First report of a carbapenemase gene blaOXA-181 harbored in a conjugative plasmid in Kluyvera sichuanensis isolated from spinach.

Antimicrobial resistance is a growing global public health concern, and carbapenem-resistant Enterobacterales (CRE) represent one of the most urgent threats due to limited treatment options. While CRE are primarily associated with healthcare settings, increasing evidence suggests that food systems may serve as additional reservoirs and transmission interfaces for clinically significant antibiotic-resistant bacteria. This study aimed to characterize CRE from vegetables, with a focus on isolates carrying carbapenem resistance genes on mobile genetic elements and evaluating their transferability to a recipient bacterium. Commercial vegetable samples were collected from retail grocery stores or farmers' markets and screened for CRE using selective enrichment and CHROMagar™ mSuperCARBA™. Antimicrobial susceptibility testing was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Whole-genome sequencing using Illumina short-read and Oxford Nanopore long-read platforms was conducted to characterize resistance genes and the plasmid sequence. Conjugation assays were performed to assess plasmid transferability to Escherichia coli DH5α. The results indicated that a carbapenem-resistant Kluyvera sichuanensis isolate (CA-CRE-C23) was recovered from a spinach sample. Genomic analysis identified a 51,477-bp hybrid ColKP3-IncX3 plasmid carrying the carbapenemase gene blaOXA-181 and quinolone resistance gene qnrS1. Conjugation experiments, disk diffusion, and sequencing confirmed successful plasmid transfer from K. sichuanensis isolate (CA-CRE-C23) to E. coli DH5α. The plasmid structure closely resembled globally disseminated IncX3 hybrid plasmids carrying blaOXA-181, which have been predominantly reported in clinical isolates. In conclusion, the detection of a transferable hybrid plasmid harboring blaOXA-181 in a fresh produce-associated K. sichuanensis isolate expands the known host range of this resistance gene and underscores the importance of food-chain surveillance in understanding the environmental dissemination of high-risk antimicrobial resistance genes.