-
ame e s (4). We ha e no p e iously obse ed such a ema k-
able inc ease a 48C wi h o he quinolones. These da a could
indica e ha mo e han one mechanism may media e he in-
acellula pene a ion o di e en quinolones. Fo o a loxa-
cin, mos da a show ha a passi e mechanism, such as ha
desc ibed o spa loxacin (4), is in ol ed.
The e ec s o phagocy osis and s imula ion o he cell mem-
b ane on he up ake o o a loxacin by human PMNs was
simila o hose p e iously obse ed wi h spa loxacin (4). The
inges ion o opsonized S. au eus cells did no a ec he in a-
cellula pene a ion o o a loxacin, bu he inges ion o op-
sonized zymosan signi ican ly inc eased he C/E a io o o a-
loxacin. Ne e heless, he up ake o o a loxacin was s ill high
unde bo h condi ions.
A ex acellula concen a ions o 0.5 and 1 mg/ml, o a-
loxacin showed ac i i y agains S. au eus g ea e han hose o
cip o loxacin and o loxacin in human PMNs. This e ec was
pa ially due o a highe in insic ac i i y o o a loxacin
agains he s ain ha we used.
In summa y, o a loxacin pene a es in o phagocy ic and
nonphagocy ic cells, eaching in acellula concen a ions se -
e al imes g ea e han he ex acellula ones, while i emains
FIG. 3. E lux o o a loxacin om human PMNs, PM s, and McCoy cells
(n53). A e incuba ion wi h 2 mg o o a loxacin pe ml o 20 min, he cells
we e washed and esuspended in an imic obial agen - ee medium. Cell-associ-
a ed o a loxacin was hen measu ed a di e en imes.
FIG. 4. Ac i i y o o a loxacin compa ed o hose o cip o loxacin and
o loxacin agains in acellula S. au eus in human PMNs (n55). Da a a e
exp essed as pe cen ages o su i ing bac e ia compa ed o ha o he con ol.
E o ba s indica e s anda d de ia ions. *, P, 0.05 compa ed o cip o loxacin and
o loxacin.
TABLE 1. E ec s o pH, cell iabili y, en i onmen al empe a u e,
and me abolic inhibi o s on he in acellula pene a ion
o o a loxacin in human PMNs
Condi ion
a
C/E a io
pH 7.2 (con ol)......................................................................11.3 60.8
pH 6 .........................................................................................16.5 60.4
b
pH 8 .........................................................................................10.3 61.0
Viable cells, 48C......................................................................62.0 614.0
b
Dead cells, 378C......................................................................14.4 64.3
Sodium luo ide ......................................................................11.1 61.7
Sodium cyanide....................................................................... 9.3 61.8
Ca bonyl cyanide m-chlo ophenyld azone.......................... 9.7 61.7
2,4-Dini ophenol ...................................................................13.4 63.5
a
Expe imen s we e ca ied ou o 20 min a an ex acellula concen a ion o
2mg/ml wi h PMNs om i e subjec s.
b
P,0.05 compa ed wi h he con ol.
276 PASCUAL ET AL. ANTIMICROB.AGENTS CHEMOTHER.
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ac i e in acellula ly in human PMNs. These p ope ies, be-
sides i s b oad spec um o ac i i y, enhance he po en ial clin-
ical use ulness o his luo oquinolone.
ACKNOWLEDGMENTS
We hank Pa icia Hidalgo and Jane Dawson o p epa a ion o he
manusc ip .
This s udy was pa ially suppo ed by P ize Labo a o ies (G o on,
Conn.).
REFERENCES
1. Buggy, B. P., D. R. Schabe g, and R. D. Swa z. 1984. In aleukocy ic se-
ques a ion as a cause o pe sis en S aphylococcus au eus pe i oni is in
con inuous ambula o y pe i oneal dialysis. Am. J. Med. 76:1035–1040.
2. Child, J., J. And es, F. Boswell, N. B enwald, and R. Wise. 1995. The in i o
ac i i y o CP 99,219, a new naph hy idone an imic obial agen : a compa i-
son wi h luo oquinolone agen s. J. An imic ob. Chemo he . 35:869–876.
3. Edels ein, P. H., M. A. Edels ein, J. Ren, R. Polze , and R. P. Gladue. 1996.
Ac i i y o o a loxacin (CP-99,219) agains Legionella isola es; in i o
ac i i y, in acellula accumula ion and killing in mac ophages, and pha ma-
cokine ics and ea men o guinea pigs wi h L. pneumophila pneumoniae.
An imic ob. Agen s Chemo he . 40:314–319.
4. Ga cı´a, I., A. Pascual, M. C. Guzman, and E. J. Pe ea. 1992. Up ake and
in acellula ac i i y o spa loxacin in human polymo phonuclea leukocy es
and issue cul u e cells. An imic ob. Agen s Chemo he . 36:1053–1056.
5. Ga cı´a, I., A. Pascual, and E. J. Pe ea. 1994. In acellula pene a ion and
ac i i y o BAY Y 3118 in human polymo phonuclea leukocy es. An imi-
c ob. Agen s Chemo he . 38:2426–2499.
6. Gi a d, A. E., D. Gi a d, T. D. Goo z, J. A. Faiella, and C. R. Cimochowski.
1995. In i o e icacy o o a loxacin (CP-99,219), a new quinolone wi h
ex ended ac i i ies agains g am-posi i e pa hogens, S ep ococcus pneu-
moniae and Bac e oides agilis. An imic ob. Agen s Chemo he . 39:2210–
2216.
7. Klempne , M. S., and B. S y . 1989. Clindamycin up ake by human neu o-
phils. J. In ec . Dis. 144:472–475.
8. Nikaido, H. 1976. Ou e memb ane o Salmonella yphimu ium: ansmem-
b ane di usion o some hyd ophobic subs ances. Biochim. Biophys. Ac a
433:118–132.
9. Pascual, A. 1995. Up ake and in acellula ac i i y o an imic obial agen s in
phagocy ic cells. Re . Med. Mic obiol. 6:228–235.
10. Pascual, A., I. Ga cı´a, and E. J. Pe ea. 1989. Fluo ome ic measu emen o
o loxacin up ake by human polymo phonuclea leukocy es. An imic ob.
Agen s Chemo he . 33:653–656.
11. Pascual, A., I. Ga cı´a, and E. J. Pe ea. 1990. Up ake and in acellula ac i i y
o an op ically ac i e o loxacin isome in human neu ophils and issue
cul u e cells. An imic ob. Agen s Chemo he . 34:277–280.
12. Pascual, A., I. Ga cı´a, and E. J. Pe ea. 1992. E ec o lome loxacin and
ema loxacin in o human neu ophils, pe i oneal mac ophages and issue
cul u e cells. Diagn. Mic obiol. In ec . Dis. 15:393–398.
13. Pascual, A., D. Tsakayama, J. Ko a ik, G. Gekke , and P. K. Pe e son. 1987.
Up ake and ac i i y o i apen ine in human pe i oneal mac ophages and
polymo phonuclea leukocy es. Eu . J. Clin. Mic obiol. 6:152–157.
14. Rena d, C., H. C. Vande Haugue, O. H. M. Claes, A. Zenebe gh, and P. M.
Tulkens. 1987. In luence o con e sion o penicillin G in o a basic de i a i e
on i s accumula ion and subcellula localiza ion in cul u ed mac ophages.
An imic ob. Agen s Chemo he . 31:410–416.
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