Gallium Nitrate Enhances Antimicrobial Activity Of Colistin Against Klebsiella Pneumoniae By Inducing ROS Accumulation

Klebsiella pneumoniae,as being a pathogen of critical clinical concern,urgently demands effective therapeutic options due to its drug resistance.The polymyxins are increasingly regarded as a last-line therapeutic option for the treatment of multidrug-resistant（MDR）Gram-negative bacterial infections.However,polymyxin resistance in K.pneumoniae has become an emerging problem.ObjectiveTo explore the effect of polymyxin on the antibacterial activity of K.pneumoniae in vitro and in vivo models in the presence of gallium nitrate（Ga（NO₃）₃）,and the molecular mechanism of the enhancement of polymyxin activity by gallium nitrate.MethodsIn this study,more than 1200 strains of K.pneumoniae collected from Anhui Province Bacterial Resistance Monitoring Center from 2017 to 2020 were re-identified using time-of-flight mass spectrometry system MALDI-TOF MS,and then AGAR method was used to measure the minimum inhibitory concentration（MIC）of 24 drugs of K.pneumoniae.The distribution and drug resistance rate of K.pneumoniae in Anhui Province were statistically analyzed by WHONET 5.6 analysis software recommended by WHO.Ten colistin resistant strains and 10 colistin sensitive strains of multidrug resistant K.pneumoniae were randomly selected as the study models.The combined effects of gallium nitrate and colistin were studied by in vitro dilution point plate test,short killing test and growth curve inhibition test.A mouse model of lung infection was used for in vivo experiments.Combined with RNA-seq and reactive oxygen species（ROS）detection,the potential mechanism of gallium nitrate inhibiting the bactericidal action of antibacterial drugs against Gram-negative bacteria was investigated.ResultsThe results showed Ga（NO₃）₃could significantly increase the antimicrobial activity of colistin,especially in iron-limiting medium.Mechanistic studies demonstrate that bacterial antioxidant activity was repressed by Ga（NO₃）₃as revealed by RNA-seq,leading to intracellular accumulation of reactive oxygen species（ROS）in K.pneumoniae,which is enhanced in the presence of colistin.Therefore,oxidative stress induced by Ga（NO₃）₃and colistin augments colistin-mediated killing of wild-type cells,which can be abolished by DMSO,an effective ROS scavenger.ConclusionsOur study indicated that the Ga（NO₃）₃had a notable impact on antimicrobial activity of colistin against K.pneumoniae,revealing the potential of Ga（NO₃）₃as a novel colistin adjuvant to improve the treatment outcome of bacterial infections.