Antibiofilm Activities of Multiple Halogenated Pyrimidines Against Staphylococcus aureus

<i>Staphylococcus aureus</i>, prevalent in hospital and community settings, forms biofilms that are highly resistant to antibiotics and immune responses, complicating treatment and contributing to chronic infections. These challenges underscore the need for novel treatments that target biofilm formation and effectively reduce bacterial virulence. This study investigates the antibiofilm and antimicrobial efficacy of novel halogenated pyrimidine derivatives against <i>S. aureus</i>, focusing on three compounds identified as potent biofilm inhibitors: 2,4-dichloro-5-fluoropyrimidine (24DC5FP), 5-bromo-2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (24DC5BPP), and 2,4-dichloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (24DC5IPP). The three active compounds are bacteriostatic. In particular, 24DC5FP at 5 µg/mL achieved a 95% reduction in hemolysis with a minimum inhibitory concentration (MIC) of 50 µg/mL. Interestingly, 24DC5FP increased cell size and produced wrinkled colonies. qRT-PCR analysis showed that 24DC5FP suppressed the gene expressions of <i>agrA</i> and <i>RNAIII</i> (quorum sensing regulator and effector), <i>hla</i> (α-hemolysin), <i>nuc1</i> (nucleases nuc1), and <i>saeR</i> (<i>S. aureus</i> virulence regulator). These findings suggest that extensive halogenation enhances the antibiofilm and antivirulence activities of pyrimidine derivatives, offering a promising strategy for combatting <i>S. aureus</i> infections, including those resistant to conventional treatments.