Published Ahead of Print 18 October 2010. 10.1128/AAC.00773-10.

2011, 55(1):349. DOI:Antimicrob. Agents Chemother. Lecuyer, Jean-Ralph Zahar and Laurent PoirelRémy A. Bonnin, Patrice Nordmann, Anaïs Potron, Hervé 

Acinetobacter baumannii-Lactamase in βExtended-Spectrum

Carbapenem-Hydrolyzing GES-Type

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 2011, p. 349–354 Vol. 55, No. 10066-4804/11/$12.00 doi:10.1128/AAC.00773-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Carbapenem-Hydrolyzing GES-Type Extended-Spectrum�-Lactamase in Acinetobacter baumannii�

Remy A. Bonnin,1 Patrice Nordmann,1* Anaïs Potron,1,2 Herve Lecuyer,2Jean-Ralph Zahar,2 and Laurent Poirel1

Service de Bacteriologie-Virologie, INSERM U914, Emerging Resistance to Antibiotics, Hopital de Bicetre,Assistance Publique/Hopitaux de Paris, Faculte de Medecine et Universite Paris-Sud, Le Kremlin-Bicetre,1 and

Laboratoire de Microbiologie Hygiene Hospitaliere, Hopital Necker-Enfants Malades AssistancePublique-Hopitaux de Paris, Paris,2 France

Received 7 June 2010/Returned for modification 30 July 2010/Accepted 12 October 2010

Acinetobacter baumannii isolate AP was recovered from a bronchial lavage of a patient hospitalized in Paris,France. A. baumannii AP was resistant to all �-lactams, including carbapenems, and produced the extended-spectrum �-lactamase (ESBL) GES-14, which differs from GES-1 by two substitutions, Gly170Ser andGly243Ala. Cloning of the blaGES-14 gene followed by its expression in Escherichia coli showed that GES-14compromised significantly the efficacy of all �-lactams, including cephalosporins, aztreonam, and carbapen-ems. The carbapenemase activity of purified GES-14 was confirmed by kinetic studies. The blaGES-14 gene waslocated into a class 1 integron structure and located onto a ca. 95-kb self-transferable plasmid. This studyidentified a very broad-spectrum �-lactamase in A. baumannii.

Acinetobacter baumannii is an opportunistic pathogen that isan important source of nosocomial infections, such as pneu-monia, septicemia, urinary tract infections, and wound infec-tions (3). Treatment of infections due to this microorganism isbecoming a serious clinical concern since A. baumannii is fre-quently resistant to multiple antibiotics (17, 18). One of themain mechanisms of resistance to �-lactam molecules in A.baumannii is the production of �-lactamases. Resistance tocarbapenems is mostly related to production of metallo-�-lactamases or carbapenem-hydrolyzing oxacillinases (26),whereas resistance to broad-spectrum cephalosporins usuallyresults from overexpression of the natural AmpC-type enzyme(4) or in sporadic cases from acquisition of extended-spectrum�-lactamases (ESBLs) in A. baumannii. Those ESBLs maycorrespond to TEM or SHV derivatives but correspond mostlyto Ambler class A �-lactamases of the VEB or PER types (15).ESBLs of the GES type are reported increasingly for Gram-negative rods, including Pseudomonas aeruginosa, Enterobactercloacae, and Klebsiella pneumoniae (8, 20, 22, 25). GES-1 pos-sesses a hydrolysis profile similar to that of other clavulanate-inhibited Ambler class A ESBLs (1), including activity againstpenicillins and broad-spectrum cephalosporins, with higher ac-tivity against ceftazidime than against cefotaxime. Also, GES-1spares cephamycins and carbapenems and is inhibited by clav-ulanate, tazobactam, and imipenem (22). However, unlikemost ESBLs, GES-1 does not hydrolyze monobactams. GES-2,which differs from GES-1 by a single Gly170Asn substitutionlocated inside the �-loop of the catalytic site, hydrolyzes ad-ditionally carbapenems, but at a low level (23). A Gly170Serchange was identified in GES-4, GES-5, and GES-6 and re-

sulted in carbapenem and cephamycin hydrolysis. GES-9,which differs from GES-1 by a Gly243Ser change, does nothydrolyze carbapenems but was shown to possess an activityagainst monobactams (15). GES-11, differing from GES-1 bytwo amino acid substitutions, including the Gly243Ala change,possesses increased activity against aztreonam and has recentlybeen identified in A. baumannii (12).

