Identification and broad dissemination of the CTX-M-14 β-lactamase in different Escherichia coli strains in the northwest area of Spain

JOURNAL OF CLINICAL MICROBIOLOGY, No . 2002, p. 4030–4036 Vol. 40, No. 11 0095-1137/02/$04.00⫹0 DOI: 10.1128/JCM.40.11.4030–4036.2002 Copy igh © 2002, Ame ican Socie y o Mic obiology. All Righ s Rese ed. Iden i ica ion and B oad Dissemina ion o he CTX-M-14 ␤-Lac amase in Di e en Esche ichia coli S ains in he No hwes A ea o Spain Ge man Bou,* Monica Ca elle, Ma ia Tomas, Delia Canle, F ancisca Molina, Ri a Mou e, Jose Ma ia Ei os, and An onio Gue e o† Se icio de Mic obiología, Complejo Hospi ala io Juan Canalejo, 15006 La Co un˜a, Spain Recei ed 22 Ap il 2002/Re u ned o modi ica ion 2 June 2002/Accep ed 5 Augus 2002 Du ing he cou se o a molecula epidemiology s udy o mechanisms o an ibio ic esis ance in he a ea se ed by ou hospi al (516,000 inhabi an s), we isola ed he gene encoding CTX-M-14 ␤-lac amase. Thi y clinical s ains (27 Esche ichia coli and 3 Klebsiella pneumoniae isola es) wi h a pheno ype o ex ended-spec um ␤-lac amase we e collec ed om Janua y o Oc obe 2001 and s udied o he p esence o he CTX-M-14 ␤- lac amase gene. By isoelec ic poin de e mina ion, PCR, and nucleo ide sequencing, we de ec ed he p esence o his gene in 17 E. coli s ains belonging o 15 di e en geno ypes (REP-PCR) causing in ec ions in 17 di e en pa ien s. Epidemiological s udies based on medical eco ds did no sugges any ela ionship be- ween he pa ien s in ec ed wi h hese E. coli s ains and, in e es ingly, 7 o 30 pa ien s ha bo ing s ains wi h ex ended-spec um ␤-lac amases ne e had con ac wi h he hospi al en i onmen be o e he clinical E. coli isola ion. Conjuga ion expe imen s e ealed ha his gene was plasmid media ed in he 17 E. coli s ains, and plasmid es ic ion agmen leng h polymo phisms showed 9 di e en pa e ns in he 17 E. coli s ains. By PCR, he sequence o he npA ansposase gene o he inse sequence ISEcp-1 was de ec ed in all he plasmids ha bo ing he CTX-M-14 gene. These esul s s ongly sugges ha plasmid dissemina ion be ween di e en E. coli s ains in addi ion o a mobile elemen ( ansposon) a ound he ␤-lac amase gene may be in ol ed in he sp eading o he CTX-M-14 gene. This s udy ein o ces he hypo hesis ha he epidemiology o he p e - alence o he ␤-lac amase genes is changing and should ale he medical communi y o he inc ease in he eme gence o he CTX-M ␤-lac amases wo ldwide. The eme gence o plasmid-media ed ex ended-spec um ␤-lac amases (ESBLs) in membe s o he amily En e obac e- iaceae has become a wo ldwide p oblem (6, 12, 21). ESBL molecula class A possesses an ex ended hyd olysis spec um owa d oxyimino-␤-lac ams and az eonam bu emains sus- cep ible o ce oxi in and ␤-lac amase inhibi o s (7). Mos ESBLs a e de i a i es o TEM-1, TEM-2, o SHV-1 enzymes; howe e , epo s desc ibing he eme gence o ␤-lac amases belonging o o he amilies, such as CTX-M and/o OXA de- i a i es, a e inc easing wo ldwide (5, 8, 10, 11, 16). CTX-M- ela ed enzymes ha e been ound in Eu ope, Sou h Ame ica, and Medi e anean coun ies (3, 13, 15, 16, 18, 21). Unlike mic oo ganisms ha bo ing TEM and SHV ␤-lac a- mases, epo s o ou b eaks caused by o ganisms ha bo ing non-TEM o non-SHV ESBL a e e y sca ce. CTX-M-14 ␤-lac amase has ecen ly been desc ibed in clinical isola es in Ko ea, China, and F ance (8, 11, 16, 19). In his communica- ion we no only epo o he i s ime he p esence o he CTX-M-14 gene in Spain bu we also show clea e idence o he dissemina ion o he gene encoding CTX-M-14 among 17 di e en pa ien s, who had no appa en con