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 work, 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 toward charged lipids. In this study, we tested this hypothesis and revised previously proposed molecular mechanisms of action. Employing a variety of techniques such as molecular dynamics, z potential with dynamic light scattering, vesicle fluctuation spectroscopy, carboxyfluorescein leakage measurement, and fluorescence trimethylammonium-diphenylhexatriene-and diphenylhexatrienebased 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 three-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. Although 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 suggests membrane disruption by influencing more discrete membrane properties.

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The spreading of antibiotic-resistant bacteria strains is one of the most serious problem in medicine to struggle nowadays. This triggered the development of alternative antimicrobial agents in recent years. One of such group is Gemini surfactants which are massively synthesised in various structural configurations to obtain the most effective antibacterial properties. Unfortunately, the comparison of antimicrobial effectiveness among different types of Gemini agents is unfeasible since various protocols for the determination of Minimum Inhibitory Concentration are used. In this work, we proposed alternative, computational, approach for such comparison. We designed a comprehensive database of 250 Gemini surfactants. Description of structure parameters, for instance spacer type and length, are included in the database. We parametrised modelled molecules to obtain force fields for the entire Gemini database. This was used to conduct in silico studies using the molecular dynamics to investigate the incorporation of these agents into model E. coli inner membrane system. We evaluated the effect of Gemini surfactants on structural, stress and mechanical parameters of the membrane after the agent incorporation. This enabled us to select four most likely membrane properties that could correspond to Gemini’s antimicrobial effect. Based on our results we selected several types of Gemini spacers which could demonstrate a particularly strong effect on the bacterial membranes.