Emergence of a High-Risk Klebsiella michiganensis Clone Disseminating Carbapenemase Genes

Abstract

Klebsiella michiganensis is emerging as an important human pathogen of concern especially strains with plasmid-mediated carbapenemase genes. The IncX3-bla _NDM-5 plasmid has been described as the primary vector for bla _NDM-5 dissemination. However, whether strains with this plasmid have any competitive edge remain largely unexplored. We characterized a bla _NDM-5-producing Klebsiella michiganensis strain (KO_408) from Japan and sought to understand the driving force behind the recent dissemination of IncX3-bla_NDM-5 plasmids in different bacterial hosts. Antibiotic susceptibility testing, conjugation, and whole-genome sequencing were performed for KO_408, a clinical isolate recovered from a respiratory culture. Fitness, stability, and competitive assays were performed using the IncX3-bla _NDM-5 plasmid, pKO_4-NDM-5. KO_408 was ascribed to a novel sequence type, ST256, and harbored resistance genes conforming to its MDR phenotype. The bla _NDM-5 gene was localized on the ~44.9 kb IncX3 plasmid (pKO_4-NDM-5), which was transferable in the conjugal assay. The acquisition of pKO_4-NDM-5 did not impose any fitness burden and showed high stability in the host cells. However, transformants with pKO_4-NDM-5 were outcompeted by their host cells and transconjugants with the IncX3-bla _OXA-181 plasmid. The genetic environment of bla _NDM-5 in pKO_4-NDM-5 has been previously described. pKO_4-NDM-5 showed a close phylogenetic distance with seven similar plasmids from China. KO_408 clustered with strains within the KoI phylogroup, which is closely associated with carbapenemase genes. This study highlights the emergence of a high-risk Klebsiella michiganensis clone harboring carbapenemase genes and affirms that the recent spread of IncX3-bla _NDM-5 plasmids might be due to their low fitness cost and stability but not their competitive prowess.

Keywords: IncX3-blaNDM-5 plasmid; Klebsiella michiganensis; KoI; fitness cost; stability.

Figure 1

Maximum-likelihood phylogenetic tree based on Klebsiella michiganensis core-genomes. The tree was rooted on the node of KoV. Bootstrap values are shown next to major nodes. Strain labels are given as the strain GenBank ID, except that of KO_408. The phylogeny of KO_408 with respect to the other K. michiganensis genomes is highlighted with a green branch color. The major phylogroups and the OXY variants in these genomes are indicated along with their sequence types. OXY variants with an identity <99% to Resfinder references are represented on the tree as new variants, whereas those with an identity >99% but not 100% identity to the reference are shown by the query result with the * numbers representing their similarity to the reference. Sequency types (STs) with two or more frequencies are shown in the figure legend and the remaining STs are described in Supplemental_data_file1. The occurrence of carbapenemase genes in these genomes is also illustrated, as well as the genome metadata.

Figure 2

Evolutionary relatedness of pKO_4-NDM-5 and other IncX3-bla_NDM-5-containing plasmids. The maximum-likelihood phylogenetic tree of these IncX3-bla_NDM-5 plasmids was constructed using MEGA X software, and their bootstrap values are shown next to the branches. The plasmid metadata are also described.

Figure 3

A circular comparison of pKO_4-NDM-5 and MH781720, an IncX3-bla_NDM-5 containing plasmid. The homology between these two plasmids is shown by the percentage identity in the figure legend, whereas the absence of or a similarity value of <70% is indicated on the circular map as a white gap.

Figure 4

Linearized comparison of bla_NDM-5 genetic environment of pKO_4-NDM-5, MH781720, and LC000627. Similar features are represented by the same color. The bla_NDM-5 gene, mobile genetic elements, and other immediate CDSs are represented by red, yellow, and purple colors, respectively.

Figure 5

Stability, growth kinetic, and competition analyses of IncX3-bla_NDM-5-containing plasmid. (A) Growth curve comparison of Escherichia coli C600, its transformant with the IncX3-bla_NDM-5 plasmid, and transconjugant with the IncX3-bla_oxa-181 plasmid. (B) Competition analysis between E. coli C600 with its transformant and transconjugant, as well as between the transformant and the transconjugant. (C) Stability analysis of IncX3-bla_NDM-5-containing plasmid. The IncX3-bla_NDM-5 plasmid persisted in E. coli Top10 after successive passaging on antibiotic-free media (12 h passage, twice daily for 100 consecutive days (200 passages) corresponding to approximately 2,400 generations of E. coli Top 10).