Abstract
Antimicrobial resistance (AMR) describes a reduction in the efficacy of antimicrobial drugs (e.g., antibiotics) against pathogens (e.g., bacteria). This is a clinical issue of growing concern and directly caused over 1.28 million deaths in 2019. As a result of increasing AMR, there is an urgent requirement for new treatment strategies to combat AMR infections, such as novel antibiotics or antibiotic adjuvants that can potentiate the activity of existing antibiotics against bacterial pathogens like methicillin-resistant Staphylococcus aureus (MRSA). MRSA has acquired resistance to all cell wall targeting β-lactam antibiotics via the SCCmec mobile genetic element. Our investigation centres on the antibiotic adjuvant potential of the zinc ionophore PBT2. While PBT2 exhibits bactericidal properties at subtherapeutic concentrations, it demonstrates the capacity to enhance the activity of β-lactam antibiotics against MRSA. However, the molecular mechanism remains unknown. To unravel this mechanism, we used unpublished transposon screening data from the MRSA Nebraska Transposon Mutant Library to characterise putative hypersensitive PBT2+Zn mutants. We hypothesised that mutants that showed hypersensitivity to PBT2+Zn could not contribute to the potentiation of oxacillin in MRSA and, therefore, the disrupted gene (or pathway) may contribute to antibiotic resistance reversal. We uncovered four candidate genes (gpmA, czrA, phoH, and pdxS) that have presumptive roles in hypersensitivity to PBT2+Zn. These four genes displayed increased cell killing in the presence of a combination of PBT2+Zn compared to untreated controls 24 hours post-treatment. To investigate these further, we utilised markerless gene knockouts to generate separate MRSA USA300 isolate TCH1516 strains lacking in these genes. While the functions of these pathways in AMR reversal are still under investigation, PBT2 administration, both alone and alongside β-lactam antibiotics, has shown antibacterial activity against MRSA and is well tolerated in humans, making it a compelling candidate for repositioning as an antibiotic adjuvant.