Journal of Antimicrobial Chemotherapy (2001) 48, 627-630
© 2001 The British Society for Antimicrobial Chemotherapy
An integron-associated ß-lactamase (IBC-2) from Pseudomonas aeruginosa is a variant of the extended-spectrum ß-lactamase IBC-1
a Laboratory of Bacteriology, Hellenic Pasteur Institute, Athens; b Department of Microbiology, Medical School, Aristotle University of Thessaloniki; c Department of Microbiology, Hippokration General Hospital, Thessaloniki; d Department of Microbiology, Medical School, University of Athens, Athens, Greece
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
|---|
An extended-spectrum ß-lactamase, IBC-2, produced by a clinical strain of Pseudomonas aeruginosa, was characterized. blaIBC-2 was found, as a gene cassette, to be the sole gene within the variable region of a class 1 integron probably located in the chromosome. IBC-2 is a variant of IBC-1 and GES-1, differing by one amino acid from each of these ß-lactamases. When expressed in Escherichia coli, IBC-2 was observed to confer resistance to ceftazidime and decreased susceptibility to other oxyimino-ß-lactams.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Most class A extended-spectrum ß-lactamases (ESBLs) described so far are variants of the penicillinases TEM-1, TEM-2 and SHV-1. However, the number of studies reporting the emergence of non-TEM, non-SHV ESBLs is growing. Recently, two related ß-lactamases, GES-1 from Klebsiella pneumoniae and IBC-1 from Enterobacter cloacae, were identified.1,2 They confer resistance to ceftazidime and are strongly inhibited by imipenem. The respective bla genes were found in the variable region of class 1 integrons, carried on self-transferable plasmids.1,2
ESBLs, while frequent in clinical strains of enterobacteria, are less common in Pseudomonas aeruginosa. To date, various TEM- and SHV-type ESBLs, together with the non-TEM, non-SHV enzymes PER-1 and VEB-1, have been found in this species.3,4 In this study we describe IBC-2, an integron-borne ESBL, produced by a P. aeruginosa clinical isolate.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Strains and plasmids
P. aeruginosa 555 was isolated from a urinary tract infection of a patient treated in the AHEPA hospital of Thessaloniki in 1998. It originates from a cluster of genetically related P. aeruginosa isolates that are resistant to carbapenems due to production of the metallo-ß-lactamase VIM-2.5 Escherichia coli JM109 was used as the host for recombinant plasmids. The plasmid pMON-38201 was used for direct cloning of PCR products.5 The plasmid pBCSK+ (Stratagene, La Jolla, CA, USA) was used as an expression vector for the bla genes.
Antibiotic susceptibility testing
Susceptibility status to various antibiotics was assessed by disc diffusion assay. MICs of ß-lactams were determined by an agar dilution technique.6
Plasmid DNA isolation and transformation experiments
Plasmids were isolated from P. aeruginosa 555 by an alkaline lysis technique2 and were used to transform E. coli JM109 competent cells either using CaCl2 or by electroporation.
Integron mapping, cloning of PCR amplicons and DNA sequencing
Integron mapping was carried out by PCR as described previously.2 To amplify that part of a class 1 integron that contains a gene cassette(s) and the common promoters, primers INT-F (5'-CGTTCCATACAGAAGCTG-3') and 3'-CS (5'-AAGCAGACTTGACCTGA-3') and the high-fidelity DNA polymerase VENT (New England BioLabs Inc., Beverly, MA, USA) were used. The amplicons were inserted into the cloning site of pMON-38201. BamHI– EcoRI fragments of the recombinants were then, in turn, cloned into the multiple cloning site of pBCSK+. The resulting recombinant plasmids were used to transform E. coli JM109. Nucleotide sequences were determined with a Sequenase 2.0 kit (US Biochemical Corp., Cleveland, OH, USA).
Isoelectric focusing
ß-Lactamases were released by ultrasonic treatment of cell suspensions. Isoelectric focusing (IEF) was performed in polyacrylamide gels containing ampholytes (pH range 3.5–9.5) (Pharmacia-LKB Biotechnology, Uppsala, Sweden). ß-Lactamases were visualized with nitrocefin (Oxoid Ltd, Basingstoke, UK).
