Double carbapenemases in Klebsiella pneumoniae blood isolates: dissemination in a single medical center via multiple plasmids and a variety of highly efficient clones

Abstract

Acquisition of multiple carbapenemase genes by Klebsiella pneumoniae (Kp) is an emerging public health threat. Here, we aim to elucidate the population structure of Kp blood isolates carrying two different carbapenemase genes and identify the mechanism facilitating their dissemination. The study was conducted in a tertiary healthcare center between 2014 and 2022. Twenty-four patients with bacteremia caused by Kp carrying two different carbapenemase genes were identified. All 24 blood isolates were analyzed by short-read genome sequences supplemented by long reads in a selected number of isolates. All isolates carried bla_KPC (23 bla_KPC-2, 1 bla_KPC-3) and bla_VIM-1 genes, along with a variety of antimicrobial resistance determinants. The isolates were clustered in six clonal lineages (ST39, ST147, ST323, ST258, ST3035, and ST340). Long-read genome sequences demonstrated that each carbapenemase gene was located in a separate group of plasmids: the bla_KPC-2 on a fusion of IncFIB(pQil) and IncFII(K) plasmids, the bla_KPC-3 on IncX3, the bla_VIM-1 on IncC, or a fusion of the IncFIB(pNDM-Mar) and IncHI1B(pNDM-MAR) plasmids. Comparison of plasmid content of eight isolates carrying a single carbapenemase gene from a previous study with eight isolates carrying two carbapenemase genes from the present study, matched by clonal lineages, revealed that the second carbapenemase gene was acquired by addition of another plasmid. Identical plasmids were found within the same lineage and across lineages. These findings suggest that dissemination of carbapenemase genes in our hospital setting was driven by multiple plasmids across a variety of highly efficient clones.

Keywords: AMR genes; Klebsiella; double carbapenemases; plasmids; sequence types.

Fig 1

Midpoint-rooted phylogeny of carbapenemase gene-carrying plasmids based on mash distances, accompanied by relevant metadata. Plasmid community networks were not computed for non-circular plasmids. The plasmids were detected in eight Klebsiella pneumoniae (Kp) isolates carrying a single carbapenemase gene from a previous study (10) and in eight Kp isolates carrying double carbapenemase genes, matched by sequence type (ST), from the present study and analyzed by long-read sequencing. Different STs, carbapenemase genes, plasmid community, circularity, and source are represented in different colors and intensities. Red denotes the presence of the Inc plasmid type.

Fig 2

Comparative genomic analysis of bla_KPC-2-carrying plasmids. The bla_KPC-2 gene is indicated in red. Flanking the bla_KPC-2 gene are genes defining the Tn4401 mobile transposon (composed of bla_KPC-2 gene, transposase gene tnpA, resolvase gene tnpR, and two insertion sequences, ISKpn6 and ISKpn7).

Fig 3

Comparative genomic analysis of selected regions of bla_VIM-1-carrying plasmids belonging to the larger bla_VIM-1 plasmid community. Numbers indicate the isolates’ code number. Red arrows represent the bla_VIM-1 gene. The blue line above the figure defines the class 1 integron region. HP, hypothetical protein.