Atin Sharma1, Rajnikant Sharma1, Tapas Bhattacharyya1, Timsy Bhando1, Ranjana Pathania2. 1. Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India. 2. Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India ranjanapathania@gmail.com rpathfbs@iitr.ac.in.
Abstract
OBJECTIVES: To decipher the function of A1S_1331, named AbaF (Acinetobacter baumannii Fosfomycin efflux), one of the primary targets of AbsR25, a small RNA of A. baumannii. METHODS: abaF was cloned in a multicopy plasmid and expressed from its native promoter in an efflux-deficient strain-Escherichia coli KAM32. Drug susceptibility, accumulation and efflux of ethidium bromide (EtBr) were determined in this strain. abaF was disrupted in A. baumannii using homologous recombination and its effect on drug susceptibility, biofilm formation and virulence was studied. Expression of abaF was followed by quantitative PCR in fosfomycin-challenged A. baumannii and fosfomycin-resistant mutants of A. baumannii. Expression of abaF in clinical strains of A. baumannii was determined by RT-PCR. RESULTS: Expression of abaF in E. coli KAM32 resulted in increased resistance to fosfomycin. Lower accumulation and higher efflux of EtBr from this strain confirmed the role of AbaF as an efflux pump. Disruption of abaF in A. baumannii caused an increase in fosfomycin susceptibility and a decrease in biofilm formation and virulence. The expression of abaF was higher in A. baumannii cells exposed to fosfomycin and in cells resistant to higher concentrations of fosfomycin. The clinically relevant strains of A. baumannii also tested positive for the expression of abaF. CONCLUSIONS: The results of this study suggest that efflux is an important mechanism of fosfomycin resistance and AbaF is involved in fosfomycin resistance in A. baumannii. AbaF also seems to play a role in biofilm formation and virulence of A. baumannii.
OBJECTIVES: To decipher the function of A1S_1331, named AbaF (Acinetobacter baumanniiFosfomycin efflux), one of the primary targets of AbsR25, a small RNA of A. baumannii. METHODS:abaF was cloned in a multicopy plasmid and expressed from its native promoter in an efflux-deficient strain-Escherichia coli KAM32. Drug susceptibility, accumulation and efflux of ethidium bromide (EtBr) were determined in this strain. abaF was disrupted in A. baumannii using homologous recombination and its effect on drug susceptibility, biofilm formation and virulence was studied. Expression of abaF was followed by quantitative PCR in fosfomycin-challenged A. baumannii and fosfomycin-resistant mutants of A. baumannii. Expression of abaF in clinical strains of A. baumannii was determined by RT-PCR. RESULTS: Expression of abaF in E. coli KAM32 resulted in increased resistance to fosfomycin. Lower accumulation and higher efflux of EtBr from this strain confirmed the role of AbaF as an efflux pump. Disruption of abaF in A. baumannii caused an increase in fosfomycin susceptibility and a decrease in biofilm formation and virulence. The expression of abaF was higher in A. baumannii cells exposed to fosfomycin and in cells resistant to higher concentrations of fosfomycin. The clinically relevant strains of A. baumannii also tested positive for the expression of abaF. CONCLUSIONS: The results of this study suggest that efflux is an important mechanism of fosfomycin resistance and AbaF is involved in fosfomycin resistance in A. baumannii. AbaF also seems to play a role in biofilm formation and virulence of A. baumannii.
Authors: Michaelle Chojnacki; Xufeng Cao; Mikaeel Young; Rebecca N Fritz; Paul M Dunman; Daniel P Flaherty Journal: ChemMedChem Date: 2020-08-13 Impact factor: 3.466
Authors: Gamal Wareth; Jörg Linde; Ngoc H Nguyen; Tuan N M Nguyen; Lisa D Sprague; Mathias W Pletz; Heinrich Neubauer Journal: Antibiotics (Basel) Date: 2021-05-11