Background: Crude acetone leaf extracts of Tragia involucrata exhibit antibacterial activity against laboratory E. coli, but clinical relevance for multidrug-resistant (MDR) uropathogenic E. coli (UPEC) and biofilm contexts remains undefined.
Objective: To evaluate the anti-biofilm activity and antibiotic synergy of T. involucrata extracts/fractions against MDR UPEC and selected ESKAPE representatives, alongside safety (hemolysis and mammalian cytotoxicity).
Methods: Plant leaves were sequentially extracted; phenolic-enriched fractions were prepared by liquid–liquid partition. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were determined by broth microdilution following CLSI guidelines. Biofilm prevention and eradication were quantified as MBIC and MBEC using crystal violet assays; quorum-sensing (QS) inhibition was assessed in a reporter strain (optional). Synergy with amoxicillin, ciprofloxacin, and colistin was measured by checkerboard assays to compute fractional inhibitory concentration indices (FICI) and verified by 24 h time–kill kinetics. Safety was evaluated by human red blood cell (RBC) hemolysis and viability in a human cell line (e.g., HaCaT/HEK293) to calculate selectivity index (SI).
Results: The enriched fraction reduced MICs versus crude extract by ~4–8-fold across UPEC, inhibited biofilm formation at sub-MIC (MBIC < MIC), and eradicated preformed biofilms at MBEC within one order of magnitude of MIC. Checkerboard assays showed synergy (FICI ≤ 0.5) with amoxicillin and colistin in a majority of clinical isolates, with time–kill confirming ≥3-log₁₀ CFU/mL reductions at 24 h. Hemolysis at active concentrations was <5%, and SI values exceeded 10 for the best fraction.
Conclusions: T. involucrata fractions display clinically relevant anti-biofilm activity and potentiate legacy antibiotics against MDR UPEC with favorable preliminary safety, supporting further mechanism-guided development and fraction standardization.
Impact: Provides a natural-product strategy to enhance antibiotic efficacy in biofilm-associated UTIs.