Affiliation:
1. 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
Abstract
Abstract
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.
Funder
British Society for Antimicrobial Chemotherapy
BBSRC New Investigator
Academy of Medical Sciences
Wellcome Trust
Government Department of Business
Energy and Industrial Strategy
British Heart Foundation
Diabetes UK Springboard
Publisher
Oxford University Press (OUP)
Subject
Infectious Diseases,Pharmacology (medical),Pharmacology,Microbiology (medical)
Cited by
1 articles.
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