ac , li ing in he no hwes pa o Spain. This CTX-M-14 gene was de ec ed in 15 di e en geno ypes o Esche ichia coli s ains causing u i- na y ac in ec ion in 17 pa ien s in an a ea o 516,000 inhab- i an s. Plasmid dissemina ion and he p esence o a pu a i e ansposon a ound he CTX-M-14 gene may accoun o he sp eading o his gene. This inding emphasizes he inc easing ole o he CTX-M ␤-lac amases in an ibio ic esis ance wo ld- wide and leads o conside a ion o empi ical ea men o in ec ions caused by E. coli s ains, especially in pa ien s com- p omised by unde lying disease o immunological s a us. MATERIALS AND METHODS Selec ion o clinical isola es and pa ien s. Be ween Janua y and Oc obe 2001, 27 E. coli and 3 Klebsiella pneumoniae clinical s ains wi h an ESBL pheno ype we e collec ed in he Depa men o Mic obiology, Juan Canalejo Hospi al, a 1,200-bed hospi al se ing a popula ion o 516,000 in he no hwes a ea o Spain. These isola es we e iden i ied by DadeMic oScan and he API 20E sys em (bioMe´ ieux, Ma cy l’E oile, F ance). Suscep ibili y es s and con i ma ion o ESBL p oduc ion. The MICs o he ␤-lac ams we e de e mined by using E- es s ips (AB Biodisk, Dal a¨gen, Swe- den). The ESBL pheno ype was de ec ed by using ESBL de ec ion E- es s ips (AB Biodisk) as speci ied by he manu ac u e . The double disk di usion es was concomi an ly used o con i m he E- es esul s. IEF assay. ␤-Lac amases we e cha ac e ized by isoelec ic ocusing (IEF) o ul asonica ed bac e ial ex ac s. Bac e ia g owing exponen ially a 37°C in Lu- ia-Be ani (LB) medium we e ha es ed, and cell- ee lysa es we e p epa ed by sonica ion. ␤-Lac amases we e analyzed by isoelec ic ocusing o cell ex ac s on polyac ylamide gels con aining ampholy es wi h a pH ange o 3 o 9 (Phas Gel; Ame sham Pha macia Bio ech AB) in a Phas Sys em appa a us (Ame sham Pha macia Bio ech AB). The ocused ␤-lac amases we e de ec ed by o e laying he gel wi h ni oce in (0.5 mg/ml) in 100 mM phospha e bu e (pH 7.0). pIs we e de e mined by compa ison wi h hose o ␤-lac amases wi h a known pI: TEM-1 (pI 5.4), SHV-1 (pI 7.6), IMP-2 (pI 8.2), and AmpC om A. baumannii (pI 9.4). DNA ex ac ion. Bac e ial ch omosomal DNA was ob ained wi h he High Pu e PCR empla e p epa a ion ki (Roche Diagnos ics Co p., Indianapolis, Ind.). The ch omosomal DNA was checked by elec opho esis in aga ose gels, * Co esponding au ho . Mailing add ess: Se icio de Mic obiol- ogía, Hospi al Ma e no In an il, Complejo Hospi ala io Juan Cana- lejo, C/Xubias de A iba 84, 15006 La Co un˜a, Spain. Phone: 34-981- 178000, ex . 21171. Fax: 34-981-178216. E-mail: ge manbou@canalejo .o g. † P esen add ess: Hospi al de la Ribe a, Alzi a, Valencia, Spain. 4030 and he concen a ions o he di e en ex ac s we e s anda dized by spec o- pho ome ic measu emen s. Conjuga ion expe imen s, plasmid pu i ica ion, cloning expe imen s, and DNA sequencing. T ans e o esis ance by conjuga ion was a emp ed using E. coli BM21 and E. coli XL-10-Gold Kan cells (S a agene, La Jolla, Cali .) as ecipien s. O e nigh ma ing expe imen s we e pe o med a 37°C, and he ansconjugan s we e selec ed on LB aga pla es supplemen ed wi h ampicillin (50 ␮g/ml) and nalidixic acid (50 ␮g/ml) o E. coli BM21 and ampicillin (50 ␮g/ml) and kanamycin (25 ␮g/ml) o E. coli XL-10-Gold Kan cells. A e wa d, ansconjugan s we e selec ed o he ce o axime esis ance pheno ype. Plasmids om di e en E. coli s ains we e pu i ied by he alkaline-lysis me hod wi h he High Pu e plasmid isola ion ki (Roche Diagnos ics Co p.). Cloning p ocedu es we e pe o med as desc ibed by Samb ook e al. (20). Re- s