Background: Bacterial infections cause a serious public health crisis due to the emergence of resistance towards multiple conventional antibacterial drugs. In particular, multidrug-resistant (MDR) Enterococcus faecium  which belongs to “ESKAPE” organisms is causing significant problems worldwide. Hence, there is an urgent need to find alternative therapies. Recently, substituted benzene guanidine compounds have been used as lead structures to discover new promising drugs in both synthetic and medicinal chemistry.
Purpose: Here we investigated the antimicrobial activity of a new substituted benzene guanidine analog, isopropoxy benzene guanidine, against Enterococci .
Material and methods: The isopropoxy benzene guanidine was synthesized by Guangzhou Insighter Biotechnology Co., Ltd and tested on both reference bacterial strain and 32 clinical MDR Enterococci  strains. The in vitro antibacterial activity was evaluated by microdilution method and kill kinetic assays. The potential antibacterial mechanism was measured by fluorescence spectrometry using fluorescent membrane potential probe 3, 3-diethyloxacarbocyanine iodide (DiOC₂ (3)).
Results: Isopropoxy benzene guanidine exhibited potent bactericidal activity against both reference strain and MDR Enterococci  isolates. The minimum inhibitory concentration (MIC) range for isopropoxy benzene guanidine was 1–4 μg/mL. Minimum bactericidal concentration (MBC) was about 2-8-fold of its MIC values. Time-kill studies showed that isopropoxy benzene guanidine provided superior bactericidal effect against reference and MDR strains within 12 hrs at 2×MIC. Furthermore, isopropoxy benzene guanidine could cause a large reduction in the magnitude of the generated membrane potential compared to that of the untreated cells.
Conclusion: The present study highlights the potent bactericidal activity of isopropoxy benzene guanidine on Enterococci  by disrupting the cell membrane potential. These findings demonstrate that isopropoxy benzene guanidine may be a good chemical lead for further medicinal chemistry and pharmaceutical development and could be used as a therapeutic agent for infectious diseases caused by MDR Enterococci .
Keywords: isopropoxy benzene guanidine, MDR Enterococci , DiOC₂ 3, cell membrane potential, bactericidal activity