Plasmid-located extended-spectrum β-lactamase genes are mostly found in Klebsiella pneumoniae (4), which is an important cause of nosocomial infections (1). In this study, we report the existence of K. pneumoniae clinical isolates producing the Ambler class A enzyme GES-1. This enzyme has been reported in Europe from K. pneumoniae (4) and Pseudomonas aeruginosa (2).
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 β-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 β-lactamase activities with pIs of 5.9 and 7.6 were detected. The β-lactamase activity of pI 7.6 corresponds to chromosomal SHV penicil-linase, and the pI value of 5.9 represented the GES-1 β-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 DH5α 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 DH5α transformants were used as templates for amplification of the blaGES-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 β-lactam antibiotics were determined with E-test strips (AB Biodisk, Solna, Sweden). For the K. pneumoniae clinical isolates, E. coli DH5α 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 blaGES-1 from P. aeruginosa Pa695 (2) and differs by a single silent mutation at position 591 from blaGES-1 described elsewhere for K. pneumoniae ORI-1 (4).
TABLE 1.
MICs of β-lactams for K. pneumoniae clinical isolates, E. coli DH5α transformants, E. coli TOP10 harboring recombinant plasmid pMFA-62, and reference strains E. coli DH5α and E. coli TOP10 with pBK-CMV
| β-Lactam | MIC (μg/ml)
|
||||
|---|---|---|---|---|---|
| K. pneumoniae clinical isolates | E. coli DH5α transformantsa | E. coli DH5α | E. coli TOP10(pMFA-62)b | E. coli TOP10(pBK-CMV) | |
| Amoxicillin | >256 | >256 | 4 | >256 | 4 |
| Amoxicillin + CLAc | 12 | 12 | 4 | 16 | 2 |
| Cefuroxime | >256 | >256 | 3 | >256 | 4 |
| Cefotaxime | 6-8 | 2 | 0.064 | 8 | 0.125 |
| Ceftazidime | 256 | 16-256 | 0.25 | >256 | 0.75 |
| Aztreonam | 3-0.75 | 0.75 | 0.125 | 1.5 | 0.094 |
| Cefepime | 2 | 0.5 | 0.047 | NDd | 0.047 |
| Imipenem | 0.25-0.387 | 0.125 | 0.19 | 0.5 | 0.25 |
E. coli DH5α harboring natural plasmids expressed GES-1 β-lactamase.
E. coli TOP10 harboring multicopy plasmid pMFA-62 produced GES-1 β-lactamase.
CLA, clavulanic acid.
ND, not determined.
The same macrorestriction pattern by pulsed-field gel electrophoresis indicated that an endemic K. pneumoniae strain producing GES-1 β-lactamase was presenting in different wards in the Hospital de Santa Maria. The persistence of these multiresistant microorganisms in the hospital may be associated with the existence of other resistance genes, inserted in multidrug-resistant integrons and/or plasmids.
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).
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