Molecular epidemiology, sequence types, and plasmid analyses of KPC-producing Klebsiella pneumoniae strains in Israel

Abstract

Sporadic isolates of carbapenem-resistant KPC-2-producing Klebsiella pneumoniae were isolated in Tel Aviv Medical Center during 2005 and 2006, parallel to the emergence of the KPC-3-producing K. pneumoniae sequence type 258 (ST 258). We aimed to study the molecular epidemiology of these isolates and to characterize their bla(KPC)-carrying plasmids and their origin. Ten isolates (8 KPC-2 and 2 KPC-3 producing) were studied. All isolates were extremely drug resistant. They possessed the bla(KPC) gene and varied in their additional beta-lactamase contents. The KPC-2-producing strains belonged to three different sequence types: ST 340 (n = 2), ST 277 (n = 2), and a novel sequence type, ST 376 (n = 4). Among KPC-3-producing strains, a single isolate (ST 327) different from ST 258 was identified, but both strains carried the same plasmid (pKpQIL). The KPC-2-encoding plasmids varied in size (45 to 95 kb) and differed among each of the STs. Two of the Klebsiella bla(KPC-2)-carrying plasmids were identical to plasmids from Escherichia coli, suggesting a common origin of these plasmids. These data indicate that KPC evolution in K. pneumoniae is related to rare events of interspecies spread of bla(KPC-2)-carrying plasmids from E. coli followed by limited clonal spread, whereas KPC-3 carriage in this species is related almost strictly to clonal expansion of ST 258 carrying pKpQIL.

FIG. 1.

The genetic relatedness of bla_KPC-2- and bla_KPC-3-producing carbapenem-resistant K. pneumoniae clones in Tel Aviv Medical Center during the years 2005 and 2006. bla_KPC alleles, sequence types, and the date of isolation are presented on the right. Isolates were clustered into five different clusters based on GelCompar Dice algorithm coefficients, which range from 0% to 100%, and using a tolerance of 1.5%, as illustrated by the scale to the left of each dendrogram. Two of the three ST 340 isolates were further analyzed.

FIG. 2.

(A) Plasmid analysis of 10 bla_KPC-2- and bla_KPC-3-carrying carbapenem-resistant K. pneumoniae clinical strains (lanes D) and their respective transformants (lanes T). C, chromosomal DNA. (B) Southern blot analysis of plasmid DNA of K. pneumoniae isolates, clinical strains (lanes D) and their respective transformants (lanes T). (C) The restriction pattern of plasmid DNA derived from K. pneumoniae transformants digested with BglII (lanes 1 to 10) and SmaI (lanes 11 to 20). PFGE types defined in Fig. 1 are presented in top parts of the panels.

FIG. 3.

A comparison of bla_KPC-2-carrying plasmids originated from two K. pneumoniae clones and two E. coli clones isolated in the same time period. Plasmid restriction analysis of transformants carrying these plasmids showed identity between the K. pneumoniae plasmids (K) and the E. coli plasmids (E). Plasmids from both organisms were digested with BglII (lanes 2 to 5), EcoRV (lanes 6 to 9), and SmaI (lanes 10 to 13) prior to electrophoresis. GeneRuler 1-kb DNA ladder (Fermentas Life Sciences), lane 1 (M); E. coli 386, lanes 2, 6, and 10; K. pneumoniae 523 PFGE type R, lanes 3, 7, and 11; E. coli 547, lanes 4, 8, and 12); K. pneumoniae 531, PFGE type, lanes 5, 9, and 13.