Contribution of IncFII and Broad-Host IncA/C and IncN Plasmids to

the Local Expansion and Diversification of Phylogroup B2 Escherichia

coli ST131 Clones Carrying blaCTX-M-15 and qnrS1 Genes
ngela Novais,a,b Diana Viana,a Fernando Baquero,a,b Javier Martnez-Botas,c,d Rafael Cantn,a,b and Teresa M. Coquea,b
Servicio de Microbiologa and CIBER en Epidemiologa y Salud Pblica (CIBERESP), Instituto Ramn y Cajal de Investigacin Sanitaria (IRYCIS) and Hospital Universitario
Ramn y Cajal, Madrid, Spaina; Unidad de Resistencia a Antibiticos y Virulencia Bacteriana asociada al Consejo Superior de Investigaciones Cientficas (CSIC), Madrid,
Spainb; Servicio de Bioqumica-Investigacin, Hospital Ramn y Cajal, IRYCIS, Madrid, Spainc; and CIBER de Fisiopatologa de la Obesidad y Nutricin (CIBEROBN), Instituto
de Salud Carlos III, Madrid, Spaind

he current pandemic spread of blaCTX-M-15 has been greatly

facilitated by high-risk clones of Escherichia coli (mainly B2
sequence type 131 [ST131]) and Klebsiella pneumoniae (6, 22) and
by IncFII plasmids with high potential for recombination (6, 17).
The recent identification of blaCTX-M-15 in other enterobacterial
species (especially Salmonella spp. or Enterobacter spp. in developing countries) and different conjugative plasmids highlights the
risk of further spread of this gene to other successful genetic backgrounds (4, 10, 14, 17, 19, 21). Differences in pulsed-field gel electrophoresis (PFGE) and virulence profiles among widespread
fluoroquinolone-resistant B2 ST131 clones carrying blaCTX-M-15
from different locations indicate frequent local processes of diversification of this lineage, with some of these variants being widely
disseminated (5, 13).
In Spain, CTX-M-15 is among the most frequent extendedspectrum -lactamase (ESBL) variants identified in ESBL-producing E. coli and increased 8-fold between 2000 and 2006 (from
0.5% to 4.04%) in hospitalized patients (5, 9). In our hospital, the
blaCTX-M-15 gene was detected for the first time in 2002 as associated with a diversity of K. pneumoniae and phylogroup D E. coli
clonal backgrounds and IncFII plasmids (15). The aim of this
study was to analyze the influence of different genetic elements on
the recent expansion of blaCTX-M-15 in our institution.
Thirty-three nonreplicate CTX-M-15-producing E. coli isolates representing 33.7% of the total ESBL-producing E. coli
isolates at Ramn y Cajal University Hospital in Madrid in
2008 were studied. This rate is higher than that observed in
recent regional (26% in Barcelona) and national (15%) surveys
in our country (5, 9). They were recovered mostly from outpatients (19/33; 57%) but also from patients located at medical
(9/33; 27%), intensive care unit (ICU) (4/33; 12%), and surgical (1/33; 3%) wards, and they were mostly obtained from
urine samples (26/33; 78.8%).
Clonal analysis performed by characterization of E. coli phylogenetic groups, PFGE, and multilocus sequence typing (6) (http:
//mlst.ucc.ie/mlst/dbs/Ecoli) established that CTX-M-15 producers belonged to a diversity of phylogroups, with B23 being the
most predominant (22/33; 66.7%; 21 ST131 and 1 ST372). The

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presence of 38 virulence factors (VFs) associated with extraintestinal pathogenic E. coli (ExPEC) was screened by PCR as previously described (12, 18). On the basis of their PFGE profiles,
ST131 isolates were grouped into two main clusters of strains
showing variable virulence (I to III) and antibiotic resistance profiles (Fig. 1). The largest cluster comprises isolates showing highly
similar PFGE profiles (EC15-2, EC15-3, and EC15-4; 80.8% homology) and a common virulence profile (fimH, iha, sat, iutA,
traT, malX, usp, fyuA, ompT) that was previously described for
ST131 (5, 13), although some of them also harbored afa/draBC
and kpsMTII (clone EC15-2, variant I; n 10) or kpsMTII-K5
(clones EC15-3 and EC15-4, variant II; n 3). Clones showing
virulence profiles designated variants I and II were identical to
others widely spread in the United Kingdom and Spain that have
been arbitrarily designated in such studies as strain A and
group II, respectively (5, 13). Virulence profile III (clones
EC15-3 and EC15-4, n 4), firstly reported in this study, adds
kpsMTII-K5, papEF, papA, papC, papGII, cnf1, and hylA to the
common profile mentioned above. Isolates of the minority cluster
(EC15-1; 4 isolates) showed 77.4% homology with the previous
strains and the virulence profile designated variant I (Fig. 1).
Other CTX-M-15 producers were identified as E. coli isolates of
the phylogroups A1 (4 ST410, 1 ST617, and 1 ST1284), B1 (1
fumC23 and 1 fumC29), and D1 (1 ST405), with some of them also
corresponding to other widespread multidrug-resistant uropathogenic lineages (particularly those from ST10, ST23, and
ST405 complexes) which are prevalent among ESBL producers in
Spain (Table 1) (5, 16).
Most blaCTX-M-15 genes were successfully transferred by broth

