Zinc eluted from siliconized latex urinary catheters decreases OprD expression, causing carbapenem resistance in Pseudomonas aeruginosa

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. 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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. VOL. 47, 2003 NOTES 2315