Investigating mechanisms of Staphylococcus aureus survival of trimethoprim-sulfamethoxazole-induced thymineless death

The antibiotic combination trimethoprim-sulfamethoxazole (SXT) is commonly used to treat diverse S. aureus infections, including those associated with cystic fibrosis (CF) pulmonary disease. SXT impairs tetrahydrofolate production, leading to DNA damage, stress response induction, and accumulation of reactive oxygen species (ROS) in a process known as thymineless death (TLD). TLD survival can occur through uptake of exogenous thymidine, countering the effects of SXT; however, a growing body of research has implicated central metabolism as another potentially important determinant of bacterial survival of SXT and other antibiotics. Here we show that (i) thymidine abundances in CF sputum were insufficient for preventing TLD of S. aureus, highlighting the importance of additional survival mechanisms; (ii) sequencing of S. aureus grown in vitro with SXT and low thymidine frequently identified adaptive mutations in genes encoding carbohydrate, nucleotide, and amino acid metabolism, supporting limited metabolism as a common survival mechanism; (iii) although intracellular ROS levels rose with SXT treatment in vitro, survival was not improved with ROS scavengers, in contrast with observations of the model organism E. coli; (iv) SXT challenge induced the SOS response, which was alleviated by added thymidine; and (v) observations with an SXT-selected isolate carrying a mutation in ptsI suggested that limitation of intracellular ATP improved survival against TLD. These results suggest that alterations in core metabolic functions, particularly those that impact ATP levels, predominate in conferring survival and persistence during SXT treatment under physiologically relevant conditions, potentially identifying novel targets for co-treatment