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Antimicrobial Agents and Chemotherapy, October 2002, p. 3286-3287, Vol. 46, No. 10
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.10.3286-3287.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Carbapenem-Resistant Pseudomonas aeruginosa in Malaysia Producing IMP-7 ß-Lactamase

Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

Received 19 November 2001/ Returned for modification 16 March 2002/ Accepted 19 July 2002

	ABSTRACT

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We have isolated and identified a carbapenem-resistant Pseudomonas aeruginosa strain from Malaysia that produces an IMP-7 metallo-ß-lactamase. This isolate showed high-level resistance to meropenem and imipenem, the MICs of which were 256 and 128 µg/ml, respectively. Isoelectric focusing analyses revealed pI values of >9.0, 8.2, and 7.8, which indicated the possible presence of IMP and OXA. DNA sequencing confirmed the identity of the IMP-7 determinant.

	TEXT

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Pseudomonas aeruginosa is a leading cause of hospital-acquired infections, giving rise to a wide range of opportunistic infections. Its high intrinsic resistance to antibiotics and ability to develop multidrug resistance pose serious therapeutic problems (5). Broad-spectrum ß-lactams, such as carbapenems, are potential drugs for the therapy of infectious caused by P. aeruginosa. However, increasing usage of carbapenems in the treatment of these infections has resulted in the development of carbapenem-resistant P. aeruginosa. Carbapenem-resistant strains producing IMP-type metallo-ß-lactamases have been reported from Asia (2, 10, 12, 13; T. H. Koh, L.-H. Sng, G. S. Babini, N. Woodford, D. M. Livermore, and L. M. C. Hall, Letter, Antimicrob. Agents Chemother. 45:1939-1940, 2001), Canada (3), and Europe (6, 11). We report our findings from screening 50 carbapenem-resistant P. aeruginosa isolates (identified with the API20NE kit; bioMerieux, Marcy l'Etoile, France) from the University of Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia, to determine the prevalence of metallo-ß-lactamases.

The isolates were screened with bla_IMP-specific primers according to the methods described by Senda et al. (10) and Arakawa et al. (1). Only 1 (Imp699) of the 50 isolates of P. aeruginosa studied was amplified with bla_IMP primers, producing a 586-bp amplicon that was sequenced. Imp699, a multidrug-resistant P. aeruginosa strain, was isolated from the peritoneal fluid of an 8-year-old child at the UMMC in December 1999. The child had developed peritonitis secondary to peritoneal dialysis. Prior to the isolation of the multidrug-resistant strain, susceptible strains of P. aeruginosa were repeatedly isolated from the peritoneal dialysate of the patient. The initial strain was sensitive to ceftazidime, carbapenems, and aminoglycosides, and subsequently a strain that was reported to be resistant to carbapenem, but sensitive to ceftazidime and aminoglycosides, was isolated. The latter was obtained prior to the isolation of the multidrug-resistant strain. Other organisms isolated from peritoneal fluid included Acinetobacter species and Candida parapsilosis. Prior to the isolation of the multidrug-resistant P. aeruginosa strain, the patient had been exposed to the antibiotics cefuroxime (parenteral), ceftriaxone, imipenem, ceftazidime, and vancomycin.

Strain Imp699 was further studied to confirm and characterize the metalloenzyme. MICs were determined by the agar dilution method of the National Committee for Clinical Laboratory Standards (7). The following antibiotics were evaluated: amikacin, cefoperazone, ceftazidime, gentamicin, and piperacillin (Sigma Chemical Co., St. Louis, Mo.); aztreonam (E. R. Squibb & Sons, Inc., Princeton, N.J.); meropenem (parenteral) (Zeneca, Macclesfield, United Kingdom); and imipenem (parenteral) (Merck Sharp & Dohme Corp., South Granville, Australia). The potencies of imipenem and meropenem in the parenteral drug preparation were taken into account when preparing the drug concentration. P. aeruginosa ATCC 27853 was used as the control strain.

Imp699 (isolated from peritoneal fluid) showed a high level of resistance to all of the antibiotics, except amikacin. The MICs of the antibiotics for Imp699 were as follows: gentamicin, 256 µg/ml; ceftazidime, >256 µg/ml; aztreonam, 32 µg/ml; amikacin, 8 µg/ml; cefoperazone, >128 µg/ml; piperacillin, 64 µg/ml; meropenem, 256 µg/ml; and imipenem, 128 µg/ml.

Crude ß-lactamase extract for isoelectric focusing was prepared according to the method of Neuwirth et al. (8). PIs of >9.0, 8.2, and 7.8 were obtained. The pI value of >9.0 given above indicated the presence of IMP. This was confirmed by PCR, by which the nucleotide sequence obtained revealed a 100% homology to the bla_IMP-7 nucleotide of P. aeruginosa from Calgary, Canada (GenBank accession no. AF318077).

To determine the sequence of the entire gene of bla_IMP-7, we designed forward and reverse primers flanking the entire coding region of bla_IMP-7. Two internal primers were designed to reconfirm the sequencing results (accession no. AF416736): a reverse primer (5'-TATAGCCGCGCTCTAC-3'), 244 bp downstream from the 5' coding region, and a forward primer (5'-TATGCATCTGAATTAACA-3'), 342 bp upstream of the 3' region of bla_IMP-7.

Plasmid DNA was extracted from the strain by two methods: the alkaline lysis method described by Sambrook et al. (9) and the method described by Kado and Liu (4). Neither method could detect the presence of any plasmid in this strain. Thus, it is possible that the gene was chromosomally encoded. Because the P. aeruginosa strains that were isolated prior to the isolation of the multidrug-resistant P. aeruginosa strains were not available for further studies, we were not able to test the hypothesis that chromosomal mutation under the pressure of antibiotics resulted in the evolution of the chromosomal gene encoding IMP-7. The spread of resistance via gene transmission is unlikely, since there have not been any reports to date on the transfer of chromosomally encoded bla_IMP in P. aeruginosa.

	ACKNOWLEDGMENTS

 
We thank Yoichi Hirakata, Department of Laboratory Medicine, Nagasaki University School of Medicine, Nagasaki, Japan, for kindly providing the bla_IMP-positive control strain.

This work was supported by IRPA grant 06-02-03-0759 and VOTE-F F0127/2001A from the Government of Malaysia and University of Malaya.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. Phone: 603-79502879. Fax: 603-79584844. E-mail: parasak{at}ummc.edu.my.

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