ic ion enzymes we e pu chased om Boeh inge (Mannheim, Ge many) and we e used as speci ied by he manu ac u e . Fo cloning he CTX-M-14 gene, plasmid DNA om ansconjugan E. coli clinical s ain 3 (see Table 2) was diges ed wi h BamHI. The esul ing agmen s we e liga ed in o pBGS18 and diges ed wi h BamHI, and he mix u e was ans- o med in o E. coli TG1 made compe en by he calcium chlo ide me hod (20). A e ans o ma ion, a ew clones g ew on LB aga pla es supplemen ed wi h kanamycin (10 ␮g/ml) and ampicillin (50 ␮g/ml). They ha bo ed an iden ical plasmid (pC) wi h an inse o abou 6 kb. These plasmids we e used as em- pla es o de e mine he nucleo ide sequence. Templa es we e sequenced on bo h s ands by he me hod o Sange e al. Sequencing was ca ied ou wi h he Taq DyeDeoxyTe mina o cycle-sequencing ki using p ime s speci ic o he coding sequence, and he sequence was analyzed in an au oma ic DNA sequence (377 ABI P ism; Pe kin-Elme ). De ec ion o he CTX-M-14 gene. Plasmid DNA om di e en E. coli s ains was used as he empla e in a PCR ampli ica ion. Ampli ica ion eac ions we e pe o med in a inal olume o 50 ␮l. Mg 2⫹ - ee PCR bu e was pu chased as a 10⫻concen a e consis ing o 500 mM KCl, 100 mM T is-HCl (pH 9.0), and 1% T i on X-100 (Pe kin-Elme , Roche Molecula Sys ems, Inc., Nu ley, N.J.) wi h 200 ␮M (each) dATP, dCTP, dGTP, and dTTP (Pe kin-Elme , Roche Molecula Sys ems, Inc.). The Mg 2⫹ concen a ion was 2.5 mM, and he p ime s we e used a 0.5 ␮M each. The p ime pai CTX-M-1 (5⬘-AACACGGATTGACCGTAT TG-3⬘) and CTX-M-2 (5⬘-TTACAGCCCTTCGGCGAT-3⬘) was used o ampli y he CTX-M-14 gene in he plasmid DNA. Ampli ica ion eac ions we e ca ied ou in an Eppendo he mal cycle (Eppendo AG, Hambu g, Ge many), wi h an ini ial dena u a ion (10 min a 94°C) ollowed by 30 cycles o dena u a ion (30 sa 94°C), annealing (30 s a 58°C), and ex ension (2 min a 72°C), wi h a single inal ex ension o 10 min a 72°C. Aliquo s (15 ␮l) o each sample we e subjec ed o elec opho esis in 1.0% aga ose gels. Ampli ied p oduc s we e de ec ed a e s aining wi h e hidium b omide (50 ␮g/ml) and pho og aphed wi h Pola oid ype 665 ilm. REP-PCR. Ampli ica ion eac ions we e pe o med in a inal olume o 50 ␮l. Mg 2⫹ - ee PCR bu e was pu chased as a 10⫻concen a e consis ing o 500 mM KCl, 100 mM T is-HCl (pH 9.0), and 1% T i on X-100 (Pe kin-Elme , Roche Molecula Sys ems, Inc.), wi h 200 ␮M (each) dATP, dCTP, dGTP, and dTTP (Pe kin-Elme , Roche Molecula Sys ems, Inc.). The Mg 2⫹ concen a ion was 3 mM, and he p ime s we e used a 0.5 ␮M each. The p ime pai REP1 (5⬘- IIIGCGCCGICATCAGGC-3⬘) and REP2 (5⬘-ACGTCTTATCAGGCCTAC- 3⬘) was used o ampli y pu a i e REP-like elemen s in he genomic bac e ial DNA (23). A o al o 500 ng o ch omosomal DNA was added o he eac ion mix u e. Ampli ica ion eac ions we e ca ied ou in an Eppendo he mal cycle , wi h an ini ial dena u a ion (10 min a 94°C) ollowed by 30 cycles o dena u a ion (1 min a 94°C), annealing (1 min a 45°C), and ex ension (2 min a 72°C), wi h a single inal ex ension o 16 min a 72°C. Aliquo s (20 ␮l) o each sample we e subjec ed o elec opho esis in 1.0% aga ose gels. Ampli ied p od- uc s we e de ec ed a e s aining wi h e hidium b omide (50 ␮g/ml) and pho o- g aphed wi h Pola oid ype 665 ilm. S ains belonging o he same DNA g oup showed iden ical o highly simila p o iles (up o wo bands di e en ). Plasmid es ic ion agmen leng h polymo phisms (RFLPs). Plasmid DNA om ansconjugan E. coli s ains 1 o 17 (see Table 2) was isola ed as desc ibed abo e. The DNA was hen diges ed wi h HindIII, and he esul ing agmen s we e loaded on a 