Unraveling the mechanism of octenidine and chlorhexidine on membranes: Does electrostatics matter?

Abstract The increasing problem of antibiotic resistance in bacteria requires the development of new antimicrobial candidates. There are several well-known substances with commercial use, but their molecular mode of action is not fully understood. In this paper, we focus on two commonly used antimicrobial agents from the detergent family – octenidine dichloride (OCT) and chlorhexidine digluconate (CHX). Both of them are reported to be agents selectively attacking the cell membrane through interaction inducing membrane disruption by emulsification. They are believed to present electrostatic selectivity towards charged lipids. In this study, we tested this hypothesis and we revised previously proposed molecular mechanisms of action. Employing a variety of techniques such as molecular dynamics, zeta potential with dynamic light scattering, vesicle fluctuation spectroscopy, carboxyfluorescein (CXF) leakage measurement and fluorescence trimethylammonium-diphenylhexatriene (TMA-DPH) and DPH based studies for determination of OCT and CHX membrane location, we performed experimental studies using two model membrane systems – zwitterionic PC and negatively charged PG (18:1/18:1):PC (16:0/18:1) 3:7, respectively. These studies were extended by molecular dynamics simulations performed on a 3-component bacterial membrane model system to further test interactions with another negatively charged lipid, cardiolipin. In summary, our study demonstrated that detergent selectivity is far more complicated than supposed simple electrostatic interactions. While OCT does disrupt the membrane, our results suggest that its primary selectivity was more linked to mechanical properties of the membrane. On the other hand, CHX did not disrupt membranes as a primary activity, nor did it show any sign of electrostatic selectivity toward negatively charged membranes at any stage of interactions, which suggest membrane disruption by influencing more discrete membrane properties.