Synergistic Antibacterial Activity Of Silver With Conventional Antibiotics Correlating With The Effect On Thioredoxin System

Objective The thiol-dependent redox systems,including thioredoxin（Trx）and glutathione（GSH）system,have been recently found to be the novel promising antibacterial targets for multidrug-resistant（MDR）bacteria treatment.We previously showed that silver act synergistically with ebselen on the direct inhibition of Trx system,and fast depletion of GSH in Gram-negative bacteria.Moreover,silver has been found by others to improve the sensitivity of bacteria to certain conventional antibiotics.Therefore,this experiment wants to improve the ability of antibiotics to kill multi-drug-resistant bacteria by using the combination of Ag⁺and common classic antibiotics.This combination of methods can provide a new theoretical basis for improving the survival rate of ICU patients in the future.Methods（1）First,the sputum was collected form the hospitalized patients,especially the ICU patients,and the strains were separated by the method of coated plate.（2）The patient’s sputum is retained and inoculated in strict accordance with the current operating procedures of clinical test in China.Trained on the separation of bacteria by automatic bacterial identification analyzer for strain identification,K-B method was used for drug sensitivity test,and the results were determined by OSIRIS susceptibility testing instrument,according to the latest version of the clinical laboratory（CLSI）developed the drug sensitivity results judgment standard to judge the test results.（3）For clinical patients,especially ICU patients with the highest infection and fatality rate,the strains were cultured,isolated and preserved.（4）Antibacterial effect of Ag⁺in combination with antibiotics（96-well plate）.（5）The bactericidal effect of combined use of antibiotics and silver:determination of intracellular reactive oxygen species and H₂O₂ generation,measurement of Trx/Trx R activity and GSH amount in classic antibiotics and silver treated E.coli cell lysates,the redox status of bacterial thioredoxin treated with silver and antibiotics was examined by Western blotting.Results（1）Combination of silver with certain antibiotics（gentamicin,kanamycin,geneticin,and tetracycline）exhibited synergistic toxicity against E.coli.（2）Ag⁺and antibiotics in combination could induce high level of ROS（p<0.0001）.（3）Ag⁺and antibiotics could disrupt bacterial Trx system.（4）Ag⁺and antibiotics could not directly disrupt bacterial GSH system.（5）Clinically isolated five most difficult-to-treat MDR Gram-negative pathogens were not highly sensitive to Ag⁺and antibiotics in combinations.Conclusion（1）ROS is a lethal factor for synergistic bactericidal effects of Ag⁺and antibiotics（2）Silver enhanced the antibacterial effects of certain antibiotics against Gram-negative bacteria through directly target bacteria Trx system strategy.