IS1216 drives the evolution of pRUM-like multidrug resistance plasmids in Enterococcus faecium

pRUM-like plasmids are commonly found in multidrug-resistant Enterococcus faecium, but the evolution of these plasmids has not been characterised in detail. When we analysed the genome sequences of two clinical E. faecium strains isolated in Birmingham, UK, we found two pRUM-like plasmids, pHHEf1 and pHHEf2. They were ~25 kb in size and shared the same 10 kb backbone but contained starkly different accessory regions that were bounded by and interspersed with the IS26 family insertion sequence IS1216. pHHEf1 contained a complete set of vancomycin resistance genes, while pHHEf2 contained aminoglycoside and erythromycin resistance genes along with an integrated small plasmid, pCOLA. It appeared that IS1216 had driven the diversification of these accessory regions. We sought to characterise the role of IS1216 in the broader evolution of pRUM-like plasmids by performing comparative analyses on 152 complete plasmid sequences from five continents. Extensive IS1216-mediated variation included backbone deletions, acquisition and loss of ten different antibiotic resistance genes, and the formation of cointegrates with plasmids of at least ten different replicon types. Cointegration events have introduced accessory segments with diverse functions, including horizontal transfer determinants and genes for bacteriocin T8. The derivations of these acquired segments highlight the impact of IS1216 in driving gene exchange between Enterococcus and Staphylococcus species. We traced the emergence of the pRUM-like lineage to a putative ancestor found in a vancomycin-sensitive ST17 E. faecium isolated in 1997. The ancestral plasmid, pCANE, includes the entire pRUM backbone with an additional 44.9 kb in place of the pRUM accessory region. The 44.9 kb segment includes putative conjugation determinants, suggesting that the emergence of the pRUM-like lineage coincided with a loss of transfer functions. We propose an IS1216-driven model for the evolution of pRUM-like plasmids, which appear to have arisen in E. faecium ST17 and contributed to the international success of CC17 as an opportunistic pathogen.