Breaking the biofilm barrier: Juglone derivatives as dual-action inhibitors and anti-quorum sensing agents

Abstract

Background

Most nosocomial infections are caused by bacteria that proliferate within quorum sensing mediated biofilms. The disruption of quorum sensing signals in biofilm-forming bacteria is the potential substitution strategy.

Purpose

The current study focuses on the evaluation of isolated juglone derivatives from Reynoutria japonica as anti-biofilm agents and enzyme inhibitors against Methicillin Resistant Staphylococcus aureus (MRSA).

Methods

The antibacterial and anti-biofilm activity of the juglone derivatives were investigated against laboratory strains MRSA using well and disc diffusion method. In addition, we evaluated the anti-biofilm inhibition potential of the juglone derivatives using a crystal violet-based assay in 96-well micro-titer plate. The suppression of S. aureus β-hemolytic, coagulase and proteolytic activity was evaluated using standard assay protocols. High Performance Liquid Chromatography (HPLC) was performed to analyze the acyl homoserine lactones (AHLs) signals degradation by juglone derivatives. Furthermore, all three ligand molecules were docked into the binding site of the S. aureus coagulase protein.

Results

All the three juglone derivatives showed potent antimicrobial activity against clinical strains of MRSA. Among these compounds, 2-ethoxy-6-acetyl-7-methyljuglone displayed potent inhibitory activity with zones of inhibition of 15 ± 0.03 and 17 ± 0.03 against MRSA respectively. In the spectrophotometric assay, we confirmed that juglone derivatives inhibit biofilm formation. All the compounds markedly inhibited the coagulase, β-hemolytic and proteolytic activity of MRSA. The application of compounds as a treatment resulted in a notable decrease in biofilm density and thickness, as demonstrated by HPLC. The untreated MRSA exhibited quorum sensing signals of N-Butanoyl-l-homoserine lactone (C4-HSL) with retention time 3.6 min and of N-hexanoyl-l-Homoserine lactone (C6-HSL) at 5.8 minutes’ vs the treated with MRSA where no peaks were observed. The docking studies and Molecular dynamics (MD) simulation results revealed that all molecules are actively binding in the target site that correlates well with the in-vitro inhibitory properties of compounds against S. aureus.

Conclusion

our results demonstrated the potential of juglone derivatives from R. japonica as a promising anti-biofilm agent and as quorum quencher for future therapy to combat multi-drug resistant infections in particular MRSA.