In vitro selection and characterization of Bacillus anthracis mutants with high-level resistance to ciprofloxacin

Area of Science:

• Microbiology
• Molecular Biology
• Pharmacology

Background:

• Bacillus anthracis is a bacterium that causes anthrax.
• Antibiotic resistance is a growing global health concern.
• Ciprofloxacin is a fluoroquinolone antibiotic used to treat bacterial infections.

Purpose of the Study:

• To investigate the genetic mechanisms conferring high-level ciprofloxacin resistance in Bacillus anthracis.
• To characterize mutations in quinolone resistance-determining regions (QRDRs) of key bacterial genes.
• To explore the role of efflux pumps in acquired antibiotic resistance.

Main Methods:

• In vitro stepwise selection of attenuated Bacillus anthracis mutants with increasing ciprofloxacin resistance.
• Determination of minimum inhibitory concentrations (MICs) for ciprofloxacin.
• Sequencing of gyrA, gyrB, and parC genes to identify mutations in QRDRs.
• Assessment of potential efflux mechanisms in resistant mutants.

Main Results:

• First-step mutants exhibited resistance due to single mutations in the gyrA QRDR.
• Second-step mutants showed higher resistance with mutations in the parC QRDR.
• Third-step mutants developed substantial resistance (MICs of 32-64 µg/ml) through additional gyrA/gyrB mutations or enhanced ciprofloxacin efflux.
• Mutants with MICs of 32 µg/ml demonstrated enhanced efflux without further target mutations.

Conclusions:

• Stepwise accumulation of mutations in gyrA and parC QRDRs is a primary driver of high-level ciprofloxacin resistance in Bacillus anthracis.
• Enhanced drug efflux represents an alternative mechanism contributing to significant ciprofloxacin resistance.
• Understanding these resistance mechanisms is crucial for developing effective anthrax treatment strategies.