Mechanisms of synergistic antibacterial and anti-biofilm effects of dihydroquercetin combined with ceftazidime against mucoid pseudomonas aeruginosa.

OBJECTIVE: This study characterized the synergistic activity of dihydroquercetin combined with ceftazidime (CAZ) against mucoid Pseudomonas aeruginosa (mPA). The regimen primarily relieves biofilm-mediated phenotypic tolerance in CAZ-susceptible isolates, rather than reversing classical genetic resistance, while also enhancing efficacy against a minor proportion of intermediate/resistant strains. We further clarified its mechanisms via membrane integrity tests, qRT-PCR of biofilm/quorum-sensing genes and untargeted metabolomics, to provide experimental basis for treating refractory biofilm-related mPA infections. METHODS: Forty-one clinical mPA isolates were screened via the K-B assay. Checkerboard FICI and time-kill assays evaluated combined antibacterial effects Mucoid EPS semi‑quantitative staining assessed mucoid phenotype. Biofilm biomass and membrane permeability were measured by crystal violet staining and ALP leakage. qRT-PCR quantified biofilm/quorum-sensing gene expression. UHPLC-Orbitrap untargeted metabolomics coupled with multivariate analysis identified differential metabolites. Statistics were analyzed using GraphPad Prism and the R package ropls. RESULTS: 87.8% of the 41 mPA isolates were ceftazidime-susceptible. Mucoid EPS semi‑quantitative staining showed dihydroquercetin‑ceftazidime co‑treatment markedly repressed mucoid EPS, particularly in resistant mPA isolates. Over 90% of strains displayed synergistic (31.7%, FICI ≤0.5) or additive (61.0%, 0.5 < FICI ≤1.0) responses to the combination. Time-kill curves showed the combination achieved a ≥ 2 log₁₀ CFU/mL reduction and suppressed early bacterial growth at 4-8 h. Single drugs weakly inhibited biofilms, whereas co-treatment sharply lowered biofilm biomass (p < 0.01). Dihydroquercetin increased membrane permeability in a time-dependent manner, with stronger effects in the combination group. qRT-PCR revealed co-treatment significantly downregulated lasR, rhlR, pslA, pelA and fliC (p < 0.05). Metabolomics confirmed the combination triggered broader metabolic reprogramming, reducing virulence metabolites and biofilm precursors while elevating citric acid, a core TCA intermediate. KEGG enrichment highlighted perturbed valine/leucine/isoleucine biosynthesis, pyruvate metabolism and purine metabolism. CONCLUSION: Dihydroquercetin synergizes with CAZ against mPA by disrupting biofilm/membrane integrity, repressing quorum-sensing and biofilm-related genes, and remodeling bacterial metabolism. This combination can treat biofilm-associated mPA infections and validates dihydroquercetin as a plant-derived antibiotic adjuvant.