Novel Insights on the Synergistic Mechanism of Action Between the Polycationic Peptide Colistin and Cannabidiol Against Gram-Negative Bacteria

Background/Objectives: Colistin (polymyxin E) has re-emerged as a last-hope treatmentagainst MDR Gram-negative pathogens due to the development of extensively drugresistantGram-negative bacteria. Unfortunately, rapid global resistance towards colistin hasemerged, which represents a major public health concern. In this context (CBD), a lipophilicmolecule derived from Cannabis sativa, exhibits antimicrobial activity mainly againstGram-positive bacteria but is generally ineffective against Gram-negative species. However,synergistic antibacterial activity between CBD and polymyxin B has been reported. Theobjective of this work is to analyze the colistin–CBD synergy against clinically relevantGram-negative isolates displaying diverse mechanisms of colistin resistance and to explorethe basis of the possible mechanism of action involved in the first steps of this synergy.Methods: Microbiological assays, minimal inhibitory concentration, cell culture, synergytests by checker board and time kill, biofilm inhibition evaluation by crystal violet and MTT,SEM (scanning electron microscopy), molecules interaction analysis by nuclear magneticresonance (NMR). Results: The colistin–CBD combination displayed synergy in colistinresistant Gram-negative bacteria and also disrupted preformed biofilms and killed bacteriawithin them. Time-kill assays revealed rapid bactericidal activity and SEM showed mildsurface alterations on bacterial outer membranes after sublethal colistin monotherapy.Furthermore, a series of sequential treatment assays on colistin-resistant Escherichia colishowed that simultaneous exposure to both compounds was required for activity, asintroducing a washing step between treatments abolished the antibacterial effect. In orderto obtain deeper insight into this mechanism, NMR analyses were performed, revealingspecific molecular interactions between CBD and colistin molecules. Conclusions: Theseresults provide evidence for the first time that both molecules engage through a specificand structurally meaningful interaction and only display synergy when acting together oncolistin-resistant bacteria.