Nucleotide sequence accession number
The GenBank accession number of the blaIBC-2 cassette is AF329699.
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
P. aeruginosa 555 was found to be resistant to all ß-lactams tested (Table
). IEF showed that it produced, apart from VIM-2, a ß-lactamase with an apparent pI of 5.8 (data not shown). Attempts to transfer ß-lactam resistance to E. coli by transformation or electroporation, using plasmid DNA preparations, were not successful. Using total DNA from P. aeruginosa 555 as template, a PCR assay specific for blaIBC-1 gave a positive result. From the results of an extended PCR analysis, a class 1 integron structure that includes blaIBC as the sole cassette in the variable region was postulated.
|
Using INT-F and 3'-CS primers and P. aeruginosa 555 DNA, a PCR product of c. 1600 bp was obtained. The amplicon was ligated into pMON-38201. A BamHI–EcoRI fragment of the recombinant DNA was then subcloned into pBCSK+ to generate pIBC2. This was used to transform E. coli JM109. The resulting strain was resistant to penicillins and ceftazidime and showed decreased susceptibility to cefotaxime and aztreonam. ß-Lactamase inhibitors restored the activities of penicillins and ceftazidime. This ß-lactam resistance pattern is similar to that conferred by IBC-1, although the MICs of oxyimino-ß-lactams were found to be lower for E. coli (pIBC2) than those for an IBC-1-producing E. coli strain tested in parallel (Table
). Double diffusion sensitivity tests were positive, indicating production of an ESBL. Also, a marked synergy was observed between oxyimino-ß-lactams and imipenem. IEF showed that E. coli (pIBC2) produced a ß-lactamase with a pI of 5.8, indistinguishable from that of a ß-lactamase in the isolate of origin (data not shown).
Nucleotide sequencing of the PCR amplicon and the cloned fragment revealed a DNA segment that contains the 5' end of intI1 followed by a 1021 bp gene cassette inserted at the attI1 recombination site. This cassette contains an open reading frame of 861 bp (blaIBC-2) and a 59-base element (59-be) that comprises 101 nucleotides and is identical to the 59-be of the blaIBC-1 cassette.2 Two putative promoters, P1 and P2, precede blaIBC-2 (Figure). The respective sequences indicate that P1 is a weak promoter. With P2, the spacing between –35 and –10 is 17 nucleotides and therefore this promoter is presumed to be functional.7
|
The polypeptide deduced from blaIBC-2 has 281 amino acids. IBC-2 possesses the motifs typical of class A enzymes,8 Thr-237 and cysteines at positions 69 and 237a. It differs by only one residue from both IBC-1 (Glu instead of Lys-98) and GES-1 (Leu instead of Ala-120).
IBC-2 together with GES-1 and IBC-1 comprise a new cluster of class A ß-lactamases with extended spectrum activities. The respective bla genes differ by only one or two nucleotides and should be considered to be alleles originating from a common ancestor. While the deduced amino acid sequences of GES/IBC ß-lactamases are somewhat divergent from the sequences of the other class A enzymes, they appear to be distant relatives of the PER-1 and VEB-1 ESBLs.1
IBC-2 confers resistance to ceftazidime and, to a lesser extent, to the other oxyimino-cephalosporins. It is inhibited by imipenem, tazobactam and clavulanic acid. As suggested for GES-1 and IBC-1, amino acid residues Cys-69, Thr-237 and Cys-237a may be involved in the interaction of IBC-2 with various ß-lactams.1,2 IBC-2 differs from IBC-1 by one amino acid (Glu instead of Lys-104), a change consistent with the more acidic pI of IBC-2. A Lys for Glu-104 substitution enhances activity of TEM ESBLs against ceftazidime and aztreonam.8 The lower level of resistance to ceftazidime shown by E. coli (pIBC2) as compared with E. coli (pIBC1) may reflect the amino acid substitution at position 104 in IBC-2. A detailed study of the hydrolytic properties of IBC-1 and IBC-2, as well as comparison of MICs for strains producing these enzymes under perfectly isogenic conditions, will clarify this point.
The blaIBC-2 was the sole gene cassette found in the variable region of a class 1 integron that is probably located in the chromosome of P. aeruginosa 555. The 59-be of the blaIBC-2 gene cassette is identical to that of the blaIBC-1 gene cassette.2 However, the associated genes within the variable regions of the IBC-1- and IBC-2-encoding integrons are different, indicating distinct phylogenies for these structures.
