Comparative genomics analysis of pTEM-2262, an MDR plasmid from Citrobacter freundii, harboring two unclassified replicons

Aim: To genetically characterize the multidrug-resistance (MDR) plasmid pTEM-2262 that could not be classified into any known incompatibility group from the clinical Citrobacter freundii isolate 2262. Materials & methods: The repA or repB deletion mutants of pTEM-2262 were constructed using the scarless Cas9-assisted recombineering system. Comparative genomic analysis of pTEM-2262 and the other four previously sequenced plasmids belonging to the same incompatibility group were performed. Results: pTEM-2262, a conjugative plasmid, harbored two unclassified replicons, repA and repB, while repB was not essential for pTEM-2262 replication. In five analyzed plasmids, their conserved backbones primarily integrated massive accessory modules at two ‘hotspots’ that were located between orf597 and orf504, and between orf393 and orf405. All the antibiotic resistance genes of pTEM-2262 were clustered in the MDR region with a complex mosaic structure. Conclusion: This study thoroughly investigates the detailed structure and genomic comparison of this unknown incompatibility group for the first time.