First identification and characterization of a plasmid co-harbouring poxtA and cfr in a clinical linezolid-resistant Staphylococcus aureus isolate

Background Linezolid remains an important treatment option for serious infections caused by Gram-positive pathogens; however, acquired oxazolidinone resistance genes carried by mobile genetic elements are an increasing clinical concern. In Staphylococcus aureus , cfr and poxtA have previously been reported together in the chromosomal mosaic transposon Tn 6349 , whereas complete plasmid-borne structures co-harbouring these genes remain poorly defined. Methods Isolate D2958 was identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry, phenotypically characterized by antimicrobial susceptibility testing, and genotypically characterized by hybrid whole-genome sequencing using Illumina NovaSeq X Plus short reads and PacBio Revio long reads. The genetic contexts of cfr and poxtA were investigated by resistance- and virulence-gene screening, comparative plasmid genomics, phylogenomic analysis, PCR, inverse PCR and Sanger sequencing. Transferability was assessed by electroporation and a donor–recipient co-culture mating assay. Results Hybrid assembly generated one circular chromosome and four circular plasmids. D2958 was a methicillin-susceptible S. aureus (MSSA) ST398 isolate. The 35,377-bp rep19b -like plasmid pD2958-B co-harboured cfr , poxtA , fexB and aacA-aphD in an IS 256 -associated cfr / aacA-aphD region and an IS 1216 -associated poxtA / fexB region. The amplicon obtained with the initial IS 1216 inverse-primer pair was reinterpreted as a product from the assembled plasmid configuration because the reverse primer had exact binding sites in both IS 1216 copies. A subsequent poxtA -targeted outward-facing PCR produced no detectable amplicon; thus, no poxtA -containing circular translocatable unit was detected under the tested conditions. Outward-facing cfr /IS 256 PCR was also negative. After electroporation of total D2958 plasmid DNA, targeted PCR detected pD2958-B but not pD2958-A, pD2958-C or pD2958-D in the representative RN4220 transformant. The transformant showed increased linezolid, chloramphenicol, clindamycin and gentamicin MICs. No transconjugants were recovered. Conclusions These findings characterize a mosaic plasmid co-harbouring cfr and poxtA in a clinical linezolid-resistant MSSA isolate and provide a complete plasmid-level example of modular oxazolidinone resistance assembly in S. aureus . Plasmid-specific PCR supports, but does not replace whole-genome confirmation of the transformant genotype.