A method for generating marker-less gene deletions in multidrug-resistant Acinetobacter baumannii
Ismawati Mohammad Amin1, Grace E Richmond, Paromita Sen
1Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore.
BMC Microbiology
|July 16, 2013
Summary
A new marker-less gene deletion method effectively removes multiple genes in multidrug-resistant Acinetobacter baumannii. This study identified the AdeIJK efflux pump as a key contributor to antimicrobial resistance in this pathogen.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Acinetobacter baumannii is a significant cause of hospital-acquired infections.
- Increasing antibiotic resistance in A. baumannii necessitates new research tools.
- Existing genetic manipulation methods are limited for multiple gene deletions.
Purpose of the Study:
- To develop a novel marker-less gene deletion method for multidrug-resistant (MDR) A. baumannii.
- To investigate the roles of AdeFGH and AdeIJK efflux pumps in MDR.
- To enable precise genetic studies in MDR A. baumannii.
Main Methods:
- Creation of a tellurite-resistant suicide vector (pMo130-TelR).
- Two-step selection process for plasmid insertion and marker-less deletion.
- Verification of gene deletions using PCR and qRT-PCR.
- Antimicrobial susceptibility testing and dye accumulation assays.
Main Results:
- Successfully deleted adeFGH and adeIJK operons individually and together in MDR A. baumannii.
- The AdeIJK efflux pump significantly contributes to antimicrobial resistance.
- The AdeFGH pump deletion showed no impact on antimicrobial susceptibility.
- Dye accumulation assays confirmed the role of AdeIJK in efflux.
Conclusions:
- The pMo130-TelR vector provides an efficient marker-less method for single and multiple gene deletions.
- This method facilitates clear definition of efflux pump contributions to MDR.
- AdeIJK is identified as a significant efflux pump in MDR A. baumannii.
