Development of resistance during antimicrobial therapy caused by insertion sequence interruption of porin genes

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 0066-4804/99/$04.0010 Ap . 1999, p. 937–939 Vol. 43, No. 4 Copy igh © 1999, Ame ican Socie y o Mic obiology. All Righ s Rese ed. NOTES De elopmen o Resis ance du ing An imic obial The apy Caused by Inse ion Sequence In e up ion o Po in Genes SANTIAGO HERNA ´NDEZ-ALLE ´S, 1 VICENTE J. BENEDI ´, 1 LUIS MARTI ´NEZ-MARTI ´NEZ, 2 A ´LVARO PASCUAL, 2 ALICIA AGUILAR, 3 JUAN M. TOMA ´S, 3 AND SEBASTIA ´N ALBERTI ´ 1 * A ´ ea de Mic obiologı´a, Depa amen o de Biologı´a, Uni e sidad de las Islas Balea es and IMEDEA (CSIC-UIB), Palma de Mallo ca, 1 Depa amen o de Mic obiologı´a, Uni e sidad de Se illa, Se ille, 2 and Depa amen o de Mic obiologı´a, Uni e sidad de Ba celona, Ba celona, 3 Spain Recei ed 10 Augus 1998/Re u ned o modi ica ion 23 No embe 1998/Accep ed 22 Janua y 1999 We ha e demons a ed by using an in i o app oach ha in e up ion o he OmpK36 po in gene by inse - ion sequences (ISs) is a common ype o mu a ion ha causes loss o po in exp ession and inc eased esis- ance o ce oxi in in Klebsiella pneumoniae. This mechanism also ope a es in i o: o 13 po in-de icien ce oxi in- esis an clinical isola es o K. pneumoniae, 4 p esen ed ISs in hei ompK36 gene. Klebsiella pneumoniae is a majo nosocomial pa hogen, and success ul chemo he apy is c i ical o he ea men o in ec- ions caused by his mic oo ganism. An ibio ic- esis an s ains eme ge wi h a iable equency, pa icula ly in he hospi al en i onmen . K. pneumoniae esis ance o expanded-spec um cephalospo ins caused by plasmid-bo ne ex ended-spec um b-lac amases is a well-documen ed example and has inc eased since he 1980s (11, 14, 17). Addi ionally, e ec s in po in ex- p ession con ibu e o inc ease he esis ance le els p o ided by he mechanism desc ibed abo e (12). We ha e desc ibed po ins OmpK36 and OmpK35 (2, 7), he K. pneumoniae homologues o Esche ichia coli po ins OmpC and OmpF, espec i ely. Clinical isola es o his species exp ess ei he OmpK36 o OmpK35 po ins o bo h (6). Recen ly, we epo ed ha loss o po ins OmpK36 and OmpK35 in ex- ended-spec um b-lac amase-p oducing clinical isola es o K. pneumoniae caused inc eased esis ance o ce oxi in and expanded-spec um cephalospo ins (3, 9). The ela ionship be ween po in loss and an imic obial esis ance has also been demons a ed in o he bac e ial species (1, 10). Howe e , he mechanisms leading o po in de iciency and subsequen an i- mic obial esis ance a e la gely unknown. The pu pose o he p esen s udy was o iden i y he mechanisms causing po in de iciency in clinical isola es o K. pneumoniae. Fo his pu - pose, we i s used an in i o app oach. We selec ed ce oxi in- esis an mu an s de i ed om s ain LB4(pSHA2) (9). S ain LB4 is a po in-de icien , ce oxi in- esis an (MIC, 128 mg/ml) clinical isola e ha e e ed o ce oxi in sensi i i y (MIC, 4 mg/ml) a e he plasmid pSHA2 ca ying he OmpK36 po in gene was cloned. Ce oxi in- esis an mu an s we e ob ained by pla ing LB4(pSHA2) on inc easing concen a ions o ce oxi in. Mu an s wi h inc eased esis ance o ce oxi in (MIC, .48 mg/ml), de e mined acco ding o he Na ional Commi ee o Clinical Labo a o y S anda ds ecommenda ions, we e selec - ed and u he cha ac e ized. The ou e memb ane p o eins (OMPs) om hese mu an s we e isola ed om bac e ial en- elopes as sodium lau yl sa cosina e-insoluble ma e ial (5) and analyzed by sodium dodecyl sul a e-polyac ylamide gel elec- opho esis. We ound ha exp ession o po in OmpK36 was comple ely abolished in hose mu an s. In o de o s udy pos- sible mu a ions a ec ing he ompK36 gene, we isola ed he pSHA2 plasmid om he ce oxi in- esis an mu an s. Plasmids we e analyzed by Sou he n blo ing (4) wi h an ompK36 p obe ob ained by PCR ampli ica ion o plasmid pSHA2 wi h p ime s U228 (59-GGTAAAAAAAACCGGATGCG-39) and L1730 (59-CGTGCTTAGAACTGGTAAACC-39), which anneal o sequences 427 bp ups eam and 1,097 bp downs eam o he ompK36 s a codon (accession no. Z33506), espec i ely. These analyses e ealed ha di e en e en s occu ed in he ompK36 gene con ained in pSHA2: poin mu a ions o small dele ions, dele ions o he en i e gene, and inse ions. These mu an plasmids de i ed om pSHA2 we e designa ed pSHA ollowed by an a bi a y numbe (Table 1). As shown in Table 1, he mos equen ypes o changes de ec ed in he mu an * Co esponding au ho . Mailing add ess: UIB-Mic obiologı´a, Ca - e e a de Valldemosa Km. 7.5, 07071-Palma de Mallo ca, Spain. Phone: 34-971-173335. Fax: 34-971-173184. E-mail: [email p o ec ed]. TABLE 1. Analysis o andomly selec ed mu an plasmids con e ing inc eased esis ance o ce oxi in (MIC, .48 mg/ml) de i ed om s ain LB4(pSHA2) Plasmid Type o mu a ion IS posi ion a pSHA119 Poin mu a ion o small dele ion pSHA124 Poin mu a ion o small dele ion pSHA125 Poin mu a ion o small dele ion pSHA128 Inse ion o IS187 pSHA123 Inse ion o IS903 8 pSHA103 Inse ion o IS524 pSHA100 Inse ion o IS26 14 pSHA146 Inse ion o IS195 pSHA149 Inse ion o IS57 pSHA151 Inse ion o IS529 pSHA101 Inse ion o IS26 77 pSHA154 Inse ion o IS903 28 pSHA157 Dele ion pSHA104 Dele ion a Values indica e he posi ions (in base pai s) o he ISs wi h espec o he ompK36 s a codon. 