抗生素持续作用下细菌传代次数对接合频率的影响

The effect of bacterial generation numbers on the conjugative efficiency under the persistent treatment of antibiotic and its possible mechanism

目的探究抗生素作用下细菌传代次数对接合频率的影响及其可能机制。方法将本实验室构建的携带庆大霉素(GM)抗性基因的质粒p UCP24T转入E.coli SM10λpir获得重组菌E.coli SM10λpir(p UCP24T)。将重组菌在含30μg/m L GM的琼脂平板上不断传代,每次挑取单个菌落传代,获得第50代和100代的E.coli SM10λpir(p UCP24T)。分别以铜绿假单胞菌PAO1、大肠埃希菌EC600和肺炎克雷伯菌A10为受体菌,第1、50、100代E.coli SM10λpir(p UCP24T)为供体菌进行接合实验,计算接合频率。采用微量肉汤稀释法检测第1、50、100代E.coli SM10λpir(p UCP24T)对GM的最低抑菌浓度(MIC)值。分别提取第1、50、100代E.coli SM10λpir(p UCP24T)的全基因组DNA,构建DNA文库并进行高通量测序,对测序结果进行生物信息学分析。结果成功获得重组菌E.coli SM10λpir(p UCP24T)。第50、100代E.coli SM10λpir(p UCP24T)与三种受体菌的接合频率较第1代均显著增高(P<0.05),第100代菌株对GM的MIC值大于2 048μg/m L,较第1和第50代菌株对GM的MIC值1 024μg/m L升高。第1、50、100代E.coli SM10λpir(p UCP24T)重组菌中携带GM抗性基因的p UCP24T质粒的拷贝数分别为601.3、1 808.2及9 190.5,呈现增高的趋势。结论在抗生素持续作用下,E.coli SM10λpir(p UCP24T)通过增加耐药质粒拷贝数使接合频率及MIC值升高,以适应抗生素的持续作用。

ObjectiveTo explore the effect of bacterial generation numbers on the conjugative efficiency under the persistent treatment of antibiotic and its possible mechanism.MethodsThe plasmid p UCP24T constructed in our laboratory which carried the gentamicin（GM）drug resistance gene was transferred into E.coli SM10λpir to generate the recombinant E.coli SM10λpir（p UCP24T）.E.coli SM10λpir（p UCP24T）was cultured on the agar plate containing 30μg/m L GM up to 50th and 100th generations.Then E.coli SM10λpir（p UCP24T）of different generation numbers were taken as the donor and PAO1（P.aeruginosa）,EC600（E.coli）and A10（K.pneumoniae）were taken as the recipient.The conjugative efficiency was calculated and the minimum inhibitory concentration（MIC）value of E.coli SM10λpir（p UCP24T）at different generation numbers was detected using micro broth dilution method.The whole genomic DNA of the bacteria strains at different generation numbers was extracted to construct DNA library.The genome was sequenced by high-throughput sequencing methods.ResultsThe recombinant E.coli SM10λpir（p UCP24T）was successfully constructed.The conjugative efficiency between 50th and 100th generation of E.coli SM10λpir（p UCP24T）and the recipient was much higher than that of the 1st generation E.coli SM10λpir（p UCP24T）（P〈0.05）.Moreover,the MIC value of the 100th generation E.coli SM10λpir（p UCP24T）was higher than 2 048μg/m L,which significantly higher than those of the 1st and50th generation（1 024μg/mL）.The copy numbers of pUCP24T of the 1st,50th and 100th generation were 601.3,1 808.2 and 9 190.5,respectively.ConclusionUnder the persistent treatment of antibiotic,E.coli SM10λpir（pUCP24T）could increase the conjugative efficiency and MIC value by the means of increasing the p UCP24T copy number to adapt to the antibiotic pressure.