LAUSR

Multidrug-resistant (MDR) bacteria are widespread throughout the world and pose an increasingly serious threat to human and animal health. Besides implementing strict measures to prevent improper antibiotic use, it remains essential that novel antibiotics must be developed. These antibiotics need to exert their activity via mechanisms different from those employed by currently approved antibiotics. In this study, we used several 5-FU analogs as chemical probes and investigated the potential of these pyrimidine analogs as antibacterial agents. Several 5-fluorouracil (5-FU) derivatives exerted potent activity against strains of Gram-positive cocci (GPC) that are sus-ceptible or resistant toward approved antibiotics, without showing cross-resistance. Furthermore, we have pro-vided evidence that the pyrimidine analogs exerted anti-GPC activity via thymineless death by inhibition of thy-midylate synthetase (Thy A) and/or inhibition of RNA synthesis. Interestingly, whole genome re-sequencing of in vitro-selected, pyrimidine analog-resistant Staphylococcus aureus mutants indicated that S. aureus strains with pyrimidine-analog resistance, induced an amino acid (AA) substitution, deletion, and/or insertion into thy-mineless-death related proteins except for ThyA, or enhanced the ThyA transcription level. Thus, S. aureus may avoid altering the ThyA function by introducing an AA substitution, suggesting that the pyrimidine analogs, which directly bind to ThyA without phosphorylation, may be more effective and show a higher genetic barrier, than the pyrimidines that depend on phosphorylation for activity. The findings of this study may assist in the future de-velopment of a novel class of antibiotics for combating MDR GPC, including methicillin-resistant S. aureus and vancomycin-resistant Enterococci.