Genomic Epidemiology of Complex, Multispecies, Plasmid-Borne blaKPC Carbapenemase in Enterobacterales in the United Kingdom from 2009 to 2014

Abstract

Carbapenem resistance in Enterobacterales is a public health threat. Klebsiella pneumoniae carbapenemase (encoded by alleles of the bla_KPC family) is one of the most common transmissible carbapenem resistance mechanisms worldwide. The dissemination of bla_KPC historically has been associated with distinct K. pneumoniae lineages (clonal group 258 [CG258]), a particular plasmid family (pKpQIL), and a composite transposon (Tn4401). In the United Kingdom, bla_KPC has represented a large-scale, persistent management challenge for some hospitals, particularly in North West England. The dissemination of bla_KPC has evolved to be polyclonal and polyspecies, but the genetic mechanisms underpinning this evolution have not been elucidated in detail; this study used short-read whole-genome sequencing of 604 bla_KPC-positive isolates (Illumina) and long-read assembly (PacBio)/polishing (Illumina) of 21 isolates for characterization. We observed the dissemination of bla_KPC (predominantly bla_KPC-2; 573/604 [95%] isolates) across eight species and more than 100 known sequence types. Although there was some variation at the transposon level (mostly Tn4401a, 584/604 [97%] isolates; predominantly with ATTGA-ATTGA target site duplications, 465/604 [77%] isolates), bla_KPC spread appears to have been supported by highly fluid, modular exchange of larger genetic segments among plasmid populations dominated by IncFIB (580/604 isolates), IncFII (545/604 isolates), and IncR (252/604 isolates) replicons. The subset of reconstructed plasmid sequences (21 isolates, 77 plasmids) also highlighted modular exchange among non-bla_KPC and bla_KPC plasmids and the common presence of multiple replicons within bla_KPC plasmid structures (>60%). The substantial genomic plasticity observed has important implications for our understanding of the epidemiology of transmissible carbapenem resistance in Enterobacterales for the implementation of adequate surveillance approaches and for control.

Keywords: Enterobacterales; KPC; carbapenemase; genomic epidemiology; long-read sequencing; outbreak analysis; short-read sequencing; whole-genome sequencing.

FIG 1

Estimated bla_KPC copy number distributions (derived from Illumina assemblies) within major species (left) and the top nineteen most common species/ST combinations (right) observed in the study. Other species were designated “other” (left) and other ST/species combinations as “other species/ST” (right). Dots represent estimated copy numbers for single isolates; boxplots represent median estimated bla_KPC copy numbers ± 1.58 × IQR/sqrt(n). Boxplots are ordered by most common species and species/ST categories, left to right, except for the other and other species/ST, assigned to the right of the plots. Kpne, Klebsiella pneumoniae; Ecol, Escherichia coli; Eclo, Enterobacter cloacae.

FIG 2

Incidence plot of bla_KPC species-ST by year-month and geography. Dots are colored by the location of isolate collection, as defined in Materials and Methods, and scaled by the number of isolates; the eight outpatient isolates were not plotted.

FIG 3

Incidence curve of species-ST in Manchester hospitals CMFT/UHSM, 2010 to 2012. Sequencing ascertainment of first-per-patient carbapenem-resistant Enterobacterales was 76% in 2010 and 59% in 2011.

FIG 4

Incidence plot of Tn4401 type and target site sequences by year-month and geography. Dots are colored by location of isolate collection, as defined in Materials and Methods, and scaled by the number of isolates; the eight outpatient isolates were not plotted.

FIG 5

Incidence plot of plasmid populations identified in isolates (replicon typing) by year-month and geography. Dots are colored by location of isolate collection, as defined in Materials and Methods, and scaled by the number of isolates; the eight outpatient isolates were not plotted. The most predominant combinations are highlighted in yellow.

FIG 6

Distribution of bla_KPC plasmid types by species-ST. Dots are colored by Tn4401/target site sequence type and scaled by the number of isolates. NA, not assigned.

FIG 7

Incidence plot of bla_KPC plasmid types identified by year-month and geography. Dots are colored by location of isolate collection, as defined in Materials and Methods, and scaled by the number of isolates; the eight outpatient isolates were not plotted. NA, not assigned.

FIG 8

Schematic of bla_KPC plasmid types and sizes identified from long-read/short-read hybrid sequencing approach by species/ST and year of collection (only 21 contigs clearly designated plasmid are represented). Closed circles denote circularized contigs (i.e., complete plasmids); replicons are denoted by colored triangles in their approximate positions in the structure. Colored triangles denote replicon types assigned to each plasmid sequence (i.e., multiple colored triangles represent multireplicon plasmids). Plasmids from isolates from the wider UK collection (i.e., collected through the national reference laboratory) are denoted with an asterisk.

FIG 9

Alignments of complete plasmid sequences harboring an IncFII(K)_1_CP000648-like replicon, including bla_KPC-negative and bla_KPC-positive sequences. All sequences were reoriented to start at IncFII for the purposes of alignment visualization. Loci of interest have been colored and annotated as shown. Shading between sequences denotes regions of homology, with light pink shading denoting areas of ≥90% nucleotide identity, dark pink areas of ≥50% nucleotide identity, and light blue areas of ≥90% nucleotide identity in reverse orientation. The order of sequences is adjusted to highlight genetic overlap between sequences but not to imply any specific direct exchange events. Annotations (i) and (ii) denote specific features highlighted in the main text.