Received 25 October 2011 Returned for modification 27 November 2011

Accepted 4 February 2012
Published ahead of print 13 February 2012
Address correspondence to Teresa M. Coque, mcoque.hrc@salud.madrid.org.
Copyright 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.06001-11

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The recent increase of CTX-M-15-producing Escherichia coli isolates in our institution was caused by diverse clonal backgrounds, including mainly B2 sequence type 131 (ST131) clones presenting variable virulence profiles but also A1 (ST617,
ST410), B1, and D1 (ST405) clones. Besides IncFII-pC15-1a, we detected multidrug-resistant IncA/C2 and IncN plasmids carrying blaCTX-M-15 and/or qnrS1. Our study highlights the diversification of highly transmissible resistant and virulent clones and
the recombinogenic potential of broad-host plasmids contributing to the expansion of genetic regions coding for multidrug resistance to other bacterial lineages.

Novais et al.

and/or filter mating assays (30/33; 91%). Characterization of plasmids by PCR-based replicon typing (3) revealed isolates containing only the IncFII (n 15) or IncA/C2 (n 2) plasmid, the IncFII
and IncN plasmids (IncFIIIncN) (n 8), or IncFIIIncA/C2
(n 4). Hybridization of S1 nuclease-digested genomic DNA
with rep and blaCTX-M-15 probes (6, 15) demonstrated the location
of blaCTX-M-15 on either the IncFII (70 to 110 kb; 93.1%) or the
IncA/C (230 kb; 6.9%) plasmid in different clones (Table 1). A
comparison of HpaI restriction patterns of CTX-M-15-encoding
IncFII plasmids showed restriction fragment length polymorphism (RFLP) patterns, arbitrarily designated pC15-A, pC15-W,
and pC15-Z, which are identical or closely related to common
worldwide-distributed variants (such as pC15-1a or pEK516,
which are in GenBank under accession numbers AY458016 and
EU935738, respectively) (Table 1) (6, 17, 22). These plasmids were
detected in B23 (ST131, ST372) and A1 (ST410, ST617, ST1284) E.
coli clones, reflecting plasmid transfer among different genetic
backgrounds. Plasmids containing multiple F replicons, such as
FII, FIA, and FIB (FIIFIAFIB) (1 ST410, 1 ST405), FIIFIB (1
fumC29-B1), or FIIFIA (2 ST131 isolates), were also detected.
The two IncA/C2 plasmids showed replicon sequences and antibiotic resistance patterns that were identical and similar, respectively, to those of other known IncA/C2 plasmids identified in
Salmonella spp. (p2039, pSN254), Yersinia spp. (pYR1, pIP1202),
and Aeromonas spp. (pRA1) (GenBank accession numbers
AM087198, CP000604, CP000602, CP000603, and FJ705807, respectively).
Characterization of the genetic environment of blaCTX-M-15 by
PCR assays (ISEcp1, IS26, orf477, blaTEM-1, blaOXA-1, and aac[6=]Ib-cr), hybridization, and sequencing (15) revealed a variable genetic context derived from the multidrug resistance region (MRR)
of the IncFII plasmid pC15-1a (17). A region containing ISEcp1blaCTX-M-15-orf477, blaTEM-1, blaOXA-1, and aac(6=)-Ib-cr was iden-

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tified among IncFII and IncA/C2 plasmids from ST131 (clones