Our study was initiated by the isolation of a carbapenem-resistant and ESBL-producing A. baumannii isolate in Paris,France, that was the source of a nosocomial dissemination.

MATERIALS AND METHODS

Bacterial strains and plasmids. Identification of A. baumannii AP was per-formed by using the API 32GN system (bioMerieux, Marcy l’Etoile, France) andwas confirmed by 16S rRNA gene sequencing as described previously (6). TheEscherichia coli TOP10 reference strain was used as a host for cloning. Thekanamycin-resistant plasmid pTOPO (Invitrogen, Cergy-Pontoise, France) wasused as a cloning vector in E. coli. A. baumannii BM4547 rifampin-resistant andE. coli TOP10 reference strains were also used as recipients for mating-out andelectroporation assays.

Antimicrobial agents and MIC determinations. The antimicrobial agents andtheir sources have been described elsewhere (19). Susceptibility testing wasperformed by disk diffusion assay (Sanofi-Diagnostic Pasteur, Marnes-la-Co-quette, France) and interpreted as described previously (5). The MICs weredetermined by Etest (AB Biodisk, Solna, Sweden) on Mueller-Hinton agar platesat 37°C (21). Production of an ESBL was searched by using synergy tests withdisks containing ceftazidime and ticarcillin-clavulanic acid and by using cloxacil-lin-containing Mueller-Hinton agar plates as described previously (21).

PCR and cloning experiments. Whole-cell DNA of A. baumannii isolate APwas extracted as described previously (19). This DNA was used as a templateunder standard PCR conditions (29) with a series of primers designed for thedetection of class A �-lactamase genes: blaTEM, blaSHV, blaPER-1, blaVEB-1,blaGES-1, and blaCTX-M (20, 21, 23, 24). Class B and D carbapenemase geneswere screened using primers specific for the blaIMP, blaVIM, blaSIM, blaOXA-23,blaOXA-40, blaOXA-58, and blaOXA-143 genes (23, 9). Since several �-lactamasegenes are part of gene cassettes which are located in class 1 integrons, primerslocated in the 5�-CS (primer INT2F, 5�-TCTCGGGTAACATCAAGG-3�) and3�-CS (primer 3�CS, 5�-AAGCAGACTTGACCTGA-3�) regions were used forPCR amplifications as described previously, with an elongation step extended to10 min (23). The obtained PCR fragment was purified with a QIAquick column(Qiagen, Courtaboeuf, France) and cloned into the pTOPO vector (Qiagen,

* Corresponding author. Mailing address: Service de Bacteriologie-Virologie-Hygiene, Hopital de Bicetre, 78 rue de General Leclerc,94275 Le Kremlin-Bicetre Cedex, France. Phone: 33-1-45-21-36-32.Fax: 33-1-45-21-63-40. E-mail: nordmann.patrice@bct.aphp.fr.

� Published ahead of print on 18 October 2010.

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Courtaboeuf, France). Recombinant plasmids were selected onto Trypticase soy(TS) agar plates containing ceftazidime (2 mg/ml) and kanamycin (30 mg/ml).The cloned DNA fragment inserted into one of the recombinant plasmids wassequenced on both strands with an Applied Biosystems sequencer (ABI 377).The nucleotide and deduced amino acid sequences were analyzed and comparedto sequences available over the Internet at the National Center for Biotechnol-ogy Information website (http://www.ncbi.nlm.nih.gov/BLAST/).

