Competitive interaction of thymol with cviR inhibits quorum sensing and associated biofilm formation in Chromobacterium violaceum

Biofilm formation associated with quorum sensing (QS) is a community behaviour displayed by many gram-negative pathogenic bacteria that provide survival advantages in hostile conditions. The inhibitors of QS interrupt bacterial communication and coordinated cell signalling for community aggregation in the biofilm. Thymol, a natural monoterpenoid, was tested against QS in Chromobacterium violaceum. As the first step, the interaction of thymol with cviR protein was investigated using in silico approach followed by validation using detailed in vitro experiments. The QS and biofilm studies were performed using the wild type of strain C. violaceum ATCC 12,472 and a mini-Tn5 mutant CV026. The MIC of thymol was established by the broth micro-dilution method, and IC₅₀ value for violacein inhibition was quantified spectrophotometrically by extracting the violacein from the treated cells. Inhibitory effect of thymol on the biofilm was quantified by the crystal violet staining method, and scanning electron microscopy (SEM) was employed for biofilm visualization. The expression of biofilm associated genes (hmsH, hmsR, pilB, and pilT) was evaluated by qRT-PCR analysis. The in silico molecular interactions of thymol with cviR exhibited a G-score of - 5.847 kcal/mol, binding with TYR-80 and SER-155 by Pi-Pi stacking and H-bond, respectively. The MIC of thymol was 160 µg/mL, and the IC₅₀ for violacein inhibition was estimated to be 28 µg/mL. The thymol treatment significantly reduced the biofilm viability and biomass by > 80% along with disruption of the well-organized biofilm architecture. QS inhibitory activity of thymol resulted in the reduction of exopolysaccharide production, swarming motility, and downregulation of biofilm-associated hmsH, hmsR, pilB, and pilT genes. This data establishes the QS inhibitory role of thymol in the biofilm formation in C. violaceum.