Antibacterial and anti-biofilm efficacy of 1,4-naphthoquinone against Chromobacterium violaceum: an in vitro and in silico investigation

Abstract

Antimicrobial resistance (AMR) is an urgent worldwide health concern, requiring the exploration for novel antimicrobial interventions. A Gram-negative bacterium, Chromobacterium violaceum, synthesizes quorum-sensing-regulated violacein pigment, develops resilient biofilms, and is often used for the screening of anti-infective drugs. The aim of this work is to assess the antibacterial and antibiofilm properties of three polyphenols: 1,4-naphthoquinone, caffeic acid, and piperine. The determination of antibacterial activity was conducted by the agar overlay and broth microdilution techniques. Analysis of membrane rupture was conducted by crystal violet uptake and β-galactosidase assay. Inhibition of biofilm was evaluated using a 96-well microtiter plate assay. Biofilm structures were visualized using light, scanning electron microscopy (SEM), and confocal laser scanning electron microscopy (CLSM). Among the phytochemicals, 1,4-naphthoquinone exhibited the highest antibacterial action (25 mm zone of inhibition). The minimum inhibitory concentration of 1,4-naphthoquinone was determined to be 405 µM. Outer and inner membrane permeability was enhanced by 52.01% and 1.28 absorbance, respectively. Violacein production was reduced by 74.85%, and biofilm formation was suppressed by 63.25% at sub-MIC levels (202.5 µM). Microscopic analyses confirmed reduced adhesion on surfaces. Hemolytic activity of 1,4-naphthoquinone showed a concentration-dependent effect, with 32.16% haemolysis at 202.5 µM. Molecular docking revealed significant interactions of 1,4-naphthoquinone with DNA gyrase followed by CviR. These findings highlight 1,4-naphthoquinone’s potent antibacterial efficacy against C. violaceum, proposing its use as a surface coating agent to prevent biofilm formation on medical devices, thereby offering a promising strategy to combat bacterial infections.