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Antimicrobial Agents and Chemotherapy, August 2005, p. 3593-3597, Vol. 49, No. 8
0066-4804/05/$08.00+0 doi:10.1128/AAC.49.8.3593-3597.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Integron-Encoded GES-Type Extended-Spectrum ß-Lactamase with Increased Activity toward Aztreonam in Pseudomonas aeruginosa
Laurent Poirel,1
Laura Brinas,1,2
Nicolas Fortineau,1 and
Patrice Nordmann1*
Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris XI, 94275 K.-Bicêtre, France,1
Area de Bioquimica y Biologia Molecular, Universidad de La Rioja, 26006 Logrono, Spain2
Received 15 February 2005/
Returned for modification 22 March 2005/
Accepted 4 May 2005

ABSTRACT
A
Pseudomonas aeruginosa strain expresses an extended-spectrum
ß-lactamase, GES-9, which differs from GES-1 by a
Gly243Ser substitution, is inhibited by clavulanic acid and
imipenem, and hydrolyzes aztreonam. The
blaGES-9 gene was located
inside a class 1 integron structure containing two copies of
a novel insertion sequence belonging to the IS
1111 family.

TEXT
Extended-spectrum ß-lactamases (ESBLs) are reported
increasingly for
Pseudomonas aeruginosa (
29), including TEM
and SHV variants, PER-1, VEB-1-like (mostly from southeast Asia)
(
9), and GES/IBC-type enzymes (
29) that have been identified
in
P. aeruginosa in France, South Africa, Greece, and Brazil
(
4,
6,
15,
23,
24). Recently, a nomenclature update has been
proposed for GES-like enzymes (
14), such as GES-5 and GES-6
from Japan (
27,
28), IBC-1, GES-3, and GES-4 from Greece (
8,
26). The
blaGES genes are part of class 1 integrons, with the
exception of a
blaGES-1 gene from a
Klebsiella pneumoniae strain
from Portugal which was embedded in a class 3 integron (
5).
P. aeruginosa DEJ was isolated from a rectal swab of a patient hospitalized at the hospital Bicêtre in March 2004 for a stroke who had not been hospitalized before and did not travel abroad. P. aeruginosa DEJ was resistant to all ß-lactams except imipenem, piperacillin (Table 1), colistin, and fosfomycin (17). A synergy between aztreonam- and clavulanic acid-containing disks suggested the production of an ESBL (22). PCR experiments performed with primers specific for ESBL-encoding genes (9) revealed that P. aeruginosa DEJ possessed a blaGES-type gene. No transfer of resistance markers to Escherichia coli or to P. aeruginosa reference strains was obtained by conjugation and transformation (20). Plasmid extraction (10) did not identify plasmids in P. aeruginosa DEJ, suggesting a chromosomal location of the blaGES-like gene.
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TABLE 1. MICs of ß-lactams for P. aeruginosa DEJ, E. coli DH10B harboring recombinant plasmid pDEJ-1 from P. aeruginosa DEJ, E. coli DH10B harboring recombinant plasmid pC1, and the E. coli DH10B reference strain
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Cloning experiments, performed as described previously (
19),
gave rise to recombinant strains with an ESBL phenotype.
E. coli DH10B (pDEJ-1) was resistant to amino- and ureido-penicillins
and to narrow- and extended-spectrum cephalosporins and was
susceptible to cephamycins and carbapenems (Table
1). In addition,
resistance to aztreonam reached a higher level than that observed
for a GES-1-producing
E. coli recombinant strain (Table
1) (
21).
A blaGES-9 gene was identified in the 5,466-bp insert of recombinant plasmid pDEJ-1. GES-9 differed from GES-1 by only a Gly-to-Ser change at Ambler position 243 (Fig. 1) (1). This substitution is located near Ambler position 240, known to be a key amino acid residue for extension of the hydrolysis profile of CTX-M-type ß-lactamases (2).
E. coli DH10B (pDEJ-1) produced a ß-lactamase with
a pI value of 5.8 according to isoelectric focusing results.
Purification of ß-lactamase GES-9 was performed as
established for GES-1 (
21). The specific activity of purified
ß-lactamase GES-9 against benzylpenicillin was 140
U/mg of protein, and its purification factor was 40-fold with
an estimated purity of >95% by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis analysis (data not shown). ß-Lactamase
GES-9 had a broad-spectrum hydrolysis profile (Table
2). The
catalytic activity (
kcat/
Km) of GES-9 for aztreonam was high
(60 mM
1 · s
1), despite a low affinity for
this substrate. Although GES-2, GES-3, and GES-4 hydrolyze imipenem
as a result of a Gly170Ser substitution, GES-9 that possesses
a Gly170 residue spared carbapenems. Activity inhibition measurements
showed that GES-9 was inhibited by clavulanic acid (0.45 µM),
tazobactam (0.5 µM), and sulbactam (0.5 µM) and
very strongly inhibited by imipenem (50% inhibitory concentration
[IC
50], 10 nM). In addition, as observed for GES-1 and VEB-1
(
21,
22), cefoxitin was an inhibitor of GES-9 activity (IC
