Old drugs learn new tricks – repurposing phenothiazines to uncover effective antimicrobial

Thioridazine (TZ) is an antipsychotic drug that acts against antibiotic resistant bacteria. The main aim of this study was to uncover the mechanism of action of TZ using Salmonella enterica serovar Thypimurium as a model bacterium. The antibacterial activity of TZ was initially determined based on its minimum inhibitory concentration (MIC). Membrane permeability assays were performed and fluorescence measured using the Ethidium Bromide accumulation assay. Salmonella was exposed to TZ and its effects on membrane potential and cell wall assessed by flow cytometry and Transmission Electron Microscopy, respectively. Effects on the bacterial proteome were assessed through 2D gel electrophoresis. Infection assasys were performed in THP-1 ad RAW 264.7 cells treated and non-treated with TZ. The MIC of TZ against Salmonella was 200 mg l−1. Our in vitro data demonstrates that TZ mechanism(s) of action involves primarily Salmonella’s membrane by affecting its permeability and potential after 15 min of exposure to TZ. At half of the MIC, and only after 15 min, TZ disrupts the bacterial membrane leading to leakage of the cellular contents and lysis of Salmonella. Proteomic profiling revealed 75 upregulated and 62 downreuglated proteins. Infected macrophages treated with sub-MIC of TZ, showed a reduction on intracellular c.f.u./mL. This may be indicative of TZ’s ability to enhance the killing activity of infected macrophages. The results obtained suggest that TZ may act in vitro by targeting the bacterial cell-envelope. Due to its effect on infected macrophages, TZ may be considered a useful adjuvant to current therapeutics.