0.8% aga ose gel. A e elec opho esis, DNA agmen s we e de ec ed by s aining wi h e hidium b omide (50 ␮g/ml) and pho og aphed wi h Pola oid ype 665 ilm. De ec ion o he gene encoding he npA ansposase. To de ec he p esence o he npA gene o he inse sequence ISEcp-1, we used he p ime pai npa1 (5⬘-GAATTCATCAATTGTATT-3⬘) and npa2 (5⬘-CAAGAAATACATACTT CAA-3⬘). Abou 10 ng o plasmid DNA was added o he eac ion mix u e. Ampli ica ion eac ions we e ca ied ou in an Eppendo he mal cycle , wi h an ini ial dena u a ion (10 min a 94°C), 30 cycles o dena u a ion (1 min a 94°C), annealing (1 min a 48°C), and ex ension (2 min a 72°C), and a single inal ex ension o 16 min a 72°C. Aliquo s (20 ␮l) o each sample we e subjec ed o elec opho esis in 1.0 % aga ose gels. Ampli ied p oduc s we e de ec ed a e s aining wi h e hidium b omide (50 ␮g/ml) and pho og aphed wi h Pola oid ype 665 ilm. RESULTS Selec ion o clinical isola es and pa ien s. Be ween Janua y and Oc obe 2001, 27 E. coli and 3 K. pneumoniae clinical s ains wi h a pheno ype sugges i e o ESBL we e selec ed. These s ains we e isola ed om 30 di e en pa ien s. One isola e pe pa ien was selec ed (Table 1). The s ains we e associa ed wi h u ina y ac in ec ions (77%), wound in ec ion (13.3%), ca he e in ec ion (3.3%), blood in ec ion (3.3%), and conjun i al in ec ion (3.3%). Mos pa ien s had unde lying diseases. Mo eo e , al hough mos o he pa ien s we e hospi- alized be o e he E. coli and/o K. pneumoniae isola ion, se en pa ien s had no hospi al admission no ed in hei medical eco ds. De e mina ion o an ibio ic suscep ibili y and ESBL p o- duc ion. MICs o he 30 clinical s ains we e de e mined by he DadeMic oScan. In addi ion, all MICs we e con i med by he E es (AB Biodisk) acco ding o he NCCLS c i e ia (14). The o e all esul s a e shown in Table 2. Fo all s ains, he amoxicillin MICs we e highe han 256 ␮g/ml; mo eo e , his concen a ion dec eased d as ically in he p esence o cla u- lanic acid, sugges ing he p esence o a class A ␤-lac amase. Rega ding b oad-spec um cephalospo ins, s ains 1 o 17 and 22 had a pheno ype sugges ing ce o aximase ␤-lac amase ac- i i y as shown by he ce o axime MICs ( he ce o axime MICs we e clea ly highe han he ce azidime MICs). In e es ingly, he K. pneumoniae isola es included in his s udy wi h an ESBL pheno ype (s ains 28 o 30) showed unique SHV-like ␤-lac amase ac i i y band by IEF. This band p obably co esponds o he SHV ch omosomal ␤-lac amase ha is consi u i ely p esen in K. pneumoniae s ains; al e na- i ely, i is an SHV ESBL indis inguishable om he ch omo- somal SHV-1. The p esence o a second an ibio ic esis ance mechanism in hese K. pneumoniae s ains canno be uled ou . IEF assay. P o ein ex ac s o he 30 clinical s ains and hei ansconjugan s ob ained by sonica ion we e esol ed by IEF (3–9). Di e en pIs co esponding o di e en ␤-lac amases we e ob ained. In e es ingly, s ains 1 o 17 yielded an iden ical pI o 8.0; in addi ion, some o hem yielded a pI o 5.4, sug- ges i e o a TEM-1-like ␤-lac amase. S ains 18 o 30 showed di e en pIs such as 5.4, 5.9, 7.4, 7.6, 7.9, 8.5, and 9.0 (Table 2). Cloning o he CTX-M-14 gene. The ce o axime MICs and pIs ob ained by IEF sugges ed he p esence o an iden ical o simila ␤-lac amase gene associa ed wi h esis ance o ce o- axime in s ains 1 o 17 (Table 2). We a emp ed o clone he ␤-lac amase gene esponsible o his selec i e esis ance o ce o axime. Fo his pu pose, s ain 3 (Table 2) was chosen o he cloning expe imen s. Fi s , we ied o show whe he he ␤-lac- amase gene was plasmid o ch omosomally media ed. A con- juga