Probiotic-derived long chain fatty acids disrupt flagellar assembly and biofilm development in Pseudomonas aeruginosa

Abstract The global rise of multidrug-resistant pathogens highlights the urgent need for alternatives to conventional antibiotics. Probiotic-based solutions have emerged as a promising strategy, yet the mechanisms underlying probiotic-pathogen interactions are still not fully understood. Here, we identified a novel mechanism by which probiotic Lactobacillus casei interferes with Pseudomonas aeruginosa (Pa) flagellar synthesis, thereby inhibiting Pa biofilm formation. RNA sequencing and bioinformatics analysis revealed that long-chain fatty acids, particularly stearic acid, produced by L. casei effectively disrupted Pa flagellar assembly. At molecular level, stearic acid suppressed the expression of fleR , a central regulator of flagellum biosynthesis, leading to substantial flagella loss and impaired motility in Pa. This interference further hindered the establishment of biofilm architecture. Beyond its effects on the flagellar assembly, L. casei also altered Pa cell surface morphology and induced strong upregulation of murA , a gene essential for peptidoglycan biosynthesis and cell wall formation. These results suggest that L. casei perturbs Pa flagella synthesis, potentially causing structural instability, which in turn triggers compensatory activation of peptidoglycan biosynthesis. Collectively, our findings reveal a new probiotic-driven mechanism that disrupts Pa biofilm development and highlight L. casei -derived metabolites as potential candidates for preventing and treating biofilm-associated infections.