A dual antibacterial action of soft quaternary ammonium compounds: bacteriostatic effects, membrane integrity, and reduced in vitro and in vivo toxicity

Abstract

Quaternary ammonium compounds (QACs) have served as essential antimicrobial agents for nearly a century due to their rapid membrane-disrupting action. However, the emergence of bacterial resistance and environmental concerns have driven interest in alternative designs, such as “soft QACs”, which are designed for enhanced biodegradability and reduced resistance potential. In this study, we explored the antibacterial properties and mechanisms of action of our newly synthesized soft QACs containing a labile amide bond within a quinuclidine scaffold. Our findings revealed that these compounds primarily exhibit a bacteriostatic mode of action, effectively suppressing bacterial growth even at concentrations exceeding their minimum inhibitory concentrations (MICs). Unlike traditional QACs, fluorescence spectroscopy and microscopy demonstrated membrane preservation during treatment, with reduced membrane integration compared to cetylpyridinium chloride (CPC), as corroborated by parallel artificial membrane permeability assays. Additionally, molecular dynamics simulations revealed “hook-like” conformations that limit lipid bilayer penetration and promote the formation of larger aggregates, reducing their effective concentration and minimizing cytotoxic effects. Interestingly, secondary antibacterial mechanisms, including inhibition of protein synthesis, were observed, further enhancing their activity. Zebrafish embryotoxicity and in vitro cytotoxicity studies confirmed significantly lower toxicity compared to CPC. By addressing limitations associated with conventional QACs, including toxicity, resistance, and environmental persistence, these soft QACs provide a promising foundation for next-generation antimicrobials. This work advances the understanding of QAC mechanisms while paving the way for safer, eco-friendly applications in healthcare, agriculture, and industrial settings.