Genomic epidemiology of a complex, multi-species plasmid-borne bla_KPC carbapenemase outbreak in Enterobacterales in the UK, 2009-2014

Abstract

Carbapenem resistance in Enterobacterales is a public health threat. Klebsiella pneumoniae carbapenemase (encoded by alleles of the blaKPC family) is one of the commonest transmissible carbapenem resistance mechanisms worldwide. The dissemination of blaKPC has historically been associated with distinct K. pneumoniae lineages (clonal group 258 [CG258]), a particular plasmid family (pKpQIL), and a composite transposon (Tn4401). In the UK, blaKPC has caused a large-scale, persistent outbreak focused on hospitals in North-West England. This outbreak has evolved to be polyclonal and poly-species, but the genetic mechanisms underpinning this evolution have not been elucidated in detail; this study used short-read whole genome sequencing of 604 blaKPC-positive isolates (Illumina) and long-read assembly (PacBio)/polishing (Illumina) of 21 isolates for characterisation. We observed the dissemination of blaKPC (predominantly blaKPC-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), blaKPC spread appears to have been supported by highly fluid, modular exchange of larger genetic segments amongst plasmid populations dominated by IncFIB (580/604 isolates), IncFII (545/604 isolates) and IncR replicons (252/604 isolates). The subset of reconstructed plasmid sequences also highlighted modular exchange amongst non-blaKPC and blaKPC plasmids, and the common presence of multiple replicons within blaKPC 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.IMPORTANCEAntimicrobial resistance is a major threat to the management of infections, and resistance to carbapenems, one of the “last line” antibiotics available for managing drug-resistant infections, is a significant problem. This study used large-scale whole genome sequencing over a five-year period in the UK to highlight the complexity of genetic structures facilitating the spread of an important carbapenem resistance gene (blaKPC) amongst a number of bacterial species that cause disease in humans. In contrast to a recent pan-European study from 2012-2013(1), which demonstrated the major role of spread of clonal blaKPC-Klebsiella pneumoniae lineages in continental Europe, our study highlights the substantial plasticity in genetic mechanisms underpinning the dissemination of blaKPC. This genetic flux has important implications for: the surveillance of drug resistance (i.e. making surveillance more difficult); detection of outbreaks and tracking hospital transmission; generalizability of surveillance findings over time and for different regions; and for the implementation and evaluation of control interventions.

Genomic surveillance of carbapenem-resistant Klebsiella in Wales reveals persistent spread of K. pneumoniae ST307 and adaptive evolution of pOXA-48-like plasmids

“…A visual comparison of pKpQIL-like plasmids from our collection, together with two published pKpQIL-like plasmids from North-West England (Stoesser et al 2020) and pKpQIL (Leavitt et al 2010), showed the high structural conservation overall ( Figure 4 ). However, two plasmids (from ARGID_31921 and ARGID_24826) had lower nucleotide similarity with pKpQIL over one half, likely as a result of recombination.…”

“…The plasmids include the original pKpQIL sequence (accession GU595196) (Leavitt et al 2010), seven from the PHW collection and two previously sequenced plasmids recovered in North-West England hospitals (Stoesser et al 2020). Open reading frames (ORFs) are depicted as arrows with resistance genes (including bla KPC ) labelled.…”

“…Notably, bla KPC-2 genes from isolates in North Wales collected from 2013 onwards were typically encoded within the Tn 4401a transposon on IncF plasmids, the same genetic background identified among bla KPC-2 isolates from North-West England. This region has experienced endemic spread of bla KPC-2 -encoding Enterobacteriaceae since 2008, driven largely by horizontal exchange of plasmids and plasmid fragments between strains and species (Stoesser et al 2020). Thus our findings likely reflect the outward expansion of this complex, multi-species, plasmid-driven outbreak to Wales via hospital networks.…”

Genetic diversity ofblaKPC-gene-containing IncF plasmids from epidemiologically related and unrelated Enterobacteriaceae

“…A recent study has stated that OXA-48 containing plasmids from outbreak-related patients could not be distinguished from similar plasmids of non-outbreak related patients based on SNP differences ( 31 ). Interestingly, other studies found bla KPC -containing plasmids to be highly variable in their nucleotide sequence within the same outbreak and even within patients ( 15,20 ). This suggests that the plasmid content could also be highly unstable in vivo during hospital outbreaks.…”

“…A pangenome was constructed, and phylogenetic distance based on gene-presence/-absence between the different plasmids was determined using Roary v1.13.2 ( 27 ). All plasmids were pairwise aligned using dnadiff , and a similarity index was calculated between the different plasmids as described by Stoesser et al ( 20 ) (Stoesser-similarity index). This similarity index can vary from 0 (completely unsimilar plasmids) to 1 (identical plasmids) and, contrary to both Roary phylogenetic distance and number of SNP differences, penalises size differences between plasmids.…”