# Molecular characterization of a clinical ST145 Klebsiella oxytoca strain co-producing KPC-2 and IMP-96 carbapenemases

## TL;DR

This study reports a rare Klebsiella oxytoca strain that produces two carbapenem resistance genes, which are located on transferable plasmids, highlighting the risk of spreading antibiotic resistance.

## Contribution

The first report of a clinical ST145 Klebsiella oxytoca isolate co-producing blaKPC-2 and blaIMP-96 resistance genes on transferable plasmids.

## Key findings

- The isolate K. oxytoca K31 is resistant to multiple antibiotics including carbapenems and ceftazidime-avibactam.
- blaKPC-2 and blaIMP-96 genes are located on IncFIB(K)-like and IncM1 plasmids, respectively, and both are transferable.
- 94.2% of 104 ST145 K. oxytoca isolates globally carry at least one carbapenem resistance gene.

## Abstract

The objective of this study was to investigate the antimicrobial resistance phenotype and genetic characteristics of a clinical ST145 Klebsiella oxytoca isolate co-producing KPC-2 and IMP-96 carbapenemases. The isolate was first identified by MALDI-TOF MS. PCR and Sanger sequencing were used to detect carbapenem resistance genes. Antimicrobial susceptibility testing was performed using broth microdilution. Whole genome sequencing was carried out using Illumina and Nanopore platforms. Conjugation experiments and comparative genomic analysis were used to assess plasmid transferability and the genetic context of resistance genes. A total of 103 K. oxytoca genome sequences were retrieved from public databases and, together with the isolate from this study, used to construct a core genome single nucleotide polymorphism (SNP)-based phylogenetic tree. The antimicrobial resistance genes carried by each strain were also analyzed. Antimicrobial susceptibility testing revealed that K. oxytoca K31 was resistant to cephalosporins, carbapenems, and ceftazidime-avibactam (MIC range: 8 to >64 µg/mL), but susceptible to amikacin, meropenem-vaborbactam, aztreonam-avibactam, eravacycline, tigecycline, and colistin. Whole genome sequencing analysis showed that the strain was classified as ST 145. The blaKPC-2 and blaIMP-96 genes were located on IncFIB(K)-like and IncM1 plasmids, respectively. Conjugation experiments confirmed that both plasmids carrying carbapenem resistance genes were transferable to recipient strain Escherichia coli J53 and conferred carbapenem resistance. Comparative genomic analysis indicated that the blaKPC-2 gene was located in the variable region of a Tn3 family transposon, whereas the blaIMP-96 gene was embedded in a gene cassette captured by the IntI1. Genomic analysis of 104 ST145 K. oxytoca isolates revealed that 94.2% (98/104) harbored at least one carbapenem resistance gene. This study is the first report of a clinical isolate of ST145 K. oxytoca co-producing blaKPC-2 and blaIMP-96. Both resistance genes are located on mobile genetic elements that can be transferred between different bacterial species, facilitating the spread of antimicrobial resistance.

Carbapenem-resistant Klebsiella oxytoca has been increasingly reported worldwide; however, isolates co-producing both class A and class B carbapenemases remain rare. This study reported a clinical ST145 K. oxytoca isolate co-harboring the blaKPC-2 and blaIMP-96 resistance genes, which exhibited high-level resistance to both carbapenems and ceftazidime-avibactam. The two carbapenemase genes were located on conjugative plasmids separately with autonomous transfer capability. Genetic context analysis revealed that both resistance genes were embedded in mobile genetic elements, which likely mediate their capture and horizontal transfer across bacterial species. The widespread distribution of such mobile elements carrying resistance genes accelerates the evolution of multidrug-resistant bacteria. Genomic analysis of global ST145 K. oxytoca strains further indicated that this sequence type represents a high-risk, multidrug-resistant clonal lineage with significant public health implications. Enhanced surveillance and screening of ST145 K. oxytoca are warranted to limit its further global spread.