Analysis of the comM gene, known as a target of AbaR resistance islands in A.baumannii, was performed by amplifying that hot spot integration site withprimers comM-3�F (5�-GATTACCTCGATAAGAGAGTG-3�) and comM-5�R(5�-TTACACGCGCCTCTCATTGAG-3�) as previously described (7).

Plasmid content, hybridizations, and mating-out assays. The plasmid DNA ofA. baumannii AP was extracted by using the Kieser method (11). E. coliNCTC50192, harboring four plasmids of 154, 66, 48, and 7 kb, was used as aplasmid size marker. Plasmid DNAs were analyzed by agarose gel electrophore-sis as described previously (21). Restricted plasmid DNA fragments were trans-ferred to a nylon membrane (Hybond N�; GE Healthcare, Orsay, France) by theSouthern technique (29). The DNAs were then UV cross-linked (Stratalinker;Stratagene). An 860-bp PCR-generated probe (primers GES-A and GES-B) wasused for hybridization of genes encoding GES-type �-lactamases.

Direct transfer of the ceftazidime resistance marker into A. baumannii BM4547was attempted by liquid mating-out assays at 37°C and by electroporation of aplasmid DNA suspension extracted from A. baumannii AP into A. baumanniiBM4547 overproducing the efflux system AdeABC and into E. coli TOP10. Selectionwas performed on agar plates supplemented with ceftazidime (2 �g/ml).

�-Lactamase purification. Cultures of E. coli TOP10 harboring recombinantplasmid pTOPO-GES-14 were grown overnight at 37°C in 4 liters of TS brothcontaining amoxicillin (50 �g/ml) and kanamycin (30 �g/ml). �-LactamaseGES-14 was purified by ion-exchange chromatography. Briefly, the bacterialsuspension was pelleted, resuspended in 40 ml of 100 mM sodium phosphatebuffer (pH 7.0), sonicated, cleared by ultracentrifugation, and treated withDNase. The extract was then dialyzed against 20 mM bis-Tris {[bis(2-hydroxy-ethyl-imino]tris(hydroxymethyl)methane}(pH 6.5) and loaded onto a preequili-brated Q-Sepharose column with the same buffer. The resulting enzyme extractwas recovered in the flowthrough and dialyzed against 20 mM Tris-HCl (pH 7.5)overnight at 4°C. This extract was then loaded onto a preequilibrated (20 mMTris-HCl [pH 7.5]) Q-Sepharose column, and the proteins were eluted with alinear NaCl gradient (0 to 0.5 M). The �-lactamase activity was eluted with NaClat a concentration of 200 mM in the same Tris-HCl buffer. Finally, fractionscontaining the highest �-lactamase activities were pooled and subsequently di-alyzed overnight against 100 mM phosphate buffer (pH 7.0). The �-lactamase

activity was determined qualitatively using nitrocefin hydrolysis (Oxoid, Dardilly,France). The protein content was measured using the Bio-Rad DC protein assay.

Kinetic studies. Kinetic measurements (kcat and Km) of purified �-lactamaseGES-14 were performed as described previously (24). The 50% inhibitory con-centration (IC50) was determined for GES-14 as the concentration of clavulanateand tazobactam that reduced the hydrolysis rate of 100 �M benzylpenicillin by50%, under conditions in which GES-14 was preincubated with various concen-trations of inhibitor for 10 min at 30°C, before the substrate was added.

Nucleotide sequence accession number. The nucleotide sequence data re-ported in this work have been deposited in the GenBank nucleotide databaseunder accession no. HM451497.

