Technical-Grade Sabinene Attenuates Biofilm Formation and Quorum Sensing-Regulated Phenotypes in Pseudomonas aeruginosa and Chromobacterium violaceum

Abstract

Recent studies have focused on developing innovative therapeutic interventions in response to the increasing antibiotic resistance reported in opportunistic pathogens such as Pseudomonas aeruginosa. Sabinene is a bicyclic monoterpene found in various essential oils. In this study, the anti-virulence activity of a technical-grade sabinene (77.6% sabinene, 22.3% β-pinene, GC purity; hereafter TGS) was evaluated against the biosensor strain Chromobacterium violaceum and a single clinical isolate of β-lactamase-producing Pseudomonas aeruginosa. The minimum inhibitory concentration (MIC) of TGS was 0.971 mg/mL. Sub-MICs of TGS (0.036 mg/mL and 0.108 mg/mL) demonstrated antibiofilm activity, with more pronounced inhibition at the higher concentration. Swarming motility, surface hydrophobicity, and cell aggregation were also reduced by TGS, while the metabolic activity of biofilm-embedded cells remained high (>89%) at sub-MIC concentrations. In addition, TGS inhibited exopolysaccharide (EPS) production by 66%, thereby disrupting biofilm matrix formation. Moreover, TGS inhibited key quorum sensing (QS)-associated virulence factors, including extracellular protease, rhamnolipids, and pyocyanin. Anti-QS activity was further corroborated phenotypically using the biosensor strain Chromobacterium violaceum. At 0.108 mg/mL, TGS inhibited violacein production (60%) and N-acyl homoserine lactone (AHL) generation. qRT-PCR analysis showed that the QS-associated genes lasI, lasR, and rhlI were downregulated following TGS treatment. These genes are required for the expression of several virulence factors, as they encode regulators and enzymes involved in QS signal transduction and AHL production. TGS exhibits promising activity as a natural anti-virulence candidate that attenuates quorum sensing and biofilm-associated virulence factors in P. aeruginosa, warranting further investigation into its therapeutic potential.