| The discovery of the plasmid-mediated polymyxins resistance gene mcr-1 in multi-drug resistant Enterobacteriaceae bacteria has rapidly attracted the attention of governments and scientists in various countries around the world,which has led to a significant reduction in the role of polymyxin as "last line of defense" for the treatment of severely resistant Gram-negative Enterobacteriaceae infections.Therefore,there is an urgent need to solve the problem of infection of multidrug resistant bacteria carrying mcr-1 in clinic.The purpose of this study was to screen out a MCR-1 activity inhibitor through the MCR-1 resistance enzyme inhibitor screening platform established in the laboratory to restore the antibacterial activity of polymyxin against multidrug resistant Enterobacteriaceae bacteria,and we succeed screening a natural compound honokiol,which can significantly restore the antibacterial activity of polymyxin in vitro and in vivo.Through the checkerboard MIC test,it was found that 16 μg/mL honokiol can significantly restore the antibacterial effect of polymyxin on MCR-1-positive E.coli,Salmonella,and Klebsiella pneumoniae.When used ≥16 μg/mL honokiol,the FICI index were less than 0.5(0.09 ± 0.00 to 0.27 ± 0.06).In addition,honokiol and polymyxin also have a significant synergistic effect in MCR-1-negative polymyxin-resistant Gram-negative Enterobacteriaceae strains.The growth curve verified that honokiol had no significant influence on the growth of MCR-1-positive Enterobacteriaceae strains under the tested concentration.It was also found that when the concentration of honokiol was less than 32 μg/mL,there was no significant cytotoxic after incubation with MH-S cells for 24 h.Further time-killing curve and combined disk test confirmed that honokiol can obviously restore the bactericidal activity of polymyxin.In this study,Western-blot and molecular dynamics simulation were used to further confirm the inhibitory mechanism of honokiol against MCR-1.Western-blot results showed that co-culture with honokiol and MCR-1-positive bacteria for 4-6 h had no significant influence on the expression of MCR-1.The results of molecular dynamics simulation showed that honokiol was directly bind to the active region of MCR-1,thereby affecting the specific binding of MCR-1 to the substrate,resulting in the inhibition of its activity to transfer phosphoethanolamine.In addition,three different mouse infection models were established to determine whether honokiol can enhance the protective effect of polymyxin on tested mice.The results of the mouse systemic infection model test found that compared with the treatment of polymyxin alone,honokiol can increase the protective rate of polymyxin to the tested mice by 40%;in the mouse thigh muscle infection model,honokiol combined with polymyxin can significantly reduce the bacterial colonization of the thigh muscle;in the mouse pneumonia model,the combination therapy can significantly reduce the lung pathological damage in mice.This study further explored the extraction and purification process of honokiol from the natural compound Magnolia officinalis.Through the established extraction process,the ideal extract of honokiol was finally obtained.The MCR-1 inhibitory activity of the extract was tested,and the results showed that the extract and polymyxin also had a significant synergistic effect.In summary,this study determined that honokiol as a MCR-1 activity inhibitor can significantly improve the antibacterial activity of polymyxin in vivo and in vitro.By establishing a complete extraction process of honokiol,it was provided raw material guarantee to continue large-scale application of honokiol in clinical treatment of severe Gram-negative bacterial infections. |
