Broad-host-range gyrase A gene probe

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Quinolone resistance in Pseudomonas aeruginosa is a significant clinical concern.
• Understanding the genetic basis of quinolone resistance is crucial for developing effective treatments.
• The DNA gyrase A subunit is a primary target for quinolone antibiotics.

Purpose of the Study:

• To investigate the expression and function of the Escherichia coli gyrase A gene in Pseudomonas aeruginosa.
• To develop a tool for detecting quinolone resistance mechanisms in P. aeruginosa.
• To differentiate between gyrase A mutations and permeability alterations as causes of quinolone resistance.

Main Methods:

• Cloning of the E. coli gyrase A gene into a broad-host-range cosmid vector (pLA2917).
• Mobilization of the resulting plasmid (pNJR3-2) into various P. aeruginosa strains, including wild-type and quinolone-resistant mutants.
• Assessment of quinolone susceptibility in engineered P. aeruginosa strains.

Main Results:

• The E. coli gyrase A gene (on pNJR3-2) conferred wild-type quinolone susceptibility on P. aeruginosa gyrA mutants.
• Intermediate susceptibility was observed in cfxA-cfxB double mutants, suggesting a role for both genes.
• pNJR3-2 did not affect quinolone susceptibility in wild-type or permeability mutants (cfxB).
• The probe successfully analyzed quinolone resistance in clinical P. aeruginosa isolates.

Conclusions:

• The E. coli DNA gyrase A gene is expressed in P. aeruginosa and functionally complements gyrA mutations.
• The pNJR3-2 plasmid serves as a valuable probe for identifying gyrase A mutations conferring quinolone resistance.
• This approach can distinguish between gyrase A-mediated resistance and resistance due to altered permeability.