In Vitro And In Vivo Antibacterial Activity And Mechanism Study Of Colistin Combined With Auranofin Against Colistin-resistant Gram-negative Bacteria

Background:Infections caused by multidrug-resistant(MDR)Gram-negative bacteria are increasingly harder to treat with existing antimicrobial agents.Colistin is often used as the last line agent to treat these infections.However,with the discovery of plasmid-borne mcr-1 and the increasing clinical application of colistin,colistin resistant(Col-R)Gram-negative bacteria show a steady increasing trend in recent years.With practically no new antibiotics being introduced on the market,this highlights the need for effective combination therapies.A promising strategy is to take advantage of the potential synergistic effect of colistin with non-antimicrobial agents to restore the therapeutic effect of colistin.This study aimed to investigate the in vitro and in vivo antibacterial activity and mechanism of colistin combined with anti-rheumatoid arthritis drug auranofin against Col-R Gram-negative bacteria.Method:(1)Minimum inhibitory concentration(MIC)of 9 antibiotics against 66 clinical isolates of Col-R Gram-negative bacteria were determined by broth dilution method,and the existence of mcr-1 and mcr-8 in these strains was detected by PCR.The fractional inhibitory concentration index(FICI)of colistin and auronofin against 66 Col-R Gram-negative bacteria was determined by checkboard method.(2)Time-killing curve was used to further evaluate the antibacterial effect of colistin combined with auronofin against 8 selected Col-R strains.Col-R strains were cultured with subinhibitory concentration of colistin monotherapy or combined with auranofin for serial passages experiments,and the MIC values for colistin was recorded to evaluate whether the combination therapy could inhibit the progress of colistin resistance or not.Scanning electron microscopy(SEM)and transmission electron microscopy(TEM)were used to observe the impact of colistin and auranofin on the morphology of Col-R strains.(3)A mouse peritoneal infection model was established to evaluate the survival rate of mice after treatment of colistin or auranofin alone or their combination.The bacterial loads in the peritoneal fluid(PF)and spleen of mice were evaluated.The inflammatory factors and inflammatory cells in PF were recorded.Light microscope was used to observe Wright-Giemsa stained PF of mice.(4)Widely targeted metabolomics method was used to explore the synergistic mechanism of colistin combined with auranofin against Col-R Klebsiella pneumoniae.Results:(1)Among the 66 Col-R Gram-negative bacteria,68.18%(45/66)were MDR strains,12 strains were mcr-1 positive and 5 strains were mcr-8 positive.(2)The results of the checkerboard assays showed that the combination of colistin and auranofin had synergistic effect against 92.86%Col-R Klebsiella pneumoniae,81.25%Col-R Escherichia coli,100%Col-R Enterobacter cloacae,Pseudomonas aeruginosa and Acinetobacter baumannii.Time-killing curves showed that colistin combined with auranofin had synergistic effect against all 8 Col-R strains,however,regrowth was observed for Escherichia coli 08-85 and Pseudomonas aeruginosa 26587 after 24 h.Serial passage assays showed that the combination significantly inhibit the increase of colistin MIC compared with colistin alone.Electron microscopy showed that the outer membrane of Klebsiella pneumoniae 18605 was significantly destroyed by the combination.Bacterial cells of Acinetobacter baumannii 13660 treated with the combination were much shorter in length than that of monotherapy.(3)In the mouse peritoneal infection model,colistin combined with auranofin treatment significantly improved the survival rate.The bacteria loads in PF and spleen,inflammatory factors(IL-1?,IL-6,TNF-?)and the number of neutrophils in PF were significantly decreased in the combination therapy group compared with monotherapy group.Wright-Giemsa stained PF showed that the inflammation in the combination therapy group was significantly improved compared with monotherapy group.(4)Metabonomics analysis revealed that colistin alone and in combination with auranofin significantly reduced the level of phospholipids and fatty acid of cell membrane at 15 min and 1.The level of Uridine diphosphate-N-acetylglucosamine(UDP-GlcNAc),an amino sugar associated with the synthesis of peptidoglycan and lipopolysaccharide,was significantly reduced by the combination.In addition,the combination significantly inhibited glycolysis,the tricarboxylic acid cycle,amino acid,peptide and nucleotide metabolism.Conclusion:(1)A high proportion of MDR strains were found in 66 Col-R strains,and part of the 66 Col-R strains were mcr-1 or mcr-8 gene positive.(2)The combination of colistin with auranofin showed synergistic effect in vitro,and could inhibit the progress of colistin resistance.(3)Colistin combined with auranofin had a good therapeutic effect on the model of mouse peritoneal infection.(4)The synergistic mechanism of colistin and auranofin is firstly caused by the destruction of cell membrane driven by colistin and then by the inhibition of several key metabolic pathways driven by the combination.