ion expe imen was pe o med using E. coli XL-1-blue as a hos , as desc ibed in Ma e ials and Me hods. T ansconju- gan s we e selec ed on LB aga pla es supplemen ed wi h kanamycin and ampicillin. Se e al ansconjugan s we e ob- ained, and IEF o he sonica ed ex ac s o he clinical s ain VOL. 40, 2002 SPREADING OF THE CTX-M-14 ␤-LACTAMASE GENE IN E. COLI 4031 and he ansconjugan s showed a pI 8.0 ac i i y band in all cases. Plasmid analysis o he clinical s ain (s ain 3) and one ansconjugan (s ain 3Tc) showed ha he ␤-lac amase gene was associa ed wi h a plasmid o ca. 40 kb (da a no shown). Mo eo e , MIC expe imen s pe o med wi h bo h pa en al (s ain 3) and ansconjugan (s ain 3Tc) isola es showed al- mos iden ical an ibio ic suscep ibili ies (Table 2), clea ly sug- ges ing ha he ␤-lac amase gene was plasmid media ed. Cloning o he CTX-M-14 gene was pe o med as desc ibed in Ma e ials and Me hods. The nucleo ide sequence o he agmen e ealed only one open eading ame wi h homology wi h ␤-lac amase genes. The CTX-M-14 open eading ame was 876 bp long and encoded a p o ein o 291 amino acid esidues. This gene has p e iously been epo ed (GenBank accession numbe s AJ416341 and AF252622). In he ups eam egion o he CTX-M-14 gene, he nucleo ide sequence o he npA gene, he ansposase gene o inse ion sequence ISEcp1, was also de ec ed (24). Sp ead o he CTX-M-14 gene in di e en E. coli s ains. To elucida e whe he he CTX-M-14 gene was plasmid media ed in all E. coli s ains, conjuga ion expe imen s we e pe o med. Rega ding he kanamycin and/o nalidixic acid suscep ibili y o he E. coli clinical s ains, wo di e en E. coli isola es we e used as hos s in he conjuga ion expe imen s, E. coli BM21 ( esis an o nalidixic acid) when he E. coli clinical s ains we e suscep ible o nalidixic acid and E. coli XL-10-Gold Kan ( e- sis an o kanamycin) when he E. coli clinical s ains we e suscep ible o kanamycin. T ansconjugan s we e ob ained in all E. coli s ains (isola es 1 o 17 in Table 2) ha yielded a ␤-lac amase ac i i y band o pI 8.0 wi h ce o aximase ac i i y, s ongly sugges ing ha in all cases he ␤-lac amase wi h ce o- aximase ac i i y was plasmid media ed. To assess he possibili y o he sp ead o his CTX-M-14 gene in he 16 emaining E. coli s ains, a PCR assay was pe - o med. Plasmid DNA ob ained om he 27 E. coli and 3 K. pneumoniae s ains was used as a empla e in a PCR am- pli ica ion wi h CTX-M-1 (5⬘-AACACGGATTGACCGTA TTG-3⬘) and CTX-M-2 (5⬘-TTACAGCCCTTCGGCGAT-3⬘) p ime s, which annealed a he p omo e and he 3⬘pa o he CTX-M-14 gene, espec i ely. In e es ingly, a posi i e PCR esul , indica ed by a band o 905 bp, was de ec ed in 17 ou o 30 clinical s ains, including s ain 3 (da a no shown). The o e all esul s showed ha he CTX-M-14 gene was dissemi- na ed among 17 E. coli s ains in he no hwes a ea o Spain. Mo eo e , nucleo ide sequence analysis o hese 17 amplicons showed o al iden i y o he CTX-M-14 nucleo ide sequence. I is wo h men ioning ha s ain 18, al hough possessing a pI 8.5 band o ␤-lac amase ac i i y, did no yield an ampli ica- ion p oduc wi h he CTX-M-1 and CTX-M-2 p ime s. Also, he an ibio ic suscep ibili y pa e n o his s ain did no show he pu a i e ce o aximase p o ile de ec ed in he o he 17 E. coli s ains. The nex s ep was o y o de e mine whe he hese 17 E. coli s ains we e gene ically ela ed. This expe imen was necessa y o dis inguish be ween wo pu a i e hypo heses: (i) one epidemic E. coli s ain has been dissemina ed among all TABLE 1. Pa ien s om whom he E. coli and K. pneumoniae s ains used in his s udy we e isola ed Pa ien da a (no., sex/age [y ]) Type o in ec ion a Unde lying disease Specimen Hospi aliza ion be o e he E. coli isola ion 1. F/81 UTI Diabe es U ine No 2. F/9 UTI Acu e pyeloneph i is U ine Yes 3. M/66 UTI P os a e umo U ine Yes 4. F/1 UTI Respi a o y ac in ec ion U ine Yes 5. F/Unknown UTI Unknown U ine Unknown 6. F/77 UTI Blood in ec ion, diabe es U ine Yes 7. F/80 UTI No U ine No 8. F/Unknown UTI Unknown U ine Unknown 9. M/67 UTI Colon cance U ine Yes 10. M/58 UTI P os a e umo U ine Yes 11. F/73 UTI HBP, b a h osis U ine Yes 12. F/75 UTI HPB, u ina y incon inence U ine No 13. F/41 UTI Unknown U ine No 14. F/57 WI S omach ulce a ion Exuda e Yes 15. F/76 UTI Type II diabe es, ch onic hepa i is U ine Yes 16. M/86 WI Unknown Exuda e Unknown 17. F/37 UTI No U ine No 18. F/73 UTI HBP, suba achnoid hemo hagia U ine Yes 19. F/32 UTI Unknown U ine Yes 20. F/51 UTI Unknown U ine Yes 21. F/73 UTI HBP U ine Yes 22. M/25 WI Unknown Exuda e No 23. F/14 CI Unknown Ca he e Unknown 24. M/79 WI Pneumonia Exuda e Yes 25. F/66 UTI Unknown U ine No 26. M/73 UTI P os a e umo U ine Yes 27. M/5 UTI Se e al UTIs U ine Yes 28. M/53 UTI Leukemia U ine Yes 29. F/1 BI Hepa i is Blood Yes 30. F/1 CoI P ema u e bi h Conjunc i al swab Yes a UTI, u ina y ac in ec ion; WI, wound in ec ion; CI, ca he e in ec ion; BI, blood in ec ion; CoI, conjunc i al in ec ion. b HBP, high blood p essu e. 4032 BOU ET AL. J. CLIN.MICROBIOL. he pa ien s, and so one ances o s ain may be esponsible o he sp eading; i implies a link be ween all he pa ien s, and epidemiological s udies mus con i m his hypo hesis; and (ii) he CTX-M-14 gene has dissemina ed among di e en E. coli s ains. The e o e, he possibili y ha a plasmid ha bo ing he CTX-M-14 gene o a mobile gene ic elemen ca ying his gene had been dissemina ed in di e en E. coli s ains canno be uled ou . A REP-PCR assay was pe o med as desc ibed in Ma e ials and Me hods. This assay showed ha mos o he E. coli s ains we e gene ically un ela ed (Fig. 1). Fi een di e en geno ypes may be assigned o he 17 E. coli clinical s ains (isola es 3 and 4 and isola es 13 and 15 yielded he same band pa e ns). This esul he e o e elimina ed he possibili y o clonal dissemina ion o one epidemic E. coli s ain. Plasmid RFLP. Pu i ied plasmids om ansconjugan s o s ains 1 o 17 (Table 2) we e diges ed wi h HindIII, and he esul ing agmen s we e sepa a ed on 0.8% aga ose gels (Fig. 2). The o e all esul s showed a simila i y be ween plasmids in s ains 1, 4, 5, 7, 9, 10, 12, 13, and 14 (RFLP pa e n I in Table 3) ob ained om he 17 E. coli s ains, hus sugges ing iden i y in he plasmids ha bo ing CTX-M-14 on hese s ains. How- e e , s ains 2, 3, 6, 8, 11, 15, 16, and 17 did no show a simila i y in hei RFLP pa e ns, and hei pa e ns we e di - e en om RFLP pa e n I. The e o e, he CTX- M-14 gene was ha bo ed in di e en plasmids on hese s ains, and so he possibili y ha a mobile gene ic elemen migh be in ol ed in he dissemina ion o he CTX-M-14 gene in hese plasmids canno be uled ou . De ec ion o he npA ansposase gene. Plasmid DNAs om he conjugan s o he 17 E. coli clinical s ains (s ains 1 o 17 in Table 2) we e subjec ed o PCR ampli ica ion wi h p ime s which annealed o he npA gene, as desc ibed in Ma e ials and Me hods. A posi i e band o 405 bp was ob ained in all he plasmid pu i ica ions (da a no shown), sugges ing ha a pu- a i e unc ional ansposon was p esen in all E. coli s ains and ha his ansposon may be in ol ed in he mobiliza ion o he CTX-M-14 gene. DISCUSSION In 1992 (1, 2) a no el ype o ESBL ha con e ed a high le el o esis ance o ce o axime