Role of Klebsiella pneumoniae LamB porin in antimicrobial resistance

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oc . 2003, p. 3332–3335 Vol. 47, No. 10 0066-4804/03/$08.00⫹0 DOI: 10.1128/AAC.47.10.3332–3335.2003 Copy igh © 2003, Ame ican Socie y o Mic obiology. All Righ s Rese ed. Role o Klebsiella pneumoniae OmpK35 Po in in An imic obial Resis ance An onio Dome´nech-Sa´nchez, 1,2 Luis Ma ínez-Ma ínez, 3,4 * San iago He na´ndez-Alle´s, 1,2 Ma ía del Ca men Conejo, 3 A ´l a o Pascual, 3,4 Juan M. Toma´s, 5 Sebas ia´n Albe í, 1,2,6 and Vicen e Ja ie Benedí 1,2 † Labo a o y o Mic obiology, Depa men o Biology, Uni e si y o he Balea ic Island, 1 IMEDEA (CSIC-UIB), 2 and Resea ch Uni , Uni e si y Hospi al Son Du e a, 6 Palma de Mallo ca, Depa men o Mic obiology, School o Medicine, Uni e si y o Se ille, 3 and Uni e si y Hospi al V. Maca ena, 4 Se ille, and Depa men o Mic obiology, Uni e si y o Ba celona, 5 Ba celona, Spain Recei ed 25 Feb ua y 2003/Re u ned o modi ica ion 21 Ap il 2003/Accep ed 12 July 2003 OmpK35 om Klebsiella pneumoniae is he homologue o Esche ichia coli OmpF po in. Exp ession o OmpK35 in K.pneumoniae s ain CSUB10R (lacking bo h OmpK35 and OmpK36) dec eased he MICs o cephalospo ins and me openem >128- old and dec eased he MICs o imipenem, cip o loxacin, and chlo am- phenicol >8- old. MIC educ ions by OmpK35 we e 4 imes (ce epime), 8 imes (ce o e an, ce o axime, and ce pi ome), o 128 imes (ce azidime) highe han hose caused by OmpK36, bu he MICs we e simila o 1 dilu ion lowe o o he e alua ed agen s. Klebsiella pneumoniae p oduces wo majo po ins (OmpK35 and OmpK36) and he quiescen po in OmpK37. De ails on OmpK36 and OmpK37 ha e been p e iously epo ed (1, 6, 10). Mos clinical isola es o K.pneumoniae lacking ex ended- spec um ␤-lac amase (ESBL) exp ess bo h OmpK35 and OmpK36 po ins, while mos ESBL-exp essing K.pneumoniae clinical isola es p oduce only OmpK36 (9). Un il now, he ew clinical isola es lacking bo h OmpK35 and OmpK36 ha e been ESBL-p oducing s ains (14). Loss o OmpK36 is ela ed o ce oxi in esis ance and in- c eased esis ance o oxyimino- and zwi e ionic cephalospo- ins in s ains p oducing ESBL and o ca bapenem esis ance in s ains p oducing plasmid-media ed AmpC- ype ␤-lac a- mase (3, 4, 13, 15). Loss o OmpK36 also esul s in a mode a e inc ease in luo oquinolone esis ance in s ains wi h al e ed opoisome ases and/o ac i e e lux o quinolones. P elimina y esul s (6) indica e ha OmpK35 allows e icien pene a ion o ce oxi in, ce o axime, and ca bapenems, bu he e has been some con o e sy on he ole o his po in in cephalospo in pene a ion in K.pneumoniae (18). De ailed s udies on he impo ance o OmpK35 in an imic obial esis- ance a e lacking. In o de o in es iga e he ole o OmpK35 in an imic obial esis ance, we cloned he ompK35 gene. Fo his pu pose, genomic DNA om K.pneumoniae s ain KT755 (19) was diges ed wi h Sau3A. F agmen s we e liga ed o cosmid pLA2917 (2) and used o ans o m Esche ichia coli DH5␣ (17). Recombinan s we e sc eened o ompK35 by PCR using p ime s U681 (5⬘-CGG TTA CGG CCA GTG GGA ATA-3⬘) and L1316 (5⬘-GAC GCA GAC CGA AAT CGA ACT-3⬘), speci ic o en e obac e ial po ins and loca ed 215 and 850 bp downs eam o he ompK36 s a codon, espec i ely (6). The sizes o PCR-ampli ied p oduc s om ompF- ype genes