The plasmidome associated with Gram-negative bloodstream infections: A large-scale observational study using complete plasmid assemblies

Abstract

Plasmids carry genes conferring antimicrobial resistance and other clinically important traits, and contribute to the rapid dissemination of such genes. Previous studies using complete plasmid assemblies, which are essential for reliable inference, have been small and/or limited to plasmids carrying antimicrobial resistance genes (ARGs). In this study, we sequenced 1,880 complete plasmids from 738 isolates from bloodstream infections in Oxfordshire, UK. The bacteria had been originally isolated in 2009 (194 isolates) and 2018 (368 isolates), plus a stratified selection from intervening years (176 isolates). We demonstrate that plasmids are largely, but not entirely, constrained to a single host species, although there is substantial overlap between species of plasmid gene-repertoire. Most ARGs are carried by a relatively small number of plasmid groups with biological features that are predictable. Plasmids carrying ARGs (including those encoding carbapenemases) share a putative 'backbone' of core genes with those carrying no such genes. These findings suggest that future surveillance should, in addition to tracking plasmids currently associated with clinically important genes, focus on identifying and monitoring the dissemination of high-risk plasmid groups with the potential to rapidly acquire and disseminate these genes.

Fig. 1. Characteristics of plasmid carriage in E. coli/K pneumoniae bloodstream infections.

A, B Number of plasmids per isolate for E. coli (A) and Klebsiella spp. (B), coloured by the number of ARG classes per isolates where MDR is ≥3, AMR 1–2 and no AMR 0. C Rarefaction curve of the number of novel plasmid groups (as defined using the Louvain-based method described above) per new plasmid sequenced stratified by size (large ≥100,000 bp, medium ≥10,000 to < 100,000 bp, small < 10,000 bp. D Number of plasmid-associated ARGs per isolate vs number of plasmids carrying at least one ARG. Isolates with only one plasmid-associated ARG (by definition, carried on one plasmid) are excluded. Source data are provided in the supplementary “Source Data” file.

Fig. 2. Phylogenetic distribution of the most common (n> = 10 members) plasmid groups (n = 33 groups) and the content of these.

The tree is a neighbour-joining tree built on Mash distances between chromosomes. Tip colours represent species/phylogroup. The black bars represent the presence or absence of plasmid groups (shown along the bottom x axis) for each isolate in the tree. The right panel shows the percentage of isolates within each of these 33 plasmid groups carrying the genes indicated (darker colours denote higher proportion of isolates carrying gene). To improve readability, gene groups have been clustered together. Source data are provided in the supplementary “Source Data” file.

Fig. 3. A Umap projection of distances (measured by gene presence/absence) between the plasmidomes of isolates (each point represents the plasmidome, i.e., all plasmid sequences of a single isolate).

These are coloured to show the variability explained by species (A)/ARG carriage (B)/year (C) and infection source (D). Source data are provided in the supplementary “Source Data” file.

Fig. 4. Plasmid network where individual plasmids (nodes) are connected by edges if they cluster in the same group using the Louvain-based methodology and coloured according to the number of classes of ARGs that they carry.

Edge thickness is drawn proportional to the Jaccard distance (see methods) between plasmids. Multi-species clusters are donated by black outlined shapes. Only plasmids groups with ≥10 members are shown.The ordering of clusters corresponds to that in Fig. 2. Labels above clusters denote the PlasmidFinder/COPLA taxnomic designations, respectively; plasmid groups are numbered consecutively from the top left. Source data are provided in the supplementary “Source Data” file.

Fig. 5. Nested genetic complexity associated with bla_CTX−M−15 mobilisation.

The “Tree” panel shows a neighbour-joining tree of Mash distances between chromosomes for isolates carrying a bla_CTX−M−15 gene. Tip colours represent species/ST/phylogroup. The chromosomal copy 1 and 2 panels show the genetic context 5000 bp up- and downstream from chromosomal copies of the bla_CTX−M−15 gene (shown in red); the plasmid copy panel shows this equivalent information for isolates carrying a plasmid-borne copy of this gene. The outlining colour in these panels shows the hierarchical cluster assignment of these flanking groups. The plasmid group panel shows group membership of plasmids carrying the bla_CTX−M−15 gene with each x axis position representing a distinct group and black bars showing the presence or absence of these for isolates in the tree. The encircled numbers denote: 1—different flanking sequences in the same ST, 2—different flanking sequences in the same plasmid group, 3—the same flanking group found in both chromosomal and plasmid contexts and 4—different plasmid groups harbouring the gene found within the same ST. Source data are provided in the supplementary “Source Data” file.

Fig. 6. Plasmids carrying carbapenemase genes are highly similar to plasmids without these genes found in Oxfordshire BSIs.

A Each horizontal bar represents a plasmid assembly either from Oxfordshire “Group 2/11” or the NCBI global dataset. Common genes are shown in colour (with blast identity between these shown from light grey to black (where the latter represents a perfect match), whereas genes unique to a given plasmid are shown in grey. B A network plot of plasmids which cluster with carbapenemase-carrying plasmids in the global network analysis. The “carbapenemase gene” grouping includes all those variants identified and classified as conferring resistance to the class “Carbapenem” in AMRFinder. Plasmids (nodes) are connected with an edge where the edge weight is ≤0.551 (see “Methods”). The thickness of the edges is displayed so that it is proportional to the edge weight. Source data are provided in the supplementary “Source Data” file.