Emergence and clonal dissemination of KPC-2- and NDM-1-coharboring <i>Citrobacter freundii</i> in China with an IncR plasmid

The rise of carbapenem-resistant <i>Enterobacteriaceae</i> coharboring KPC-2 and NDM-1 poses a significant public health threat. KPC-2-NDM-1-<i>Citrobacter freundii</i> is rarely reported in clinical settings. In this study, we report the largest cohort of eight KPC-2-NDM-1-<i>C. freundii</i> 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- <i>C. freundii</i> belonging to ST523, exhibiting a close relationship characterized by 45 single nucleotide polymorphisms. Whole-genome sequencing and plasmid analysis confirmed the presence of <i>bla</i>_KPC-2 and <i>bla</i>_NDM-1 on an IncR plasmid named pC275-2 with 46,050 bp. The <i>bla</i>_NDM-1 was integrated within the <i>bla</i>_NDM-1 related region, which includes ΔIS<i>Aba125</i>, <i>bla</i>_NDM-1, <i>ble-MBL</i>, <i>trpF</i>, <i>dsbD</i>, and IS<i>CR1</i>, while the <i>bla</i>_KPC-2 gene was located on a novel non-Tn<i>4401</i> genetic element. The <i>bla</i>_KPC-2 genetic architectures containing <i>bla</i>_KPC-2 differed from classical structures, underscoring the ongoing evolution of these genetic elements.IMPORTANCEOur study described the largest cohort to date of eight ST523 KPC-2-NDM-1-<i>C. freundii</i> isolated from children with urinary tract infections. The cooccurrence of KPC-2, a serine <i>β</i>-lactamases, and NDM-1, a metallo-β-lactamase on an IncR plasmid pC275-2 from a clinical carbapenem-resistant <i>C</i>. <i>freundii</i>. The conserved insertion structure mediated the <i>bla</i>_NDM-1, and the propagation of <i>bla</i>_KPC-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-<i>C. freundii</i> 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-<i>C. freundii</i>s.