ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2003, p. 2313–2315 Vol. 47, No. 7
0066-4804/03/$08.00⫹0 DOI: 10.1128/AAC.47.7.2313–2315.2003
Copy igh © 2003, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
Zinc Elu ed om Siliconized La ex U ina y Ca he e s Dec eases
Op D Exp ession, Causing Ca bapenem Resis ance
in Pseudomonas ae uginosa
M. Ca men Conejo,
1
* Isabel Ga cía,
1
Luis Ma ínez-Ma ínez,
1
Leand o Picabea,
2
and A
´l a o Pascual
1
Depa men o Mic obiology, School o Medicine, Uni e si y o Se ille,
1
and
Fo ensic Science Ins i u e,
2
Se ille, Spain
Recei ed 3 Janua y 2003/Re u ned o modi ica ion 21 Feb ua y 2003/Accep ed 26 Ma ch 2003
The ac i i ies o ca bapenems agains Pseudomonas ae uginosa dec eased in he p esence o siliconized la ex
u ina y ca he e s (SLUCs). This e ec was associa ed wi h he loss o Op D. The zinc ha elu ed om SLUCs
is esponsible o his phenomenon. We ha e ound ha zinc exe s a nega i e e ec on he exp ession o Op D,
he po in esponsible o ca bapenem en y in o P. ae uginosa.
Pseudomonas ae uginosa is an impo an cause o u ina y
ac in ec ions in pa ien s wi h u ina y ca he e s (17). This
o ganism is able o colonize he su ace o he ca he e , o m-
ing a bio ilm ha in e e es wi h he ac i i ies o an imic obial
agen s and hos de ense mechanisms (7, 13). In i o P. ae ugi-
nosa adhe es mo e e icaciously o siliconized la ex u ina y
ca he e s (SLUCs) han o o he plas ic ma e ials (7, 10, 11).
SLUCs elu e subs ances ha can be used as nu ien s by his
o ganism (10). Mo eo e , he elua e om SLUCs dec eases
he ac i i ies o ca bapenems agains P. ae uginosa (16). The
las e ec is no due o d ug inac i a ion o inc eased be a-
lac amase ac i i y bu is associa ed wi h changes in he ou e
memb ane p o ein (OMP) p o ile, ha is, he loss o an Op D-
like p o ein and he exp ession o a new OMP o abou 50 kDa.
This phenomenon is e e sible, which indica es ha , by means
o some unknown mechanism, he elua e egula es he physi-
ology o P. ae uginosa a he han selec s o mu an s (9).
Op D is a p o ein whose p ima y ole is he passi e up ake
o basic amino acids ac oss he ou e memb ane o P. ae ugi-
nosa, bu i o ms po es ha a e also pe meable o ca bapen-
ems (4, 21, 22). Op D loss educes he ac i i ies o ca bapen-
ems agains hese bac e ia (18, 21). Op D exp ession is e y
much in luenced by en i onmen al condi ions. Di e en amino
acids such as a ginine, his idine, glu ama e, and alanine
s ongly induce he exp ession o his po in when hey a e he
only sou ces o ca bon o ni ogen in he en i onmen (14).
Weak a oma ic acids like ace yl salicyla e o benzoa e educe
he exp ession o Op D (15, 20). None o se e al p e iously
iden i ied o ganic compounds in SLUC elua es, howe e , a ec
Op D exp ession o he suscep ibili ies o P. ae uginosa o
ca bapenems (9).
This s udy was unde aken in o de o in es iga e which o
he SLUC componen s a e esponsible o he dec eased ac-
i i ies o ca bapenems agains P. ae uginosa g own in he
p esence o SLUCs.
To in es iga e he p esence o di e en elemen s in u ina y
ca he e s, 0.5-cm segmen s o SLUCs ( wo-way pedia ic sili-
cone-coa ed la ex Foley ca he e 8FR/CH; Kendall Co., Kan-
ga , Malaysia) we e analyzed by a pa icle-induced X- ay emis-
sion (PIXE) echnique (8). A e emo al o he o ganic
ma ix, elemen al analysis was ca ied ou wi h 2.5-MeV p o-
ons gene a ed by a Tandem Van de G a accele a o
(9SDH-2; NEC, Middle on, Wis.). The induced X ays we e
simul aneously de ec ed wi h a 350-␮m- hick Myla abso be
(Dupon , Wilming on, Del.) wi h an LEGe and Si(Li) de ec o .
The spec a ob ained by he PIXE echnique we e analyzed
wi h an AXIL pe sonal compu e (Canbe a Packa d, Zelik,
Belgium). Zinc was he mos abundan elemen ound in all he
samples, ollowed by silicon, phospho us, sul u , and chlo ine.