a e di e en om hose o o he po in genes (da a no shown). One clone ca ying a plasmid, designa ed pSHA15, p oduced an amplicon o he desi ed size. Ou e memb ane p o eins (OMPs) we e isola ed as desc ibed p e iously (1, 13). Wes e n blo analysis o OMPs was pe o med on Immobilon P il e s (Millipo e, Bed o d, Mass.) using an i-OmpK35 an ibody (di- lu ed 1:1,000) and alkaline phospha ase-labeled goa an i- ab- bi immunoglobulin G (dilu ed 1:5,000) (13). OMP p o iles o E.coli DH5␣ca ying pSHA15 exhibi ed a band wi h he same mobili y as ha o OmpK35 exp essed by K.pneumoniae KT755 (da a no shown). The exp ession o OmpK35 was down egula ed in a high-osmola i y cul u e me- dium, as occu s wi h he OmpF-like po ins (9), and OmpK35 eac ed wi h an i-OmpK35 in immunoblo expe imen s (da a no shown). The OmpK35 p o ein exp essed by he E.coli clone was ex ac ed by po in ex ac ion me hods based on he ypsin esis ance o po ins and hei s ong nonco alen asso- cia ion wi h he pep idoglycan, and i also e ained i s hea modi iabili y. The ompK35 gene o K.pneumoniae KT755 was sequenced (EMBL da abase accession no. AJ011501). The amino acid sequence o OmpK35 was aligned wi h he sequences o o he en e obac e ial po ins (5, 7) (Fig. 1) on he basis o he con- se a ion o he ␤-s ands and some key esidues ha a e well conse ed in po ins: Lys16, A g38, Glu58, A g75, Asp106, Glu110, and A g126. OmpK35 is an OmpF homologue and p esen s a ypical 16 ␤-s and s uc u e, wi h eigh sho pe iplasmic u ns and eigh ex acellula loops o a iable leng hs. OmpK35 loop 3, which de ines he size o he ans- memb ane po e in o he po ins, ex ends inside he ba el and is he mos conse ed loop and con ains only one mo e esidue han OmpF and OmpC om E.coli and OmpK36 om K. pneumoniae. Fo suscep ibili y es ing expe imen s, he ompK35 gene was cloned in pWSK30 (20) and endowed wi h a kanamycin esis- * 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: 34-955008287. Fax: 34-954377413. E-mail: [email p o ec ed]. † Deceased. 3332 on July 27, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om ance casse e om pCS12 (8) o gi e pSHA16K. The pSHA16K plasmid was cloned in o he po in-de icien K.pneu- moniae CSUB10R clinical isola e and in o he clonally ela ed (3) isola e K.pneumoniae CSUB10S (exp essing OmpK36). The exp ession o OmpK35 does no in e e e wi h ␤-lac a- mase ac i i y in CSUB10R, as de e mined spec opho ome i- cally wi h c ude supe na an s om sonica ed cells as he en- zyme sou ce (15) (da a no shown). MICs o ce oxi in (Sigma, Mad id, Spain), ce o e an (Zen- eca, Mad id, Spain), ce azidime (Glaxo, Ba celona, Spain), ce o axime (Sigma), ce epime (B is ol-Mye s Squibb, Mad id, Spain), ce pi ome (Hoechs Ma ion-Roussel, Romain ille, F ance), imipenem (Me ck Sha p & Dohme, Mad id, Spain), me openem (Zeneca), cip o loxacin (Sigma), clina loxacin (Pa ke-Da is, Ann A bo , Mich.), amikacin (Sigma), gen ami- cin (Sigma), e acycline (Sigma), and chlo amphenicol (Sig- ma) o s ains CSUB10S and CSUB10R and OmpK35-ex- p essing ansconjugan s de i ed om hese wo s ains (Table 1) we e de e mined by mic odilu ion, acco ding o Na ional Commi ee o Clinical Labo a o y S anda ds (NCCLS) guide- lines (16). An imic obial agen MICs o K.pneumoniae CSUB10R con aining plasmids pKSK ( ec o ) and pSHA25K (OmpK36) we e also