ABSTRACT
A Pseudomonas aeruginosa strain expresses an extended-spectrum β-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 IS1111 family.
Extended-spectrum β-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 β-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.
MICs of β-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
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 β-lactamases (2).
Comparison of the amino acid sequence of β-lactamase GES-9 of P. aeruginosa DEJ to that of other GES enzymes. Names correspond to an update of the GES nomenclature, with GES-1 (21), GES-2 (24), GES-3 (26), GES-4 (26), GES-5 (27), and GES-6 (28), and include name changes for IBC-1 and IBC-2, named here GES-7 (8) and GES-8 (15), respectively. Numbering of β-lactamases is according to Ambler et al. (1). The vertical arrow indicates the putative cleavage site of the leader peptide of the mature β-lactamases. The amino acid residues that are part of the omega loop of Ambler class A β-lactamases are underlined.
E. coli DH10B (pDEJ-1) produced a β-lactamase with a pI value of 5.8 according to isoelectric focusing results. Purification of β-lactamase GES-9 was performed as established for GES-1 (21). The specific activity of purified β-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). β-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 [IC50], 10 nM). In addition, as observed for GES-1 and VEB-1 (21, 22), cefoxitin was an inhibitor of GES-9 activity (IC50, 1 μM).
Kinetic parameters of purified β-lactamase GES-9 compared to those of GES-1 and GES-2
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, ISPa21 (Fig. 2A). The ISPa21 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 ISPa11 identified in P. aeruginosa and 32% with that of IS1111 (18), indicating that ISPa21 belonged to the IS1111 family. Uncommonly for IS elements, the IRs of ISPa21 were not located at its termini, and no target site duplication was identified on each side of ISPa21 (Fig. 2A). In the ISPa21 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 IS5075 (7) and IS4321 (25), other members of the IS1111 family, and also for IS1383 from Pseudomonas putida (13, 16). Sequence analysis of the ISPa21 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 IS1111 family (18).
Features of insertion sequence ISPa21. Analysis of the sites of insertion (A) of the ISPa21 element in the 59-be of blaGES-9 and (B) of the aadA gene cassette. The 59-be sequences are indicated in italics. The stop codons of the blaGES-9 and aacA4 genes are indicated by an asterisk. The inverted repeats left (IRL) and right (IRR) of ISPa21 are shaded in gray, and the nucleotides that belong to ISPa21 but that are not part of the IRs are underlined. (C) Putative promoter sequences driving the expression of the transposase gene of ISPa21 in its circular form compared to those of IS1111 and IS4321 belonging to the same family. The base that may originate from either the left or the right end of the IS is shown in boldface, according to the observations made by Partridge et al. (18). In this configuration, the −35 and −10 promoter sequences are separated by 16 bp.
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 ISPa21 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).
Schematic map representing integron structures identified in association with blaGES-like genes. The 59-be's are indicated by black circles. The horizontal arrows indicate the transcription orientations. The integron In109 structure identified in this study is indicated (A), as well as (B) the blaGES-5-containing class 1 integron identified on pKGB525 (27), (C) the blaGES-2-containing class 1 integron identified on pLAP-1 (24), (D) the blaGES-1-containing class 1 integron identified on pTK-1 (21), and (E) the blaGES-1-containing class 3 integron identified on p22K9 (5).
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 ISPa21 element was identified inside its 59-be (Fig. 1). Again, no duplication of the target site of the ISPa21 insertion was noticed (Fig. 2B). Analysis of the insertion sites of ISPa21 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 β-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
- Received 15 February 2005.
- Returned for modification 22 March 2005.
- Accepted 4 May 2005.
- American Society for Microbiology
