Plasmid-Mediated Carbapenem-Hydrolyzing β-Lactamase KPC-2 in Klebsiella pneumoniae Isolate from Greece

The emergence and dissemination of Enterobacteriaceae isolates harboring carbapenemases in various geographic regions represent a significant threat to the management of nosocomial infections. Carbapenem-hydrolyzing β-lactamases can be metallo β-lactamases, expanded-spectrum oxacillinases, or Ambler class A enzymes (8, 9). The class A KPC β-lactamases hydrolyze all β-lactams except cephamycins. They are increasingly reported in data from studies of isolates of enterobacterial species (mostly Klebsiella pneumoniae species) collected in the United States, especially in New York City hospitals (1, 2, 4, 9, 12). Outside of the United States, single KPC-producing isolates containing members of the Enterobacteriaceae or containing Pseudomonas aeruginosa strains have been reported from France, China, and Colombia, but an epidemic situation has also been reported from Israel (5-7, 10, 11). We report here a K. pneumoniae isolate from Greece that produced β-lactamase KPC-2.

On 17 July 2007, a 22-year-old woman was hospitalized in the intensive care unit of the hospital of Heraklion, Greece, for severe cranial trauma with coma (Glasgow coma scale score, 7) and multiple cranial and costal fractures subsequent to a traffic accident. On 10 August, she was initially transferred to the orthopedic ward and then to the intensive care unit of the hospital of Meaux, France. The day of her admission, bacteriological screening and wound samples revealed the presence of panresistant Acinetobacter baumannii (susceptible only to colistin) and of K. pneumoniae GR, which was resistant to all antibiotics except gentamicin according to disk diffusion susceptibility testing results (3). The patient was treated locally with antiseptics and fortunately did not experience a systemic infection with K. pneumoniae requiring antibiotic treatment. No other A. baumannii or K. pneumoniae isolates with similar antibiotic resistance patterns were recovered from the hospital during this same period of time.

The MICs of β-lactams tested by the Etest method (AB BIODISK, Solna, Sweden) and interpreted according to CLSI standards (3) for K. pneumoniae GR showed values for expanded-spectrum cephalosporins and carbapenems only slightly modified after the addition of clavulanic acid (Table 1). A crude β-lactamase extract of that isolate showed significant carbapenem hydrolyzing activity, as measured spectrophotometrically (0.2, 0.06, and 0.05 μmol of imipenem, meropenem, and ertapenem/min/mg of total protein, respectively) (6, 8).

TABLE 1.

MICs of β-lactams for K. pneumoniae GR, its E. coli transconjugant TcGR, and an E. coli recipient strain

β-Lactam(s)a	MIC (μg/ml)
	K. pneumoniae GR	E. coli J53 TcGR	E. coli J53
Amoxicillin	>256	>256	4
Amoxicillin + CLA	48	24	4
Ticarcillin	>256	>256	2
Ticarcillin + CLA	>256	>256	2
Piperacillin	>256	128	1
Piperacillin + TZB	>256	128	1
Cephalothin	>256	>256	4
Cefoxitin	64	4	2
Cefotaxime	>32	4	0.06
Cefotaxime + CLA	32	0.38	0.06
Ceftazidime	>256	4	0.06
Ceftazidime + CLA	96	1	0.12
Aztreonam	>256	16	0.03
Aztreonam + CLA	>256	1.5	0.03
Cefepime	>256	1.5	0.03
Imipenem	12	1.5	0.12
Imipenem + CLA	0.75	0.25	0.12
Meropenem	6	0.38	0.03
Meropenem + CLA	3	0.094	0.03
Ertapenem	12	0.38	0.03
Ertapenem + CLA	6	0.094	0.03
Tigecycline	3	0.5	0.5
Gentamicin	1.5	0.064	0.064
Colistin	128	0.38	0.38

^aCLA, clavulanic acid at a fixed concentration of 2 μg/ml; TZB, tazobactam at fixed concentration of 4 μg/ml.

Plasmid analysis detected a ca. 75-kb self-conjugative plasmid that conferred a β-lactam resistance pattern for Escherichia coli transconjugants that was consistent with the presence of a slightly clavulanic acid-inhibited carbapenemase (Table 1). A β-lactamase extract from a transconjugant culture subjected to analytical isoelectric focusing (8) identified two β-lactamases with pI values of 5.4 and 6.8. PCR experiments using primers for detection of Ambler class A, class D, and class B β-lactamase genes (6, 8) followed by sequencing identified β-lactamase genes coding for carbapenemase KPC-2 (pI 6.8) and the narrow-spectrum TEM-1 (pI 5.4). Then, using a series of successive PCR primers, the 2.8-kb sequences surrounding the bla_KPC-2 gene were found to be identical to those surrounding the same bla_KPC-2 gene in a K. pneumoniae isolate from New York (6).

Up to now, carbapenemases of the KPC type seemed to be limited to Israel in this Mediterranean region. This is the first evidence of the identification of carbapenemases of the KPC type in Greece, a country located near Israel. It remains to be determined to what extent the spread of KPC-type enzymes contributes to the high level of carbapenem resistance that until now had been thought to occur only in the presence of class B carbapenemases (VIM-type enzymes).