ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2010, p. 4026–4028
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 54, No. 9
Association of the Extended-Spectrum ?-Lactamase Gene blaTLA-1
with a Novel ISCR Element, ISCR20?
Beatrice Berc ¸ot,1,2Laurent Poirel,1Jesus Silva-Sanchez,3and Patrice Nordmann1*
Service de Bacte ´riologie-Virologie, INSERM U914 “Emerging Resistance to Antibiotics,” Ho ˆpital de Bice ˆtre,
Assistance Publique-Ho ˆpitaux de Paris, Faculte ´ de Me ´decine et Universite ´ Paris-Sud, Le Kremlin-Bice ˆtre,
France1; Service de Bacte ´riologie-Virologie, Ho ˆpital Lariboisiere, Assistance Publique-Ho ˆpitaux de Paris,
Faculte ´ de Me ´decine et Universite ´ Paris Diderot, Faculte ´ de Me ´decine, Paris VII, France2; and
Departamento de Resistencia Bacteriana, Instituto Nacional de Salud Publica, Centro de
Investigaciones Sobre Enfermedades Infecciosas, Cuernavaca, Morelos, Mexico3
Received 19 January 2010/Returned for modification 12 May 2010/Accepted 20 June 2010
The blaTLA-1gene encoding an extended-spectrum ?-lactamase was identified in 11 enterobacterial isolates
from Mexico City, Mexico. This gene was located on different plasmids and plasmid types with different sizes
and incompatibility groups. It was associated with a novel insertion sequence, ISCR20, encoding a putative
transposase that shared only 20% amino acid identity with the most closely related transposase of ISCR1. The
ISCR20 element provided specific promoter sequences for expression of the blaTLA-1gene.
Although the majority of Ambler class A extended-spectrum
?-lactamases (ESBLs) identified in the family Enterobacteria-
ceae worldwide are mostly of the TEM, SHV, and CTX-M
types, some “minor” ESBLs have been reported, such as VEB-
like, GES-like, BES-1, SFO-1, and TLA-1 enzymes (5). The
blaTLA-1gene was first reported in 2000 from an Escherichia
coli clinical isolate from Mexico (8). From April 2000 until
February 2002, the blaTLA-1gene was detected from an epi-
demic Klebsiella pneumoniae clone in Mexico, and both the
blaTLA-1and blaSHV-5ESBL genes were located on the same
plasmid (1). The blaTLA-1gene was identified on a 150-kb
conjugative plasmid named pRZA92, but its genetic context
had not been further characterized (8). Considering that many
ESBL genes may be transposon or integron associated, the aim
of this study was to characterize the genetic elements at the
possible origin of acquisition of the blaTLA-1gene from isolates
of members of the family Enterobacteriaceae recovered from
several hospitals in Mexico City, Mexico.
The 150-kb plasmid pRZA92 of E. coli R170 (8) was ex-
tracted by using the Qiafilter plasmid purification maxikit
(Qiagen, Courtaboeuf, France) and partially sequenced using
the primers indicated in Table 1. A sequence of 5,923 bp was
identified through a primer-walking approach and contained
several open reading frames (ORFs) (Fig. 1). Upstream of the
blaTLA-1gene, another gene (named orf432) was identified; this
gene encoded a 432-amino-acid protein sharing 86% sequence
identity with an uncharacterized orf430 gene (83% amino acid
identity) located in a class 1 integron from a Proteus mirabilis
isolate recovered from Mediterranean herring gulls in Italy
(GenBank accession number DQ520941) (3). The orf432 gene
encoded a putative transposase belonging to the IS91 family of
transposases that includes the newly described ISCR elements.
The Orf432 transposase shared 20%, 21%, and 18% amino
acid identity with the transposases encoded by the ISCR1,
ISCR2, and ISCR3 elements, respectively. Detailed analysis
allowed us to identify the orf432 gene as part of a novel
ISCR element, termed ISCR20 according to the official no-
is 1,705 bp long and possesses a G?C content of 47.1% (Fig.
1). A putative oriIS sequence was identified 255 bp
downstream of the stop codon of the transposase gene and
shared 10 identical bp (5?ACTGATAGGAACTGTCATTTC3?