50,
1 µM).
Sequence analysis of the 5.4-kb insert of plasmid pDEJ-1 revealed
that the
blaGES-9 gene was part of a cassette, preceded by an
intI1 integrase gene of a class 1 integron that was named In109.
The 59-base-pair element (59-be) sequence of the
blaGES-9 gene
cassette was interrupted by a novel insertion sequence (IS)
element, IS
Pa21 (Fig.
2A). The IS
Pa21 element was 1,374-bp long
and possessed 13-bp-long perfect inverted repeats (IRs). Its
transposase shared 35% amino acid identity with that of IS
Pa11 identified in
P. aeruginosa and 32% with that of IS
1111 (
18),
indicating that IS
Pa21 belonged to the IS
1111 family. Uncommonly
for IS elements, the IRs of IS
Pa21 were not located at its termini,
and no target site duplication was identified on each side of
IS
Pa21 (Fig.
2A). In the IS
Pa21 sequence, 7 bp separate the
IR from the left-hand end of the element (IRL) and 3 bp separate
the IR from the right-hand end (IRR). A similar observation
has been made for IS
5075 (
7) and IS
4321 (
25), other members
of the IS
1111 family, and also for IS
1383 from
Pseudomonas putida (
13,
16). Sequence analysis of the IS
Pa21 element revealed that
its transposase-encoding gene was likely expressed under the
control of a promoter consisting of a 35 region located
inside the right-hand end of the IS and a 10 region created
by the fusion of the right- and left-hand terminal sequences
of the IS in its circular form (Fig.
2C) as observed for other
elements of the IS
1111 family (
18).
Downstream of the
blaGES-9 gene cassette, part of the
aacA4 gene cassette encoding an AAC(6')-Ib aminoglycoside acetyltransferase
was identified in plasmid pDEJ-1 (Fig.
3). PCR mapping identified
the complete
aacA4 gene cassette and its downstream-located
sequences (Fig.
3). Detailed analysis revealed that IS
Pa21 may
provide promoter sequences for the expression of the
aacA4 gene.
Indeed, a 35 (TTGGCC) motif and a 10 (TTTCAT)
motif separated by 17 bp were able to constitute an efficient
promoter (Fig.
3).
The third cassette contained the
orfD gene encoding a putative
protein of unknown function previously identified in class 1
integrons in
Aeromonas salmonicida and in
Enterobacter aerogenes (
3,
12). The fourth and last cassette was the
aadB gene cassette
that encodes an aminoglycoside 2'-
O-adenylyltransferase conferring
resistance to gentamicin (
3). Surprisingly, a second copy of
the IS
Pa21 element was identified inside its 59-be (Fig.
1).
Again, no duplication of the target site of the IS
Pa21 insertion
was noticed (Fig.
2B). Analysis of the insertion sites of IS
Pa21 in In109 revealed that it targeted identical nucleotide motifs
in the 59-be of
blaGES-9 and of the
aadB gene cassettes (Fig.
2B).
Analysis of the sequences located upstream of blaGES-9 revealed that the integrase gene was truncated by the insertion of transposon Tn5393C (Fig. 3). Sequencing of pDEJ-1 identified the 78-bp long inverted IRL and part of the tnpA transposase gene of this transposon previously identified in the R plasmid pRAS2 from the fish pathogen Aeromonas salmonicida (11). PCR mapping revealed that Tn5393C was entire in P. aeruginosa DEJ, including the strA and strB streptomycin resistance genes. In addition, the 3' end of the integrase gene was present at the right-hand end of Tn5393C, suggesting that its insertion occurred independently inside the intI1 gene.
Conclusion.
This study emphasizes the spread of GES-type ESBLs in P. aeruginosa with further identification in France. A novel GES-type ß-lactamase with a broad spectrum hydrolysis profile extended to aztreonam was identified, its gene being located in a class 1 integron different from the other GES-positive integrons (Fig. 3).
Nucleotide sequence accession number.
The nucleotide and protein sequences of the In109 integron content have been registered in GenBank under accession no. AY920928.

ACKNOWLEDGMENTS
This work was funded by a grant from the Ministère de
l'Education Nationale et de la Recherche (UPRES-EA3539), Université
Paris XI, France, and by the European Community (6th PCRD, LSHM-CT-2003-503-335).
L.P. is a researcher for the INSERM, Paris, France. L.B. was
a recipient of the Sociedad Espanola de Enfermedades Infecciosas
y Microbiologia Clinica (SEIMC).
We thank C. Torres for constant support.

FOOTNOTES
* Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre cedex, France. Phone: 33-1-45-21-36-32. Fax: 33-1-45-21-63-40. E-mail:
nordmann.patrice{at}bct.ap-hop-paris.fr.


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Antimicrobial Agents and Chemotherapy, August 2005, p. 3593-3597, Vol. 49, No. 8
0066-4804/05/$08.00+0 doi:10.1128/AAC.49.8.3593-3597.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
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