Evaluation of phytochemical-mediated inhibition of the efflux pump in Pseudomonas aeruginosa PAO1.

BACKGROUND: Natural bioactive compounds derived from plants have gained considerable attention due to their potential therapeutic and medicinal applications. In the context of antimicrobial resistance, these compounds are being explored as potential efflux pump inhibitors (EPIs) and antibiotic adjuvants to enhance antimicrobial efficacy. Their low toxicity and broad availability make them promising candidates for the development of alternative therapeutic strategies against multidrug-resistant (MDR) pathogens such as Pseudomonas aeruginosa. METHODS: This study evaluated the efflux pump inhibitory activity of selected phytochemicals Mirificin, Forsythoside A, Salvianolic acid A, Epigallocatechin gallate, and Epicatechin gallate against Pseudomonas aeruginosa. A range of in vitro analyses were employed, including biofilm inhibition assays, fluorescence-based efflux assays (using Ethidium Bromide, N-phenyl-1-naphthylamine and Hoechst dye accumulation), advanced imaging techniques and qRT-PCR assays to assess their efflux pump inhibitory potential comprehensively. RESULTS: The results demonstrated a dose-dependent inhibition of biofilm formation, with more than a 75% reduction in biofilm biomass observed at higher treatment concentrations. The tested phytochemicals significantly enhanced the intracellular accumulation of both Ethidium Bromide and Hoechst dye, indicating effective inhibition of efflux pump activity. Among the tested compounds, Salvianolic acid A (at 100 µM) exhibited the highest inhibitory activity, achieving a level comparable to the positive control, PAβN. RT-PCR analysis confirmed this activity at the transcriptional level, showing significant downregulation of the efflux pump genes mexA, mexB, and oprM, along with the quorum sensing genes rhlI and rhlR, following treatment with Salvianolic acid A. CONCLUSIONS: Collectively, these results position Salvianolic acid A and the other phytochemicals evaluated here as promising adjuvant candidates for tackling multidrug-resistant Pseudomonas aeruginosa, and warrant their continued investigation as adjunctive therapeutic options alongside conventional antibiotics.