The global prevalence and emergence of enterococcal pRUM-like multi-drug resistance (MDR) plasmids in Australia is of significant clinical concern, as they can transfer high-level vancomycin resistance (via the vanA determinant) to clinical isolates of vancomycin-sensitive enterococci and methicillin-resistant Staphylococcus aureus; vancomycin is the first-line antimicrobial used to treat infections caused by these important ESKAPE group pathogens. Surprisingly, despite the importance of pRUM-like MDR plasmids, little is known about the transmission mechanisms that they utilise to maintain and spread resistance. Therefore, we are using the vancomycin resistance plasmid pJEG40 (a 37.6 kb pRUM-like plasmid), which was identified in an Australian Enterococcus faecium clinical isolate, as a model to understand how the fundamental vertical transmission mechanism, replication, is controlled at the molecular level. Results to date will be discussed, including identification of a novel regulatory network comprised of a transcriptional repressor and an antisense RNA-inhibited pseudoknot activation mechanism.