EC15-1 and EC15-3) and non-ST131 isolates. IS26 was found upstream of blaCTX-M-15 on IncFII plasmids in the EC15-2 clone (exhibiting virulence profile I), a configuration similar to that observed in the strain A found in the United Kingdom and recently
detected in North Spain (5, 13). Interestingly, two different ST131
isolates harbored two copies of blaCTX-M-15, located either on the
IncFII (80 kb; reppC15-1a) and IncN (55 kb; reppKP96) or on the
IncFII (80 kb; reppC15-1a) and IncA/C (150 kb; repp2039) plasmids
(Table 1). Our results and the detection of different fragments of
this MRR in a diversity of plasmid groups from E. coli (IncFII,
IncI), K. pneumoniae (IncR, IncFIIk), or Salmonella (IncA/C2, IncHI2) and on chromosomes of different species reflect the recombinogenic potential of this region, which can be further
spread by widespread clones (4, 6, 10, 14, 19).
The qnrS1 gene was detected among 48.5% of CTX-M-15 E.
coli producers, a rate similar to that obtained in another survey
from Mexico (44.9%) (20) and higher than that observed in other
studies from areas as distant as Europe or North Africa (2 to 16%)
(2, 7, 8). This gene was frequent among B2 ST131 isolates (13/21;
62%) but also present among E. coli clones, such as A-ST410 (22),
located on the IncFII (n 5), IncN (40 to 55 kb, n 3), or IncA/C
(150 kb, n 1) plasmid and occasionally carrying blaCTX-M-15,
that are predominant in Spain and other countries. Previous studies have identified the presence of qnrS1 on different groups of
plasmids, including IncN, IncX, and ColE among non-ESBL-producing isolates from Salmonella (11); here we report for the first
time the identification of a qnrS1 gene in a blaCTX-M-15-carrying
IncA/C2 plasmid in an E. coli isolate.
This study highlights the local diversification of globally spread
and highly transmissible clones of ST131 with an increasing number of virulence and antibiotic resistance genes. The recombinogenic potential of broad host plasmids might contribute to the

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FIG 1 PFGE profiles of XbaI-digested genomic DNA of representative CTX-M-15-producing ST131 isolates included in this study. The dendrogram was
generated using the unweighted-pair group method using average linkages (UPGMA) algorithm based on the Dice similarity coefficient (1.5% optimization;
1.5% tolerance), and the vertical dotted line shows the cutoff value of 85%. The isolates possess a common pool of virulence genes (fimH, iha, sat, iutA, traT, malX,
usp, fyuA, ompT) and differ in the presence of afa/draBC and kpsMTII (profile I), kpsMTII-K5 (profile II), and kpsMTII-K5, papEF, papA, papC, papGII, cnf1, and
hylA (profile III). Isolation dates are shown by the day followed by the month. a, absence of kpsMTII. Gene definitions: fimH, type 1 fimbriae; iha, iron-regulated
gene homologue adhesin; sat, secreted autotransporter toxin; iutA, ferric aerobactin receptor; traT, serum survival associated; malX, pathogenicity-associated
island (PAI) marker; usp, uropathogen-specific protein; fyuA, yersiniabactin receptor; ompT, outer membrane protease; afa/draBC, Dr antigen-specific adhesin;
kpsMTII, group II capsular polysaccharide; K5, variant of kpsMTII capsule; papEF, P fimbriae minor tip pilins; papA, P fimbriae major subunit, pyelonephritis
associated; papC, P fimbriae assembly; papGII, papG variant, pyelonephritis associated; cnf1, cytotoxic necrotizing factor 1; and hlyA, alpha-hemolysin.

ST131 E. coli Clones Encoding CTX-M-15 and QnrS1

TABLE 1 Genetic context of recent blaCTX-M-15-carrying E. coli clones and plasmids from Hospital Universitario Ramn y Cajal

Phylogroup ST (CC)a
B23

B1

D1

Inc replicon
content (kb)b

Presence of gened
RFLPc

blaTEM-1 blaOXA-1 aac(6)-Ib-cr qnrS1e Resistance profileh

pC15-A /

pC15-A

ST131

EC15-1a (2),
FII (70-85)
EC15-1b (1)
EC15-1a (1)
FII (85), N (45)

ST131

EC15-2a (1)

ST131
ST131

EC15-2a (4),
FII (70-85), Nf
EC15-2b (1)
(30-55)
EC15-2b (2)
FII (80)

ST131

EC15-2b (2)

FII (80), A/C (140)

ST131
ST131

EC15-3a (1)
EC15-3a (1)

ST131
ST131

FII (75-80)
FII (80) () FIA,
A/C
FII (75), N (30)g
FII (75-80)

ST131

EC15-3b (1)
EC15-4a (1),
pC15-A /
EC15-4b (2)

EC15-4a (1)
FII (80), A/Cf (150)

ST372

EC15-8 (1)

FII (80), 40

pC15-W

ST617 (ST10)

EC15-7 (1)

FII (80), 40

pC15-W

ST1284

EC15-4 (2)

FII (70-80)

pC15-Z

ST1284

EC15-4 (1)

A/C (230)

ND

ST410 (ST23)
ST410 (ST23)

EC15-13 (1)
EC15-11 (1)

