The Effect Of Auranofin And Polymyxin Combined With Vorinostat Against Multidrug-resistant Gram-negative Bacteria And The Resistance Mechanism Of Auranofin

Objective 1.Investigate the combination of auranofin and the antineoplastic vorinostat for gram-negative bacteria in vitro.2.Study the combination of polymyxin B and vorinostat against MDR Gram-negative bacteria in vitro and in vivo.3.Find the mutation site of the target gene trx B in auranofin-resistant Staphylococcus aureus strains,which were induced and screened.Materials and Methods 1.For five Gram-negative bacteria,the results of the susceptibility testing and the checkerboard dilution method were used to observe the synergistic effect in the combination of auranofin and vorinostat.2.Four Gram-negative bacteria and two MDR isolates were selected,the results of the susceptibility testing and the checkerboard dilution method were used to observe the synergistic effect in the combination of polymyxin and vorinostat.3.In vitro through the time-killing kinetic test to observe antibacterial activity in two combinations(ARF and SAHA,PMNB and SAHA),compared to the monotherapy.4.SEM was used for detecting the cell morphologic changes of A.baumannii strain in the combination of PMNB and SAHA.5.To measure intracellular ROS generation,the fluorescent probe carboxy-H2DCFDAwas used for observing in the combination of PMNB and SAHA.Added an effective radical scavenger and used to study antibacterial effects in combination.6.The G.mellonella infection model was built to evaluate the antibacterial activity of polymyxin B combined with vorinostat in vivo.7.In the high concentration of auranofin environment,S.aureus RN450 resistant to auranofin was induced,and the change of MIC was confirmed by the susceptibility testing.Multiple passages in auranofin-resistant strains were used to observe whether the resistance of auranofin was stable.8.Through extracting the bacterial genome,primer design,PCR amplification of the target gene,purification of PCR production,high-throughput sequencing,the nucleotide sequence of the target gene is aligned to analysis and clarify the mutation site leading to drug resistance.Results 1.The combination of auranofin and vorinostat,as well as the other combination of polymyxin and vorinostat all showed good synergistic effects for Escherichia coli,Acinetobacter baumannii,Klebsiella pneumoniae,and Pseudomonas aeruginosa in vitro.2.In vitro through the time-killing kinetic test,a significantly increase in antibacterial activity was observed compared to the monotherapy in all the combinations.3.Cellular morphology study showed the change of membrane and disruption of the integrity in the combination of PMNB and SAHA.4.ROS assay showed significantly more oxidative stress in combination group than in polymyxin B and vorinostat monotherapy groups.5.In animal models,the combination of PMNB and SAHA group showed a higher survival rate than that of monotherapy groups.6.The MICs of five auranofin-resistant S.aureus RN450 strains were significantly increased.Through subculture,the auranofin resistance did not disappear,and the MIC results remained consistent.7.We first found two different point mutations in trx B gene from AR strains.A single point mutation was analyzed in the nucleotide sequence from G to T at the 13 th position,resulting in the change in amino and the other point mutation from G to A at nucleotide 139.Conclusions 1.Auranofin combined with SAHA showed good antibacterial activity against gram-negative bacteria,and provided a theoretical basis for further study of the in vivo model and preparation of later clinical trials.2.Polymyxin B combined with SAHA showed good antibacterial activity against MDR Gram-negative bacteria,and provided a theoretical basis for further study of the mice model and preparation of later clinical trials.3.We supposed that polymyxin B destroys the integrity of the outer membrane,letting vorinostat into the bacterial cell,which causes oxidative stress and enhances the generation of ROS.4.Two point mutations,respectively changing the aspartic acid at the 5th amino acid to tyrosine,and the glutamic acid at the 47 th amino acid to lysine,which led to the loss of negative charges.