The optrA, cfr(D) and vanA genes are co-located on linear plasmids in linezolid- and vancomycin-resistant enterococcal clinical isolates in Italy.

Objectives: To characterize the optrA-, cfr(D)- and vanA-carrying linear plasmids detected in three MDR enterococcal clinical isolates.

Methods: Enterococcus faecium (868), E. faecium (1001) and Enterococcus faecalis (2048), which were linezolid- and vancomycin-resistant due to the presence of optrA, cfr(D) and vanA genes, were tested for their susceptibility to several antibiotics. Characterization of the genetic elements carrying antibiotic resistance genes and ST determination were achieved using WGS data. The plasmid topology was evaluated by S1-PFGE. Resistance gene transferability was assessed by filter-mating experiments.

Results: The linezolid- and vancomycin-resistant enterococci also showed resistance to tedizolid, chloramphenicol, tetracycline, erythromycin, ampicillin and levofloxacin. Both E. faecium 868 and E. faecium 1001 belonged to ST80 (included in clade A1), whereas E. faecalis 2048 was associated with ST6. WGS analysis revealed a plasmid co-localization of the optrA, cfr(D) and vanA genes. optrA was carried by Tn6674-like or Tn7695-like transposons; cfr(D) was associated with a truncated guaA gene, both flanked by IS1216 with opposite polarity; vanA was found on a Tn1546-like transposon containing IS1542 and IS1251 transposases. PFGE of S1 nuclease-treated and untreated DNAs displayed the linear topology of optrA-, cfr(D)- and vanA-harbouring plasmids. Only E. faecium 868 was able to transfer linezolid and vancomycin genes to an enterococcal recipient.

Conclusions: To the best of our knowledge this is the first report on the occurrence of a linear plasmid in E. faecalis. Linear plasmids can play a key role in the spread of oxazolidinone and glycopeptide resistance with serious consequences for public health.