A high-efficiency scar-free genome-editing toolkit for Acinetobacter baumannii.
A high-efficiency scar-free genome-editing toolkit for Acinetobacter baumannii.
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DOI：
10.1093/jac/dkac328
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发表时间：
2022-11-28
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--
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中科院分区：
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目前用于鲍曼不动杆菌的诱变工具留下选择标记或残留序列，或涉及繁琐的反选择和筛选步骤。此外，它们通常适用于模型菌株，而不是MDR临床分离株。开发适用于MDR A中染色体和质粒修饰的无疤痕基因组编辑工具。鲍曼不动杆菌AB 5075。我们通过删除多药外排泵craA、cmlA5和耐药岛2（RI2）以及治愈质粒p1AB5075并组合这些突变来证明我们的适应性基因组编辑系统的效率。然后，我们通过纸片扩散试验表征了突变体与WT相比对不同抗生素（即氯霉素、阿米卡星和妥布霉素）的敏感性，并确定了每种菌株的MIC。我们成功地将基因组编辑方案适用于A。鲍曼不动杆菌AB 5075，实现了接近100%的双重组频率，并且常规地确保在10个工作日内构建突变体。此外，我们表明CraA和p1AB 5075都参与了氯霉素耐药性，而RI 2和p1AB 5075在对阿米卡星和妥布霉素的耐药性中发挥作用。我们已经开发了一个通用的和高效的基因组编辑工具，为A。鲍曼不动杆菌。我们已经证明它可以用于修饰染色体和天然质粒。通过挑战该方法，我们显示了CraA和p1AB5075在抗生素耐药性中的作用。
The current mutagenesis tools for Acinetobacter baumannii leave selection markers or residual sequences behind, or involve tedious counterselection and screening steps. Furthermore, they are usually adapted for model strains, rather than for MDR clinical isolates. To develop a scar-free genome-editing tool suitable for chromosomal and plasmid modifications in MDR A. baumannii AB5075. We prove the efficiency of our adapted genome-editing system by deleting the multidrug efflux pumps craA, cmlA5 and resistance island 2 (RI2), as well as curing plasmid p1AB5075, and combining these mutations. We then characterized the susceptibility of the mutants compared with the WT to different antibiotics (i.e. chloramphenicol, amikacin and tobramycin) by disc diffusion assays and determined the MIC for each strain. We successfully adapted the genome-editing protocol to A. baumannii AB5075, achieving a double recombination frequency close to 100% and routinely securing the construction of a mutant within 10 working days. Furthermore, we show that both CraA and p1AB5075 are involved in chloramphenicol resistance, and that RI2 and p1AB5075 play a role in resistance to amikacin and tobramycin. We have developed a versatile and highly efficient genome-editing tool for A. baumannii. We have demonstrated it can be used to modify both the chromosome and native plasmids. By challenging the method, we show the role of CraA and p1AB5075 in antibiotic resistance.
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