RESULTS AND DISCUSSION

Susceptibility testing. A. baumannii AP was isolated from abronchial sample of a 34-year-old patient hospitalized in anintensive care unit at the Necker-Enfants Malades UniversityHospital (Paris, France) in January 2010. A. baumannii AP wasresistant to penicillins, �-lactamase inhibitor/penicillin combi-nations, broad-spectrum cephalosporins, aztreonam, and car-bapenems (Table 1). Synergy between ceftazidime and clavu-lanic acid observed with media supplemented with cloxacillinsuggested the production of an ESBL. In addition, A. bauman-nii AP was resistant to aminoglycosides, fluoroquinolones, sul-fonamides, and tetracycline and showed susceptibility only tocolistin and rifampin.

Identification of naturally occurring �-lactamase genes. A.baumannii AP harbored the naturally occurring blaOXA-91 gene,also known as blaOXA-Ab-24 (27). No ISAba1 element precededthat blaOXA-Ab-24 gene or the intrinsic blaampC gene in A. bau-mannii AP, likely indicating that both genes were not overex-pressed. In addition, PCR and sequencing showed that the comMgene was intact in A. baumannii AP, thus ruling out the presenceof an AbaR resistance island, at least at that location.

TABLE 1. MICs of �-lactams for A. baumannii isolate AP, A. baumannii BM4547(pAP) (transconjugant), A. baumannii BM4547, E. coliTOP10(pTOPO-GES-14), and E. coli TOP10 reference straina

�-Lactam

MIC (�g/ml)

A. baumanniiisolate AP

A. baumanniiBM4547(pAP)

A. baumanniiBM4547

E. coli TOP10(pTOPO-GES-14) E. coli TOP10

Amoxicillin �256 �256 16 �256 2Amoxicillin � CLA 128 128 16 32 2Ticarcillin �256 �256 8 �256 2Ticarcillin � CLA 128 128 8 16 2Piperacillin �256 �256 4 �256 1Piperacillin � TZB 128 128 4 128 1Cephalothin 256 256 256 256 4Cefuroxime 256 256 64 256 2Cefoxitin 256 256 256 64 2Cefotaxime �32 �32 �32 4 0.06Ceftazidime �256 �256 4 �256 0.12Ceftazidime � CLA 64 32 ND 8 NDCefepime 32 32 16 0.5 0.06Cefpirome 256 256 64 0.5 0.06Aztreonam 128 128 32 1 0.03Aztreonam � CLA 64 32 ND 0.12 NDMeropenem 16 16 0.5 0.25 0.02Doripenem 8 4 0.25 0.25 0.02Imipenem 32 8 0.25 1 0.12Imipenem � CLA 8 2 ND 0.25 ND

a CLA, clavulanic acid (4 �g/ml); TZB, tazobactam (4 �g/ml); ND, not determined.

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Cloning and sequencing of the ESBL gene. Using whole-cellDNA of A. baumannii AP as a template, preliminary PCRdetection of class B and class D carbapenem-hydrolyzing �-lac-tamase genes failed (data not shown), but a PCR amplificationwas obtained with blaGES-1-specific primers. Since blaGES-like

genes are known to be integron encoded, consensus primers5�-CS and 3�-CS for class 1 integrons were used, and PCRamplification gave an 8-kb DNA fragment that was sub-sequently cloned into vector pTOPO and expressed inE. coli TOP10. The resulting recombinant strain E. coliTOP10(pTOPO-GES-14) displayed an ESBL phenotype withhigh-level resistance to ceftazidime and reduced susceptibility toaztreonam, cefoxitin, and carbapenems (Table 1). In addition, itwas resistant to chloramphenicol, trimethoprim, kanamycin, to-bramycin, streptomycin, and spectinomycin but remained suscep-tible to sulfonamides. Sequence analysis of the cloned DNA frag-ment revealed an 864-bp-long open reading frame encoding a287-amino-acid preprotein designed as GES-14, differing by onesingle substitution from GES-5 (Gly243Ala) and from GES-11(Gly170Ser) and by two amino acid substitutions (Gly170Ser andGly243Ala) from GES-1 (Fig. 1). The Gly170Ser substitutionfirstly identified in GES-5 is known to confer to that enzyme ahigher catalytic efficiency toward carbapenems and cephamycins(2, 30), although the Gly243Ala substitution firstly identified inGES-9 increases the catalytic activity of that enzyme toward az-treonam (25). Therefore, the susceptibility pattern observed forE. coli TOP10(pTOPO-GES-14) that showed reduced suscepti-bility to cephamycins, monobactams, and carbapenems in addi-tion to oxyimino-cephalosporins was similar to those previouslyobserved. This suggested that GES-14 might combine both

