The antimicrobial killing mechanism of octenidine (OCT), a well-known antiseptic is poorly understood. We recently reported its interaction with Gram-negative bacteria by insertion of OCT into the outer and cytoplasmic membrane of Escherichia coli, resulting in a chaotic lipid rearrangement and rapid disruption of the cell envelope. Its action primarily disturbs the packing order of the hydrophobic moiety of a lipid, which consequently might result in a cascade of multiple effects at a cellular level. Here, we investigated OCT's impact on two different Gram-positive bacteria, Enterococcus hirae and Bacillus subtilis, and their respective model membranes. In accordance with our previous results, OCT induced membrane disorder in all investigated model systems. Electron and fluorescence microscopy clearly demonstrated changes in cellular structure and membrane integrity. These changes were accompanied by neutralization of the surface charge in both E. hirae and B. subtilis and membrane disturbances associated with permeabilization. Similar permeabilization and disordering of the lipid bilayer was also observed in model membranes. Furthermore, experiments performed on strongly versus partly anionic membranes showed that the lipid disordering effect induced by OCT is a result of maximized hydrophobic over electrostatic forces without distinct neutralization of the surface charge or discrimination between the lipid head groups. Indeed, mutants lacking specific lipid head groups were also susceptible to OCT to a similar extent as the wild type. The observed unspecific mode of action of OCT underlines its broad antimicrobial profile and renders the development of bacterial resistance to this molecule less likely. IMPORTANCE OCT is a well-established antiseptic molecule routinely used in a large field of clinical applications. Since the spread of antimicrobial resistance has restricted the use of antibiotics worldwide, topically applied antiseptics like OCT, with a broad spectrum of antimicrobial activity and high safety profile, gain increasing importance for effective infection prevention and therapy. To eliminate a wide spectrum of disease-causing microorganisms, a compound's antiseptic activity should be unspecific or multitarget. Our results demonstrate an unspecific mechanism of action for OCT, which remained largely unknown for years. OCT disturbs the barrier function of a bacterial cell, a function that is absolutely fundamental for survival. Because OCT does not distinguish between lipids, the building blocks of bacterial membranes, its mode of action might be attributed to all bacteria, including (multi)drug-resistant isolates. Our results underpin OCT's potent antiseptic activity for successful patient outcome.
Keywords: Bacillus subtilis; Enterococcus hirae; Gram-positive bacteria; cell membrane; lipid mutants; membrane activity; mode of action; octenidine.