金诺芬与伏立诺他联合对革兰阴性菌抗菌活性的影响及金诺芬作用位点的研究

Effect of auranofin combined with antineoplastic vorinostat on bactericidal activities against gram-negative bacteria and study on the target of auranofin

目的明确金诺芬对革兰阴性菌抗菌活性的作用靶点,探究金诺芬与伏立诺他联合对革兰阴性菌抗菌活性的影响。方法采用缺失硫氧还蛋白还原酶(thioredoxin reductase,TrxR)菌株明确靶点基因,通过药物敏感试验,棋盘微量稀释法,以及时间-杀菌动力学试验来观察金诺芬与伏立诺他的联合对大肠埃希菌、鲍曼不动杆菌、肺炎克雷伯菌、铜绿假单胞菌和多耐药鲍曼不动杆菌是否具有协同作用。分别观察大肠埃希菌BW25113 (wild type,WT)菌株敲除表达Trx的相关基因(trxA、trxB、trxC),表达谷胱甘肽(glutathione, GSH)的基因(gor),对金诺芬药物的敏感结果,以及金诺芬对ΔtrxC、Δgor的时间-杀菌动力学影响,并使用回补菌株(C-trxA、C-trxB、C-trxC、C-gor)来验证,明确基因目标。结果药物敏感试验结果显示,金诺芬对大肠埃希菌BW25113和肺炎克雷伯菌ATCC 43816菌株的最低抑菌浓度(minimum inhibitory concentration,MIC)均为64 mg/L,对铜绿假单胞菌PA14的MIC为128 mg/L,对鲍曼不动杆菌ATCC 17978和AB5075的MIC为32 mg/L;伏立诺他对所有菌株的MIC均为512 mg/L。金诺芬联合伏立诺他对大肠埃希菌BW25113、鲍曼不动杆菌ATCC 17978、多耐药鲍曼不动杆菌AB5075、肺炎克雷伯杆菌ATCC 43816、铜绿假单胞菌PA14的分级抑菌浓度指数(fractional inhibitory concentration index,FICI)分别约为0.313、0.375、0.375、0.375和0.375,所有值均<0.5,表现为协同作用。在缺乏不同基因的敲除菌株药物敏感结果中显示,金诺芬对ΔtrxC的MIC由64 mg/L上升至256 mg/L,对Δgor的MIC下降至16 mg/L,对ΔtrxA、ΔtrxB的MIC未发生改变。金诺芬对回补菌株(C-trxC, C-gor)与对大肠埃希菌BW25113表现出相同的抗菌活性,细菌数量减少约1.8 lg菌落形成单位(colong formins units, CFU)/mL;而金诺芬对ΔtrxC的抗菌活性明显降低,细菌数量减少< 1 lg CFU/mL;在Δgor中,细菌数量减少4.6 lg CFU/mL,抗菌活性明显增加。结论金诺芬对抗革兰阴性菌的潜在靶点基因可能是trxC,为临床上治疗多耐药的革兰阴性菌提供新的思路和途径。

Objective To find the target of auranofin with the antibacterial activity against gram-negative bacteria and to investigate the effect of the combination of auranofin and vorinostat on the antibacterial activity against gram-negative bacteria. Methods The strains of E. coli lacking thioredoxin reductase (TrxR) was used to find the target gene. The potential synergies of the combination of auranofin and vorinostat for E. coli strain, A. baumannii strain, P .aeruginosa strain, K. pneumonia strain and multidrug-resistant (MDR) A. baumannii strain were evaluated using susceptibility tests, micro-dilution checkerboard tests and time-kill studies. The genes related to Trx (trxA, trxB, trxC) and the gene expressed glutathione (gor) of E. coli BW25113 strains (WT) were separately knocked out to observe the effect of auranofin on minimum inhibitory concentration (MIC) and the time-kill kinetics of ΔtrxC and Δgor. Furthermore, the complemented strains (C-trxA, C-trxB, C-trxC, C-gor) were used to verify and define the genetic targets. Results According to the results of susceptibility tests, MICs of auranofin were 64 mg/L for E. coli strain BW25113 and K. pneumonia strain ATCC 43816, 128 mg/L for P. aeruginosa strain PA14 and 32 mg/L for both A. baumannii strain ATCC 17978 and A. baumannii strain AB5075. However, MICs of vorinostat are 512 mg/L for all isolates. The fractional inhibitory concentration indexes (FICIs) of the combination of auranofin and vorinostat for E. coli strain BW25113, A. baumannii strain ATCC 17978, MDR A. baumannii strain AB5075, K. pneumonia starin ATCC 43816 and P. aeruginosa strain PA14 were 0.313, 0.375, 0.375, 0.375, and 0.375, respectively, with all values <0.5, which showed synergy. In susceptibility tests of knockout strains, MICs of auranofin for ΔtrxC increased from 64 mg/L to 256 mg/L, decreased to 16 mg/L for Δgor, and no changes for ΔtrxA and ΔtrxB. Auranofin showed the same antibacterial activities against the complemented strains (C-trxC, C-gor) and E. col BW25113, which decreased by about 1.8 lg colong formins units (CFU)/mL of bacterial counts. However, the antibacterial activity of auranofin was significantly reduced for ΔtrxC, and decreased by<1 lg CFU/mL of bacterial counts. For Δgor, bacterial counts decreased 4.6 lg CFU/mL, and the antibacterial activity markedly increased. Conclusions The potential target gene of auranofin against gram-negative bacteria could be trxC, which provides new ideas and methods for the clinical treatment of multidrug-resistant gram-negative bacteria.