PDF(Pubmed)
Abstract：
This study investigated resistance evolution mechanisms of conjugated plasmids and bacterial hosts under different concentrations of antibiotic pressure. Ancestral strain ECNX52 was constructed by introducing the blaNDM-5-carrying IncX3 plasmid into E. coli C600, and was subjected to laboratory evolution under different concentrations of meropenem pressure. Minimal inhibitory concentrations and conjugation frequency were determined. Fitness of these strains was assessed. Whole genome sequencing and transcriptional changes were performed. Ancestral host or plasmids were recombined with evolved hosts or plasmids to verify plasmid or host factors in resistance evolution. Role of the repA mutation on plasmid copy number was determined. Two out of the four clones (EM2N1 and EM2N3) exhibited four-fold increase in MIC when exposed to a continuous pressure of 2 μg/mL MEM (1/32 MIC), by down regulating expression of outer membrane protein ompF. Besides, all four clones displayed four-fold increase in MIC and higher conjugation frequency when subjected to a continuous pressure of 4 μg/mL MEM (1/16 MIC), attributing to increasing plasmid copy number generated by repA D140Y (GAT→TAT) mutation. Bacterial hosts and conjugative plasmids can undergo resistance evolution under certain concentrations of antimicrobial pressure by reducing the expression of outer membrane proteins or increasing plasmid copy numbers.
摘要：
■这项研究调查了在不同浓度的抗生素压力下，共轭质粒和细菌宿主的抗性进化机制。
■祖先菌株ECNX52是通过将携带blaNDM-5的IncX3质粒引入大肠杆菌C600而构建的，并在不同浓度的美罗培南压力下进行实验室进化。测定最小抑制浓度和结合频率。评估这些菌株的适合性。进行全基因组测序和转录改变。祖先宿主或质粒与进化的宿主或质粒重组，以验证抗性进化中的质粒或宿主因子。确定repA突变对质粒拷贝数的作用。
■当暴露于2μg/mLMEM（1/32MIC）的连续压力时，四个克隆中的两个（EM2N1和EM2N3）的MIC增加了四倍，通过下调外膜蛋白ompF的表达。此外,当经受4μg/mLMEM（1/16MIC）的连续压力时，所有四个克隆的MIC增加了四倍，并且缀合频率更高，归因于repAD140Y(GAT→TAT)突变产生的质粒拷贝数增加。
■细菌宿主和接合质粒可以通过减少外膜蛋白的表达或增加质粒拷贝数，在一定浓度的抗菌压力下进行抗性进化。
■祖先菌株ECNX52是通过将携带blaNDM-5的IncX3质粒引入大肠杆菌C600而构建的，并在不同浓度的美罗培南压力下进行实验室进化。测定最小抑制浓度和结合频率。评估这些菌株的适合性。进行全基因组测序和转录改变。祖先宿主或质粒与进化的宿主或质粒重组，以验证抗性进化中的质粒或宿主因子。确定repA突变对质粒拷贝数的作用。
■当暴露于2μg/mLMEM（1/32MIC）的连续压力时，四个克隆中的两个（EM2N1和EM2N3）的MIC增加了四倍，通过下调外膜蛋白ompF的表达。此外,当经受4μg/mLMEM（1/16MIC）的连续压力时，所有四个克隆的MIC增加了四倍，并且缀合频率更高，归因于repAD140Y(GAT→TAT)突变产生的质粒拷贝数增加。
■细菌宿主和接合质粒可以通过减少外膜蛋白的表达或增加质粒拷贝数，在一定浓度的抗菌压力下进行抗性进化。
