| Bacterial resistance has now become a worldwide problem that needs to be urgently solved which was accompanied by the irrational use of antibiotics.We have found that less and less new antibiotics developed and put into clinical usage and bacteria are becoming more resistant to different types of antibiotics,but the resistance mechanism is becoming more and more complicated.The emergence of severely resistant bacteria in the clinic forced us to reconsider the use of an ancient antibiotic of polymyxin.However,the emergence of plasmid carried mobile colistin resistance gene of mcr-1 astonished us because it caused polymyxin which as the “last line of defense” was breached.If a “superbug” carrying genes such as mcr-1 and new delhi metallo-?-lactamases-1(ndm-1)appears in the clinic,no effective antibiotics will be available,and the consequences will be unimaginable.It was reported that the polymyxin resistant gene of mcr-1 was located on plasmid in 2015.Since being reported for the first time,mcr-1 gene has been reported in more than 40 countries and regions around the world.It can be seen that mcr-1 has become a global epidemic.The full length of mcr-1 gene sequence was 1626 bp,and the ratio of guanine and cytosine was 49%.The results of amino acid sequence homology analysis showed that the similarity between mcr-1 gene and the gene of phosphoethanolamine transferase was about 60%.The transferase has the ability to transfer phosphoethanolamine groups(PEtN)to lipid A on the outer membrane lipopolysaccharide of bacteria,which changes the electronegativity of the outer membrane of bacteria.Polymyxin antibiotics cannot be bound to LPS therefor losing its bactericidal activity.The rapid and widespread of MCR-1 will lead to a further deepening of the resistance of bacteria to polymyxins,and the scope of resistance will further expand.In recent years,there have been more and more reports of drug-resistant enzyme inhibitors combined with antibiotics for the treatment of severe drug-resistant bacteria infections.As a new kind of adjuvant drug to resist the infection of drug-resistant bacteria,drug-resistant enzyme inhibitors have attracted more and more attentions of researchers.Evaluation of the synergistic effect of the inhibitor of drug-resistant enzyme in combination with antibiotics in vitro was a necessary prerequisite for clinical trials.In this study,we have screened several inhibitors of MCR-1 with potential synergistic effects from the natural compound library of our laboratory by an improved checkerboard method.Among them,genistein was used as the main research target to carry out the mechanism of confirmation.Genistein has been reported to have a variety of biological activities with less toxic and side effects,and good medicinal value.Up to now,no commercial genistein product has been put into clinical usage,and its research as an MCR-1 resistant enzyme inhibitor has not been reported so far.In this study,a checkerboard method MIC test,a time-killing assays,a growth curve,Western blot test and a drug Antimicrobial susceptibility test were used to verify the synergistic effect of the combination of genistein and polymyxin,and genistein can significantly improve the antibacterial activity of polymyxin against all mcr-1 positive test strains.It had no significant effect on the growth of different MCR-1 positive bacteria at 32 ?g/mL of Genistein.Western blot analysis confirmed that genistein did not increase the antibacterial activity of polymyxin by inhibiting the expression of MCR-1 resistant enzymes.In this study,the laboratory constructed strain E.coli W3110(pUC19-mcr-3)(MCR-1 mutant)was verified by the synergistic test of polymyxin and genistein,and it was also found that genistein can significantly improve the antibacterial activity of polymyxin against MCR-3 positive bacteria.In addition,It was also determined that genistein has no potential cytotoxicity to cells of various origins at a concentration of 32 ?g/mL of genistein through LDH detection tests and adhesion tests.Genistein enhances the protective effect of polymyxins on cell damage caused by bacteria.A mouse pneumonia model was established by using mcr-1 positive K.pneumoniae,and the different combinations of genistein and polymyxin E were used for treatments.It was determined whether genistein can enhance the protective effect of polymyxin E on mice by detecting the mortality,bacterial loading in target-organs,inflammation factors and pathological changes.The results showed that genistein(50 mg/kg)or polymyxin E(10 mg/kg)did not significantly increase the mortality of the mice,did not improve the degree of lung tissue lesions and did not decrease the number of colony colonies in lung tissues when compared with the infection group.The survival rate of mice was significantly improved after treated with genistein(50 mg/kg)in combination with polymyxin E(10 mg/kg).And the pathological changes have been greatly improved.Therefore,the combination of genistein and polymyxin has significant synergistic effects in vivo and the medicinal value of genistein needs to be further developed.In this study,Molecular Docking was used to explain the interaction and structure-activity relationship between genistein and MCR-1 resistant enzymes and confirmed several important binding sites of GLY-282,THR-283,TYR-287,ASN-482 and ARG-490 the binding sites between genistein and MCR-1.The simulation results were verified by amino acid residue mutations.Genistein bond to the vicinity of the active region of MCR-1,inhibited the function of MCR-1 thereby restoring the antibacterial activity of polymyxin against mcr-1 positive bacteria.In addition,in this study,the interaction between genistein and MCR-1 was further verified by a hydrophobicity test and an MPNP(modified rapid polymyxin Nordmann / Poirel,MPNP)detection test.In summary,genistein,as an inhibitor of MCR-1,can significantly improve the antibacterial activity of polymyxin antibiotics against Enterobacteriaceae carrying MCR-1 resistant enzymes in vitro / in vivo.And genistein was a new complementary drug for clinical application to provid protection against severely antibiotic-resistant Enterobacteriaceae infections. |
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