KPC-2

The rise of carbapenem-resistant Enterobacteriaceae coharboring KPC-2 and NDM-1 poses a significant public health threat. KPC-2-NDM-1- Citrobacter freundii is rarely reported in clinical settings. In this study, we report the largest cohort of eight KPC-2-NDM-1- C. freundii isolated from children with urinary tract infections. Comprehensive characterization, including antimicrobial susceptibility testing, plasmid conjugation, whole-genome sequencing, and comparative genomics, was conducted for these clinical strains. Antimicrobial susceptibility testing indicated that all strains were resistant to meropenem [minimal inhibitory concentration (MICs) ≥ 8 µg/mL] and imipenem (MICs > 8 µg/mL). Genotyping and comparative genomics analyses identified the eight KPC-2-NDM-1- C. freundii belonging to ST523, exhibiting a close relationship characterized by 45 single nucleotide polymorphisms. Whole-genome sequencing and plasmid analysis confirmed the presence of blaKPC-2 and blaNDM-1 on an IncR plasmid named pC275-2 with 46,050 bp. The blaNDM-1 was integrated within the blaNDM-1 related region, which includes ΔIS Aba125 , blaNDM-1 , ble-MBL , trpF , dsbD , and IS CR1 , while the blaKPC-2 gene was located on a novel non-Tn 4401 genetic element. The blaKPC-2 genetic architectures containing blaKPC-2 differed from classical structures, underscoring the ongoing evolution of these genetic elements.

Our study described the largest cohort to date of eight ST523 KPC-2-NDM-1- C. freundii isolated from children with urinary tract infections. The cooccurrence of KPC-2, a serine β -lactamases, and NDM-1, a metallo-β-lactamase on an IncR plasmid pC275-2 from a clinical carbapenem-resistant C . freundii . The conserved insertion structure mediated the blaNDM-1 , and the propagation of blaKPC-2 gene with a new genetic background using IncR plasmid in clinical settings promotes the emergence of superbugs necessitating vigilant monitoring. Our research detected that ST523 KPC-2- NDM-1- C. freundii were disseminated in a children’s hospital. The potential spread of an IncR plasmid within the hospital raises concerns about the pandemic potential of this clone that produces two carbapenemases: KPC-2 and NDM-1. Further investigations will be necessary to control and prevent the spread of KPC-2-NDM-1- C. freundii s.

Klebsiella pneumoniae, a Gram-negative bacterium, poses a significant public health threat due to its resistance to various antibiotics, including β-lactams and carbapenems. This resistance is mainly due to the production of Klebsiella pneumoniae carbapenemases (KPCs). The issue of KPC-2 and its variant, KPC-3, by K. pneumoniae strains, results in resistance to the substrate imipenem and β-lactamase inhibitors. Using Schrodinger software, we performed a high-throughput virtual screening of 374 compounds from the ChemDiv natural compound library in this study, targeting KPC-2 and KPC-3. The top compounds were identified using Extra Precision (XP) mode. Molecular dynamics simulations (MDS) were performed for 500 ns using GROMACS. Among the compounds, N075-0013 and N098-0051 for KPC-2 and N025-0014 and N099-0011 for KPC-3 exhibited binding energies ranging from -5.40 to -7.01 kcal/mol against both KPC-2 and KPC-3. The complexes formed with these compounds remained stable in their dynamic environments, suggesting their potential as effective inhibitors of KPC-2 and KPC-3. These results underscore the potential therapeutic promise of these compounds, justifying further in vitro and in vivo validation for their development as inhibitors of Klebsiella pneumoniae carbapenemases.