Finally, aces o coppe , i anium, ch omium, and manganese
we e also de ec ed.
The elua es om he SLUCs we e also in es iga ed o he
p esence o zinc. Fo his pu pose, elua es we e p epa ed by
incuba ing segmen s o SLUCs in s e ile ca ion-adjus ed Muel-
le -Hin on b o h (MH) a 37°C o 24 h ( ou 1-cm segmen s
pe millili e o medium). The zinc con en s in MH and in he
elua es om he SLUCs we e de e mined by induc i ely cou-
pled plasma a omic emission spec ome y wi h a sequen ial
mul ielemen ins umen (ARL 3410; FISONS Ins umen s,
Valencia, Cali .) (3). The analyses we e pe o med in duplica e
and we e epea ed in h ee di e en ba ches o media. The zinc
con en s in MH and he elua e we e 0.29 ⫾0.1 and 36.8 ⫾3.2
␮g/ml, espec i ely.
The MICs o imipenem (IPM; Me ck Sha p & Dohme,
Mad id, Spain) o P. ae uginosa PAO1 and i s Op D-de icien
mu an we e de e mined by a mic odilu ion assay acco ding o
he guidelines o NCCLS (12). The media used we e MH, he
elua e om he SLUCs, and MH supplemen ed wi h zinc ac-
e a e dihyd a e (Sigma, Mad id, Spain) o achie e inal con-
cen a ions o 73.6, 36.8, 18.4, 11.2, 5.7, 3.0, 1.65, and 0.97 ␮g
o zinc/ml o medium. Zinc concen a ions in MH lowe han
5.7 ␮g/ml did no a ec he ac i i y o IPM agains P. ae ugi-
nosa PAO1, while zinc concen a ions be ween 5.7 and 18.9
␮g/ml caused a ou old inc ease in he MIC. Finally, when he
zinc concen a ion in MH was equal o ha ound in he elua e
* 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: 34-954552862. Fax: 34-954377413. E-mail: cconejo
@us.es.
2313
(36.8 ␮g/ml), he ac i i ies o IMP we e he same in bo h MH
and he elua e (8 ␮g/ml). On he o he hand, he MICs o IPM
o he Op D-de icien mu an we e he same (8 ␮g/ml) in all
he media es ed.
The e ec o zinc on he OMP p o ile o P. ae uginosa g own
in MH, elua e, o zinc-supplemen ed MH ( inal zinc concen-
a ion, 36.8 ␮g/ml) was e alua ed. OMPs we e p epa ed as
desc ibed p e iously (5); sepa a ed by sodium dodecyl sul a e-
polyac ylamide gel elec opho esis (SDS-PAGE) wi h he
bu e s desc ibed by Laemmli (6), wi h 10% (w / ol) ac yl-
amide and 0.1% (w / ol) bisac ylamide in he unning gel; and
s ained wi h Coomassie blue. Bo h P. ae uginosa PAO1 and i s
Op D-de icien mu an showed he same OMP p o iles when
hey we e g own in ei he he elua e o zinc-supplemen ed MH
(Fig. 1). The Op D-like p o ein and he 50-kDa p o ein ex-
p essed in he elua e and in zinc-supplemen ed MH we e u -
he analyzed by ma ix-assis ed lase deso p ion–ioniza ion
ime-o - ligh (MALDI-TOF) mass spec ome y a e in-gel
ypsin diges ion. Diges ion o he p o eins (in he gel) was
pe o med as desc ibed p e iously (19), wi h mino modi ica-
ions. The diges ed p o eins we e analyzed on a Re lex III
MALDI-TOF mass spec ome e (B uke -F anzen Analy ic
GmbH, B emen, Ge many) equipped wi h he SCOUT sou ce
in he posi i e ion e lec o mode. The ion accele a ion ol age
was 20 kV. The equipmen was i s ex e nally calib a ed wi h
p o ona ed mass signals om a pep ide mix u e co e ing he
1,000- o 4,000-m/z ange, and hen e e y spec um was in e -
nally calib a ed by using signals a ising om ypsin au op o-
eolysis. Analysis o yp ic agmen s om he OMP los in he
elua e and in zinc-supplemen ed MH showed ha hey
ma ched Op D ( o me ly Op D2), and he new OMP ex-
p essed in hose media was iden i ied as Op D3, as in e ed
om he PAO genome (PA2505; h p://pseudomonas.com).
The exp ession o Op D3 seemed o be un ela ed o he
dec eased suscep ibili ies o he wo P. ae uginosa s ains o
ca bapenems when hey we e g own in he elua e, as i was
exp essed by bo h s ains when hey we e g own in he elua e.
