Quantification of Violacein in <em>Chromobacterium violaceum</em> and Its Inhibition by Bioactive Compounds

Bacteria communicate through a system known as quorum sensing (QS), which allows them to coordinate their behavior in response to changes in cell density. This process involves the production, secretion, and detection of small extracellular signaling molecules, usually called autoinducers (AI). QS regulates a wide range of genes and functions, including biofilm formation and dispersal, swarming motility, production of virulence factors, antibiotic resistance, and bioluminescence, among others. QS inhibition has become a promising antivirulence strategy against antibiotic-resistant bacteria as it exerts a lower selective pressure compared to traditional antibiotics. In the bacterium Chromobacterium violaceum ATCC 12472, the QS proteins CviI/CviR, which are homologous to the LuxI/LuxR-type QS proteins of Aliivibrio fisheri, control many genes, including those responsible for producing the purple pigment violacein. C. violaceum has been widely used as a biosensor strain in QS inhibition studies, particularly the inhibition of violacein production. Many compounds have the potential to inhibit QS by binding to QS proteins, either LuxI or LuxR homologues, disrupting QS circuits. This protocol describes the method for quantifying violacein produced by C. violaceum ATCC 12472 and its inhibition by bioactive compounds at concentrations that do not affect bacterial growth. The assay described here demonstrates a significant reduction in violacein production by the tested compounds. This study highlights the potential for using this protocol to screen promising candidates in QS inhibition research.