Abstract
BACKGROUND: To explore the plasmid characteristics and transfer mechanisms of an extensive drug resistant (XDR) clinical isolate, Citrobacter portucalensis L2724hy, co-producing bla (SFO-1), bla (NDM-1), and bla (KPC-2). METHODS: Species confirmation of L2724hy was achieved through 16S rRNA sequencing and Average Nucleotide Identity (ANI) analysis. Antimicrobial susceptibility testing (AST) employed the agar dilution and micro broth dilution methods. Identification of resistance genes was carried out by PCR and whole-genome sequencing (WGS). Essential resistance gene locations were verified by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and southern hybridization experiments. Subsequent WGS data analysis delved into drug resistance genes and plasmids. RESULTS: The confirmation of the strain L2724hy as an extensive drug-resistant Citrobacter portucalensis, resistant to almost all antibiotics tested except polymyxin B and tigecycline, was achieved through 16S rRNA sequencing, ANI analysis and AST results. WGS and subsequent analysis revealed L2724hy carrying bla (SFO-1), bla (NDM-1), and bla (KPC-2) on plasmids of various sizes. The uncommon ESBL gene bla (SFO-1) coexists with the fosA3 gene on an IncFII plasmid, featuring the genetic environment IS26-fosA3-IS26-ampR-bla (SFO-1)-IS26. The bla (NDM-1) was found on an IncX3 plasmid, coexisting with bla (SHV-12), displaying the sequence IS5-IS3000-IS3000-Tn2-bla (NDM-1)-ble-trpF-dsbD-cutA-gros-groL, lacking ISAa125. The bla (KPC-2) is located on an unclassified plasmid, exhibiting the sequence Tn2-tnpR-ISKpn27-bla (KPC-2)-ISKpn6-korC. Conjugation assays confirmed the transferability of both bla (NDM-1) and bla (KPC-2). CONCLUSION: We discovered the coexistence of bla (SFO-1), bla (NDM-1), and bla (KPC-2) in C. portucalensis for the first time, delving into plasmid characteristics and transfer mechanisms. Our finding highlights the importance of vigilant monitoring of drug-resistance genes and insertion elements in uncommon strains.