Functional cloning and characterization of a multidrug efflux pump, mexHI-opmD, from a Pseudomonas aeruginosa mutant
Hiroshi Sekiya1, Takehiko Mima, Yuji Morita
1Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama, 700-8530, Japan.
Abstract:
We isolated mutant YM644, which showed elevated resistance to norfloxacin, ethidium bromide, acriflavine, and rhodamine 6G, from Pseudomonas aeruginosa YM64, a strain that lacks four major multidrug efflux pumps. The genes responsible for the resistance were mexHI-opmD. Elevated ethidium extrusion was observed with cells of YM644 and YM64 harboring a plasmid carrying the genes. Disruption of the genes in the chromosomal DNA of YM644 made the cells sensitive to the drugs.
Insights
Researchers identified the mexHI-opmD genes in Pseudomonas aeruginosa responsible for multidrug resistance. This discovery helps understand how bacteria develop resistance to antibiotics like norfloxacin.
Area of Science:
- Microbiology
- Molecular Biology
Background:
- Pseudomonas aeruginosa is a significant opportunistic pathogen.
- Multidrug efflux pumps play a crucial role in bacterial resistance to antimicrobial agents.
- Understanding novel resistance mechanisms is vital for combating infections.
Purpose of the Study:
- To identify the genetic basis of multidrug resistance in a specific Pseudomonas aeruginosa mutant.
- To characterize the role of identified genes in conferring resistance to various antimicrobial compounds.
Main Methods:
- Isolation and characterization of a multidrug-resistant Pseudomonas aeruginosa mutant (YM644).
- Genetic analysis to identify the genes responsible for the observed resistance phenotype.
- Plasmid-based complementation and gene disruption experiments to confirm gene function.
- Measurement of ethidium bromide extrusion to assess efflux pump activity.
Main Results:
- A mutant strain, YM644, derived from a strain lacking major efflux pumps, exhibited high resistance to norfloxacin, ethidium bromide, acriflavine, and rhodamine 6G.
- The mexHI-opmD gene cluster was identified as responsible for this elevated drug resistance.
- Expression of mexHI-opmD in a susceptible strain conferred resistance and increased ethidium bromide extrusion.
- Disruption of mexHI-opmD in the resistant mutant restored sensitivity to the tested drugs.
Conclusions:
- The mexHI-opmD gene cluster constitutes a novel multidrug efflux system in Pseudomonas aeruginosa.
- This system contributes significantly to the resistance of Pseudomonas aeruginosa to a range of antimicrobial agents.
- Targeting the mexHI-opmD efflux system could be a potential strategy to overcome multidrug resistance in this pathogen.
