Abstract
Linezolid is a critical last-resort antimicrobial for multidrug-resistant Enterococcus faecium, particularly against vancomycin-resistant lineages where therapeutic options are severely limited. While resistance has historically arisen through de novo chromosomal mutations, the global emergence of transferable resistance mechanisms threatens to render more infections untreatable. Here, we characterise a recent (2023-2024) hospital-associated outbreak of linezolid-resistant E. faecium in Queensland, Australia. Although the cohort comprised a variety of sequence types, the outbreak was primarily driven by the clonal expansion of an ST80 lineage carrying the plasmid-borne poxtA-Ef gene. Standard short-read genomic surveillance failed to resolve the genetic context of the resistance determinant. However, long-read sequencing revealed that poxtA-Ef was carried within a novel transposon, Tn8026, situated on a linear plasmid. Structural analysis defined Tn8026 as a unique element flanked by IS1678 and the novel insertion sequence ISEfa26. Furthermore, we identified an instance of Tn8026 integration into the chromosome, providing functional evidence of its mobility and capacity for stabilisation within the genome. Global genomic screening demonstrated that Tn8026 significantly predates the local outbreak, identified in a historical Norwegian isolate from 2012, indicating a long-standing yet unrecognised global reservoir. Phylogenomic analysis provided strong evidence that the linear plasmid was imported from the Indian subcontinent, initiating a chain of silent dissemination in eastern Australia where the lineage circulated undetected prior to clinical recognition. Crucially, we also confirmed the presence of the linear plasmid in Enterococcus gallinarum, demonstrating its capacity to mobilise transmissible linezolid resistance across enterococcal species boundaries. These findings emphasise the need for detailed long-read-based surveillance of mobile genetic elements, with a particular focus on identifying linear plasmids that are often overlooked.