bu no o ce azidime was iden i ied in some membe s o En e obac e iaceae. This new amily o plasmid-media ed ESBLs o Amble class A was TABLE 2. MICs o he clinical s ains included in his s udy Isola e no. pI MIC (␮g/ml) o a : AMX AMC FOX AT CTX CTX-CL CAZ CAZ-CL IMP MRP CEP 1 5.4, 8.0 ⬎256 4 8 4 24 0.094 1.5 0.25 0.25 0.016 2 2 8.0 ⬎256 4 2 2 24 0.047 1.0 0.094 0.25 0.016 2 3 8.0 ⬎256 6 4 3 16 0.064 1.0 0.25 0.25 0.016 2 3 (Tc) b 8.0 ⬎256 4 4 3 32 0.047 1.0 0.125 0.125 0.032 4 4 5.4, 8.0 ⬎256 4 2 0.75 16 0.023 0.5 ⬍0.064 0.094 0.012 1.5 5 5.4, 8.0 ⬎256 4 12 2 48 0.094 1.0 0.125 0.125 0.023 4 6 5.4, 8.0 ⬎256 4 4 2 24 0.047 0.75 0.125 0.19 0.016 2 7 8.0 ⬎256 4 2 2 24 0.032 1.0 0.064 0.19 0.012 1.5 8 8.0 ⬎256 4 4 2 16 0.064 1.0 0.125 0.125 0.016 4 9 5.4, 8.0 ⬎256 8 24 8 ⬎128 ⬎1.0 6.0 4.0 0.25 0.023 4 10 5.4, 8.0 ⬎256 6 12 2 64 0.094 1.0 0.125 0.125 0.016 4 11 5.4, 8.0 ⬎256 4 3 2 32 0.047 1.0 0.064 0.25 0.016 4 12 8.0 ⬎256 4 4 2 64 0.047 1.0 0.094 0.19 0.016 4 13 8.0 ⬎256 4 4 4 ⬎128 0.047 1.0 0.094 0.125 0.012 2 14 8.0 ⬎256 4 4 2 48 0.064 1.5 0.19 0.125 0.012 4 15 8.0 ⬎256 4 3 1.5 ⬎128 0.047 1.0 0.125 0.19 0.016 4 16 5.4, 8.0 ⬎256 4 4 2 16 0.064 1.0 0.19 0.19 0.023 2 17 8.0 ⬎256 4 4 2 24 0.064 1.0 0.125 0.125 0.012 2 18 5.4, 8.5 ⬎256 4 2 8 24 ⬎1⬎32 ⬎4 0.25 0.032 0.38 19 5.4 ⬎256 16 16 24 0.5 0.19 ⬎32 0.75 0.25 0.032 2 20 5.4 ⬎256 2 1 0.5 ⬍0.016 ⬍0.016 12 0.064 0.19 0.023 0.032 21 5.4 ⬎256 4 12 6 0.5 ⬍0.016 ⬎32 0.19 0.125 0.023 0.75 22 9.0 ⬎256 4 2 4 ⬎256 0.047 6 0.094 0.125 0.016 8 23 9.0 ⬎256 12 ⬎256 ⬎256 ⬎256 ⬎1.0 ⬎32 ⬎4 0.125 0.064 ⬎128 24 9.0 ⬎256 12 ⬎256 ⬎256 ⬎256 ⬎1.0 ⬎32 ⬎4 0.125 0.047 128 25 5.9 ⬎256 4 2 1 2 0.094 2 0.19 0.5 0.032 0.094 26 5.4, 5.9 ⬎256 4 8 6 0.19 0.047 ⬎32 0.125 0.125 0.016 0.5 27 7.6 ⬎256 8 4 0.125 0.064 0.032 4 0.19 0.19 0.032 0.5 28 c 7.4 ⬎256 4 1 6 24 ⬍0.016 ⬎32 0.19 0.19 0.032 3 29 c 7.9 ⬎256 4 12 6 0.5 0.064 ⬎32 0.19 0.125 0.023 0.75 30 c 7.4 ⬎256 6 2 1 1 0.047 1.5 0.125 0.19 0.032 0.094 E. coli TG1 3 3 2 0.047 0.023 0.023 0.064 0.064 0.125 0.032 0.094 E. coli TG1 (pC) d 8.0 ⬎256 6 2 48 ⬎256 0.064 4 0.125 0.19 0.023 64 a AMX, amoxicillin; AMC, amoxicillin-cla ulanic acid; FOX, ce oxi in; AT, az eonam; CTX, ce o axime; CTX-CL, ce o axime-cla ulanic acid; CAZ, ce azidime; CAZ-CL, ce azidime-cla ulanic acid; IMP, imipenem; MRP, me openem; CEP, ce epime. b This isola e co esponds o he ansconjugan o clinical s ain 3. c This isola e co esponds o K. pneumoniae. d E. coli ans o med wi h plasmid pC, which ca ies he CTX-M-14 gene. VOL. 40, 2002 SPREADING OF THE CTX-M-14 ␤-LACTAMASE GENE IN E. COLI 4033 assigned o he ce o aximase (CTX-M) amily. Ce o aximases (CTX-M) ha e been de ec ed mainly in Sou h Ame ica, Eas - e n Eu ope, Japan, Spain, Kenya, and F ance (3, 13, 15, 17, 22). The amily o he CTX-M- ype ESBLs comp ises se e al membe s belonging o ou majo phylogene ic ees on he basis o hei amino acid sequence simila i ies (4): he CTX- M-1 g oup (CTX-M-1, CTX-M-3, and CTX-M-10), he CTX- M-2 g oup (CTX-M-2, Toho-1, CTX-M-4, CTX-M-5, and CTX-M-6), he CTX-M-9 g oup (CTX-M-9 and Toho-2), and he CTX-M-8 g oup. The CTX-M-14 ␤-lac amase is an Ala231-Val mu an o CTX-M-9; he e o e, i may be assigned o he CTX-M-9 g oup. We ha e de ec ed he CTX-M-14 ␤-lac amase o he i s FIG. 1. REP-PCR o he 17 E. coli s ains and 2 gene ically un ela ed E. coli con ols (C1 and C2). The s ain numbe s a e shown abo e he gel. Mw (molecula size ma ke s) co esponds o he 1-kb DNA ladde (P omega, Madison, Wis.). FIG. 2. Plasmid RFLP ob ained by HindIII diges ion wi h plasmid pu i ica ions o he ansconjugan s o he 17 E. coli s ains. The s ain numbe s a e shown abo e he gel. Mw (molecula size ma ke s) co esponds o he lambda DNA HindIII diges and bac e iophage ⌽174 HaeIII diges (Fynnzymes OY). 