Screening Antibacterial Compounds Using C. violaceum

The initial screening of compound library was conducted using two strains of C. violaceum as model bacterium, and two assays (violacein extraction and resazurin staining) in parallel as recently optimized in [26 (link)]. For the screening assays, overnight cultures of C. violaceum were diluted in LB broth supplemented with yeast extract (LBY) (0.1% w/v, Sigma Aldrich, St. Louis, MO, USA). Culture of C. violaceum CV026 was further supplemented with 0.5 µM N-(β-ketocaproyl)-l-Homoserine lactone (3-O-C₆-(l)-HSL, Cayman chemicals, Ann Arbor, MI, USA) to induce the violacein production. Bacteria (10⁶ CFU/mL) was exposed to compounds (400 µM) in a total volume of 200 µL in 96-microtiter well plates (Nunc, Roskilde, Denmark), and incubated at 27 °C, with shaking at 200 rpm for 24 h. DMSO was added in untreated control wells and bacteria-free wells with only LBY were included controls. Additionally, autoinducer-free wells containing only bacteria were included in C. violaceum CV026 plates. Plates were prepared in duplicates. Quercetin dihydrate (Carl Roth GmbH, Karlsruhe, Germany) at 400 µM was used as positive control in violacein extraction assay, and azithromycin (Cayman chemicals, Ann Arbor, MI, USA) at 10 µM in viability assay. Both control compounds were prepared in DMSO. DMSO concentration was 2.5% throughout the experiments.