de e mined o compa ison. Exp ession o OmpK35 in K.pneumoniae CSUB10R e- duced (Fig. 2) he MICs o all agen s es ed wo o mo e imes. The highes educ ions (ⱖ128- old) we e obse ed o cepha- mycins, oxyimino-cephalospo ins, zwi e ionic cephalospo ins, and me openem. Signi ican educ ions (ⱖ8- old) we e also no ed o imipenem, cip o loxacin, and chlo amphenicol. The lowes educ ions we e ob ained o hose agen s o which K. FIG. 1. Compa ison by alignmen o he deduced OmpK35 sequence om K.pneumoniae wi h he sequences o OmpF and OmpC om E.coli and OmpK36 om K.pneumoniae a ailable in GenBank, EMBL, and DDBJ. Seconda y s uc u e mo i s a e desc ibed on he basis o he c ys al s uc u e o OmpK36. The numbe ing is based on he ma u e OmpK36. Conse ed amino acids (shaded and boxed) and gaps in oduced o maximize alignmen (hyphens) a e indica ed. VOL. 47, 2003 NOTES 3333 on July 27, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om pneumoniae CSUB10R was al eady suscep ible: clina loxacin, e acycline, amikacin, and gen amicin. Exp ession o OmpK36 in CSUB10R also dec eased he MICs o all an imic obial agen s es ed, excep clina loxacin, o alues simila o he MICs o he ela ed clinical isola e CSUB10S. MIC educ- ions caused by OmpK35 exp ession we e 4 imes (ce epime), 8 imes (ce o e an, ce o axime, and ce pi ome), o 128 imes (ce azidime) highe han hose caused by OmpK36 exp es- sion. These esul s, howe e , do no necessa ily mean ha OmpK35 should be conside ed speci ic o hese agen s, as exp ession o OmpK36 also signi ican ly educed hei MICs. MIC educ ions caused by OmpK35 we e he same (me o- penem, amikacin, gen amicin, and e acycline) o 1 dilu ion s ep lowe (ce oxi in, imipenem, cip o loxacin, clina loxacin, and chlo amphenicol) han hose caused by OmpK36. Exp ession o OmpK35 in K.pneumoniae CSUB10S, leading o he simul aneous exp ession o he wo majo po ins o K. pneumoniae (Fig. 2), esul ed in he MICs o he e alua ed agen s being he same o one dilu ion s ep highe han hose agains he ans o man exp essing only OmpK35. Exp ession o bo h OmpK35 and o a lesse ex en OmpK36 dec eases he MICs o cip o loxacin o K.pneumoniae CSUB10R (which con ains a Se 83Phe change in he A subuni o DNA gy ase and exp esses ac i e e lux o luo oquinolones [12]), indica ing ha bo h po ins allow pene a ion o his d ug. Po in exp ession was minimally ele an o he ac i i y o clina loxacin, a luo oquinolone much mo e ac i e agains CSUB10R han cip o loxacin. OmpK35 and OmpK36 exp es- sion also dec eased he MICs o e acycline and chlo amphen- icol. These da a suppo he gene al ole o bo h po ins as hyd ophilic po es. MICs o aminoglycosides did no signi i- can ly change a e po in exp ession, p esumably because o he pene a ion o hese agen s by po in-independen pa h- ways. Mos ESBL-p oducing K.pneumoniae s ains lack OmpK35 (11). Loss o his po in may be one o he ac o s con ibu ing o an imic obial esis ance in ESBL-p oducing K.pneumoniae and may a o he selec ion o addi ional mechanisms o e- sis ance, including loss o OmpK36 and/o ac i e e lux (14). OmpK35 is no no mally exp essed in high-osmola i y me- dia, which may esul in ep ession o i s exp ession in K. pneumoniae in i o. This may be o he apeu ic impo ance because o he limi ed en ance o ce ain an imic obial agen s in K.pneumoniae. New s udies on po in exp ession in K.pneu- moniae g own in i o a e needed. This wo k was suppo ed in pa by g an s om he Fondo de In es igaciones Sani a ias o he Minis e io de Sanidad y Consumo o Spain o L.M.