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. In addi ion, we ale he medical communi y o
he inc ease o hese ␤-lac amases wo ldwide.
ACKNOWLEDGMENT
We exp ess ou g a i ude o Luis de Ra ael o English co ec ions.
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TABLE 3. REP-PCR pa e n, de ec ion o he CTX-M-14 gene and
RFLP o he plasmids o he ansconjugan s o
he clinical s ains used in his s udy
Isola e
no. pI REP-PCR
pa e n
PCR
CTX-M-14
T ansconjugan plasmid
RFLP pa e n
1 5.4, 8.0 1 ⫹I
2 8.0 2 ⫹II
3 8.0 3 ⫹III
4 5.4, 8.0 3 ⫹I
5 5.4, 8.0 4 ⫹I
6 5.4, 8.0 5 ⫹IV
7 8.0 6 ⫹I
8 8.0 7 ⫹V
9 5.4, 8.0 8 ⫹I
10 5.4, 8.0 9 ⫹I
11 5.4, 8.0 10 ⫹VI
12 8.0 11 ⫹I
13 8.0 12 ⫹I
14 8.0 13 ⫹I
15 8.0 12 ⫹VII
16 5.4, 8.0 14 ⫹VIII
17 8.0 15 ⫹IX
18 5.4, 8.5 ND
a
⫺ND
19 5.4 ND ⫺ND
20 5.4 ND ⫺ND
21 5.4 ND ⫺ND
22 9.0 ND ⫺ND
23 9.0 ND ⫺ND
24 9.0 ND ⫺ND
25 5.9 ND ⫺ND
26 5.4, 5.9 ND ⫺ND
27 7.6 ND ⫺ND
28 7.4 ND ⫺ND
29 7.9 ND ⫺ND
30 7.4 ND ⫺ND
a
ND, no done.
VOL. 40, 2002 SPREADING OF THE CTX-M-14 ␤-LACTAMASE GENE IN E. COLI 4035
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4036 BOU ET AL. J. CLIN.MICROBIOL.