937 on Augus 1, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://aac.asm.o g/Downloaded om plasmids we e inse ions. Among 14 andomly selec ed mu- an s, 9 (64.3%) p esen ed an inse ion. De ailed cha ac e iza- ion o he na u e and posi ion o he inse ed DNA was ca - ied ou by sequencing he mu an plasmids. DNA sequencing was pe o med wi h p ime s U681 (59-CGGTTACGGCCAG TGGGAATA-39) and L1316 (59-GACGCAGACCGAAATC GAACT-39), which ep esen DNA sequences 230 and 345 bp downs eam o he ompK36 s a codon, and p ime s U228 and L1730. We de ec ed ou di e en inse ion sequences (ISs; IS26,IS5,IS903, and IS1) loca ed in di e en si es o he ompK36 gene. Fu he mo e, wi h p obes speci ic o he iden- i ied ISs (ob ained by elu ion o an IS in e nal es ic ion agmen om he co esponding mu an plasmid), we e i ied by Sou he n blo analysis ha he ISs we e p esen in many copies in he ch omosome o he o iginal s ain LB4 (da a no shown). Since esis ance o ce oxi in in labo a o y-de i ed mu an s was dependen on mu a ions o he OmpK36 po in gene, p e- dominan ly inse ion o ISs, we in es iga ed whe he his ype o mechanism also occu ed in i o. Fo his pu pose, we am- pli ied he en i e ompK36 gene om wo pai s o s ains iso- la ed om wo pa ien s be o e and a e an imic obial he apy: s ains LB1 and LB4 (9) and s ains CMD1 and CMD2 (19). LB4 and CMD2 a e po in-de icien ce oxi in- esis an clinical isola es de i ed om he po in-su icien , ce oxi in-sensi i e s ains LB1 and CMD1, espec i ely. Addi ionally, we cha ac- e ized he OmpK36 po in gene in 11 addi ional po in-de i- cien K. pneumoniae clinical isola es: s ains HUSR2/94, C1, LB66, LB68, LB73, CSUB2, CSUB8R, CSUB9R, CSUB10R, CSUB11, and CSUB12. Analysis o he amplicons wi h he ompK36 gene p obe de- ec ed inse ions, seen as inc eases in he size o ompK36,in some o hese clinical isola es (Fig. 1A). In addi ion, we ob- se ed in s ain HUSR2/94 a dele ion on he OmpK36 po in gene (Fig. 1A, lane 2). Fu he cha ac e iza ion o he abo e- desc ibed inse ions was ob ained by sequencing he PCR am- plicons. We iden i ied he p esence o IS102 in s ain CMD2, and we iden i ied inse ions wi h a high deg ee o sequence iden i y o IS5in he amplicons om s ains LB73, CSUB2, and CSUB11. These ISs we e loca ed in di e en posi ions wi hin he ompK36 coding egion (Fig. 1B). These esul s dem- ons a e ha po in loss due o inse ions o ISs wi hin he po in genes is a mechanism ha ope a es in i o in pa ien s unde - going an ibio ic ea men . I has been sugges ed ha a low le el o andom ansposi- ion migh help cells o adap o en i onmen al changes and o inc ease he su i al a e (18), and i is also well known ha ISs can in e up o al e gene exp ession. The e a e many exam- ples in he li e a u e whe e ISs ha e been in ol ed in esis ance o ce ain an ibio ics by c ea ing no el p omo e s o an imi- c obial esis ance genes (8, 15). Howe e , o ou knowledge, his is he i s epo whe e an imic obial esis ance is caused by in e up ion o po in genes by ISs. Mechanisms o po in loss and inc eased esis ance o imipenem we e also s udied in Pseudomonas ae uginosa by Yoneyama and Nakae by using an in i o app oach (20): only dele ions in he Op D2 po in gene we e obse ed. Howe e , we ha e shown ha , bo h in i o and in i o, ISs cause po in de iciency in K. pneumoniae. ISs a e widesp ead in all bac e ial species s udied, and, al hough hei p esence has no been co ela ed wi h an imic obial esis ance, hey ha e also been ound in E. coli po ins (13, 16). Thus, in e up ion o po in genes by ISs is he e o e no an exclusi e phenomenon o K. pneumoniae. Fu he s udies will be e- qui ed o de e mine he impo ance o his mechanism in o he bac e ial species. This wo k was suppo ed by g an s om he Comisio´n In e minis- e ial de Ciencia y Tecnologı´a (CICYT). S.H.A. and S.A. we e sup- po ed by a p edoc o al ellowship and a pos doc o al con ac om CICYT, espec i ely. We hank J. A. M. an de Klunde (Uni e si y Hospi al Leiden), G. Jacoby (Lahey Hi chcock Clinic), and J. 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