银离子对细菌整合子捕获耐药性基因盒频率的抑制作用

Inhibition of silver ion on the frequency of bacterial integron trapping drug resistance gene cassette

目的讨论银离子对大肠埃希菌BL21(DE3)宿主中第一类整合子整合频率的影响。方法将pUCINT及pACINAD两种重组质粒依次转化大肠埃希菌BL21(DE3),命名为HS2。实验组分别使用含0.3μg/ml、0.6μg/ml和0.8μg/ml银离子LB液体培养基,对照组使用普通LB液体培养基,银离子通过硝酸银提供,培养条件为37℃培养24 h。菌株培养完成后均用实时聚合酶链反应(qPCR)测定发生重组反应的整合子拷贝数和总的整合子拷贝数,二者比值即为整合频率。同时通过3次独立表型筛选法分析整合频率变化并利用质谱分析菌株蛋白质表达情况。结果qPCR测定的对照组HS2菌株的整合频率为1.79×10^(-5)(1.44×10^(-5),3.13×10^(-5)),实验组中0.3μg/ml银离子组整合频率为2.07×10^(-5)(1.49×10^(-5),2.67×10^(-5)),0.6μg/ml银离子组为2.25×10^(-6)(1.47×10^(-6),4.54×10^(-6)),0.8μg/ml银离子组为1.69×10^(-6)(0.22×10^(-6),3.08×10^(-6))。实验组中0.6μg/ml银离子组与0.8μg/ml银离子组整合频率明显低于对照组,差异具有统计学意义(P<0.01),但0.3μg/ml银离子组整合频率与对照组比较差异无统计学意义。3次独立表型筛选法测定结果与qPCR结果一致且质谱分析得出经银离子作用的菌株蛋白质表达存在差异。结论一定浓度的银离子对细菌整合子捕获耐药性基因盒频率存在抑制作用。

Objective To analyze the effects of silver ion on the integration frequency of the class 1 integron in Escherichia coli(E.coli)BL21(DE3)host.Methods Two recombinant plasmids,pUCINT and pACINAD,were successively transformed into E.coli BL21(DE3)to construct HS2 strains.Three experimental groups were set up using 0.3μg/ml,0.6μg/ml and 0.8μg/ml silver ion LB liquid medium,while control group used common LB liquid medium.Silver ion was supplied by silver nitrate and HS2 strains were cultured at 37℃for 24 h.The copy number of cointegrates and the total copy number of integrons in each group were detected by real-time polymerase chain reaction(qPCR),and the ratio of them was the integration frequency.Changes in the integration frequency were analyzed by three independent phenotypic screening method and the protein expression in HS2 strains was analyzed by mass spectrometry.Results The integration frequency in HS2 strains in the control group and three experimental groups(0.3μg/ml,0.6μg/ml and 0.8μg/ml silver ion)was 1.79×10^(-5)(1.44×10^(-5),3.13×10^(-5)),2.07×10^(-5)(1.49×10^(-5),2.67×10^(-5)),2.25×10^(-6)(1.47×10^(-6),4.54×10^(-6))and 1.69×10^(-6)(0.22×10^(-6),3.08×10^(-6)),respectively.The integration frequency in the 0.6μg/ml and 0.8μg/ml silver ion groups was significantly lower than that in the control group(P<0.01),but there was no significant difference between the 0.3μg/ml silver ion group and the control group.Results of three independent phenotypic screening method were consistent with those obtained by qPCR.Mass spectrometry analysis showed that there were differences in protein expression in HS2 strains between the control group and the experimental groups.Conclusions Silver ion at a certain concentration had an inhibitory effect on the frequency of drug resistance gene cassette captured by bacterial integron.