Cyclic diguanylate (c-di-GMP) is a central biofilm regulator in Pseudomonas aeruginosa, where increased intracellular levels promote biofilm formation and antibiotic tolerance. Targeting the c-di-GMP network may be a promising anti-biofilm approach, but most strategies studied so far aimed at eliminating surface-attached biofilms, while in vivo P. aeruginosa biofilms often occur as suspended aggregates. Here, the expression profile of c-di-GMP metabolism-related genes was analysed among 32 P. aeruginosa strains grown as aggregates in synthetic cystic fibrosis sputum. The diguanylate cyclase SiaD proved essential for auto-aggregation under in vivo-like conditions. Virtual screening predicted a high binding affinity of echinacoside towards the active site of SiaD. Echinacoside reduced c-di-GMP levels and aggregate sizes and potentiated tobramycin activity against aggregates in >
80% of strains tested. This synergism was also observed in P. aeruginosa-infected 3-D alveolar epithelial cells and murine lungs, demonstrating echinacoside's potential as an adjunctive therapy for recalcitrant P. aeruginosa infections.