A high-efficiency scar-free genome-editing toolkit for Acinetobacter baumannii

Area of Science:

• Microbiology
• Genetics
• Molecular Biology

Background:

• Current mutagenesis tools for Acinetobacter baumannii are inefficient, leaving markers or requiring tedious steps.
• Existing tools are often adapted for model strains, not clinically relevant multidrug-resistant (MDR) isolates.

Purpose of the Study:

• To develop a scar-free genome-editing tool for chromosomal and plasmid modifications in MDR A. baumannii AB5075.
• To establish an efficient and rapid method for genetic manipulation in clinical A. baumannii isolates.

Main Methods:

• Adapted a genome-editing system for A. baumannii AB5075.
• Deleted multidrug efflux pumps (craA, cmlA5) and resistance island 2 (RI2), and cured plasmid p1AB5075.
• Assessed antibiotic susceptibility of generated mutants using disc diffusion assays and determined minimum inhibitory concentrations (MICs).

Main Results:

• Achieved a scar-free genome-editing protocol with a double recombination frequency near 100% in A. baumannii AB5075.
• Successfully constructed mutants within 10 working days.
• Demonstrated that CraA and p1AB5075 contribute to chloramphenicol resistance, while RI2 and p1AB5075 are involved in amikacin and tobramycin resistance.

Conclusions:

• Developed a versatile and highly efficient scar-free genome-editing tool for A. baumannii.
• The tool effectively modifies both chromosomal and native plasmid DNA.
• Elucidated the roles of specific genes and plasmids in conferring antibiotic resistance in A. baumannii.