Genomic epidemiology of carbapenem-resistant Enterobacterales at a New York City hospital over a 10-year period reveals complex plasmid-clone dynamics and evidence for frequent horizontal transfer of bla KPC

Abstract

Transmission of carbapenem-resistant Enterobacterales (CRE) in hospitals has been shown to occur through complex, multifarious networks driven by both clonal spread and horizontal transfer mediated by plasmids and other mobile genetic elements. We performed nanopore long-read sequencing on CRE isolates from a large urban hospital system to determine the overall contribution of plasmids to CRE transmission and identify specific plasmids implicated in the spread of bla _KPC (the Klebsiella pneumoniae carbapenemase [KPC] gene). Six hundred and five CRE isolates collected between 2009 and 2018 first underwent Illumina sequencing for genome-wide genotyping; 435 bla _KPC-positive isolates were then successfully nanopore sequenced to generate hybrid assemblies including circularized bla _KPC-harboring plasmids. Phylogenetic analysis and Mash clustering were used to define putative clonal and plasmid transmission clusters, respectively. Overall, CRE isolates belonged to 96 multilocus sequence types (STs) encoding bla _KPC on 447 plasmids which formed 54 plasmid clusters. We found evidence for clonal transmission in 66% of CRE isolates, over half of which belonged to four clades comprising K. pneumoniae ST258. Plasmid-mediated acquisition of bla _KPC occurred in 23%-27% of isolates. While most plasmid clusters were small, several plasmids were identified in multiple different species and STs, including a highly promiscuous IncN plasmid and an IncF plasmid putatively spreading bla _KPC from ST258 to other clones. Overall, this points to both the continued dominance of K. pneumoniae ST258 and the dissemination of bla _KPC across clones and species by diverse plasmid backbones. These findings support integrating long-read sequencing into genomic surveillance approaches to detect the hitherto silent spread of carbapenem resistance driven by mobile plasmids.

Figure 1.

Population structure of CRE isolates. Number and proportion of CRE isolates harboring bla_KPC (n = 460) that belonged to each (A) bacterial species and (B) ST. Doubles refer to STs that include two isolates; singleton STs consist of only one isolate. (C) Diversity of plasmids harboring bla_KPC. As above, the term doubles refers to plasmid clusters that include two plasmids. The small proportion of plasmids that did not share a secondary MOB-cluster designation with any other plasmid are denoted as nonclustering.

Figure 2.

Comparison of most frequently identified plasmids from this study with publicly available plasmids including those with widespread distribution. Alignment of representative plasmids from each of the largest plasmid clusters against similar, publicly available plasmid genomes. (A) AA057; (B) AA068; (C) AA050 and AA057; (D) AA073; and (E) AA030. For all plasmid annotations, bla_KPC is shown in pink and Tn4401-associated genes in teal, other antibiotic resistance genes in green, and other genes in blue.

Figure 3.

Plasmid distribution across different CRE STs. This Sankey plot demonstrates the relationship between isolated STs and plasmid clusters found in this collection; bacterial species are designated by color, demonstrating the host range for each plasmid cluster. STs consisting of only one isolate are labeled as singletons (S). Bar sizes correspond to the number of plasmids in each ST and plasmid cluster.

Figure 4.

Clade and plasmid distribution in K. pneumoniae ST258 and E. hormaechei ST171 isolates. For (A) ST258 and (B) ST171 phylogenetic trees, the annotations denote the bla_KPC allele (inner ring) and associated Tn4401 subtype (second ring) as well as the genetic location of bla_KPC (chromosome and plasmid cluster; outer rings) for each isolate. Clade designations refer to groups of isolates within each ST with pairwise genetic distances of ≤25 SNPs.

Figure 5.

Top 10 plasmids demonstrating the greatest host diversity. (A) Plasmid clusters found in the greatest number of isolate STs. (B) Analysis of pairwise mash distances between plasmid host genomes corresponding to within-strain and species and across species genetic differences demonstrated the potential for distribution of these plasmids to diverse hosts.

Figure 6.

Distribution of bla_KPC-harboring plasmids and non-bla_KPC-harboring plasmids across ST258 clades. Phylogenetic trees for isolates belonging to (A) clade A1 and (B) clade A3, with corresponding distribution of bla_KPC, Tn4401 subtype, and CRE plasmid cluster as shown previously, as well as results of MOB-clustering analysis including both bla_KPC-harboring and non-bla_KPC-harboring plasmids.