Molecular Mechanism About Reversing Colistin Resistance Of Niclosamide In Salmonella

Colistin(COL)are a class of cationic polypeptide antibiotics,which kill gram-negative pathogens(GNB)through disruption of membrane permeability via polar and hydrophobic interactions.Despite having serious nephrotoxicity and neurotoxicity,COL have become the last-resort treatment options for multidrug-resistant(MDR)and extensive drug resistance(XDR)bacterial infections.The overuse of antibiotics and the emergence and spread of superbugs carrying the mobile COL resistance gene(mcr)have become the most serious and urgent threat to healthcare and veterinary clinics.New therapeutic strategies are urgently required to combat MDR GNB.The combination of two or more drugs,especially antibacterial agents with non-antibacterial adjuvants is a promising approach,can slow the evolution of resistance,and can facilitate the use of lower doses of each drug,thus reducing side effects and adverse reactions.Niclosamide(NCL)exhibits no activity against most Gram-negative pathogens.Nevertheless,it was recently reported that in vitro coadministration of NCL and COL can overcome COL resistance in GNB.While these findings suggest that NCL may hold cryptic antibiotic potential,the molecular mechanism about reversing COL resistance has been hitherto unknown.This study combination of COL and NCL was used to demonstrate the synergistic reversal of clinical Salmonella resistance and the molecular mechanisms were investigated and analysed in depth,with a view to providing a scientific basis for the development of combination drug regimens for the clinical control of COL-resistant bacterial infection.In this study,we found that NCL has no antibacterial activity alone against Salmonella in vitro,and the MIC was 1024 μg/m L.NCL at 1 μg/m L,the COL MIC fold change ranged from 64 to 8192;NCL at 4μg/m L,COL MIC fold chang ranged from 128 to 65536.COL at 2 μg/m L,the NCL MIC decreased from 1024 μg/m L to0.25 μg/ m L ~ 0.00391 μg/m L.The combaintion COL and NCL can reduce MIC of Salmonella below the sensitive fold point,which indicates that NCL increases the antimicrobial activity of COL with specificity.The checkerboard assays showed that FICI of combination of COL and NCL for 19 clinical drug-resistant strains and 1standard strain was < 0.5,showing synergistic effect,and synergistic effect accounted for 100%.The results of time-kill curves also showed that compared with the COL monotherapy group,the number of colonies in the combined group was ≥ 2log10 CFU/m L,which further proved that the combination of the two drugs had a synergistic effect on salmonella.The emergence of resistance/serial passage assay indicates that after serial passages of 30 days compared with COL monotherapy group,the combination group could significantly delay the resistance of Salmonella.Forthermore,it was found that the dose-effect relationship between NCL and COL did not appear dose-dependent,and the dose-effect relationship curve showed a polyphase.When combined with other antibiotics fixed NCL at the same concentration,the MIC fold change was 2 fold,indicating that NCL had certain specificity for reversing the resistance of COL.Previous phenotypes proved that NCL could reverse the COL resistance of Salmonella,and this study continued to study the molecular mechanism of NCL reversing COL resistance of Salmonella from the perspective of membrane-related mechanisms,efflux pumps,oxidative damage and relative expression of mcr-1.The fluorescence assays results: the combined use of COL and low doses of NCL resulted in increasing fluorescence intensity of NPN and PI,which indicated COL and NCL combination could not only enhance outer membrane permeability and also disrupt the integrity of bacterial cell membranes of salmonella.The combined use of COL and NCL resulted in increasing fluorescence intensity of Et Br,inhibiting the efflux pump activity,increasing intracellular drug accumulation.When the efflux pump genes tol C and the global regulatory gene sox S were absent,NCL alone showed antibacterial activity against Salmonella,and the binding energy was calculated to be-3.77 and-3.39 kcal/mol by molecular docking simulating with the docking of Acr B and Tol C protein to NCL,indicating that both had good binding stability.The combined use of COL and NCL resulted in significantly decreasing fluorescence intensity of Di SC3(5)and ATP detection reagent luminescence intensity,indicating that the combination of the two drugs could lead to the dissipation of the trans-membrane proton gradient and the decrease of ATP level in bacteria,so that the activity of efflux pump was inhibited.The changes of reactive oxygen species(ROS)in cells were observed by DCFH-DA fluorescence staining.The results showed that the combination of the two drugs could induce a significant increase in intracellular ROS,and then accelerate the oxidative damage of the cell,leading to the death of the cell.The results of RT-PCR showed that the combination of COL and NCL could reduce the relative expression level of mcr-1 to reduce the resistance of Salmonella.To test the efficacy of the combination therapy in vivo a mouse infection model was established.The result showed that COL combined with NCL increased the survival rate of infected mice.The mortality rate of control group,COL and NCL monotherapy group reached 100%.Compared with COL monotherapy group,the survival rate of mice in 5 mg/kg NCL + 5 mg/kg COL group was the highest survival rate of 50%.In addition,the combined treatment of the two drugs could significantly reduce the bacterial load in the liver and spleen of mice.Compared with the COL monotherapy group,the bacterial load in the liver and spleen of mice in the 5 mg/kg NCL + 5 mg/kg COL group was the lowest and showed highly significant differences.The results showed that COL combined with NCL had obvious synergistic effect in vivo.In conclusion,NCL can reverse COL resistance in Salmonella both in vivo and in vitro.The molecular mechanism for reversing drug resistance is mainly through destroying the integrity of cell membrane,inhibiting efflux pump activity,inducing the production of reactive oxygen species and reducing the relative expression of mcr-1.Our results suggest that the combination of NCL and COL has the potential to treat COL resistant salmonella infections,and may provide a new combination therapy against COL resistant salmonella infections.