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

Abstract

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.