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

Abstract

Mutants of attenuated Bacillus anthracis with high-level ciprofloxacin resistance were isolated using a three-step in vitro selection. Ciprofloxacin MICs were 0.5 micro g/ml for first-step mutants, which had one of two gyrA quinolone resistance-determining region (QRDR) mutations. Ciprofloxacin MICs were 8 and 16 microg/ml for second-step mutants, which had one of three parC QRDR mutations. Ciprofloxacin MICs for third-step mutants were 32 and 64 microg/ml. Mutants for which MICs were 64 microg/ml had one of two additional mutations within the gyrA QRDR or one of two mutations within the gyrB QRDR. Mutants for which MICs were 32 microg/ml had no additional target modifications but showed evidence of enhanced ciprofloxacin efflux.

FIG. 1.

Effect of INF271 on the susceptibility of third-step, high-level CIP-resistant B. anthracis mutants. S2-1 is the parent strain for the third-step mutants; S3-1, -3, -4, -5, -7, -12, -14, -15, and -17 are third-step mutants with no additional gyrase or topoisomerase mutations. S3-6, -8, -11, and -16 are third-step mutants with additional gyrase mutations. ΔΔ, Bacillus subtilis with inactivated blt and bmr transporter genes; ΔΔNA, B. subtilis strain ΔΔ with a functional NorA transporter from the plasmid expression vector pBEV.