Prodigiosin inhibits Acinetobacter baumannii biofilm formation by disrupting metabolic pathways and downregulating biofilm-associated genes

Acinetobacter baumannii (Ab), a common opportunistic pathogen, forms biofilms that enhance antibiotic resistance and environmental tolerance, posing challenges for clinical treatment. Prodigiosin (PG), a natural red pigment with broad-spectrum antimicrobial activity, has unclear mechanisms against Ab and its biofilms. This study investigated PG (derived from Serratia marcescens CM01) for its antibacterial/antibiofilm effects on Ab and explored its molecular mechanisms through phenotypic and wholegenome transcriptomic analyses.

Seven clinical Ab strains and one reference strain were studied. Antimicrobial susceptibility was assessed by disk diffusion; PG's minimum inhibitory concentrations (MICs) were determined via microbroth dilution; antibiofilm activity (0.1 to 3.0 μg/mL PG) was quantified by crystal violet staining; synergistic effects of tigecycline were evaluated via checkerboard assay. Under minimum biofilm inhibitory concentration (MBIC), PG's impact on extracellular polymeric substances (EPS) was measured using phenol-sulfuric acid (exopolysaccharides) and bicinchoninic acid (extracellular proteins) assays. Hydrogen peroxide (H₂O₂) sensitivity and cell surface hydrophobicity (CSH) were assessed post-MBIC PG treatment. RNA sequencing (RNA-Seq) analyzed differentially expressed genes (DEGs) in Ab reference strain exposed to 1/2 MIC PG.

Six of seven clinical Ab strains exhibited multidrug resistance. Prodigiosin (PG) demonstrated minimum inhibitory concentrations (MICs) of 64 to 128 μg/mL against all eight strains. Biofilm formation was inhibited at PG concentrations of 0.1 to 1.0 μg/mL (inhibition rate >50%). Synergy testing with tigecycline yielded fractional inhibitory concentration indices (FICI) of 0.5 to 1.0, indicating additive effects. PG treatment at minimum biofilm inhibitory concentration (MBIC) significantly reduced exopolysaccharide content (from 28.77 μg/mL to 14.90 μg/mL; P<0.05), extracellular protein production (from 0.192 to 0.164; P<0.05), and cell surface hydrophobicity [from (41.31±6.51)% to (31.61±7.18), P<0.05]. Bacterial survival under hydrogen peroxide stress decreased from 6.7×10⁶ CFU/mL to 1.5×10⁴ CFU/mL (P<0.05). RNA sequencing revealed 1342 differentially expressed genes (|Log₂FC|>1, P<0.05) with 737 downregulated and 605 upregulated, showing significant enrichment in amino acid metabolism, peptidoglycan biosynthesis, and energy metabolism pathways. Key biofilm-associated genes were substantially downregulated.

PG inhibits Ab growth and biofilm formation by disrupting amino acid, peptidoglycan and energy metabolic pathways, downregulating biofilm-related genes, reducing EPS production, diminishing bacterial stress resistance, and ultimately impeding biofilm development.