Breaking Staphylococcus aureus biofilms with the antimicrobial lipopeptide humimycin and its synthetic analogs

The growing burden of biofilm-associated infections and antibiotic resistance driven by Staphylococcus aureus underscores the urgent need for new antimicrobial agents and new strategies. To address this challenge, antimicrobial lipopeptides like humimycins have emerged as promising candidates for combating Staphylococcus aureus biofilms. In this study, humimycin A 1 and five structural analogs ( 2 - 6 ) were synthesized via solid-phase peptide synthesis and evaluated for antimicrobial and antibiofilm activity against S. aureus ATCC 29213. Minimum inhibitory concentrations (MIC) and minimum biofilm inhibitory concentrations (MBIC) values were determined using broth microdilution and crystal violet staining assays, respectively. Scanning electron microscopy (SEM) was used to visualize changes in biofilm morphology, while sliding motility assays assessed the impact on virulence-associated bacterial movement. The newly introduced analogs displayed MIC values ranging from 2 to 16 µg/mL, with analog 6 showing the strongest activity. In biofilm inhibition assays, analogs 5 and 6 exhibited MBIC of 4 µg/mL and 2 µg/mL, respectively, up to 16-fold lower than the parental compound. Treatment of mature biofilms with analogs 3–6 at four times the MIC led to biomass reductions ranging from 74.8 % to 89.8 %. Scanning electron microscopy confirmed disrupted biofilm architecture following treatment. Furthermore, analogs 4 and 6 significantly inhibited sliding motility, a virulence factor linked to colonization. Resistance development was not observed after 10 serial passages under sub-MIC treatments. These findings highlight the therapeutic potential of synthetic humimycin analogs as precision antibiofilm agents against S. aureus infections. • Synthetic humimycin analogs showed good antimicrobial activity against S. aureus . • The analogs are able to inhibit biofilm formation and disrupt mature biofilm. • The sliding motility of S. aureus was also inhibited by the synthetic lipopeptides. • Low emergence of resistance was observed after several expositions to the analogs.