Molecular epidemiology of KPC-producing Klebsiella pneumoniae isolates in the United States: clonal expansion of multilocus sequence type 258

Abstract

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae have become more common in the United States and throughout the world. We used pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to examine the molecular epidemiology of KPC-producing K. pneumoniae isolates sent to the Centers for Disease Control and Prevention (CDC) for reference testing from 1996 to 2008. A dominant strain, sequence type 258 (ST 258), was found and likely accounts for 70% of the CDC's K. pneumoniae PFGE database. Isolates with PFGE patterns related to ST 258 were identified in 10 of the 19 U.S. states currently reporting KPC-producing K. pneumoniae, in addition to one isolate from Israel. KPC subtyping and analysis of the surrounding genetic environment were subsequently performed on 23 representative isolates. Thirteen isolates identified as ST 258 possessed either bla(KPC-2) or bla(KPC-3) and some variability in the Tn4401 element upstream of the bla(KPC) gene. Escherichia coli DH10B was successfully transformed by electroporation with KPC-encoding plasmid DNA from 20 of the 23 isolates. Restriction analysis of plasmid DNA prepared from transformants revealed a diversity of band patterns, suggesting the presence of different plasmids harboring the bla(KPC) gene, even among isolates of the same ST.

FIG. 1.

Representative dendrogram of the CDC's KPC-producing Klebsiella pneumoniae PFGE database (n = 248). Black markers have been placed on the major branches of the dendrogram to indicate divergence of further branches; these markers show a shared percentage of similarity of PFGE pattern for the isolates associated with each branch of the dendrogram (seen on the right).

FIG. 2.

The dendrogram is based on similarity of PFGE patterns from 23 representative K. pneumoniae isolates. The table to the right illustrates results from bla_KPC sequence analysis and MLST, along with PCR and sequence analysis of the nonconserved region of the Tn4401 element (16).

FIG. 3.

HindIII digests of bla_KPC-containing plasmid DNA from E. coli transformants. State (or country) of origin, MLST type, and Tn4401 deletions are shown at the top of each lane. Lanes 1 and 22 contain a 1-kb ladder size standard.

FIG. 4.

Map of the United States identifying states that currently have known KPC-producing Enterobacteriaceae (shaded in gray) (n = 33). States that have KPC-producing K. pneumoniae isolates identified by this study as MLST ST 258 are marked with a target symbol. We also identified ST 258 in an isolate from Israel. These data are based on a passive reporting system that relies on isolates voluntarily sent to the CDC for KPC testing. Thus, states on this map that are not marked to indicate known KPC-producing Enterobacteriaceae (white) have neither reported nor voluntarily sent isolates to the CDC.