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Structured synopsisBackgroundThe 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 to multidrug resistant (MDR) clinical isolates.ObjectivesTo develop a scar-free genome editing tool suitable for chromosomal and plasmid modifications in MDR A. baumannii AB5075.MethodsWe prove the efficiency of our adapted genome editing system by deleting the multidrug efflux pumps craA and cmlA5, as well as curing plasmid p1AB5075. We then characterised the antibiotic sensitivity phenotype of the mutants compared to the wild type for chloramphenicol, tobramycin and amikacin by disc diffusion assays and determined their minimum inhibitory concentration for each strain.ResultsWe successfully adapted the genome editing protocol to A. baumannii AB5075, achieving a double recombination frequency close to 100% and securing the construction of a mutant within 10 work days. Furthermore, we show that the ΔcraA has a strong sensitivity to chloramphenicol, tobramycin and amikacin, whereas the ΔcmlA5 mutant does not show a significant decrease in viability for the antibiotics tested. On the other hand, the removal of p1AB5075 produced an increased sensitivity to tobramycin and amikacin.ConclusionWe have adapted a highly efficient genome editing tool for A. baumannii and proved that craA has a broader substrate range than previously thought. On the other hand, whereas cmlA5 is annotated as a chloramphenicol efflux pump and is encoded within an aminoglycoside resistance island, it does not provide resistance to any of those compounds.