The discovery of blaIBC/GES alleles on three independent occasions from pathogenic microorganisms indicates that the genes may be widespread. IBC-1 was detected in an E. cloacae clinical strain that was also isolated in Thessaloniki. Therefore, a survey for GES/IBC producers in the hospitals of that area is warranted.
|
Notes |
|---|
* Correspondence address. Laboratory of Antimicrobial Agents, Department of Microbiology, Medical School, National University of Athens, M. Asias 75, Athens 115, Greece. Tel: +30-1-778-5638; Fax: +30-1-770-9180; E-mail: Ltzouvel{at}cc.uoa.gr
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
1 . Poirel, L., Le Thomas, I., Naas, T., Karim, A. & Nordmann, P. (2000). Biochemical sequence analyses of GES-1, a novel class A extended-spectrum ß-lactamase, and the class1 integron In52 from Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 44, 622–32.
2
.
Giakkoupi, P., Tzouvelekis, L. S., Tsakris, A., Loukova, V., Sofianou, D. & Tzelepi, E. (2000). IBC-1, a novel integron-associated class A ß-lactamase with extended-spectrum properties produced by an Enterobacter cloacae clinical strain. Antimicrobial Agents and Chemotherapy 44, 2247–53.
3
.
Nordmann, P. & Naas, T. (1994). Sequence analysis of PER-1 extended-spectrum ß-lactamase from Pseudomonas aeruginosa and comparison with class A ß-lactamases. Antimicrobial Agents and Chemotherapy 38, 104–14.
4 . Naas, T., Poirel, L., Karim, A. & Nordmann, P. (1999). Molecular characterization of In50, a class 1 integron encoding the gene for the extended-spectrum ß-lactamase VEB-1 in Pseudomonas aeruginosa. FEMS Microbiology Letters 176, 411–9.[Web of Science][Medline]
5
.
Mavroidi, A., Tsakris, A., Tzelepi, E., Pournaras, S., Loukova, V. & Tzouvelekis, L. S. (2000). Carbapenem-hydrolysing VIM-2 metallo-ß-lactamase in Pseudomonas aeruginosa from Greece. Journal of Antimicrobial Chemotherapy 46, 1041–2.
6 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Testing for Bacteria that Grow Aerobically—Second Edition: Approved Standard M7-A4. NCCLS, Wayne, PA.
7 . Levesque, C., Brassard, S., Lapointe, J. & Roy, P. H. (1994). Diversity and relative strength of tandem promoters for the antibiotic-resistance genes of several integrons. Gene 142, 49–54.[Web of Science][Medline]
8 . Matagne, A., Lamotte-Brasseur, J. & Frere, J.-M. (1998). Catalytic properties of class A ß-lactamases: efficiency and diversity. Biochemical Journal 330, 581–98.
Received 20 December 2000; returned 22 March 2001; revised 4 April 2001; accepted 16 July 2001
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Frase, Q. Shi, S. A. Testero, S. Mobashery, and S. B. Vakulenko Mechanistic Basis for the Emergence of Catalytic Competence against Carbapenem Antibiotics by the GES Family of {beta}-Lactamases J. Biol. Chem., October 23, 2009; 284(43): 29509 - 29513. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. D. Lister, D. J. Wolter, and N. D. Hanson Antibacterial-Resistant Pseudomonas aeruginosa: Clinical Impact and Complex Regulation of Chromosomally Encoded Resistance Mechanisms Clin. Microbiol. Rev., October 1, 2009; 22(4): 582 - 610. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Strateva and D. Yordanov Pseudomonas aeruginosa - a phenomenon of bacterial resistance J. Med. Microbiol., September 1, 2009; 58(9): 1133 - 1148. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Walther-Rasmussen and N. Hoiby Class A carbapenemases J. Antimicrob. Chemother., September 1, 2007; 60(3): 470 - 482. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Poirel, T. Naas, and P. Nordmann Pyrosequencing as a Rapid Tool for Identification of GES-Type Extended-Spectrum {beta}-Lactamases. J. Clin. Microbiol., August 1, 2006; 44(8): 3008 - 3011. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Wang, P. Cai, D. Chang, and Z. Mi A Pseudomonas aeruginosa isolate producing the GES-5 extended-spectrum {beta}-lactamase J. Antimicrob. Chemother., June 1, 2006; 57(6): 1261 - 1262. [Full Text] [PDF]
