High-Risk Lineages of Hybrid Plasmids Carrying Virulence and Carbapenemase Genes

Abstract

Background/Objectives: Carbapenem-resistant Enterobacterales (CRE) are a global health threat due to their high morbidity and mortality rates and limited treatment options. This study examines the plasmid-mediated transmission of virulence and antibiotic resistance determinants in carbapenem-resistant Klebsiella pneumoniae (Kpn) and Escherichia coli (E. coli) isolated from Russian hospitals. Methods: We performed short- and long-read whole-genome sequencing of 53 clinical isolates (48 Kpn and 5 E. coli) attributed to 15 genetic lineages and collected from 21 hospitals across nine Russian cities between 2016 and 2022. Results: The plasmid analysis identified 18 clusters that showed high concordance with replicon typing, with all clusters having a major replicon type. The majority of plasmids in the IncHI1B(pNDM-MAR)/IncFIB(pNDM-Mar)-like cluster (79.16%) carried both antibiotic resistance genes (e.g., bla_NDM-1 and bla_OXA-48) and virulence factors (VFs) such as siderophore genes. We hypothesized that hybrid plasmids could play a critical role in the dissemination of antibiotic resistance genes and VFs. Comparative analyses with global plasmid databases revealed high-risk lineages of hybrid plasmids that are predominantly spread throughout Russia at present. Conclusions: Our findings underscore the importance of monitoring plasmid backbones for clinical management, surveillance, and infection control activities.

Keywords: Enterobacterales; carbapenem resistance; plasmids; virulence.

Figure 1

Prevalence of VFs and carbapenemase genes in the identified clusters of plasmids. The clusters of the plasmids were named according to the prevalent replicon gene in this cluster, as shown on the right side of the figure. Only those clusters in which VFs and carbapenemase genes were identified are included in the figure. Genes are grouped by their classification into VFs (blue) or carbapenemase genes (red). Gray shading shows the prevalence of each gene within each cluster. The bar chart on the right side indicates the number of plasmids in each cluster. iuc = iucABCD and iutA genes; iro = iroBCDN genes.

Figure 2

(A) Evolution of IncHI1B(pNDM-MAR)/IncFIB(pNDM-Mar)-like plasmids with/without aerobactin. From top to bottom: alignment of plasmids carrying aerobactin and ARGs (pVKpST512_3035, pVKpST512_3048), plasmids carrying aerobactin (pVKpST147_8, pVKpST395_1456), and one plasmid without the aerobactin cluster (pVKpST512_3061). ARGs, rmpA2, tellurium resistance genes (ter), and the aerobactin gene cluster (iuc) with surrounding MGE are marked at the top. (B) Comparison of upstream and downstream regions of rmp associated with different mobile variants. Arrows represent coding sequences, and those corresponding to genes of interest are labeled. Arrows are colored according to gene clusters, and shading corresponds to regions of similarity (sequence identity ≥ 30%) as identified by clinker. The following were identified: 3035 stands for plasmid pVKpST512_3035, 3048 for pVKpST512_3048, 8 for pVKpST147_8, 1456 for pVKpST395_1456, and 3061 for pVKpST512_3061.

Figure 3

Plasmid similarity network of IncHI1B(pNDM-MAR)/IncFIB(pNDM-Mar)-like plasmids from cluster 2 and the “Global dataset”. For the visual representation of the obtained table with source plasmid node IDs (identification), target plasmid node IDs, and the Jaccard coefficient between them, a graph and a layout for it were generated using the package igraph, where a layout describes the vertical and horizontal placement of nodes when plotting a particular graph structure. Each circle in the figure represents a plasmid, and it is connected with an edge to another node (plasmid) if the Jaccard coefficient between them is greater than 0.8 (see Section 4). Each node (plasmid) is colored according to various features: the assigned cluster (A); the country of origin (B); the sequence of IncHI1B replicon (C); carried replicons, where “+” indicates that a plasmid coded one or more replicons (D); mobility (E); and ARGs (F).

Figure 4

Evolution of plasmids from cluster A with/without aerobactin from our data and the “Global dataset”. From top to bottom: alignment of canonical virulent plasmid pLVPK carrying aerobactin (AY378100), a plasmid carrying aerobactin from our data (86 stands for pVKpST86_86), and plasmid 2 isolate INF331 without the aerobactin cluster from Australia (NZ_LR890425). Replicons, rmpA, rmpA2, salmochelin (iro), and aerobactin gene clusters are marked at the top. Arrows are colored according to gene clusters, and shading corresponds to regions of similarity (sequence identity ≥ 30%) as identified by clinker.