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

Publisher: Oxford University Press Country of Publication: England NLM ID: 7513617 Publication Model: Print Cited Medium: Internet ISSN: 1460-2091 (Electronic) Linking ISSN: 03057453 NLM ISO Abbreviation: J Antimicrob Chemother Subsets: MEDLINE

Publication: 1997- : London : Oxford University Press
Original Publication: London, New York, Academic Press.

Acinetobacter baumannii*; Amikacin/pharmacology ; Anti-Bacterial Agents/pharmacology ; Chloramphenicol ; Tobramycin ; Drug Resistance, Multiple, Bacterial/genetics ; Microbial Sensitivity Tests

Background: 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.
Objectives: To develop a scar-free genome-editing tool suitable for chromosomal and plasmid modifications in MDR A. baumannii AB5075.
Methods: 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.
Results: 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.
Conclusions: 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.
(© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

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BSAC-2018-0095 British Society for Antimicrobial Chemotherapy; United Kingdom WT_ Wellcome Trust; BB/V007823/1 United Kingdom BB_ Biotechnology and Biological Sciences Research Council

84319SGC3C (Amikacin)
0 (Anti-Bacterial Agents)
66974FR9Q1 (Chloramphenicol)
VZ8RRZ51VK (Tobramycin)

Date Created: 20221010 Date Completed: 20221130 Latest Revision: 20221212

20240829

PMC9704439

10.1093/jac/dkac328

36216579