Fingolimod as a potent anti-Staphylococcus aureus: pH-dependent cell envelope damage and eradication of biofilms/persisters.

Background: The urgent need for new antibacterial drugs has driven interest in repurposing therapies to combat Gram-positive biofilms and persisters. Fingolimod, an Food and Drug Administration (FDA)-approved drug for multiple sclerosis, shows bactericidal activity, particularly against Methicillin-resistant Staphylococcus aureus (MRSA) and biofilm-related infections. With a well-documented safety profile and strong translational potential, it aligns with World Health Organization's goals for antimicrobial repurposing. However, the action mode and mechanism of Fingolimod against gram-positive bacteria remain elusive.

Methods: This study utilized clinical Staphylococcus aureus (S. aureus), Enterococcus faecalis (E. faecalis), Streptococcus agalactiae (S. agalactiae). And their susceptibility to Fingolimod and other antibiotics was tested via Minimum Inhibitory Concentration (MIC) assays. Biofilm inhibition and hemolytic activity were evaluated using crystal violet staining, Confocal Laser Scanning Microscopy (CLSM), and hemolysis assays, respectively, while the effect of phospholipids on Fingolimod efficacy was assessed with checkerboard assays. Membrane permeability and integrity were measured using SYTOX green staining and transmission electron microscopy. Whole-genome sequencing was performed on Fingolimod-resistant S. aureus isolates to identify Single Nucleotide Polymorphisms (SNPs) linked to resistance.

Results: Our data indicated that Fingolimod exerted bactericidal activity against a wide spectrum of gram-positive bacteria, including S. aureus, E. faecalis, S. agalactiae. Moreover, Fingolimod could significantly eliminate the persisters, inhibit biofilm formation and eradicate in-vitro mature biofilms of S. aureus. The mechanism by which Fingolimod rapidly eradicated S. aureus involved a pH-dependent disruption of bacterial cell permeability and envelope integrity. Concomitantly, exogenous supplementation of phospholipids in the culture medium resulted in a dose-dependent increase in the MIC of Fingolimod. Specifically, the addition of 64 μg/mL of cardiolipin (CL) and phosphatidylethanolamine (PE) completely nullified the bactericidal activity of Fingolimod at a concentration of 4 times the MIC. After four months of Fingolimod exposure, the MIC values of S. aureus showed a slight increase, indicating that it is not prone to developing drug resistance.

Conclusion: Fingolimod exhibits bactericidal activity against diverse gram-positive bacteria, with remarkable effects on S. aureus (including MRSA), disrupting bacterial cell structural integrity in a pH-dependent way and eradicating biofilms and persisters of S. aureus.