FII (80)
FII (85), N (45)

pC15-A
ND

ST410 (ST23)

EC15-6 (2)

FII (110), FIA, FIB, ND

NTf (140)

fumC23

EC15-10 (1)

A/C (230)

ND

fumC29

EC15-9 (1)

FII, FIB (NI)

ND

CIP, GEN, KAN, NET, TOB,

STR, AMK, TET, SUL, SXT
CIP, NAL, STR, TET

FII, FIA, FIB (NI)

ND

ND

ND

ND

ND

CIP, GEN, TOB, AMK, SUL, SXT

ST405 (ST405) EC15-12 (1)

FIIFIA (90)

ND

CIP, NAL, GEN, KAN, TOB,

AMK, TET, SUL, SXT
CIP, NAL, GEN, KAN, TOB,
AMK, TET, SUL, SXT
CIP, NAL, (GEN), (KAN), TOB,
(TET), SUL, SXT, TMP
CIP, NAL, (GEN), (KAN), TOB,
(TET), SUL, SXT, TMP
CIP, NAL, (GEN), (KAN), TOB,
(TET), SUL, SXT, TMP
CIP, NAL, (GEN), (KAN), TOB,
(TET), SUL, SXT, TMP
CIP, NAL, (TOB)
CIP, NAL, (TOB)
CIP, NAL, (TOB)
CIP, NAL, (GEN), KAN, TOB,
(STR), TET, (SUL), TMP
CIP, NAL, (GEN), KAN, TOB,
(STR), TET, (SUL), TMP
CIP, GEN, KAN, TOB, TET
CIP, NAL, GEN, KAN, TET, SUL,
SXT
CIP, NAL, GEN, KAN, TOB,
TET, SUL, SXT
CIP, GEN, KAN, NET, TOB,
STR, AMK, TET, SUL, SXT,
TMP
CIP, NAL, KAN, TOB, SXT
CIP, NAL, GEN, KAN, TOB,
TET, SUL, SXT
CIP, NAL, GEN, KAN, NET,
TOB, STR, (AMK), TET, SUL,
SXT, TMP

CC, clonal complex.

b
Plasmids were identified in transconjugants (or wild-type strains in the absence of transfer). Positive hybridization signals obtained with the blaCTX-M-15 and rep probes appear in
bold. Transferability is indicated by underlining. NT, not typed. NI, not identified.
c
pC15-A, pC15-W, and pC15-Z were highly related by RFLP. ND, not done.
d
Antibiotic resistance genes were searched for in transconjugants (or wild-type strains in the absence of transfer)., presence; , absence; /, variable presence.
e
qnrS1 was identified within the IncN, IncFII, and occasionally IncA/C plasmid types.
f
A second copy of blaCTX-M-15 was identified in this plasmid type.
g
This isolate was a producer of both CTX-M-15 and SHV-12.
h
Drug names in parentheses indicate variable resistance profile. Abbreviations: AMK, amikacin; CIP, ciprofloxacin; GEN, gentamicin; KAN, kanamycin; NAL, nalidixic acid; NET,
netilmicin; STR, streptomycin; SUL, sulfonamide; TET, tetracycline; TOB, tobramycin; TMP, trimethoprim; SXT, trimethoprim-sulfamethoxazole.

expansion of multidrug resistance platforms among both globally

successful clones and other bacterial lineages, as is being observed
in developing and developed countries (10, 14, 19, 21). In a general context, our scenario illustrates the frequent trade-off between the four main processes (the four Ps) involved in the spread
and diversification of antibiotic resistance (1): (i) penetration in
microbial ecosystems of highly effective pathogenic clones and
plasmids, (ii) promiscuity of genetic traits involved in antibiotic

May 2012 Volume 56 Number 5

resistance by lateral gene transfer, (iii) plasticity of genetic vehicles

and platforms, taking advantage of highly recombinogenic sequences, and (iv) persistence and maintenance of different pathogenic multidrug-resistant E. coli high-risk clones and globally distributed plasmids (IncFII, IncA/C, IncN). Such interplay of
evolutionary processes, detectable at the local level, suggests that
the wide penetration of common clones and plasmids in large
geographical areas drives local effective population sizes, resulting

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A1

ST131

PFGE type(s)
(no. of
isolates)

Novais et al.

in similar effects on diversification and spread of antibiotic resistance at the local scale.
ACKNOWLEDGMENTS
This work was supported by research grants from the European Commission
(HEALTH-F3-2008-223031, PIEF-GA-2009-255512, and PAR-241476).
ngela Novais was supported by a Marie Curie Intra-European Fellowship
within the 7th European Community Framework Programme (PIEF-GA2009-255512).

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