[the identical bases are shown in boldface type and under-
lined]) with the 19-bp consensus sequence reported for the
other ISCRs (5?XXGTATAGGAAGTTCAAACGC3?) (10)
(Fig. 1). A putative terIS sequence forming a hairpin structure
(5?GGACCCGCACGCAGGGTGTT3? [the boldfaced and
underlined nucleotides indicate the complementary nucleo-
tides forming the hairpin structure]) was also evidenced 136 bp
upstream of the transposase start codon. The transposase of
ISCR20 shared an overall significant degree of identity with
members of the IS91 family transposases especially in the five
functional domains previously described (Fig. 2). A tyrosine
residue at position 218, which has been shown to be critical for
the transposase activity of IS91 and speculated to be also
critical for those of the ISCR elements, was present in motif IV
of the ISCR20 transposase.
The blaTLA-1gene was followed by part of a gene encoding
a methyl-accepting chemotaxis protein that shares 58% amino
acid identity with that found in Desulfovibrio magneticus RS-1
(GenBank accession number YP_002952376). A group II in-
tron, containing an orf458 gene, was identified at the left ex-
tremity of the ISCR20 element (Fig. 1). The orf458 gene en-
coded a putative reverse transcriptase and maturase sharing
69% amino acid identity with those of Vibrio cholerae (GenBank
accession number ABV21790). Analysis of the nucleotide se-
quence surrounding the blaTLA-1gene did not yield other inser-
tion sequences that could be part of a transposon or any class 1
* Corresponding author. Mailing address: Service de Bacte ´riologie-
Virologie, Ho ˆpital de Bice ˆtre, 78 rue du Ge ´ne ´ral Leclerc, 94275 Le
Kremlin-Bice ˆtre Cedex, France. Phone: 33-1-45-21-36-32. Fax: 33-1-
45-21-63-40. E-mail: firstname.lastname@example.org.
?Published ahead of print on 28 June 2010.
A collection of 11 TLA-1-producing Enterobacteriaceae clin-
ical strains (3 E. coli strains [including E. coli R170], 1 K.
pneumoniae strain, and 7 Enterobacter cloacae strains) isolated
from different samples (urine, blood, and tracheal fluid sam-
ples) from 11 patients hospitalized over a 10-year period since
1991 from several hospitals in Mexico City, Mexico, were ret-
rospectively analyzed. These isolates were selected among clin-
ical ESBL-producing enterobacterial isolates on the basis of
two criteria: their ESBL profile and their production of a
?-lactamase with an isoelectric point of 9.0. By DNA restric-
tion with XbaI endonuclease, followed by pulsed-field gel elec-
trophoresis (PFGE) analysis, we showed that most of the iso-
lates were not clonally related, with four distinct E. cloacae
PFGE types and three distinct E. coli PFGE types, suggesting
a diffusion of blaTLA-1-positive plasmids. The blaTLA-1gene
was transferred from seven of those distinct PFGE types to E.
coli J53 by conjugation performed as described previously (6).
Together with the expression of an ESBL phenotype, the E.
coli transconjugants were resistant to trimethoprim, sulfameth-
oxazole, and tetracycline, and some of these transconjugants
were resistant to amikacin (n ? 5) or gentamicin (n ? 1).
Plasmid analysis performed by the Kieser technique (4) al-
lowed us to visualize several plasmids in all the clinical isolates
but only a single plasmid from each E. coli transconjugant.
Southern blot hybridization with a blaTLA-1-specific probe in-
dicated that this ESBL gene was located on a single plasmid,
being of either ca. 110 or 150 kb. PCR-based replicon typing
(PBRT) analysis performed on the E. coli transconjugants (2)
showed that the plasmids carrying the blaTLA-1gene belonged
to the IncA/C, IncL/M, or IncN incompatibility group, corre-
sponding to the 150-, 150-, and 110-kb plasmids, respectively,
highlighting that three different plasmid types were at the or-
igin of the diffusion of the blaTLA-1gene.
Except for E. coli isolate R2915 and its transconjugant, PCR
amplification identified in all cases the same ISCR20 element
upstream of the blaTLA-1gene. It has been suggested that the
promoter of the blaTLA-1gene was located 71 bp from the
translational start site by researchers using a computer-based
promoter analysis (8). However, by using a 5? rapid amplifica-
tion of cDNA end PCR system (Invitrogen, Cergy-Pontoise,
France) and primers TLA-1-5?-TR1 to TLA-1-5?-TR3 (Table
1), the initiation site of transcription of the blaTLA-1gene was
mapped from E. coli R170. Promoter sequences were located
144 bp upstream of the translational start codon, correspond-
ing to the ?35 promoter sequence TTGACA separated by the
17-bp distance from the ?10 promoter sequence TTAAAG.
