Carbapenem-resistant strain of Klebsiella oxytoca harboring carbapenem-hydrolyzing beta-lactamase KPC-2

Abstract

We investigated a Klebsiella oxytoca isolate demonstrating resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The MICs of both imipenem and meropenem were 32 microg/ml. The beta-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. Isoelectric focusing studies demonstrated five beta-lactamases with pIs of 8.2 (SHV-46), 6.7 (KPC-2), 6.5 (unknown), 6.4 (probable OXY-2), and 5.4 (TEM-1). The presence of the bla(SHV) and bla(TEM) genes was confirmed by specific PCR assays and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the beta-lactamase with a pI of 6.7, Klebsiella pneumoniae carbapenemase-2 (KPC-2), was encoded on an approximately 70-kb conjugative plasmid that also carried SHV-46, TEM-1, and the beta-lactamase with a pI of 6.5. The bla(KPC-2) determinant was cloned in E. coli and conferred resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of KPC-2 showed a single amino acid difference, S174G, when compared with KPC-1, another carbapenem-hydrolyzing beta-lactamase from K. pneumoniae 1534. Hydrolysis studies showed that purified KPC-2 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and aztreonam. KPC-2 had the highest affinity for meropenem. The kinetic studies revealed that KPC-2 was inhibited by clavulanic acid and tazobactam. An examination of the outer membrane proteins of the parent K. oxytoca strain demonstrated that it expressed detectable levels of OmpK36 (the homolog of OmpC) and a higher-molecular-weight OmpK35 (the homolog of OmpF). Thus, carbapenem resistance in K. oxytoca 3127 is due to production of the Bush group 2f, class A, carbapenem-hydrolyzing beta-lactamase KPC-2. This beta-lactamase is likely located on a transposon that is part of a conjugative plasmid and thus has a very high potential for dissemination.

FIG. 1.

(A) Isoelectric focusing patterns of cell lysates from carbapenem-resistant strains. The gel was stained with nitrocefin. Lanes 1 to 4, cell lysates prepared from strains producing TEM-3 (pI 6.3), TEM-1 (pI 5.4), SHV-5 (pI 8.2), and MIR-1 (pI 8.4), respectively; lane 5, the imipenem-resistant E. coli HB101 transformant containing a 70-kb plasmid from 3127; lane 6, an imipenem-resistant E. coli HB101 transconjugant of 3127; lane 7, K. oxytoca 3127. (B) Isoelectric focusing patterns of cell lysates prepared from carbapenem-resistant clones of K. oxytoca 3127. Lane 1, strain producing SHV-46 (pI 8.2); lanes 2 to 3, clones of strain 3127; lane 4, an imipenem-resistant E. coli HB101 transconjugant of K. oxytoca 3127; lane 5, E. coli DH5α containing the bla_KPC-1 clone. The pIs of the β-lactamases were calculated by using the known pIs of TEM-1 (5.4), TEM-3 (6. 3), SHV-5 (8.2), KPC-1 (6.7), and MIR-1 (8.4).

FIG. 2.

Alignment of the partial sequence of the protein encoded upstream of KPC-2 (GenBank accession number AAO53444.1) to IstB-like proteins (6); IS21 putative ATP-binding protein (GenBank accession number P15026), putative IS100 transposase from Y. pestis C092 (GenBank accession number NP_395401), putative transposase from Y. pestis (GenBank accession number AAC44982), and putative transposition helper protein from S. enterica subsp. enterica serotype Cubana (GenBank accession number AAM10642).

FIG. 3.

NuPAGE gel and Western blot analysis of OMPs of K. oxytoca 3127 and a carbapenem-susceptible control strain, K. pneumoniae ATCC 13883. (A) NuPAGE gel analysis of OMPs. Lane 1, molecular mass markers; lane 2, OMPs prepared from K. oxytoca 3127; lane 3, OMPs prepared from K. pneumoniae ATCC 13883. (B) Western blot analysis of OMPs performed with anti-OMPK36 antisera. (C) Western blot analysis of OMPs performed with anti-OMPK35 antisera. Molecular mass is indicated in gels to the left of each panel.