Emergence and Inter- and Intrahost Evolution of Pandrug-Resistant Klebsiella pneumoniae Coharboring tmexCD1-toprJ1, blaNDM-1, and blaKPC-2

Abstract

Klebsiella pneumoniae is capable of acquiring various exogenous genetic elements and subsequently conferring high antimicrobial resistance. Recently, a plasmid-mediated RND family multidrug efflux pump gene cluster, tmexCD1-toprJ1, was discovered in K. pneumoniae. In this study, we analyzed tigecycline-resistant K. pneumoniae isolates from patients from surveillance from 2017 to 2021. In addition to phenotype detection, including growth curves, plasmid transferability and stability, hypermucoviscosity, biofilm formation, and serum survival, by whole-genome sequencing, we analyzed the phylogenetic relationships of the isolates harboring tmexCD1-toprJ1 and discovered the composition of plasmids carrying tmexCD1-toprJ1. In total, we discovered that 12 tigecycline-resistant isolates from 5 patients possessed tmexCD1-toprJ1, designated sequence type 22 (ST22) and ST3691. An ST11 isolate harbored a partial tmexD1, and a complete toprJ1 (tmexC1 was lost) was tigecycline sensitive. All the ST22 tigecycline-resistant isolates coharbored tmexCD1-toprJ1, bla_NDM-1, and bla_KPC-2. tmexCD1-toprJ1 was encoded by a novel IncU plasmid in ST22 and an IncFIB/HI1B plasmid in ST3691, which presented differences in mobility and stability. Interestingly, isolates from the same patients presented heteroresistance to tigecycline, not only among isolates from different specimens but also those from the same sample, which might be attributed to the differential expression of tmexCD1-toprJ1 due to the dynamic genetic heterogeneity caused by relocating tmexCD1-toprJ1 close to the replication origin of plasmid. Here, we reported the emergence of K. pneumoniae isolates coharboring tmexCD1-toprJ1, bla_NDM-1, and bla_KPC-2. The results highlight the impact of in vivo genetic heterogeneity of tmexCD1-toprJ1-carrying elements on the in vivo variation of tigecycline resistance, which might have notable influences on antimicrobial treatment. IMPORTANCE Pandrug-resistant (PDR) Klebsiella pneumoniae poses a great challenge to public health, and tigecycline is an essential choice for antimicrobial treatment. In this study, we reported the emergence of PDR K. pneumoniae coharboring tmexCD1-toprJ1, bla_NDM-1, and bla_KPC-2, which belongs to ST22 and ST3691. By whole-genome analysis, we reconstructed the evolutionary map of the ST22 ancestor to become the PDR superbug by acquiring multiple genetic elements encoding tmexCD1-toprJ1 or bla_NDM-1. Importantly, the genetic contexts of tmexCD1-toprJ1 among the ST22 isolates are different and present with various mobilities and stabilities. Furthermore, we also discovered the heterogeneity of tigecycline resistance during long-term infection of ST22, which might be attributed to the differential expression of tmexCD1-toprJ1 due to the dynamic genetic heterogeneity caused by relocating tmexCD1-toprJ1 close to the replication origin of plasmid. This study tracks the inter- and intrahost microevolution of the superbug PDR K. pneumoniae and highlights the importance of timely monitoring of the variation of pathogens during antimicrobial treatment.

Keywords: heterogeneity; pandrug resistance; tmexCD1-toprJ1; whole-genome sequencing; within-host evolution.

FIG 1

Phylogenetic relationship and molecular characteristics of tigecycline-resistant K. pneumoniae strains in this study. (A) Phylogenetic tree (left), carriage of resistance genes (middle), and types of plasmid replicons (right) of our ST22 K. pneumoniae and published ST22 genomes. The colored block represents the presence of each element. (B) Carriage of resistance genes (left) and types of plasmid replicons (right) of our ST3691 K. pneumoniae and published ST22 genomes. The colored block represents the presence of each element. (C) SNPs among tigecycline-resistant ST22 using PEKP3116 as a reference. The mutation positions and bases (reference base, position, mutant) are marked on the top of the heatmap. The colored blocks represent the existence of mutations.

FIG 2

Sequence alignments of tmexCD1-toprJ1-bearing IncU plasmids of tigecycline-resistant ST22 K. pneumoniae within lineage A (A) and lineage B (B). The matched regions between two sequences are displayed by light blue blocks, and the identities are marked. The arrows represent the genes related to resistance and transfer (red, AMR; green, integrase recombinase and transposase; purple, transfer associated; dark blue, plasmid replication; gray, other functions).

FIG 3

Genetic contexts and distribution of tmexCD1-toprJ1-bearing elements. (A) Genetic contexts and alignments of tmexCD1-toprJ1-bearing elements. The matched regions between two sequences are displayed by light blue blocks, and the identities are marked. The arrows represent the genes related to resistance and transfer (red, AMR; blue, integrase recombinase and transposase; gray, other functions). The plasmid names, types of plasmid replicons, sequence types of the K. pneumoniae strains, and sources of the strains are marked.

FIG 4

Circular sketch map and alignments of plasmids harboring bla_NDM-1 (A and B), bla_KPC-2 (C and D), and heavy metal resistance genes (E and F) from ST22 K. pneumoniae strains. The matched regions between two sequences are displayed by light blue blocks, and the identities are marked. The arrows represent the genes related to resistance and transfer (red, AMR; green, integrase recombinase and transposase; purple, transfer associated; dark blue, plasmid replication; orange, heavy metal resistance; gray, other functions).

FIG 5

Inter- and intrahost microevolution of ST22 K. pneumoniae. (A) Evolutionary history of ST22 K. pneumoniae strains. The evolutionary sketch map was constructed by chromosomal SNPs first, and then the variations in plasmids and clinical information were used to illustrate the details. The number of SNPs and loss or acquisition of plasmids between two strains are marked. The plasmid composition is marked beside the strain names. The sequence variations of plasmids are also illustrated. (B) Sketch map exhibiting the occurrence of inversion caused by inverted repeats comprising IS26, qnrS1, and ISKpn19.

FIG 6

Heteroresistance of ST22 K. pneumoniae within patient 2 (A) and patient 4 (B). The use of antimicrobial agents, isolation, MIC, expression of tmextCD1-toprJ1, and OD₅₉₀ values of culture for 12 h are displayed.