Simila esul s we e ound o P. ae uginosa s ains wi h di e -
en le els o exp ession o he AmpC be a-lac amase, Op D,
and MexA-MexB-Op M and we e e en ound o hose s ains
wi hou changes in ca bapenem suscep ibili y (M. C. Conejo,
L. Ma ínez-Ma ínez, I. Ga cía, and A. Pascual, Abs . 12 h
Eu . Cong . Clin. Mic obiol., abs . P1402, 2002). The exp es-
sion o Op D3 migh be a compensa o y change ha allows he
en y o amino acids in o P. ae uginosa when Op D2 is e-
p essed.
Di e en in es iga o s ha e epo ed ha he concen a ion
o zinc in MH signi ican ly a ec s he suscep ibili y o P. ae ugi-
nosa o IPM (1, 2). The eason o his obse a ion was un-
known. We ha e ound ha zinc exe s a nega i e e ec on he
exp ession o Op D2, he po in esponsible o ca bapenem
en y in o P. ae uginosa. New s udies a e in p og ess in o de o
e alua e he ole o zinc in he egula ion o OMP exp ession
in P. ae uginosa. The in i o ele ance o he loss o Op D om
P. ae uginosa s ains o ming bio ilms on SLUCs needs o be
e alua ed.
We hank Ma ía Dolo es Ynsa (Cen o Nacional de Acele ado es,
Pa que Tecnolo´gico Ca uja 93, Se ille, Spain) o de e mina ions by
he PIXE echnique and Juan An onio Ocan˜a (Depa men o Ana-
ly ical Chemis y, Uni e si y o Se ille) o measu emen o he zinc
concen a ions in he media. The P o eomics Facili y o he Cen o
Nacional de Bio ecnología de Mad id (CNB) is acknowledged o i s
help wi h he MALDI-TOF mass spec ome y analysis. We a e g a e-
ul o Jose´Luis Ma ínez (CNB) o help ul ad ice h oughou his
wo k.
This wo k was suppo ed by he Di eccio´n Gene al de In es igacio´n
del Minis e io de Ciencia y Tecnología (p ojec SAF2000-035).
REFERENCES
1. Coope , G. L., A. Louie, A. L. Bal ch, R. C. Chu, R. P. Smi h, W. J. Ri z, and
P. Michelsen. 1993. In luence o zinc on Pseudomonas ae uginosa suscep i-
bili ies o imipenem. J. Clin. Mic obiol. 31:2366–2370.
2. Daly, J. S., R. A. Dodge, R. H. Glew, D. T. Soja, B. A. DeLuca, and S. Hebe .
1997. E ec o zinc concen a ion in Muelle -Hin on aga on suscep ibili y o
Pseudomonas ae uginosa o imipenem. J. Clin. Mic obiol. 35:1027–1029.
3. Floyd, M. A., A. A. Halouma, R. W. Mo ow, and R. B. Fa a . 1985. Rapid
mul ielemen analysis o wa e samples by sequen ial ICP-AES. Am. Lab.
17:84.
4. Huang, H., and R. E. Hancock. 1993. Gene ic de ini ion o he subs a e
selec i i y o ou e memb ane po in p o ein Op D o Pseudomonas ae ugi-
nosa. J. Bac e iol. 175:7793–7800.
5. Ko¨hle , T., M. Miche´a-Hamzehpou , U. Henze, N. Go oh, K. Cu y, and J. C.
Peche` e. 1997. Cha ac e iza ion o MexE-MexF-Op N, a posi i ely egula ed
mul id ug e lux sys em o Pseudomonas ae uginosa. Mol. Mic obiol. 23:343–
354.
6. Laemmli, U. K. 1970. Clea age o s uc u al p o eins du ing he assembly o
he head o bac e iophage T4. Na u e (London) 227:680–685.
7. Lo´pez- Lo´pez, G., A. Pascual, L. Ma ínez-Ma ínez, and E. J. Pe ea. 1991.
E ec o a siliconized la ex u ina y ca he e on bac e ial adhe ence and
human neu ophil ac i i y. Diagn. Mic obiol. In ec . Dis. 14:1–6.
8. Maenhau , W., and K. G. Malmq is . 1993. Pa icle-induced X- ay emission
analysis, p. 719–809. In R. E. Van G ieken and A. A. Ma kowicz (ed.),
Handbook o X- ay spec ome y, p ac ical spec oscopy se ies, ol. 14. Ma -
cel Dekke , Inc., New Yo k, N.Y.
