Multi‐Halogenated Indoles as Antimicrobial and Antivirulence Agents Against Drug‐Resistant <i>Staphylococcus aureus</i>

The emergence of multidrug-resistant Staphylococcus aureus requires the development of novel agents that can target both planktonic cells and persistent biofilms. In this study, a library of multi-halogenated indoles was evaluated for antibacterial, antibiofilm and antivirulence activities against S. aureus including methicillin-resistant strains. Two lead compounds, 6-bromo-4-iodoindole and 4-bromo-6-chloroindole, exhibited potent bactericidal activity (MIC = 20-30 μg/mL) comparable to the antibiotic gentamicin, effectively inhibited biofilm formation and persister formation and suppressed key virulence haemolysis. These effects were associated with intracellular ROS generation and transcriptional downregulation of quorum-sensing genes of agrA and RNAIII and virulence genes of hla and nuc1. Also, 6-bromo-4-iodoindole synergised with aminoglycoside tobramycin and gentamicin, significantly reducing their effective MICs. Notably, these two multi-halogenated indoles did not induce drug resistance for 20 days while gentamicin rapidly increased drug resistance. Cytotoxicity assays in HepG2 cells and phytotoxicity tests confirmed a favourable safety profile. Structure-activity relationship identified multi-halogenation at the C4, C5, C6 and C7 positions of indole as favourable for enhanced activities and also suggested that more halogens could improve the activities. This study highlights multi-halogenated indoles as promising multi-target antimicrobial agents with potential therapeutic and environmental applications against S. aureus, including drug-resistant and biofilm-forming strains.