|
|
|
|
|
|
D. L. Paterson and R. A. Bonomo Extended-Spectrum {beta}-Lactamases: a Clinical Update Clin. Microbiol. Rev., October 1, 2005; 18(4): 657 - 686. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Poirel, L. Brinas, A. Verlinde, L. Ide, and P. Nordmann BEL-1, a Novel Clavulanic Acid-Inhibited Extended-Spectrum {beta}-Lactamase, and the Class 1 Integron In120 in Pseudomonas aeruginosa Antimicrob. Agents Chemother., September 1, 2005; 49(9): 3743 - 3748. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Poirel, L. Brinas, N. Fortineau, and P. Nordmann Integron-Encoded GES-Type Extended-Spectrum {beta}-Lactamase with Increased Activity toward Aztreonam in Pseudomonas aeruginosa Antimicrob. Agents Chemother., August 1, 2005; 49(8): 3593 - 3597. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. H. Lee, S. H. Jeong, J.-i. Wachino, Y. Arakawa, L. Poirel, and P. Nordmann Nomenclature of GES-Type Extended-Spectrum {beta}-Lactamases Antimicrob. Agents Chemother., May 1, 2005; 49(5): 2148 - 2150. [Full Text] [PDF]
|
|
|
|
|
|
F. Pasteran, D. Faccone, A. Petroni, M. Rapoport, M. Galas, M. Vazquez, and A. Procopio Novel Variant (blaVIM-11) of the Metallo-{beta}-Lactamase blaVIM Family in a GES-1 Extended-Spectrum-{beta}-Lactamase-Producing Pseudomonas aeruginosa Clinical Isolate in Argentina Antimicrob. Agents Chemother., January 1, 2005; 49(1): 474 - 475. [Full Text] [PDF]
|
|
|
|
|
|
G. F. Weldhagen Rapid Detection and Sequence-Specific Differentiation of Extended-Spectrum {beta}-Lactamase GES-2 from Pseudomonas aeruginosa by Use of a Real-Time PCR Assay Antimicrob. Agents Chemother., October 1, 2004; 48(10): 4059 - 4062. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. F. Weldhagen Sequence-Selective Recognition of Extended-Spectrum {beta}-Lactamase GES-2 by a Competitive, Peptide Nucleic Acid-Based Multiplex PCR Assay Antimicrob. Agents Chemother., September 1, 2004; 48(9): 3402 - 3406. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-i. Wachino, Y. Doi, K. Yamane, N. Shibata, T. Yagi, T. Kubota, and Y. Arakawa Molecular Characterization of a Cephamycin-Hydrolyzing and Inhibitor-Resistant Class A {beta}-Lactamase, GES-4, Possessing a Single G170S Substitution in the {Omega}-Loop Antimicrob. Agents Chemother., August 1, 2004; 48(8): 2905 - 2910. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-i. Wachino, Y. Doi, K. Yamane, N. Shibata, T. Yagi, T. Kubota, H. Ito, and Y. Arakawa Nosocomial Spread of Ceftazidime-Resistant Klebsiella pneumoniae Strains Producing a Novel Class A {beta}-Lactamase, GES-3, in a Neonatal Intensive Care Unit in Japan Antimicrob. Agents Chemother., June 1, 2004; 48(6): 1960 - 1967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Correia, F. Boavida, F. Grosso, M. J. Salgado, L. M. Lito, J. M. Cristino, S. Mendo, and A. Duarte Molecular Characterization of a New Class 3 Integron in Klebsiella pneumoniae Antimicrob. Agents Chemother., September 1, 2003; 47(9): 2838 - 2843. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. F. Weldhagen, L. Poirel, and P. Nordmann Ambler Class A Extended-Spectrum {beta}-Lactamases in Pseudomonas aeruginosa: Novel Developments and Clinical Impact Antimicrob. Agents Chemother., August 1, 2003; 47(8): 2385 - 2392. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||