Molecular Mechanism Of Reversing Synergistically Resistance Of Salmonella To Colistin By The Combination Of Tetrandrine And EDTA

Colistin(COL)is a cationic polypeptide antibacterial drug,which is the last line of defense for the treatment of multidrug-resistant(MDR)bacteria,especially carbapenem-resistant Enterobacteriaceae infections.With the growth of colistin use,bacterial colistin resistance is increasing worldwide and has now become the focus of global attention.It is extremely difficult to develop new antibacterial drugs with new targets of action,and combination drugs to reverse resistance are cost-effective strategies to combat MDR bacteria.Combination therapy can take advantage of the synergistic effect between drugs,greatly improve the antibacterial efficacy,overcome bacterial resistance,and slow down the development of drug resistance.In this study,tetrandrine(TET),a traditional Chinese medicine active monomer with the function of inhibiting efflux pump,and EDTA,a metal chelator,were used in combination with colistin to observe their synergistic reversal of colistin resistance in Salmonella,and to study its molecular mechanism,in order to provide a scientific basis for clinical combination drug control of drug-resistant Salmonella infection.In vitro combined drug susceptibility test found that 1/4×MIC TET could reduce the minimum inhibitory concentration(MIC)of COL to isolated Salmonella from0.25~4 μg/m L to 0.008~4 μg/m L,the colistin MIC fold change ranging from 1to≥256,1/4×MIC EDTA can reduce the MIC of COL to isolated Salmonella from0.25~4 μg/m L to 0.008~2 μg/m L,the colistin MIC fold change ranging from 2 to128,and make 83.33% and 77.78% of Salmonella were reversed from colistin resistance to sensitivity,respectively.The reversal rate of 1/4×MIC TET+1/4×MIC EDTA+COL was as high as 100%,and the colistin MIC fold change ranging from 4 to 2048,which was much larger than the sum of the reduction multiple when TET and EDTA were combined with COL alone,showing a significant synergistic reversal effect.The results of the checkerboard test showed that the average FIC values of TET+COL and EDTA+COL against drug-resistant strains were 0.50 and 0.47,respectively,showing synergistic antibacterial effects.The time-kill curve measurement showed that the combination of 1/4×MIC TET+1/4 MIC EDTA+1/2×MIC COL directly killed bacteria in 4 hours,which was 8.39 log10(CFU/m L)lower than that of COL single drug group.By constructing a mouse intraperitoneal infection model for in vivo verification,the results showed that the combination of TET +EDTA+COL can significantly reduce the number of bacteria in the spleen and liver of mice by 3.25 and2.76 log10(CFU/g),respectively,indicating that the combination also has a synergistic effect in vivo.In this study,the molecular mechanism of the synergistic reversal effect of TET+ EDTA + COL on mcr-1 positive Salmonella was mainly studied from the aspects of cell membrane permeability,efflux pump activity and oxidative damage.The results showed that the fluorescence intensity of the fluorescent probe NPN was significantly enhanced when TET + EDTA + COL was combined,while the fluorescence intensity of the fluorescent probe PI did not change significantly,indicating that their combined effect led to a significant increase in the permeability of the outer cell membrane,and does not affect the stability of the intracellular membrane.At the same time,the scanning electron microscope images directly and vividly confirmed the damage effect of TET and EDTA combined with COL on bacterial outer membrane.It was detected by the fluorescent probe method that the combination of TET+EDTA+COL could cause the fluorescence intensity of the fluorescent probes Di SC3(5)and BCECF-AM to increase,indicating that their combination can not only destroy the transmembrane potential,but also destroy the p H value gradient,and thus completely destroy the proton motive force of Salmonella.The results of the efflux pump activity detection showed that the fluorescence intensity of the fluorescent probe Et Br in the TET+COL and TET+EDTA+COL groups was significantly increased,while the fluorescence intensity of the EDTA+COL group had no significant change,indicating that the presence of TET could significantly inhibit the activity of bacterial efflux pump.The changes of reactive oxygen species(ROS)in the bacterial cells were observed by DCFH-DA fluorescence staining,and the results showed that their combination could induce a substantial increase in intracellular ROS,which in turn accelerated the oxidative damage of the bacterial cells,resulting in bacterial cell death.In this study,RT-PCR technology was used to detect the effect of TET + EDTA +COL combination on the transcription level of colistin resistance gene mcr-1 and efflux pump related genes acr A,acr B,tol C and the positive regulator gene mar A.The results showed that their combination decreased the m RNA relative expression level of mcr-1 gene by 3.58 times,and the m RNA relative expression level of efflux pump related genes acr A,acr B,tol C and mar A by 2.50,5.79,1.85 and 3.40 times,respectively.The interaction mechanism between TET and MCR-1 protein was elucidated by molecular docking,and the binding site between TET and MCR-1 was confirmed.Among them,the key amino acid residues that mediate the binding of MCR-1 to TET were Tyr476,Leu419 and Ala420.TET directly binds to the active region of MCR-1through hydrogen bonds,resulting in changes in protein conformation,affecting the specific binding of MCR-1 to substrates,and thus inhibiting the biological activity of MCR-1.For the first time,tetrandrine was found to act as an inhibitor of MCR-1.At the same time,molecular docking between TET and MCR-1 mutant proteins(MCR-1.2,MCR-1.3 and MCR-1.6)was also performed,and the binding free energies were-7.82,-7.02 and-7.23 kcal/mol,respectively,indicating that TET and MCR-1 mutant protein also had good binding stability.In conclusion,TET and EDTA combined with COL can synergistically reverse Salmonella colistin resistance in vitro and in vivo.The molecular mechanism of synergistic reversal of Salmonella colistin resistance by TET combined with EDTA is mainly to enhance outer membrane permeability,inhibit bacterial efflux pump activity,promote oxidative damage and down-regulate mcr-1 and efflux pump Acr AB-Tol C expression of related genes.Our findings demonstrate that TET and EDTA,as novel colistin adjuvants,highlight the great potential of non-antibiotic drugs against colistinresistant Salmonella,and provide a basis for the development of a combined treatment regimen for colistin-resistant Salmonella infections.