The β-lactam adjuvant guanosine potentiates anti-folate antibiotics and pyrimidine synthesis inhibitors by depleting thymidine in methicillin-resistant <i>Staphylococcus aureus</i>

Efforts to improve the effectiveness of existing interventions for antimicrobial-resistant (AMR) infections include identifying new ways to overcome resistance to licensed antibiotics using adjuvants or deploying antibiotics in novel combinations. Although antibiotics targeting the bacterial cell wall (e.g., β-lactams) and folate metabolism (e.g., trimethoprim-sulfamethoxazole, TMP-SMX) remain cornerstones of modern healthcare, resistance to both classes poses an ongoing therapeutic challenge. We recently demonstrated that purine nucleosides can act as potent antibiotic adjuvants, restoring β-lactam susceptibility in methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). Here, we show that the β-lactam adjuvant guanosine significantly reduces intracellular thymidine levels in MRSA and potentiates the activity of antifolate antibiotics (TMP-SMX) as well as the pyrimidine antimetabolites 5-fluorouracil (5-FU) and 5-fluorouridine (5-FUrd). Incorporation of oxacillin into guanosine-antifolate or guanosine-pyrimidine analogue combinations further enhanced killing of both planktonic and biofilm-associated MRSA. Thymidine depletion was accompanied by elevated intracellular reactive oxygen species (ROS) and dissipation of membrane potential, providing mechanistic insight into the bactericidal effects of these combinations. Together, these findings demonstrate that guanosine expands MRSA susceptibility beyond β-lactams to include additional clinically relevant antimicrobial drug classes commonly used to treat bacterial infections.