Antibiofilm activity of Plumbagin against Staphylococcus aureus

Abstract

In chronic infections caused by Staphylococcus aureus, biofilm is a major virulence factor. In Staphylococcus aureus biofilms, bacteria are embedded in a matrix of extracellular polymeric substances and are highly tolerant to antimicrobial drugs. However, the lack of effective solutions to inhibit biofilm formation remains a challenge, and the mechanism of inhibition of biofilm formation targeting extracellular polymeric substances is unclear. The aim of the present study was to investigate the inhibitory mechanisms of Plumbagin against Staphylococcus aureus biofilms formation by affecting secretion of extracellular polymeric substances using the high-content screening. Our results showed Plumbagin (16 µg/mL) inhibited biofilm formation, revealing a significant reduction in both biomass and bacterial metabolic activity, and disrupted the biofilm structure, leading to a significant decrease in both biological volume and average thickness (P ≤ 0.01). High-content screening imaging indicated that the Plumbagin treatment induced alterations in the extracellular polymeric substances of Staphylococcus aureus biofilm, significantly reducing the quantities of extracellular polysaccharide, proteins and extracellular DNA. Interestingly, extracellular DNA within the matrix was found to be the most sensitive to Plumbagin treatment. Extracellular DNA formation was significantly inhibited at a concentration of 4 µg/mL, whereas the inhibition of extracellular polysaccharide and proteins required a higher concentration of 8 µg/mL. Overall, these results demonstrated the inhibitory effects of Plumbagin on Staphylococcus aureus biofilm formation and extracellular polymeric substances secretion, suggesting that extracellular DNA may be a potential target for the anti-biofilm activity of Plumbagin. These findings will provide new insights into the mode of action of Plumbagin in treating infections caused by Staphylococcus aureus biofilms.

Keywords: Staphylococcus aureus; Biofilm; Extracellular polymeric substance; High-content screening; Plumbagin.

Fig. 1

MIC determination and growth curve construction. (A) The MIC of PLB against planktonic S. aureus. (B) The effect of PLB on growth of S. aureus. (C) The growth curve of the biofilm from 0 h to 48 h. Data were presented as the mean ± standard deviation.

Fig. 2

Effect of PLB on S. aureus biofilm formation. (A) Effect of PLB on the biofilm biomass of S. aureus as quantified by CV staining. (B) The viability of biofilm cells of S. aureus was determined by XTT assay. (C) Light microscope images at 40 times magnification. (D) The HCS Z-stack maximum projections, 3D reconstructions and merge images of S. aureus biofilm stained with the Film Tracer™ LIVE/DEAD biofilm kit. (E) COMSTAT image analysis. Data were presented as the mean ± standard deviation. **Indicated P ≤ 0.01 compared to control.

Fig. 3

Effect of PLB on the EPS components of S.aureus biofilms. (A) HCS images of PIA, protein and eDNA in the biofilm formation assay. (B) The quantitative analysis of PIA production in each sample was determined by measuring the intensity of green fluorescence using Image J software. The date was presented as the PIA production in each layer of the biofilm (1 μm sections). Total PIA production in biofilms was presented as the area under the curve. (C) The quantitative analysis of protein production in each sample was determined by measuring the intensity of red fluorescence using Image J software. The data are presented as the protein production in each layer of the biofilm (1 μm sections). Total protein production in biofilms was presented as the area under the curve. (D) The concentration of eDNA was assessed using the Quant-iT™ PicoGreen^® dsDNA Assay Kit. (E) The percentage of EPS components (PIA, protein, eDNA) in biofilms treated with various concentrations of PLB compared to the untreated control was calculated. Data were expressed as mean ± standard deviation. **Indicates a significant difference P ≤ 0.01 compared to control.