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Antimicrobial Agents and Chemotherapy, April 2003, p. 1481-1482, Vol. 47, No. 4
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.4.1481-1482.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Outbreak of GES-1 ß-Lactamase-Producing Multidrug-Resistant Klebsiella pneumoniae in a University Hospital in Lisbon, Portugal

	LETTER

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Between February 1999 and 2001, 30 K. pneumoniae clinical isolates were collected from different patients, distributed among several wards (surgery and medical services and in different intensive care units) at the Hospital de Santa Maria, Lisbon, Portugal.

Twenty-four isolates were identified from urine, four were identified from respiratory tract samples (three from sputum and one from bronchial exudate), and the remaining two isolates were found in blood and pus.

Antibiotic susceptibility testing by disk diffusion (3) suggested the presence of an extended-spectrum ß-lactamase. Synergies were observed among clavulanic acid-amoxicillin, cefotaxime, aztreonam, and cefepime. All isolates were resistant to clavulanic acid, ceftazidime, cefuroxime, gentamicin, kanamycin, netilmicin, nalidixic acid, and norfloxacin. They were susceptible to imipinem and cefepime and presented reduced susceptibilities to amikacin, cefotaxime, and aztreonam.

The analysis of genomic DNA, digested with XbaI and resolved by pulsed-field gel electrophoresis (1), revealed the same macrorestriction pattern among all isolates, classified as indistinguishable according to the work of Tenover et al. (6).

On the isoelectric focusing gel two ß-lactamase activities with pIs of 5.9 and 7.6 were detected. The ß-lactamase activity of pI 7.6 corresponds to chromosomal SHV penicil-linase, and the pI value of 5.9 represented the GES-1 ß-lactamase.

Plasmid extraction, performed according to the alkaline lysis method (5), revealed plasmids with molecular sizes ranging from 3 to 23 kb. From five selected K. pneumoniae strains we obtained Escherichia coli DH5 transformants more resistant to ceftazidime than to cefotaxime and aztreonam and harboring a plasmid with a molecular size of ca. 9 kb. Under standard PCR conditions, plasmid DNA preparations from K. pneumoniae and E. coli DH5 transformants were used as templates for amplification of the bla_GES-1 gene with primers GES-1A and GES-1B (4). All isolates revealed an 864-bp PCR product. The resulting amplicon was cloned into the SmaI site of pBK-CMV. The E. coli TOP10 harboring pMFA-62 was selected for subsequent analysis and sequencing. MICs of ß-lactam antibiotics were determined with E-test strips (AB Biodisk, Solna, Sweden). For the K. pneumoniae clinical isolates, E. coli DH5 transformants, and E. coli TOP10 harboring recombinant plasmid pMFA-62, the cefuroxime and ceftazidime MICs were >256 µg/ml, and the MIC ranges (in micrograms per milliliter) were 3 to 0.75 for aztreonam and 6 to 8 for cefotaxime (Table 1). The nucleotide sequence of the cloned fragment revealed 100% identity with bla_GES-1 from P. aeruginosa Pa695 (2) and differs by a single silent mutation at position 591 from bla_GES-1 described elsewhere for K. pneumoniae ORI-1 (4).

View this table: [in this window] [in a new window]   TABLE 1. MICs of ß-lactams for K. pneumoniae clinical isolates, E. coli DH5 transformants, E. coli TOP10 harboring recombinant plasmid pMFA-62, and reference strains E. coli DH5 and E. coli TOP10 with pBK-CMV

	ACKNOWLEDGMENTS

 
This work was funded by grants "Programa de Investigação Integrada" from the University of Lisbon and ADEIM (Associação para o Desenvolvimento do Ensino e Investigação da Microbiologia).

	REFERENCES

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1. Barroso, H., A. Freitas-Vieira, L. M. Lito, J. Melo Cristino, M. J. Salgado, H. Ferreira Neto, J. C. Sousa, G. Soveral, T. Moura, and A. Duarte. 2000. Survey of Klebsiella pneumoniae producing extended-spectrum ß-lactamases at a Portuguese hospital: TEM-10 as endemic enzyme. J. Antimicrob. Chemother. 45:611-616.[Abstract/Free Full Text]
2. Dubois, V., L. Poirel, C. Marie, C. Arpin, P. Nordmann, and C. Quentin. 2002. Molecular characterization of a novel class 1 integron containing bla_GES-1 and a fused product of aac(3)-Ib/aac(6')-Ib' gene cassettes in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 46:638-645.[Abstract/Free Full Text]
3. National Committee for Clinical Laboratory Standards. 2000. Methods fordisk antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A5. National Committee for Clinical Laboratory Standards, Wayne, Pa.
4. Poirel, L., I. Le Thomas, T. Naas, A. Karim, and P. Nordmann. 2000. Biochemical sequence analyses of GES-1, a novel class A extended-spectrum ß-lactamase, and the class 1 integron In52 from Klebsiella pneumoniae. Antimicrob. Agents Chemother. 44:622-632.[Abstract/Free Full Text]
5. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
6. Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.[Medline]

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