Repurposing Non-Antibiotic Drugs Auranofin and Pentamidine in Combination to Combat Multidrug-Resistant Gram-Negative Bacteria

Infections caused by multidrug-resistant (MDR) bacteria, especially MDR Gram-negative bacteria, have posed a great challenge to healthcare systems globally. To address the shortage of effective antibiotics against MDR Gram-negative bacterial infections, two non-antibiotic drugs - auranofin (rheumatoid arthritis drug) and pentamidine (antiprotozoal drug) - are being repurposed to treat MDR Gram-negative bacteria by a combination approach.Chequerboard microdilution assay was used to determine the interaction of auranofin and pentamidine against drug-susceptible and MDR Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii and Klebsiella pneumoniae). Fluorescence microscopy, scanning electron microscopy and inductively coupled plasma mass spectrometry were used to explore the mechanism of synergistic antibacterial effect.These two non-antibiotic drugs displayed a strong synergistic antibacterial effect, with the fraction inhibitory concentration index ranging 0.094-0.506. The MIC of auranofin reduced by as much as ≥ 1024-fold when combined with pentamidine at sub-MIC. Fluorescence and inductively coupled plasma mass spectrometry analyses revealed that bacterial membrane disruption caused by pentamidine treatment at sub-MIC led to an increased intracellular auranofin content with the combination treatment. The enhanced auranofin uptake in bacteria resulted in efficient bacterial killing. More importantly, the auranofin/pentamidine combination slowed down auranofin resistance development in clinically isolated MDR bacteria (Klebsiella pneumoniae) more than the combination of auranofin and colistin, which is a last-line antibiotic with a membrane-lytic antibacterial mechanism.The combination of non-antibiotic drugs with complementary antibacterial mechanisms provides a potentially promising approach to discover new antibacterial drugs and delay drug resistance development.