1 Department of Medical Microbiology and Immunology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178; 2 Baltimore Veterans Administration Medical Center, Baltimore, MD, USA
Received 28 June 2002; returned 23 October 2002; revised 13 December 2002; accepted 14 December 2002
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Abstract |
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Keywords: imipenem, meropenem, ertapenem, ß-lactamase, carbapenemase
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Introduction |
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In this report, we describe four clinical isolates of K. pneumoniae that produce the novel class A carbapenem-hydrolysing enzyme KPC-2. The isolates were obtained from patients at the Baltimore Veterans Administration Medical Center, MD, USA, between July 1998 and November 1999.
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Materials and methods |
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The four strains (UMM3, UMM12, UMM21 and UMM30) were isolated from samples of blood, sputum and urine (two patients) from four patients in July 1998 and January, June and November 1999. They were identified as K. pneumoniae by API 20E (bioMérieux, Marcy-lÉtoile, France), and one strain (UMM3) was confirmed by 16S rRNA analysis (MIDI LABS, Newark, DE, USA).
Antimicrobial susceptibility testing
Antibiotic susceptibilities were determined by overnight microdilution MIC testing using dehydrated custom-prepared panels provided by Dade MicroScan, Inc. (Sacramento, CA, USA) and by TREK Diagnostic Systems, Inc. (Westlake, OH, USA). Results were interpreted according to current NCCLS breakpoints and recommendations.7
Disc diffusion susceptibilities were determined by NCCLS methodology7 on MuellerHinton agar plates (Oxoid LTD, Basingstoke, UK) using BD Biosciences discs (Sparks, MD, USA).
Microbiological assay
A modification of the bioassay of Masuda et al.8 was performed using solutions containing 50 mg/L imipenem and 50 mg/L ertapenem on MuellerHinton agar plates that had been inoculated with a lawn of Escherichia coli ATCC 25922.
Conjugal transfer experiments
Direct transfer of carbapenem resistance into E. coli strains C600N (nalidixic acid resistant) and J53 Azir (sodium azide resistant) was attempted by a filter mating procedure. Transconjugant selection was performed on MuellerHinton agar containing nalidixic acid (25 mg/L), ertapenem (8 mg/L) and ceftazidime (2 mg/L) for the transfers to strain C600N, and sodium azide (200 mg/L), ertapenem (8 mg/L) and ceftazidime (2 mg/L) for transfer to strain J53 Azir.
Isoelectric focusing and ß-lactamase assays
The crude ß-lactamase extracts were subjected to analytical isoelectric focusing (IEF) on an ampholine polyacrylamide gel (pH range 3.59.5) to assess isoelectric points (pIs) and general inhibitor characteristics as described previously.9 Inducibility of the UMM3 isolate by imipenem (1 mg/L) and cefoxitin (2 mg/L) was investigated by the broth induction method10 and also by the disc approximation method using BBL discs containing cefoxitin and imipenem as inducing agents, and ceftriaxone, aztreonam, cefepime and ceftazidime as substrates.11 ß-Lactamase activity and inhibition characteristics were determined by spectrophotometric hydrolysis assay12 using 100 µM imipenem as substrate and the following inhibitors: 1.0 M NaCl, 1 M tazobactam and 0.25 M EDTA. The total protein content was measured with the Bio-Rad Protein assay kit (Bio-Rad, Hercules, CA, USA).
Molecular investigations
Gene identification was investigated by PCR using primers designed from the NMC-A, IMI-1 and KPC-1 sequences. PCR products were generated using primers designed from the blaKPC-1 nucleotide sequence.6 Primers used for amplification were forward primer KPC1F (5'-GCTACACCTAGCTCCACCTTC-3') and reverse primer KPC1R (5'-GCATGGATTACCAACCACTGT-5'). Products were generated and sequenced at least twice, as described previously.13
PFGE
The relatedness of the K. pneumoniae isolates was analysed by PFGE using XbaI as described previously.14
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Results |
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Although the above-mentioned tests involving ceftriaxone, cefepime and aztreonam tested alone and in combination with clavulanate were suggestive of ESBL production, it was interesting that the NCCLS ESBL confirmatory disc tests were positive for only the strain that produced the SHV-5-like ESBL (Table 2).
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Conjugation studies resulted in the transfer of reduced carbapenem susceptibility from three of the four isolates. IEF and plasmid profile results for the E. coli transconjugants indicated that the KPC-2 enzyme was transferred by conjugal transfer of large molecular weight plasmids. Using a set of primers specific for the blaKPC-1 gene, PCR-amplified products were generated and the resulting sequence was compared with the KPC-1 gene, with the sequence analysis indicating a point mutation (A to G) at nucleotide position 650. This point mutation resulted in the amino acid substitution of serine to glycine that distinguishes KPC-2 from KPC-1 (GenBank nucleotide sequence accession number AY034847).
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Discussion |
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At present, there are no clinical outcome data to assess the implications of KPC-1 or KPC-2 for carbapenem therapy. Determining the clinical significance of these enzymes is likely to prove difficult because they appear to be rare and microbiologists are unlikely to recognize their occurrence unless they carefully evaluate reductions in carbapenem susceptibility. The capability to detect enzymes such as KPC-2 requires the qualities of a good microbiologist: careful observation, experience and judgement. In the case of the KPC-2-producing isolates, failure to observe the reduced susceptibility to carbapenems (MICs > 1 mg/L), combined with certain test results suggestive of ESBL production, could prove very misleading to an inexperienced microbiologist.
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Acknowledgements |
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Footnotes |
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References |
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