Emergence of Klebsiella pneumoniae ST258 with KPC-2 in Poland

Plasmidic KPC β-lactamases in gram-negative pathogens are of the highest clinical and epidemiologic concern, conferring resistance to all β-lactams, including carbapenems (15, 18). The main KPC producer is Klebsiella pneumoniae, but other species are being identified as well (15). Strains with KPCs spread rapidly and cause outbreaks; in hospitals on the east coast of the United States and in Israel, they have already become endemic (2, 3, 10, 14, 17). Recently, more countries have reported the presence of these organisms, in some cases as a result of importation (15).

On 19 May 2008, a 56-year-old patient with no travel history was admitted to a cardiology ward in a Warsaw hospital (H1) with ischemic cardiomyopathy and several comorbidities. He had been transferred from another hospital with pneumonia of unknown etiology and was treated empirically with ceftriaxone and ciprofloxacin until 28 May. The eradication of pneumonia was asserted by clinical criteria. However, without a bladder catheter, the patient developed a urinary tract infection caused by K. pneumoniae (isolate 2337/08) on 23 May. With the imipenem MIC for the isolate being 2 μg/ml, the organism was classified by the Vitek 2 Expert system (bioMérieux, Marcy l'Etoile, France) as being resistant and having the “extended-spectrum β-lactamase-plus-carbapenemase” phenotype. The next such isolate (2338/08) was recovered from urine collected on 28 May, 2 days after the start of treatment with amikacin. Environmental sampling, conducted on the same day in the patient's room, yielded a similar K. pneumoniae isolate (2641/08) from a basin. On 3 June, the eradication of the urinary tract infection by amikacin was proven through clinical criteria and the results of microbiological examination, and the patient was discharged without having been tested for fecal carriage. On 6 June, he was admitted to a cardiology center (H2) for heart transplantation. At the time of admission, samples of his urine, stool, and sputum were analyzed. K. pneumoniae (isolate 2639/08) was cultured only from the stool specimen. The patient was kept under strict isolation without antimicrobial treatment, and his stool was examined each day during the first week and then twice a week during the remaining hospitalization. The stool samples were analyzed by standard procedures for enteric organisms, after which species identification was performed with the ATB ID32E test (bioMérieux) and susceptibility testing with drugs including imipenem, meropenem, and ertapenem was carried out by disk diffusion according to the CLSI guidelines (6). On 27 June, the sample was finally free of K. pneumoniae, and this result was confirmed in several subsequent analyses. On 17 July, the patient, disqualified for heart transplantation, was discharged from center H2 without symptoms of infection.

Analyzed by Etest (AB Biodisk, Solna, Sweden), the isolates showed multidrug resistance, including reduced susceptibilities to carbapenems (Table 1). In the spectrophotometric assay (17), products of bacterial sonication hydrolyzed imipenem in an EDTA-independent manner. Isoelectric focusing (1) revealed β-lactamases with a pI of 7.6 (probably the SHV-like chromosomal enzymes) and three others with pIs of 8.2, ∼6.8, and 5.4. Since pI ∼6.8 specifies the KPC-2/KPC-3 β-lactamases, a PCR (13) and sequencing were performed, which identified the bla_KPC-2 genes (19). PCR mapping revealed that they resided on one of the Tn4401 transposon variants, Tn4401a (12). Mating was performed with Escherichia coli A15 Rif^r (1); transconjugants were selected with 100 μg/ml rifampin (rifampicin) and 0.5 μg/ml imipenem or 2 μg/ml ceftazidime. On the imipenem plate, only isolate 2641/08 gave a transconjugant with KPC-2 and the pI 5.4 enzyme, identified by PCR and sequencing (9) as TEM-1. In contrast, on the ceftazidime plates, all isolates yielded transconjugants which had only the pI 8.2 enzyme, identified by PCR and sequencing (9) to be extended-spectrum β-lactamase SHV-12. Plasmid DNA from the isolates, purified with the Qiagen plasmid midi kit (Qiagen, Hilden, Germany), was used for electroporation of E. coli DH5α and then analyzed on plates with 0.5 μg/ml imipenem. Transformants with KPC-2 and TEM-1 were obtained for all isolates. PstI fingerprinting (9) showed three plasmid molecules in each isolate, two of which segregated individually into SHV-12-producing transconjugants and KPC-2/TEM-1-producing transformants (Fig. 1). Sizes of the plasmids were evaluated by calibration of the fingerprint bands and by pulsed-field gel electrophoresis (PFGE) analysis of total DNA digested with nuclease S1 (16). Plasmids with the bla_SHV-12 gene and with the bla_KPC-2 and bla_TEM-1 genes were ∼40 and ∼110 kb, respectively, whereas those likely to be without bla genes were ∼200 kb. The PCR-based replicon typing (5) yielded products only with primers specific for the replicon FIIA_S, carried on Salmonella virulence plasmids (5). Moreover, this method worked only with DNA from the K. pneumoniae isolates and not with that from the transconjugants and transformants, indicating that the amplicons corresponded to the plasmids likely to be without bla genes. Sequencing of the amplicons revealed their ca. 97% identity to fragments of repA genes on plasmids pKPN3 and pKPN4 from K. pneumoniae MGH78578 (GenBank accession numbers CP000648 and CP000649) and only ca. 79% identity to plasmid pSLT from Salmonella enterica serovar Typhimurium LT2 (11). K. pneumoniae isolates were indistinguishable by PFGE analysis of their DNA cuts with the XbaI restriction enzyme (9). Multilocus sequence typing (8; www.pasteur.fr) classified them as sequence type 258 (ST258), recently identified for KPC producers in Norway and Sweden, mostly of Greek and Israeli origins (16).

