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

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