Vancomycin-resistant Enterococci (VRE) are a major concern in Australian healthcare settings, and account for approximately 45% of Enterococcus faecium bloodstream infections. High-level vancomycin resistance is driven by the acquisition of the vanA determinant in association with the transposable element Tn1546, which is commonly located on pRUM-like multidrug resistance (MDR) plasmids. These large MDR plasmids replicate autonomously and can be transferred to other bacteria, promoting the spread of MDR phenotypes. However, replication mechanisms of MDR plasmids in E. faecium are poorly understood, despite being critical to plasmid survival and resistance gene dissemination.
To better understand MDR plasmid replication in E. faecium strains, we used the vanA pRUM-like plasmid pJEG40 as a model. Whole genome sequence analysis identified a replication initiation gene belonging to the repA_N family, as well as adjacent parA and parB genes likely comprising a type IB partitioning system. The functionality of repA was confirmed by constructing a mini replicon that carried repA with and without the adjacent partition locus, par. Although still able to replicate, the absence of par led to inefficient replication, suggesting that it may play an important role in replication or copy number regulation.
RNAseq analysis was performed on strains carrying pJEG40 and the mini replicon (par + repA) to determine the positions and abundance of transcripts produced in the par-rep region and identify putative promoters. Two distinct promoters for the repA gene,PrepA-1and PrepA-2, and an antisense RNA (PrnaI) were identified. The importance of the various promoters to plasmid replication were investigated by generating promoter knockouts and their impact on repA expression and plasmid copy number was determined using reporter gene constructs and droplet digital PCR (ddPCR), respectively.
Our findings indicate that antisense RNA serves as a regulator of replication, since disrupting the PrnaI promoter resulted in a change in plasmid copy number. Furthermore, individual knockout mutations of PrepA-1 and PrepA-2 led to a reduction in copy number, and current experiments are exploring the importance of RNAI interaction with the repA transcripts. Eventually, understanding the mechanisms of plasmid transmission and replication in these pRUM-like plasmids may help combatting combat antibiotic resistance in healthcare settings.