Antibacterial and anti-virulence activity of juglone against Enterococcus faecalis compared with chlorhexidine

We compared the antimicrobial and antivirulence activities of juglone, a plant-derived naphthoquinone, with chlorhexidine (CHX), a widely used dental antiseptic, against Enterococcus faecalis, a pathogen associated with persistent oral and endodontic infections.

Antimicrobial efficacy was evaluated using well diffusion, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays. Possible interactions between juglone and CHX were investigated using well diffusion and checkerboard methods. Antivirulence activity was assessed by measuring secreted enzymes (caseinase, gelatinase, and hemolysin), biofilm formation, metabolic activity, extracellular polymeric substance (EPS) production, and disruption of mature biofilms. Oxidative stress responses were examined through lactate dehydrogenase (LDH) release, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, and total antioxidant capacity (T-AOC).

Juglone showed antibacterial activity with MICs in a similar range to CHX; however, no synergistic interaction between the two agents was observed. Juglone strongly inhibited caseinase, gelatinase, and hemolysin, whereas CHX showed no effect on caseinase and weaker inhibition of the others. CHX more effectively reduced early biofilm formation, metabolic activity, and mature biomass, whereas both agents similarly decreased EPS production and viability during biofilm development. Notably, in preformed biofilms, juglone at 16 ×MIC caused greater killing (∼4-log₁₀ CFU) than CHX (∼2-log₁₀). Both compounds increased LDH release and reduced SOD activity and T-AOC, while juglone uniquely decreased MDA levels, indicating additional redox-modulating effects.

Juglone impairs E. faecalis virulence by reducing enzymatic activity, oxidative stress tolerance, and biofilm viability, with stronger effects on established biofilms, highlighting its potential for further investigation in oral infection management.