GES-5 and GES-9 hydrolysis spectra, thus defining that enzymeas the first �-lactamase hydrolyzing significantly all �-lactams.

Biochemical properties of GES-14. The pI value of GES-14was determined to be 5.8. After a purification process, thepurity of the enzyme was estimated to be 90% according tosodium dodecyl sulfate-polyacrylamide gel electrophoresis(data not shown). Kinetic parameters of GES-14 confirmed itsbroad-spectrum activity against all �-lactams, including, in par-ticular, oxyimino-cephalosporins, monobactams, and carbap-enems (Table 2). The hydrolysis rates of GES-14 for penicillins

FIG. 1. Alignment of the amino acid sequences of �-lactamases GES-1, GES-2, GES-5, GES-6, GES-7 (IBC-1), GES-9, GES-11, GES-13, andGES-14. Boldface amino acids are conserved residues among Ambler class A �-lactamases. The amino acids of the omega loop are underlined.

TABLE 2. Kinetic parameters for �-lactamase GES-14a

Substrate Km (�M) RelativeV

max(%)b kcat (s�1) kcat/Km

(mM�1 s�1)

Benzylpenicillin 200 100 18 90Amoxicillin 130 37 7 55Ticarcillin 200 0.7 0.1 0.5Piperacillin 500 495 90 180Cephalothin 4,800 9 1.6 0.5Cephaloridine 4,000 108 20 5Cefoxitin 163 40 0.9 5.4Cefotaxime 4,300 58 10 2.5Ceftazidime �5,000 �1,000 20 1Cefepime 1.3 1 0.2 154Aztreonam �5,000 �1,000 3.5 0.5Ertapenem 2 0.3 0.05 25Meropenem 2 0.3 0.05 25Imipenem 2 0.4 0.1 50

a Data are the means of three independent experiments. Standard deviationswere within 10% of the means.

b Values are relative to those of benzylpenicillin, which were set at 100.

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were similar to those of GES-1 and GES-2 (except for pip-eracillin) but were lower than those observed for GES-9, whichpossesses a serine at position 243 (25) (Fig. 1; Table 2). Thehydrolysis rates of GES-14 for cephalosporins were similar tothose found for GES-1 and GES-2. However, the very highmaximum rate of metabolism (Vmax) value obtained for thatsubstrate confirmed that it was efficiently hydrolyzed byGES-14 (Table 2). GES-14 also efficiently hydrolyzed imi-penem, with a kcat/Km value 4-fold higher than that of GES-2.In addition, GES-14 hydrolyzed meropenem and ertapenem atrates similar to those for imipenem. This is in accordance withprevious findings obtained for GES-5 (2) demonstrating thatthe serine residue at position 170 conferred significant carbap-enemase activity to GES derivatives. The hydrolysis of aztreo-nam was also significant despite a low affinity (Km � 5,000 �M)for this substrate. GES-14 exhibited relatively low catalyticefficiency for some substrates compared to many other class A�-lactamases, such as TEM or SHV derivatives, but conferredhigh resistance to �-lactams. This discrepancy might be re-lated to a high level of expression of the blaGES-14 gene in A.baumannii AP, in accordance with the identification of astrong promoter configuration (see below).