-M. and V.J.B and Plan Nacional de I⫹D (Minis e io de Ciencia y Tecnología) o J.M T. A.D.-S. was suppo ed by a p edoc- o al ellowship om he Minis e io de Educacio´n y Cul u a o he Spanish go e nmen . REFERENCES 1. Albe í, S., F. Rod íguez-Quin˜ones, T. Schi me , G. Rummel, J. M. Toma´s, J. P. Rosenbusch, and V. J. Benedí.1995. A po in om Klebsiella pneu- moniae: sequence homology, h ee-dimensional model, and complemen binding. In ec . Immun. 63:903–910. 2. Allen, L. N., and R. S. Hanson. 1985. Cons uc ion o b oad-hos - ange cosmid cloning ec o s: iden i ica ion o genes necessa y o g ow h o Me hy- lobac e ium o ganophilum on me hanol. J. Bac e iol. 161:955–962. 3. A danuy, C., J. Lin˜a es, M. A. Domínguez, S. He na´ndez-Alle´s, V. J. Benedí, and L. Ma ínez-Ma ínez. 1998. Ou e memb ane p o iles o clonally e- la ed Klebsiella pneumoniae isola es om clinical samples and ac i i ies o cephalospo ins and ca bapenems. An imic ob. Agen s Chemo he . 42:1636– 1640. 4. B ad o d, P. A., C. U ban, N. Ma iano, S. J. P ojan, J. J. Rahal, and K. Bush. 1997. Imipenem esis ance in Klebsiella pneumoniae is associa ed wi h he combina ion o ACT-1, a plasmid-media ed AmpC ␤-lac amase, and he loss o an ou e memb ane p o ein. An imic ob. Agen s Chemo he . 41:563– 569. 5. Cowan, S. W., T. Schi me , G. Rummel, M. S eie , R. Ghosh, R. A. Paup i , J. N. Jansonius, and J. P. Rosenbusch. 1992. C ys al s uc u es explain unc ional p ope ies o wo E.coli po ins. Na u e 358:727–733. 6. Dome´nech-Sa´nchez, A., S. He na´ndez-Alle´s, L. Ma ínez-Ma ínez, V. J. Benedí, and S. Albe í.1999. Iden i ica ion and cha ac e iza ion o a new po in gene o Klebsiella pneumoniae: i s ole in ␤-lac am an ibio ic esis ance. J. Bac e iol. 181:2726–2732. 7. Du zle , R., G. Rummel, S. Albe í, S. He na´ndez-Alle´s, P. Phale, J. Rosen- busch, V. Benedí, and T. Schi me . 1999. C ys al s uc u e and unc ional FIG. 2. Sodium dodecyl sul a e-polyac ylamide gel elec opho e ic analysis o OMPs om K.pneumoniae s ain CSUB10R (lane 1) and clones de i ed om CSUB10R ca ying plasmids pSHA25K (lane 3) and pSHA16K (lane 4). K.pneumoniae isola e CSUB10S and i s clone ca ying pSHA16K a e also shown in lanes 2 and 5, espec i ely. Po ins exp essed by each s ain a e indica ed abo e each lane. The posi ions o LamB (whi e a owhead) and OmpA (black a owhead) homo- logues o K.pneumoniae a e indica ed o he le o lane 1. TABLE 1. MICs o an imic obial agen s agains K. pneumoniae s ains wi h di e en pa e ns o po in exp ession S ain Po in(s) MIC (␮g/ml) a FOX CTT CAZ CTX FEP PIR IPM MEM CIP CLX AMK GEN TET CHL CSUB10S OmpK36 2 0.06 256 8 2 4 0.125 0.03 0.5 0.06 1 4 2 16 CSUB10R None 128 32 ⬎512 512 512 512 1 4 4 0.125 1 8 4 64 CSUB10R/pSHA16K OmpK35 1 0.03 2 0.5 0.125 0.5 0.125 0.03 0.25 0.06 0.5 4 1 8 CSUB10R/pSHA25K OmpK36 2 0.125 256 4 0.5 4 0.25 0.03 0.5 0.125 0.5 4 1 16 CSUB10S/pSHA16K OmpK35 and OmpK36 1 0.03 4 1 0.125 0.5 0.125 0.03 0.5 0.06 1 4 1 8 CSUB10R/pKSK None 128 16 ⬎512 256 128 256 1 4 2 0.125 1 8 2 64 a Abb e ia ions: FOX, ce oxi in; CTT, ce o e an; CAZ, ce azidime; CTX, ce o axime; FEP, ce epime; PIR, ce pi ome; IPM, imipenem; MEM, me openem; CIP; cip o loxacin; CLX, clina loxacin; AMK, amikacin; GEN, gen amicin; TET, e acycline; CHL, chlo amphenicol. 