Division of Biosciences, Department of Life Sciences, Centre of Inflammation Research and Translational Medicine, College of Health and Life Sciences, Brunel University London, Uxbridge, UB8 3PH, UK.
J Antimicrob Chemother. 2022 Nov 28;77(12):3390-3398. doi: 10.1093/jac/dkac328.
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.
目前用于鲍曼不动杆菌的诱变工具会留下选择标记或残留序列，或者涉及繁琐的反向选择和筛选步骤。此外，它们通常适用于模式菌株，而不是多药耐药临床分离株。
开发一种无疤痕的基因组编辑工具，适用于多药耐药鲍曼不动杆菌 AB5075 的染色体和质粒修饰。
我们通过删除多药外排泵 craA、cmlA5 和抗性岛 2（RI2）以及消除质粒 p1AB5075，并结合这些突变，证明了我们改编的基因组编辑系统的效率。然后，我们通过圆盘扩散试验比较了突变体与 WT 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应于鲍曼不动杆菌 AB5075，实现了近 100%的双重组频率，并常规在 10 个工作日内构建突变体。此外，我们表明 CraA 和 p1AB5075 均参与氯霉素耐药，RI2 和 p1AB5075 参与阿米卡星和妥布霉素耐药。
我们开发了一种用于鲍曼不动杆菌的多功能、高效基因组编辑工具。我们已经证明它可以用于修饰染色体和天然质粒。通过对该方法进行挑战，我们表明 CraA 和 p1AB5075 在抗生素耐药性中发挥作用。