9. Ma ínez-Ma ínez, L., A. Pascual, M. C. Conejo, L. Picabea, and E. J.
Pe ea. 1999. Resis ance o Pseudomonas ae uginosa o imipenem induced by
elua es om siliconized la ex u ina y ca he e s is ela ed o ou e memb ane
p o ein al e a ions. An imic ob. Agen s Chemo he . 43:397–399.
10. Ma ínez-Ma ínez, L., A. Pascual, and E. J. Pe ea. 1990. E ec o h ee
plas ic ca he e s on su i al and g ow h o Pseudomonas ae uginosa. J. Hosp.
In ec . 16:311–318.
11. Ma ínez-Ma ínez, L., A. Pascual, and E. J. Pe ea. 1991. Kine ics o adhe -
ence o mucoid and non-mucoid Pseudomonas ae uginosa o plas ic ca he-
e s. J. Med. Mic obiol. 34:7–12.
12. Na ional Commi ee o Clinical Labo a o y S anda ds. 2000. Me hods o
dilu ion an imic obial suscep ibili y es s o bac e ia ha g ow ae obically,
5 h ed. App o ed s anda d M7-A5. Na ional Commi ee o Clinical Labo-
a o y S anda ds, Wayne, Pa.
13. Nickel, J., I. Ruseska, J. B. W igh , and J. W. Cos e on. 1985. Tob amycin
esis ance o Pseudomonas ae uginosa cells g owing as a bio ilm on u ina y
ca he e ma e ial. An imic ob. Agen s Chemo he . 27:619–624.
14. Ochs, M. M., C. D. Lu, R. E. Hancock, and A. T. Abdelal. 1999. Amino
acid-media ed induc ion o he basic amino acid-speci ic ou e memb ane
po in Op D om Pseudomonas ae uginosa. J. Bac e iol. 181:5426–5432.
15. Ochs, M. M., M. P. McCuske , M. Bains, and R. E. W. Hancock. 1999.
Nega i e egula ion o he Pseudomonas ae uginosa ou e memb ane po in
Op D selec i e o imipenem and basic amino acids. An imic ob. Agen s
Chemo he . 43:1085–1090.
16. Pascual, A., L. Ma ínez-Ma ínez, E. Ramí ez de A ellano, and E. J. Pe ea.
FIG. 1. SDS-PAGE showing OMP p o iles o P. ae uginosa g own
in MH (lanes 2 and 5), elua es om SLUCs (lanes 3 and 6), and
zinc-supplemen ed MH ( inal zinc concen a ion, 36.8 ␮g/ml) (lanes 4
and 7). Lanes: 1, molecula weigh ma ke s; 2 o 4, OMP p o ile o
PAO1; 5 o 7, OMP p o ile o Op D-nega i e PAO1. Whi e a ow,
Op D2; black a ow, Op D3. Numbe s on he le a e in kilodal ons.
2314 NOTES ANTIMICROB.AGENTS CHEMOTHER.
1993. Suscep ibili y o an imic obial agen s o Pseudomonas ae uginosa a -
ached o siliconized la ex u ina y ca he e s. Eu . J. Clin. Mic obiol. In ec .
Dis. 12:761–766.
17. Pollack, M. 1990. Pseudomonas ae uginosa, p. 1980–2002. In G. L. Mandell,
J. E. Benne , and R. Dolin (ed.), P inciples and p ac ice o in ec ious dis-
eases, 4 h ed. Chu chill Li ings one, New Yo k, N.Y.
18. Quinn, J. P., E. J. Dudek, C. A. DiVicenzo, D. A. Lucks, and S. A. Le ne .
1986. Eme gence o esis ance o imipenem du ing he apy o Pseudomonas
ae uginosa in ec ions. J. In ec . Dis. 154:289–294.
19. She chenko, A., M. Wilm, O. Vo m, and M. Mann. 1996. Mass spec ome ic
sequencing o p o eins sil e -s ained polyac ylamide gels. Anal. Chem. 68:
850–858.
20. Sumi a, Y., and M. Fukasawa. 1993. T ansien ca bapenem esis ance in-
duced by salicyla e in Pseudomonas ae uginosa associa ed wi h supp ession o
ou e memb ane p o ein D2 syn hesis. An imic ob. Agen s Chemo he . 37:
2743–2746.
21. T ias, J., and H. Nikaido. 1990. Ou e memb ane p o ein D2 ca alyzes
acili a ed di usion o ca bapenems and penems h ough he ou e mem-
b ane o Pseudomonas ae uginosa. An imic ob. Agen s Chemo he . 34:52–
57.
22. T ias, J., and H. Nikaido. 1990. P o ein D2 channel o Pseudomonas ae ugi-
nosa ou e memb ane has a binding si e o basic amino acids and pep ides.
J. Biol. Chem. 265:15680–15684.
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