Inhibition studies, carried out by measuring the IC50s,showed that the catalytic activities of GES-14 and GES-2 weresimilarly inhibited by clavulanic acid (Table 3).

Genetic support of blaGES-14 gene and transfer of �-lactamresistance. Plasmid DNA analysis showed that A. baumanniiAP harbored a 95-kb plasmid (data not shown). Mating-outassays gave the A. baumannii BM4547 strain harboring naturalplasmid pAP, which harbored the blaGES-14 gene and showed

an ESBL phenotype. It was also resistant to carbapenems,indicating that acquisition of ESBL GES-14 together with ef-flux overproduction (a property of A. baumannii BM4547) ledto resistance to carbapenems. In addition, that transconjuganthad acquired resistance to chloramphenicol, tetracycline, andaminoglycosides. Attempts to transfer the blaGES-14-carryingnatural plasmid pAP into the E. coli TOP10 recipient strain byconjugation and electroporation failed. This may indicate thatplasmid pAP possesses a host replication range that excludesE. coli. Southern hybridization performed with a blaGES-14-specific probe confirmed that this gene was located on a 95-kbplasmid in A. baumannii AP (data not shown).

Genetic environment of the blaGES-14 gene. Sequencing ofthe entire insert of recombinant plasmid pTOPO-GES-14 re-vealed that blaGES-14 was present in the form of a gene cas-sette, preceded by an intI1 integrase gene of a class 1 integronthat was named In125. Detailed analysis of the sequences lo-cated upstream of blaGES-14 revealed that a 238-bp fragmentwas missing compared to the In52 integron carrying theblaGES-1 gene as the first cassette (22) (see Fig. 3). Conse-quently, the integrase gene upstream of the blaGES-14 gene wastruncated in its 5� extremity, resulting in the lack of the first 28N-terminal amino acids of the corresponding protein. Thissuggested that this integrase was not functional here. In addi-tion, the entire attI1 recombination site was absent, togetherwith the first 14 bp of the blaGES-14 gene cassette (Fig. 2).Noteworthy, in silico analysis showed that the same deletionshad occurred upstream of the blaGES-11 gene in A. baumanniiBM4674 (Fig. 2) (14). Overall, the In125 integron appears tobe a stable genetic structure that cannot acquire or lose itscassette content. The attI1 deletion might be the consequenceof a recombination event that had been mediated by the inte-grase itself. Indeed, it has been demonstrated already that theclass 1 integron integrase may induce some nonspecific recom-bination events in the attI1 region, with a preferential G(T/A)Ttarget site that fits with the sequence where the deletion hasoccurred in In125 (Fig. 3) (28).

As a consequence of the deletion of the attI sequence, no P2

promoter was identified for gene cassette expression in In125.However, the Pc promoter was still present and, as a conse-quence of that deletion, was located very close to the blaGES-14

TABLE 3. IC50s of clavulanic acid and tazobactam for�-lactamases GES-14, GES-1, and GES-2a

InhibitorIC50 (�M) for:

GES-14 GES-1b GES-2b

Clavulanic acid 1 5 1Tazobactam 0.15 2.5 0.5

a Data are the means of three independent experiments. Standard deviationswere within 10% of the means.

b Data for GES-1 and GES-2 IC50s are from references 22 and 23, respectively.

FIG. 2. Schematic map representing integron structures harboring the blaGES-14 (A) and blaGES-11 (B) genes. The genes and their correspondingtranscriptional orientations are indicated by horizontal arrows. The 59-base elements are indicated by black circles.

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start codon (the codon being only 31 bp from the �10 pro-moter sequence of Pc). Analysis of the �35 and �10 Pc pro-moter sequences of the In125 Pc promoter (being �35TTGACA and �10 TGGACA) showed that it corresponded tothe so-called pPcH2 hybrid promoter possessing relativestrength (10, 12, 16). All those features, e.g., a strong promoterconfiguration that is located at a short distance from the tran-scription initiation site, suggest that the blaGES-14 gene is ex-pressed here at a high level. Since the integrase gene locatedimmediately upstream of blaGES-14 was truncated and since theusual attI1 was missing here, it is likely that this integron isdefective.

