Using metabolomic tools to bioprospect the antibacterial activity of a seaweed endophyte co-cultured with biofilm-forming bacteria Pseudomonas aeruginosa

The increasing prevalence of bacterial biofilms and their tolerance to antimicrobial treatment highlight the need for new anti-biofilm agents. Microbial co-culture offers a promising strategy for inducing otherwise poorly expressed secondary metabolites; however, the metabolic responses to fungal–bacterial interactions and their contribution to antimicrobial activity remain insufficiently understood. This study investigated whether co-culturing the algal endophytic fungus Dendryphiella salina with the biofilm-forming bacterium Pseudomonas aeruginosa could induce bioactive metabolites with anti-biofilm activity.

An integrated workflow combining multivariate data analysis, Nuclear Magnetic Resonance (NMR) spectroscopy, High-Performance Liquid Chromatography–High-Resolution Mass Spectrometry (HPLC–HRMS), and antimicrobial and anti-biofilm assays was used to characterise co-culture-induced metabolic changes and identify bioactive metabolites. Optimised co-culture conditions were selected based on metabolic profiles and biological activity and subsequently scaled up for chemical investigation. Medium-pressure liquid chromatography fractionation of the crude extract generated fourteen fractions, nine of which exhibited significant anti-biofilm activity against methicillin-resistant Staphylococcus aureus (MRSA). OPLS-DA, HRMS-based dereplication, and 1D/2D NMR spectroscopy enabled the annotation of several metabolites, including linoleic acid, 2-(2-hydroxyphenyl)thiazole, 2-phenyl-4,5-dihydrothiazol-4-ol, aeruginoic acid, dihydroaeruginoic acid, and a mono-rhamnolipid.

The mono-rhamnolipid demonstrated the strongest overall activity, with a minimum inhibitory concentration (MIC) of 6.5 µg/mL, minimum biofilm prevention concentration (MBPC) of 5.0 µg/mL, and minimum biofilm inhibitory concentration (MBIC) of 7.2 µg/mL. A mixture of 2-phenyl-4,5-dihydrothiazol-4-ol and dihydroaeruginoic acid inhibited planktonic growth, biofilm formation, and established biofilms by 95.3%, 98.9%, and 85.1%, respectively. Overall, fungal–bacterial co-culture induced substantial metabolic and biological changes and facilitated the discovery of anti-MRSA metabolites. Notably, anti-biofilm activity was predominantly associated with bacterial secondary metabolites, suggesting that microbial interactions can redirect secondary metabolism towards bioactive compounds.