Anti-biofilm and anti-virulence activity of a rare actinobacteria Nocardia sp. EMB45 against Pseudomonas aeruginosa

Pseudomonas aeruginosa forms resilient, antibiotic-tolerant biofilms, with quorum sensing (QS) as a central regulator of biofilm formation and virulence. In this study, we report Nocardia sp. EMB45, isolated from termite nest soil, as a novel source of antibiofilm metabolites. The crude extract inhibited P. aeruginosa PAO1 biofilm formation with a minimum biofilm inhibitory concentration (MBIC) of 6 mg/mL. Morphological analyses by FE-SEM and AFM revealed disrupted biofilm architecture, while confocal microscopy showed sparse and less dense biofilms composed predominantly of viable cells. Mechanistic studies demonstrated QS inhibition using the biosensor Chromobacterium violaceum CV026, suggesting interference with AHL-mediated signalling. The extract markedly reduced QS-regulated phenotypes, including exopolysaccharide production and virulence factors. qRT-PCR analysis demonstrated strong suppression of QS genes, with lasI, lasR, rhlI, and rhlR showing ~ 14- to 20-fold downregulation relative to untreated controls. GC–MS analysis identified 22 metabolites, among which compound C1 (1,2-oxathiane,6-dodecyl-,2,2-dioxide) and compound C4 (Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)-) exhibited the highest binding affinities toward LasR and RhlR, respectively, in molecular docking and dynamics simulations. These findings highlight Nocardia sp. EMB45 is a promising and underexplored source of antibiofilm metabolites with potential applications in anti-virulence therapy against P. aeruginosa.