Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A beta-lactamase, KPC-3, in a New York Medical Center

Abstract

From April 2000 to April 2001, 24 patients in intensive care units at Tisch Hospital, New York, N.Y., were infected or colonized by carbapenem-resistant Klebsiella pneumoniae. Pulsed-field gel electrophoresis identified a predominant outbreak strain, but other resistant strains were also recovered. Three representatives of the outbreak strain from separate patients were studied in detail. All were resistant or had reduced susceptibility to imipenem, meropenem, ceftazidime, piperacillin-tazobactam, and gentamicin but remained fully susceptible to tetracycline. PCR amplified a blaKPC allele encoding a novel variant, KPC-3, with a His(272)-->Tyr substitution not found in KPC-2; other carbapenemase genes were absent. In the outbreak strain, KPC-3 was encoded by a 75-kb plasmid, which was transferred in vitro by electroporation and conjugation. The isolates lacked the OmpK35 porin but expressed OmpK36, implying reduced permeability as a cofactor in resistance. This is the third KPC carbapenem-hydrolyzing beta-lactamase variant to have been reported in members of the Enterobacteriaceae, with others reported from the East Coast of the United States. Although producers of these enzymes remain rare, the progress of this enzyme group merits monitoring.

FIG. 1.

PFGE analysis of XbaI-digested genomic DNA from carbapenem-resistant isolates of K. pneumoniae. Lane 1 contains a concatemer of phage λ DNA used as a molecular size marker. Isolates representing the predominant outbreak strain are shown in lanes 3, 4, 7 to 11, 13, and 18. Other lanes show isolates unrelated to the outbreak strain; the types represented in lanes 5 and 6 and in lanes 12 and 14 were related organisms, each isolated from two or more patients.

FIG. 2.

Comparison of KPC enzyme sequences. The predicted 24-residue cleavable signal peptide is shaded.

FIG. 3.

Plasmid profiles (A) and a Southern blot hybridized with a bla_KPC probe (B) of K. pneumoniae CL 5761 (lanes 2) and a representative bla_KPC-3-containing E. coli electrotransformant (lanes 3). Lanes 1 show the 94-kb plasmid R100.1 (22, 31) as a guide to molecular size.

FIG. 4.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of OMPs extracted from carbapenem-resistant K. pneumoniae isolates. Lanes 1 and 12 show protein molecular size markers as indicated in kilodaltons to the right of the gel (Bio-Rad). Other lanes contain control strain CL 15245 (lanes 2 and 3), CL 5761 (lanes 4 and 5), CL 5762A (lanes 6 and 7), CL 5762B (lanes 8 and 9), and CL 5763 (lanes 10 and 11). Extracts in lanes 2, 4, 6, 8, and 10 were prepared from cells grown in Luria-Bertani broth (high osmolarity); those in lanes 3, 5, 7, 9, and 11 were prepared from cells grown in NB (low osmolarity). The arrow indicates the band corresponding to the inducible porin OmpK35.