4034 BOU ET AL. J. CLIN.MICROBIOL. ime in Spain. This ␤-lac amase has p e iously been desc ibed in China, Ko ea, and F ance (8, 11, 16, 19). As desc ibed abo e, he CTX-M-14 gene is plasmid medi- a ed; indeed, we de ec ed i in he plasmids o he 15 di e en geno ypes o E. coli s ains. The plasmid inge p in s yielded 9 di e en pa e ns; he e o e, 9 o he 17 p o iles we e iden ical. This esul sugges s ha he sp eading o his plasmid among di e en E. coli s ains was impo an in he dissemina ion o he CTX-M-14 gene. Howe e , in he o he eigh E. coli s ains, di e en plasmids we e de ec ed, as shown by es ic- ion analysis. This esul implies some kind o mobiliza ion o he CTX-M-14 gene in he di e en E. coli s ains in addi ion o he ho izon al ans e . The analysis o he CTX-M-14 up- s eam egion showed he p esence o he ansposase gene ( npA) o he inse ion sequence named ISEcp-1 in all plasmids de ec ed in he E. coli s ains. I has p e iously been sugges ed ha he ISEcp-1 elemen could be associa ed wi h he mobili y o he CTX-M genes (11). The e o e, he possibili y ha he ISEcp-1 inse ion sequence can be associa ed wi h he sp ead- ing o he CTX-M-14 genes in ou E. coli s ains canno be uled ou . In ac , his is he only gene ic elemen we ha e ound associa ed wi h he high dissemina ion o he CTX-M-14 gene. These 17 E. coli s ains (15 geno ypes) caused in ec ions in 17 pa ien s. In 15 pa ien s he E. coli s ain ha bo ing CTX- M-14 caused u ina y ac in ec ions whe eas in he o he wo hey caused wound in ec ions. Fu he mo e, mos pa ien s had unde lying diseases and, in e es ingly, no hospi al admission was no ed in he medical eco ds o 7 pa ien s. Thus, on he basis o hese da a, we may assume he possible exis ence o ou pa ien s wi h bac e ial s ains causing in ec ions ha bo ing an ibio ic esis ance mechanisms. The e o e, he da a p esen - ed he e aise he hypo hesis ha mic oo ganisms wi h an ibi- o ic esis ance mechanisms can be p esen in pa ien s in he communi y. Ano he in e es ing issue is ha no appa en con- ac s be ween he 17 pa ien s occu ed. Indeed, mos inpa ien s we e admi ed o di e en hospi al wa ds o he hospi al com- plex. The ou pa ien s li e in di e en a eas a away om each o he . Wi h he cu en da a, how can we explain he p esence o up o 62% o he ESBL-p oducing s ains as ha bo ing he CTX-M-14 gene? Se e al epo s ha e been published desc ib- ing he de ec ion o CTX-M- ype enzymes in di e en a eas wo ldwide. He e we epo a p ac ical s udy showing ha CTX-M- ype ␤-lac amases a e highly dissemina ed; indeed, hey a e eplacing he TEM and SHV de i a i e ␤-lac amases (9). The easons why his is happening emain o be elucida ed. In a hospi al en i onmen , sp eading be ween pa ien s can be easy when a bac e ium wi h an an ibio ic esis ance mech- anism has been es ablished, especially i his mechanism is associa ed wi h mobile gene ic elemen s. Howe e , we epo he e ha se en pa ien s om whom bac e ial s ains ha bo ing ESBL we e isola ed ne e had con ac wi h he hospi al en i- onmen be o e he clinical E. coli isola ion. Fu he epidemi- ological s udies need o be pe o med o explain hese da a. In summa y, we ha e ound a high p e alence o he CTX- M-14 ␤-lac amase in he no hwes a ea o Spain. On he basis o hese da a, he empi ical adminis a ion o ce o axime in se ious in ec ions should be a oided in o de o p e en clinical complica ions. 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