Pathogenic bacteria cause infectious diseases, mainly when the host (humans, animals, and plants) are colonised by bacteria, especially in its biofilm stage, where it is known to cause chronic infections. Biofilms are associated with resistance to antimicrobial agents, including antibiotics, antiseptics, detergents, and other therapeutic approaches. Antimicrobial resistance (AMR) is one of the biggest public health challenges of our time and is termed a ‘silent pandemic’ by the United Nations. Biofilm formation, pathogenicity and the associated AMR are regulated through a bacterial cell-to-cell communication system termed “Quorum Sensing (QS)’. As the bacterial cells sense the fluctuations in their population, they biosynthesise and secrete the signalling molecules called autoinducers (AI). In gram-negative, the signalling molecules are primarily homoserine lactones (AHL) whereas in gram-positive the signalling molecules are autoinducing peptides. The AI binds to receptor and regulator proteins in the bacterial cells to activate the complete QS system, which controls the regulations of various genes that are essential for the biosynthesis of virulence factors, extracellular biopolymers (EPS) production, biofilm formation and bacterial fitness.

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The production of pyocyanin by Pseudomonas aeruginosa increases its virulence, fitness and biofilm formation. Pyocyanin is also a redox molecule and we hypothesize that ascorbic acid being an antioxidant will interact with pyocyanin. The main objective of this study was to investigate the potential interaction of ascorbic acid with pyocyanin, and also to investigate the impact of ascorbic acid in combination with Furanone-30 on quorum sensing and biofilm formation of P. aeruginosa. When incubated with ascorbic acid, hyperchromic and hypsochromic shifts in pyocyanin absorbance peaks at 385 nm and 695 nm were observed. In the presence of dehydroascorbic acid and citric acid, these shifts were absent, indicating that the intrinsic antioxidant property of ascorbic acid was probably essential in binding to pyocyanin. NMR spectroscopy showed shifts in 1H NMR pyocyanin peaks between 8.2 to 5.8 ppm when incubated in the presence of ascorbic acid. Density Functional Theory (DFT) supported potential interactions between the –CH2OH or –OH moieties of ascorbic acid with the –C=O moiety of pyocyanin. The pyocyanin-ascorbic acid complex impaired pyocyanin binding to DNA. Ascorbic acid combined with furanone-30 elevated quorum-sensing inhibition in P. aeruginosa, which was directly associated with significantly reduced P. aeruginosa virulence, adhesion, aggregation and biofilm formation and enhanced antibiotic-mediated bacterial killing. This study demonstrated that the antioxidant ascorbic acid directly binds to pyocyanin, modulates its structure and results in disruption of biofilm formation and associated tolerance to antibiotics.