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Title: A high-efficiency scar-free genome-editing toolkit for <i>Acinetobacter baumannii</i>
Authors: de Dios, R
Gadar, K
McCarthy, RR
Issue Date: 11-Oct-2022
Publisher: Oxford University Press
Citation: de Dios, R., Gadar, K. and McCarthy, R.R. (2022) 'A high-efficiency scar-free genome-editing toolkit for <i>Acinetobacter baumannii</i>, Journal of Antimicrobial Chemotherapy, 77 (12), pp. 3390 - 3398. doi: 10.1093/jac/dkac328.
Abstract: Copyright © The Author(s) 2022. 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.
Description: Data availability: All plasmids are available through request to the corresponding author. Supplementary data: Figures S1 to S10, Tables S1 and S2, Text S1 and File S1 are available as Supplementary data at JAC Online, available at:
ISSN: 0305-7453
Other Identifiers: ORCID iDs: Rubén de Dios; Ronan R McCarthy
Appears in Collections:Dept of Life Sciences Research Papers

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