3334 NOTES ANTIMICROB.AGENTS CHEMOTHER. on July 27, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om cha ac e iza ion o OmpK36, he osmopo in o Klebsiella pneumoniae. S uc- u e Fold Des. 7:425–434. 8. Elhai, J., and C. P. Wolk. 1988. A e sa ile class o posi i e-selec ion ec o s based on he non iabili y o palind ome-con aining plasmids ha allows cloning in o long polylinke s. Gene 68:119–138. 9. He na´ndez-Alle´s, S., S. Albe í, D. Al a ez, A. Dome´nech-Sa´nchez, L. Ma - ínez-Ma ínez, J. Gil, J. M. Toma´s, and V. J. Benedí.1999. Po in exp ession in clinical isola es o Klebsiella pneumoniae. Mic obiology 145:673–679. 10. He na´ndez-Alle´s, S., S. Albe í, M. D. Al a ez, L. Ma ínez-Ma ínez, J. Gil, J. M. Toma´s, and V. J. Benedí.1995. Isola ion o FC3–11, a bac e iophage speci ic o he Klebsiella pneumoniae po in OmpK36, and i s use o he isola ion o po in-de icien mu an s. Can. J. Mic obiol. 41:399–406. 11. He na´ndez-Alle´s, S., M. Conejo, A. Pascual, J. M. Toma´s, V. J. Benedí, and L. Ma ínez-Ma ínez. 2000. Rela ionship be ween ou e memb ane al e - a ions and suscep ibili y o an imic obial agen s in isogenic s ains o Kleb- siella pneumoniae. J. An imic ob. Chemo he . 46:273–277. 12. Ma ínez-Ma ínez, L., I. Ga cía, S. Balles a, V. J. Benedí, S. He na´ndez- Alle´s, and A. Pascual. 1998. Ene gy-dependen accumula ion o luo oquino- lones in quinolone- esis an Klebsiella pneumoniae s ains. An imic ob. Agen s Chemo he . 42:1850–1852. 13. Ma ínez-Ma ínez, L., S. He na´ndez-Alle´s, S. Albe í, J. M. Toma´s, V. J. Benedí, and G. A. Jacoby. 1996. In i o selec ion o po in-de icien mu an s o Klebsiella pneumoniae wi h inc eased esis ance o ce oxi in and expand- ed-spec um cephalospo ins. An imic ob. Agen s Chemo he . 40:342–348. 14. Ma ínez-Ma ínez, L., A. Pascual, M. C. Conejo, I. Ga cía, P. Joyanes, A. Dome´nech-Sa´nchez, and V. J. Benedí.2002. Ene gy-dependen accumula- ion o no loxacin and po in exp ession in clinical isola es o Klebsiella pneumoniae and ela ionship o ex ended-spec um ␤-lac amase p oduc ion. An imic ob. Agen s Chemo he . 46:3926–3932. 15. Ma ínez-Ma ínez, L., A. Pascual, S. He na´ndez-Alle´s, D. Al a ez-Díaz, A. I. Sua´ ez, J. T an, V. J. Benedí, and G. A. Jacoby. 1999. Roles o ␤-lac- amases and po ins in ac i i ies o ca bapenems and cephalospo ins agains Klebsiella pneumoniae. An imic ob. Agen s Chemo he . 43:1669–1673. 16. 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. 17. Samb ook, J., E. F. F i sch, and T. Mania is. 1989. Molecula cloning: a labo a o y manual, 2nd ed. Cold Sp ing Ha bo Labo a o y P ess, Cold Sp ing Ha bo , N.Y. 18. Siu, L. K. 2001. Is OmpK35 speci ic o ce azidime pene a ion? An imi- c ob. Agen s Chemo he . 45:1601–1602. 19. Toma´s, J. M., V. J. Benedí, B. Ciu ana, and J. Jo e. 1986. Role o capsule and O an igen in esis ance o Klebsiella pneumoniae o se um bac e icidal ac i i y. In ec . Immun. 54:85–89. 20. Wang, R. F., and S. R. Kushne . 1991. Cons uc ion o e sa ile low-copy- numbe ec o s o cloning, sequencing and gene exp ession in Esche ichia coli. Gene 100:195–199. VOL. 47, 2003 NOTES 3335 on July 27, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om