Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae

Abstract

A Klebsiella pneumoniae isolate showing moderate to high-level imipenem and meropenem resistance was investigated. The MICs of both drugs were 16 microg/ml. The beta-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. The strain was also resistant to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing studies demonstrated three beta-lactamases, with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). The presence of bla(SHV) and bla(TEM) genes was confirmed by specific PCRs and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the beta-lactamase with a pI of 6.7, KPC-1 (K. pneumoniae carbapenemase-1), was encoded on an approximately 50-kb nonconjugative plasmid. The gene, bla(KPC-1), was cloned in E. coli and shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of the novel carbapenem-hydrolyzing beta-lactamase, KPC-1, showed 45% identity to the pI 9.7 carbapenem-hydrolyzing beta-lactamase, Sme-1, from Serratia marcescens S6. Hydrolysis studies showed that purified KPC-1 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and monobactams. KPC-1 had the highest affinity for meropenem. The kinetic studies also revealed that clavulanic acid and tazobactam inhibited KPC-1. An examination of the outer membrane proteins of the parent K. pneumoniae strain demonstrated that the strain does not express detectable levels of OmpK35 and OmpK37, although OmpK36 is present. We concluded that carbapenem resistance in K. pneumoniae strain 1534 is mainly due to production of a novel Bush group 2f, class A, carbapenem-hydrolyzing beta-lactamase, KPC-1, although alterations in porin expression may also play a role.

FIG. 1

Isoelectric focusing patterns of carbapenem-resistant K. pneumoniae 1534. The gel was stained with nitrocefin, which is specific for β-lactamases. Lane 1, cell lysate prepared from the imipenem-resistant E. coli DH5α containing the bla_KPC-1 gene on pBR322-catI; lane 2, cell lysate prepared from imipenem-resistant E. coli HB101 that was transformed with K. pneumoniae 1534 DNA; lane 3, cell lysate prepared from K. pneumoniae 1534; lane 4, cell lysates prepared from strains producing SHV-2 (pI of 7.6), TEM-3 (pI of 6.3), and TEM-1 (pI of 5.4); lane 5, cell lysates prepared from strains producing TEM-1 (pI of 5.4) and SHV-4 (pI of 7.8); lane 6, cell lysates prepared from strains producing TEM-12 (pI of 5.25), TEM-10 (pI of 5.6), SHV-3 (pI of 6.8), and SHV-2 (pI of 7.6). The extra bands between pIs 6.7 and 5.4 are presumably degradation products of KPC-1 (lanes 1 to 3). The pIs of the β-lactamases were calculated by using the known pIs of TEM-12 (pI of 5.25), TEM-1 (pI of 5.4), TEM-10 (pI of 5.6), TEM-3 (pI of 6.3), SHV-3 (pI of 6.8), SHV-2 (pI of 7.6), and SHV-4 (pI of 7.8).

FIG. 2

(A) Inhibition assay with imipenem (IPM); (B) inhibition assay with meropenem (MEM). Disks: 1, lysate of K. pneumoniae 1534 (parent strain); 2, lysate of E. coli HB101/pBR322-catI; 3, disk control with imipenem (A) or meropenem (B); 4, lysate of K. pneumoniae ATCC 13883; 5, lysate of E. coli HB101/pBR322-catI-bla_KPC-1 clone.

FIG. 3

(A) Plasmid profiles of K. pneumoniae 1534 and an E. coli transformant on 0.85% agarose gel. Lane 1, plasmid RP4 (57-kb); lane 2, plasmid pDK9 (165-kb); lane 3, total DNA prepared from an E. coli HB101 transformant; lane 4, total DNA isolated from K. pneumoniae 1534; lane 5, plasmid R1 (97-kb). Chr., chromosomal. (B) Southern blot of the gel shown in panel A after hybridization with a 1,010-bp bla_KPC-1-specific probe. Lane 1, RP4 DNA; lane 2, pDK9 DNA; and lane 5, R1 DNA served as negative controls for the probe. DNA isolated from K. pneumoniae 1534 (lane 4) was used as a positive control. Lane 3, DNA isolated from an E. coli HB101 transformant of K. pneumoniae 1534 DNA that was imipenem resistant.

FIG. 4

Nucleotide and deduced amino acid sequences of the novel class A carbapenemase KPC-1 isolated from K. pneumoniae 1534. The −10 and −35 regions of the putative promoter are underlined. The transcription start site indicated by the mRNA primer extension study is marked as +1. RBS indicates a potential ribosome-binding site (62). The HindIII recognition site and the conserved amino acid residues for class A carbapenemases are underlined (26, 35, 36). The start and stop codons for bla_KPC-1 are marked with asterisks (59).

FIG. 5

Alignment of the amino acid sequence of KPC-1 with that of Sme-1 from S. marcescens S6 (41), Nmc-A from E. cloacae NOR1 (42), and IMI-1 from E. cloacae (57). Dashes indicate the gaps that were inserted to optimize the alignment. The numbering is from Ambler et al. and Sykes (1, 67). The conserved domains of class A β-lactamases are underlined (12, 26, 35, 36, 55, 63, 66). The residues suggested to play a critical role for carbapenemase activity are marked by asterisks and underlined (63, 66). The positions where the KPC-1 sequence diverges from these conserved residues (positions 105 and 237) are indicated in italics.

FIG. 6

Dendrogram showing similarity of 20 β-lactamases. The dendrogram was constructed by using DNASIS for Window's multiple alignment option (Higgins-Sharp). Sixteen of the β-lactamases are class A enzymes, CARB-3 from P. aeruginosa (27), PSE-1 from P. aeruginosa (24), SHV-1 from E. coli (39), LEN-1 from K. pneumoniae (2), TEM-1 from E. coli (65), MEN-1 from E. coli (6), OXY-1 from Klebsiella oxytoca (19), CITDI from C. diversus (50), YENT from Y. enterocolitica (61), Nmc-A from E. cloacae (42), IMI-1 from E. cloacae (57), Sme-1 from S. marcescens (41), L2 from Stenotrophomonas maltophilia (71), ROB -1 from Haemophilus influenzae (31), and BRO-1 from Moraxella catarrhalis (D. Beaulieu, L. Piche, T. R. Parr, Jr., K. Roeger-Lawry, P. Rosteck, and P. H. Roy, β-lactamase BRO-1 precursor [penicillinase], gi:2497581, Gen Bank, 1996); IMP-1 from S. marcescens (48) and Cfi-A from Bacteroides fragilis (69) were included as representatives of class B (metallo-β-lactamases); ACT-1 from K. pneumoniae (7) represents class C, AmpC β-lactamases; and OXA-1 from E. coli (49) represents class D enzymes.

FIG. 7

SDS-PAGE and Western blot analysis of OMPs of K. pneumoniae 1543 and two carbapenem-susceptible control strains. (A) SDS-PAGE analysis of OMPs. Lane 1, molecular mass markers; lane 2, OMPs prepared from K. pneumoniae ATCC 13883; lane 3, OMPs prepared from K. pneumoniae 1534; lane 4, OMPs prepared from K. pneumoniae 37. (B) Western blot analysis of OMPs using anti-OmpK36 antisera. Lane 1, OMPs prepared from ATCC 13883; lane 2, OMPs prepared from K. pneumoniae 1534; lanes 3 and 4, molecular mass markers in kilodaltons. (C) Western blot analysis of OMPs using anti-OmpK35 antisera. Lane 1, OMPs prepared from ATCC 13883; lane 2, OMPs prepared from K. pneumoniae 1534; lane 3, molecular mass markers in kilodaltons. (The OmpK35 antibody cross-reacts with OmpK36 [22].)