TABLE 1.

Susceptibilities of the K. pneumoniae isolates and of the E. coli transconjugants obtained in the study

Isolate(s)	MICa (μg/ml) of:
	AMP	AMC	PIP	TZP	CAZ	CTX	FEP	IPM	MEM	AMK	GEN	CIP	TET	TGC	CST
Clinical isolates	>256	>256	>256	>256	>256	128	>32	2-3	3-4	12-16	1-1.5	>32	32-48	3	0.38-0.75
KPC-2/TEM-1-producing transconjugant 2641/08	>256	24	96	48	3	2	1.5	3	0.75	1.5	0.25	0.023	2	0.38	0.25
SHV-12-producing transconjugants	>256	4-6	32-48	1-1.5	16	1.5-2	0.5	0.25	0.047	1-1.5	0.19-0.25	0.016	1.5-2	0.38-0.5	0.19-0.25
E. coli A15	3	4	1.5	1	0.25	0.047	0.047	0.25	0.032	1	0.19	0.023	2	0.38	0.25

^aAbbreviations: AMK, amikacin; AMC, amoxicillin-clavulanic acid; AMP, ampicillin; CAZ, ceftazidime; CIP, ciprofloxacin; CST, colistin; CTX, cefotaxime; FEP, cefepime; GEN, gentamicin; IPM, imipenem; MEM, meropenem; PIP, piperacillin; TET, tetracycline; TGC, tigecycline; TZP, piperacillin-tazobactam. In the case of β-lactam antibiotics, the MICs shown are the values indicated by zones of confluent growth around Etest strips; the clinical isolates and the E. coli transformants with KPC-2 produced scattered colonies inside the growth inhibition zones (15).

FIG. 1.

PstI (Fermentas, Vilnius, Lithuania) fingerprinting of plasmid DNA from the K. pneumoniae clinical isolates, E. coli DH5α transformants producing KPC-2 and TEM-1 (T), and E. coli A15 transconjugants producing SHV-12 (R). Molecular size markers: M1, λ DNA cut by HindIII, and M2, GeneRuler 1-kb DNA ladder (Fermentas). DNA sizes to the left correspond to the GeneRuler 1-kb DNA ladder, whereas those to the right correspond to λ DNA cut by HindIII.

This first report on KPC producers in Poland documents their continuing spread. The strain belonged to a widespread clonal group with single-locus variants ST11, ST258, and ST270 (7; www.pasteur.fr). The previous ribotyping- and PFGE-based observations of the high clonality of KPC-producing K. pneumoniae (4, 14, 17) and the multilocus sequence typing data from this and the Scandinavian work (16) suggest a prominent role for this group in KPC dissemination.