Downstream of the blaGES-14 gene, the aacA4 gene cassetteencoding the AAC(6�)-Ib aminoglycoside acetyltransferasewas identified. It was followed by the dfrA7 gene cassette en-coding resistance to trimethoprim and then by the qacE�1gene cassette specific for the 3� extremity of class 1 integrons.

On the left-hand extremity of the In125 sequence, part of thetnpM gene encoding the modulation transposase protein that isitself part of the Tn21 transposon module vehiculating class 1integrons was identified (Fig. 2). The tnpM gene was truncatedin its 3� extremity. Then, the two streptomycin resistance genesstrA and strB previously identified in Tn5393-like transposonswere identified (13). Interestingly, downstream of that struc-ture, another class 1 integron containing three gene cassetteswas identified. The first and third cassettes corresponded, re-spectively, to aadB and aadA2, both encoding resistance tostreptomycin and spectinomycin. The second cassette encodeda CmlA5-like protein (98% amino acid identity) conferringresistance to chloramphenicol (Fig. 2). At the 3� extremity ofthat integron, neither the qacE�1 nor sul1 gene was found,since the genes have been replaced by the insertion of the strAand strB genes.

Overall, and likely as a result of homologous recombination,a large structure that corresponds to a fusion of two class 1integrons has been identified and contains a total of eightresistance genes.

Conclusion. This study characterized a novel carbapenem-hydrolyzing GES-type �-lactamase from A. baumannii. Thehydrolysis spectrum of GES-14 includes cephamycins and car-bapenems and therefore corresponds to that of the first GES-

type enzyme, and its substrate profile includes all �-lactams, asa consequence of two amino acid substitutions.

To the best of our knowledge, GES-14 is the first identified�-lactamase hydrolyzing significantly all �-lactams, since boththe Ambler class A KPC-2 and the class B carbapenemases donot hydrolyze cephamycins and monobactams, respectively.The blaGES-14 gene was located on a plasmid, which underlinesits diffusion potency, at least among Acinetobacter spp. Thatplasmid harbored a large array of resistance genes encodingresistance to several antibiotic families and therefore repre-sents one of the few examples of plasmid-mediated multidrugresistance in A. baumannii, since recent studies tend to asso-ciate multidrug resistance mostly to the chromosomally locatedAbaR resistance islands.

ACKNOWLEDGMENTS

We thank B. Bercot for contributive discussions.This work was funded partially by a grant from the INSERM (U914)

and the Ministere de l’Education Nationale et de la Recherche(UPRES-EA3539), Universite Paris XI, France, and mostly by grantsfrom the European Community (TROCAR, HEALTH-F3-2008-223031 and TEMPOtest-QC, HEALTH-2009-241742) and from theINSERM (U914).

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7. Fournier, P. E., D. Vallenet, V. Barbe, S. Audic, H. Ogata, L. Poirel, H.Richet, C. Robert, S. Mangenot, C. Abergel, P. Nordmann, J. Weissenbach,D. Raoult, and J. M. Claverie. 2006. Comparative genomics of multidrugresistance in Acinetobacter baumannii. PLoS Genet. 2:e7.

FIG. 3. Comparative structures of the sequences located upstream of the blaGES-14 and blaGES-1 genes. The 238-bp nucleotide sequence thatis deleted in In125 containing blaGES-14 (A) but present in In52 containing blaGES-1 (B) is represented. The sequences of the promoters areunderlined, and the attI1 recombination site is italicized. The �35 and �10 sequences of the Pc and P2 promoters are underlined, and the startcodons of the blaGES genes are in bold. The core site sequence of the blaGES-1 gene cassette is boxed.

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