Antimicrobial Resistance Gene Transfer in MRSA: Novel Mechanisms, Barriers, and Environmental controls

Antimicrobial resistant bacteria, and especially the problematic multi-drug resistant (MDR) bacteria, are a threat to all healthcare, limiting our ability to prevent and treat infection. MDR bacteria acquire and maintain multiple resistances, even without selective pressure in their natural habitat, which is usually the colonised human host. While much is known about gene transfer mechanisms, relatively little is known about how AMR gene transfer is controlled, regulated and stably maintained in MDR bacterial pathogens. The aim of this project is to investigate newly discovered genes in the MDR pathogen methicillin-resistant Staphylococcus aureus (MRSA) that control the horizontal gene transfer (HGT) and stability of AMR. MRSA is the most common cause of AMR death globally. In the last 20 years, increasingly MDR-MRSA clonal types have emerged on multiple occasions causing new clinical problems (e.g. Healthcare MRSA, Community MRSA, Livestock MRSA), while we wait for expected further resistance and epidemiological combinations to establish and spread (e.g. Vancomycin Resistant MRSA, Livestock MRSA in humans), potentially affecting all aspects of healthcare and medicine. In these populations, resistance genes to nearly all classes of antimicrobials are carried on relatively unstable and individual mobile genetic elements. These AMR genes are also highly mobile, as human and animal carriage studies indicate they are exchanged between populations at high frequency during colonisation, with only a few individual cells displaying full MDR phenotypes. MRSA benefits from HGT to acquire useful DNA that protects population survival upon new environmental exposures, such as antimicrobial exposure. However, MRSA also needs barrier systems to protect from foreign DNA (such as lytic bacteriophage) that can be harmful. Known barrier mechanism examples include restriction-modification systems and CRISPR. However, successful MDR-MRSA clones have additional uncharacterised barrier mechanisms which likely play a key role in their survival and successful spread. These barriers of HGT also make MRSA particularly difficult to manipulate genetically in the laboratory. Using a transposon library screen and a novel co-culture gene transfer assay (CoGTRA) to assess AMR gene transfer and stability, we have identified several new barrier gene pathways that control successful HGT and located many of them to a new putative mobile genetic element, the S. aureus Transfer Island (SauTI). In addition, several SauTI genes are predicted to respond to environmental triggers. In this project we will Define the SauTI element and its distribution and evolution in successful MRSA populations. Characterise newly identified genes that control SauTI mobility, AMR HGT and stability Identify how AMR HGT is reduced or enhanced by environmental conditions. Our results will Identify how to optimise gene transfer assays to build better tools for the genetic manipulation of aureus and potentially other cells (biotechnology). Identify targets and strategies to manipulate AMR gene acquisition and stability in models of MDR-MRSA evolution relevant to the colonisation habitat Identify markers for diagnostics or epidemiological studies to identify patterns of evolutionary adaptation and risk of the emergence of new MDR-MRSA clones. Together the results from these studies will advance our understanding of how MDR pathogens acquire and stably maintain AMR genes in the absence of selection, generating insights into evolution, epidemiology and how this might be manipulated in the laboratory, in patients or the environment to reduce the incidence of MDR-MRSA infection.