Antibiotic resistance is a rising global threat, with an increasing number of resistance pathogens occurring in hospitals and in the community1. The last novel class of antibiotic discovered was lipopeptides back in the 1980s2. A major barrier to the discovery of novel compounds is bacteria’s ability to continuously evolve resistance mechanisms and transfer this ability to other bacterial populations. Therefore, new treatment options are needed without promoting resistance to new compounds. Phyto-cannabinoids, such as cannabidiol (CBD), show significant potential as a novel line of therapeutics. CBD has potent activity against a wide range of Gram-positive pathogens including methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant S. aureus with a minimum inhibitory concentration of 1-4 μg/mL3. Furthermore, our research group has demonstrated that resistance to CBD is unlikely to occur through a 20 day serial passage experiment using S. aureus and Staphylococcus saprophyticus 3. CBD can rapidly disrupt the bacterial membrane, however the precise target or mechanism isn’t well characterised, and this project aims to elucidate this in a Gram-positive bacterial model. Previous mode of action assays have indicated that CBD could have a novel mechanism of action3. Whole genome sequencing of 20-day serial passaged isolates will be used to identify variation in the genome of S.aureus and S.saprophyticus in response to CBD treatment. Complementation of genes altered during CBD treatment will be used as proof of concept. A transposon directed insertion site (TraDIS) sequencing library of S. aureus and S.saprophyticus mutants will be generated to confirm or disprove the involvement of these genes in susceptibility to CBD. Finally, the localisation and the effects of subinhibitory levels of CBD on the bacterial membrane will be evaluated using CBD-fluorescent probes in confocal microscopy, and single cell microfluidics. The microscopy characterisation information together with bioinformatics will enable us to elucidate the antibacterial mechanisms of action of CBD, and therefore contribute to the characterisation of phytocannabinoids as a novel class of antimicrobials.