Real-time Activity Monitoring And Inhibition Of Metallo-?-lactamase And Drug-resistant Bacteria

Drug resistance caused by overdose and irregular use of antibiotics has been a serious threat to public health and a major concern to the world health organization.One of the main mechanisms of bacterial resistance is the production of Metallo-?-lactamases(M?Ls).Therefore,it has great application value to real-time monitor the active and inhibition of M?Ls.In this paper,the following four parts are carried out from the aspects of M?Ls activity monitoring,inhibition analysis,structure analysis and so on.A simple and non-destructive UV-Vis method was developed for the real-time monitoring the inhibition of M?Ls activity in vivo of resistant bacteria,and its wide applicability was demonstrated by the selection of multiple antibiotics and clinical bacteria for monitoring.The IC₅₀ values of the two known inhibitors of EDTA and ATAA for L1 in vivo were 1.6and 18.9?M,respectively.The inhibitory data obtained from the UV-Vis real-time monitor are more consistent with their real biological functions in the natural environment.Through the real-time monitoring of imipenem hydrolyzed by ImiS,it was found that the hydrolysate of imipenem could inhibit ImiS,and then two components P1 and P2 were obtained from separating the hydrolysate.Kinetic assays revealed that P1 exhibited a broad-spectrum inhibition against VIM-2,NDM-1,ImiS,and L1 with IC₅₀ values of 8–32?M,however,P2 showed no inhibition.Additionally,P1 showed synergistic antibacterial efficacy against drug-resistant E.coli producing VIM-2,NDM-1,ImiS,and L1,in combination with antibiotics,restoring 16–32-fold and 32–128-fold the efficacies of imipenem and cefazolin,respectively.In addition,our study shows that the inhibition of ImiS by imipenem hydrolysate is due to the component P1.This work provides a new way to study the hydrolysis mechanism of antibiotics and the inhibition of M?Ls.In order to avoid the influence of diamagnetism and colorlessness on spectroscopic characterization of Zn(?)ions in natural M?Ls,the spectroscopic study of Co(?)ion-substituted M?Ls has been carried out for many years,but it can not be determined whether or not EDTA can be eliminated during the preparation of apo-M?Ls,which has a great influence on the results of subsequent studies.We developed an ITC method to monitor the content of EDTA in the preparation of apo-M?Ls.Thermodynamic signals were distinguished by using the different affinity of Zn(?)ion with EDTA and apo-M?Ls(preferential binding to EDTA).The three proteins of L1,NDM-1 and ImiS were monitored for the preparation of apo-M?Ls,respectively.The ITC method was able to accurately identify the presence of EDTA or not.This method can provide more accurate thermodynamic information for the study of the properties and mechanism of M?Ls based on the advantages of wide range of measurement,high sensitivity and accuracy.NMR is the only method which can be used to determine the three-dimensional structure of biomolecules in solution at atomic resolution in the macromolecular structure characterization technique.It is an important means of structural characterization to evaluate the interaction of proteins with small molecules by ¹³C and ¹⁵N labeled.The purified ¹³C,¹⁵N double labeled ImiS and L1 were obtained in this part.The changes of HSQC spectra after the action of different concentrations of inhibitor YSK-1a and ImiS were compared,and it was determined that there was a concentration-dependent irreversible binding between YSK-1a and ImiS.