The membrane-active polyaminoisoprenyl compound NV716 re-sensitizes Pseudomonas aeruginosa to antibiotics and reduces bacterial virulence

Infections caused by Pseudomonas aeruginosa are difficult to threat for many reasons. Among them, P. aeruginosa shows intrinsic or acquired resistance to several antibiotics. It expresses virulence factors and adopts specific lifestyles, like biofilms, non-responsive to antibiotics.

Behind acquired resistance mechanisms, P. aeruginosa also shows intrinsic resistance to many antibiotic classes due to the low permeability of its outer membrane and/or to the expression of efflux pumps. An appealing strategy therefore consists in combining antibiotics with adjuvants that can improve their accumulation inside the bacteria by increasing the outer membrane permeability and/or inhibiting efflux.  Here, we show that the polyaminoisoprenyl compound NV716, synthesized at the Aix Marseille Université (Membranes et Cibles thérapeutiques (MCT), INSERM, Marseille, France) markedly increases the activity of several antibiotics against P. aeruginosa in planktonic cultures or in biofilms. NV716 binds to the LPS, displacing Mg2+ ions that are necessary for the stability of the LPS layer, and increasing the permeability of the outer membrane at sub-inhibitory concentrations. This is accompanied by an enhanced accumulation of the antibiotic inside the bacteria, as shown for rifampicin or ciprofloxacin.   In parallel NV716 also improves the penetration of the same antibiotics in biofilms, and, as a consequence, it increases their anti-biofilm activity. 


But NV716 also shows a series of unanticipated additional effects that reinforce its interest as an adjuvant to antibiotics. First, it prevents the selection of resistance to ciprofloxacin or rifampicin in serial passage experiments while being him-self incapable to select stable resistance using the same experimental approach.  A transcriptomic analysis of bacteria exposed to NV716 highlighted  an overexpression of genes involved in LPS synthesis and a downregulation of genes involved in virulence. At the phenotypic level, this translates in an inhibition of bacterial motility, of biofilm formation, or of the production of rhamnolipids, elastase or pyocyanin. All together, these properties confer also to NV716 and antivirulence activity.

Although we did not uncover the precise molecular target of NV716 in P. aeruginosa, we nevertheless progressed in the elucidation of the physiological processes it can affect in bacteria as well as in the demonstration of their consequences for antibiotic activity and bacterial virulence. The promising profile of activity we evidenced here already at sub-inhibitory concentrations, coupled with its previously demonstrated lack of toxicity in vitro at microbiologically-active concentrations, encourages further research in pertinent in-vivo models of infections in order to better delineate its potential as an adjuvant to current antibiotics. In a broader context, this works confirms that membrane-targeting approaches are promising strategies that can revive antibiotic activity, including against persistent forms of infections like biofilms or intracellular survival.

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