This result showed that the blaTLA-1gene expression was
driven by the ISCR20 element. One pair of outward primers
chosen at each end of the ISCR20 insertion sequence was used
to detect the circular form of this novel ISCR associated with
the blaTLA-1gene. The amplifications performed using strain
R170 and its transconjugant as templates failed.
Nineteen ISCR elements with weak structural relationship
(18 to 96%) have been found to be associated with different
families of antibiotic resistance genes. As demonstrated with
the transposition model of the IS1294 elements (9), it is pro-
posed that a transposase encoded by the ISCR has the ability to
cotranspose DNA adjacent to its terminal terIS sequence
through a rolling-circle (RC) transposition mechanism (10). It
has been suggested that the mobilization by ISCR elements of
adjacent DNA sequences likely involving sequences located
TABLE 1. Sequences of primers used for detection of the blaTLA-1
gene and its circular form with ISCR20 and for mapping
the genetic environment
Primer name Primer sequence Positiona
aPositions of the primers indicated in Fig. 1. All the primers were designed in
FIG. 1. Schematic representation of the sequences surrounding the blaTLA-1gene in E. coli X170. The positions of primers shown in Table 1
are indicated by arrows; primers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 are primers TLA-1F, TLA-1R, TLA-1-5?-TR1,
TLA-1-5?-TR2, TLA-1-5?-TR3, IS91-5?-TR1, IS91-5?-TR4, IS91-5?-TR5, IS91-5?-TR6, IS91-5?-TR7, TLA-1-3?-TR1, TLA-1-3?-TR4, TLA-1-3?-
TR5, TLA-1-3?-TR6, IS91-3?-extR, TLA-1-3?extF, IS91-5?-extR, IS91-3?extF, ISCR20F, maturase F, and methyl R, respectively. Genes are shown
by large arrows, and the transcription orientations of the genes are indicated. The putative origin of replication, oriIS, and termination of
transposition, terIS, of the ISCR20 element are indicated by a solid black circle and a black hairpin, respectively.
VOL. 54, 2010GENETIC ENVIRONMENT OF blaTLA-1
only at their left extremities and the frequent observation of Download full-text
resistance genes bracketed by two copies of ISCR may be the
consequence of secondary recombination events (10). This has
been recently shown with the identification of the blaVEB-1a
ESBL gene flanked by two copies of ISCR2 in Acinetobacter
baumannii (7). Here, no ISCR20 element was identified down-
stream of the blaTLA-1gene, casting doubt on the involvement
of that element in the mobilization process of the blaTLA-1
gene. Further work is needed to understand the process that
gave rise to the mobilization of the blaTLA-1gene.
Besides describing a novel ISCR element, our study indi-
cates its involvement in the expression of the blaTLA-1gene and
its likely role in its acquisition. In addition, we showed here
that the dissemination of the blaTLA-1gene may have already
occurred in Mexico and that it was related to different plasmid
backbones. Further screening of TLA-1-positive isolates
should be performed at least in neighboring countries, such as
countries in Central America and in the southern United
States. We showed a heterogeneity of plasmid backgrounds,
supporting the hypothesis that the blaTLA-1gene was very likely
harbored by a mobile genetic structure.
Nucleotide sequence accession number. The nucleotide se-
quence reported in this study has been deposited in the
GenBank database under accession number GU441460.
This work was funded mostly by a grant from the INSERM (U914)
and by grants from the European Community (TROCAR, HEALTH-
F3-2008-223031) and from the Ministe `re de l’Education Nationale et
de la Recherche (UPRES-EA3539), Universite ´ Paris XI, France.
We thank M. Toleman for providing us with an ISCR number.
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FIG. 2. Comparison of the sequence motifs of ISCR20 and those of the IS91 family transposases. The sequences (with their GenBank accession
numbers shown in parentheses) shown are as follows: ISCR1 (FJ187822), ISCR2 (AY055428), ISCR3 group, including ISCR3, ISCR4, ISCR6,
ISCR14, and ISCR16 (FJ183463), ISCR7 (AJ250371), ISCR8 (AF028594), ISCR15 (AM998375), and ISCR19 (EU503121). The five motifs found
within IS91 group elements are indicated. The amino acid residues conserved in all IS91 group elements are shown boxed. Gaps introduced to
maximize alignment are indicated by dashes.
4028 BERC ¸OT ET AL.ANTIMICROB. AGENTS CHEMOTHER.