Restoring Multidrug-Resistant Escherichia coli Sensitivity to Ampicillin in Combination with (-)-Epigallocatechin...

Multidrug-resistant (MDR) bacteria, especially Escherichia coli, are a major contributor to healthcare-associated infections globally, posing significant treatment challenges. This study explores the efficacy of (-)-epigallocatechin gallate (EGCG), a natural constituent of green tea, in combination with ampicillin (AMP) to restore the effectiveness of AMP against 40 isolated MDR E. coli strains. Antimicrobial activity assays were conducted to determine the minimum inhibitory concentrations (MIC) of EGCG using the standard microdilution technique. Checkerboard assays were employed to assess the potential synergistic effects of EGCG combined with AMP. The pharmacodynamic effects of the combination were evaluated through time-kill assays. Outer membrane disruption was analyzed by measuring DNA and protein leakage and with assessments using N-phenyl-1-naphthylamine (NPN) and rhodamine 123 (Rh123) fluorescence dyes. Biofilm eradication studies involved biofilm formation assays and preformed biofilm biomass and viability assays. Scanning electron microscopy (SEM) was used to examine changes in cellular morphology. The results indicated that EGCG demonstrated activity against all isolates, with MICs ranging from 0.5 to 2 mg/mL, while AMP exhibited MIC values between 1.25 and 50 mg/mL. Importantly, the EGCG-AMP combination showed enhanced efficacy compared to either treatment alone, as indicated by a fractional inhibitory concentration index between 0.009 and 0.018. The most pronounced synergy was observed in 13 drug-resistant strains, where the MIC for EGCG dropped to 8 µg/mL (from 1 mg/mL alone) and that for AMP to 50 µg/mL (from 50 mg/mL alone), achieving a 125-fold and 1000-fold reduction, respectively. Time-kill assays revealed that the bactericidal effect of the EGCG-AMP combination occurred within 2 h. The mechanism of EGCG action includes the disruption of membrane permeability and biofilm eradication in a dose-dependent manner. SEM confirmed that the combination treatment consistently outperformed the individual treatments. This study underscores the potential of restoring AMP efficacy in combination with EGCG as a promising strategy for treating MDR E. coli infections.