Sesquiterpene farnesol inhibits recycling of the C55 lipid carrier of the murein monomer precursor contributing to increased susceptibility to P-lactams in methicillin-resistant Staphylococcus aureus

Background and objectives: The sesquiterpene farnesol, a natural plant metabolite, is known to intensify the effect of antimicrobial agents. However, the mode of action of its antimicrobial synergism has remained poorly understood. In this study, we investigated farnesol's synergistic effects on commonly used antimicrobials, beta-lactams in particular, to explore its potential inhibitory effect on cell wall synthesis. Methods: We investigated farnesol's effects on: (i) antimicrobial susceptibilities of methicillin-susceptible and -resistant Staphylococcus aureus (MSSA and MRSA) to ampicillin, oxacillin, cefoxitin, bacitracin, teicoplanin, amikacin, ciprofloxacin and clarithromycin by MIC determination using the Etest; (ii) penicillin-binding protein PBP2' (2a) expression by western-blot analysis; (iii) beta-lactamase secretion and activity by in vivo and in vitro farnesol inhibition assays; (iv) staphyloxanthin production by thin-layer chromatography (TLC); and (v) cell wall synthesis by [C-14]GlcNAc (where GlcNAc stands for N-acetylglucosamine) and [C-14] mevalonate incorporation assays, and TLC-based lipid extract profile analysis. Results: Farnesol induced variable degrees of increased susceptibility to all antimicrobials except clarithromycin in both MSSA and MRSA. A remarkable increase in susceptibilities to ampicillin, oxacillin and cefoxitin was observed in both MRSA strains, N315 and COL, whereas a moderate increase in susceptibility to bacitracin was observed in all the strains. Although no apparent suppression of PBP2' expression was observed, beta-lactamase secretion and beta-lactamase activity were significantly reduced by farnesol. In addition, farnesol completely suppressed staphyloxanthin production. Farnesol reduced the incorporation of GlcNAc, but significantly increased that of mevalonate. Farnesol induced accumulation of C-55-PP, lipid I and lipid II. Conclusions: Farnesol increased beta-lactam susceptibility of MRSA by inhibition of cell wall biosynthesis through reduction of free C-55 lipid carrier with subsequent retardation of murein monomer precursor transport across the cell membrane.