The Reversal Mechanism Of Closantel On Colistin Resistance In Escherichia Coli

Colistin(COL)is a cationic peptide antibacterial drug that is a last resort for the treatment of multi-drug resistant(MDR)negative bacterial infections.With the increasing use of COL,the problem of COL resistance in bacteria has become imminent and poses a threat to human health.In recent years,the development of antimicrobial drugs with new targets of action has been difficult.Compared with new antibacterial development,the search for antibiotic adjuvants in combination with antimicrobial drugs is a more cost-effective strategy.The anthelmintic closantel(CST),an oxidative phosphorylation uncoupling agent,acts mainly on the worm mitochondria,inhibiting the mitochondrial phosphorylation process,and can be used as a potential COL adjuvant.In this study,the reversal effect and the quantitative-effect relationship closantel on colistin resistance in E.coli were confirmed,and the molecular mechanism of reversal of colistin resistance by CST was investigated to provide a scientific basis for the clinical treatment of colistin-resistant(COL-R)E.coli infection.The antimicrobial activity of CST alone was poor,while 1 or 4 μg/mL of CST in combination with COL reduced the MIC of COL by 2 to 32 times and converted92.86 % of E.coli from resistance to sensitivity.The results of the chessboard drug sensitivity test showed that the combination of CST and COL had synergistic effects on COL-R strains with FIC values of 0.125～0.252,and showed additive effects on COL-S strains with FIC values of 0.500.Meanwhile,the minimum reversal concentration(MRC)of CST was 0.5～4 μg/mL when 2 μg/mL COL was present,which was 256～4096-fold lower compared with the MIC of CST alone,showing good synergistic effects.The reversal effect of CST on COL-R E.coli showed a unique quantitative-effect relationship.Within a certain range,the effect became stronger as the CST concentration increased,and after reaching a certain level,the effect remained unchanged or even diminished.At certain concentrations,closantel only synergized with colistin and did not show synergistic effects with other antibiotics.The results of time-kill curves showed that 1 or 4 μg/mL of CST in combination with 1/2×MIC COL had a synergistic effect on COL-R E.coli but not on COL-S E.coli within 24 h,which was also consistent with the results of the drug sensitivity test.Resistance development studies showed that 1 or 4 μg/mL of CST in combination with COL for 30 days of continuous passaging resulted in a small or no increase in MIC compared to COL alone,which could effectively inhibit bacterial resistance development.We further constructed a mouse intraperitoneal infection model for in vivo testing,and the results showed that the combination of CST and COL could significantly improve the survival rate of mice,as well as significantly reduce the bacterial load in the liver and spleen of mice,and the combination of CST and COL exhibited good therapeutic effects in vivo.The molecular mechanism of reversal of colistin resistance by closantel and its quantitative-effect relationship were further investigated in this study in terms of cell membrane permeability,efflux pump related functions and oxidative damage.The results of the fluorescence test showed that the combination of CST and COL could significantly enhance the fluorescence intensity of NPN,while there was no significant change in the fluorescence intensity of PI,which indicated that the combination of the two drugs could damage the outer cell membrane and increase the permeability of the outer membrane,but had no effect on the inner cell membrane.Meanwhile,the combination of the two drugs could significantly decrease the fluorescence intensity of the fluorescent probe DiSC3(5),indicating that the combination of CST and COL could disrupt the proton motive force(PMF)mainly by interfering with the pH gradient.The efflux pump activity assay revealed that the combination of CST and COL resulted in a significant increase in the fluorescence intensity of the fluorescent probe Et Br,and the quantitative-effect relationship was the same as the phenotypic results,indicating that the combination of the two drugs could effectively inhibit the efflux pump activity and also be the main molecular mechanism of reversal of COL resistance by CST.Meanwhile,the dissipation of PMF further reduced ATP synthesis,but the combination of CST with COL at high doses resulted in an increase in ATP synthesis,the process that may be related to changes in the respiratory chain.Oxidative damage is also an important pathway of antibiotic action,and fluorescence staining of DCFH-DA can be used to observe the changes of ROS.The combination of CST and COL can induce ROS overproduction,and the quantitative-effect relationship is the same as the phenotypic results,indicating that the combination of the two drugs can cause oxidative damage,which in turn causes cell death,and also is the main molecular mechanism of CST to reverse COL resistance.The effect of CST in combination with COL on plasmid-mediated expression of colistin resistance gene mcr-1 was examined using RT-PCR.The combination of the two drugs reduced the relative mRNA expression of mcr-1 by 4.26-fold,indicating that the combination of the two drugs could significantly reduce the relative expression of mcr-1.Meanwhile,computer simulated molecular docking further explored the interaction relationship between CST and MCR-1 protein.The results revealed that CST interacted with Lys341 and Asp337,the key amino acid residues on MCR-1,through hydrogen bonding,affecting the altered spacial conformation of MCR-1 protein and inhibiting the biological activity of MCR-1 protein.In summary,CST can reverse COL resistance in E.coli with a unique quantitative-effect relationship.The combination of the two drugs can reverse COL resistance in E.coli through the combined effects of damage to the outer membrane,inhibition of efflux pump activity and induction of oxidative damage.Combining the molecular mechanism with the phenotypic quantitative-effect relationship,we found that inhibition of efflux pump activity and induction of oxidative damage are the main molecular mechanisms reverse COL resistance by CST.Also,the combination of CST and COL also down-regulated the relative expression of mcr-1,and CST could directly interact with MCR-1 protein.Overall,CST is an effective adjuvant for colistin that provides a new therapeutic strategy for the clinical treatment of COL-R E.coli infection and also reveals the great potential of non-antibiotic drugs in combination with antibiotics to combat bacterial resistance.