Type II topoisomerase mutations in Bacillus anthracis associated with high-level fluoroquinolone resistance.
OBJECTIVE
To identify and characterize the mechanisms of high-level fluoroquinolone resistance in two strains of Bacillus anthracis following serial passage in increasing concentrations of fluoroquinolones.


METHODS
Fluoroquinolone-resistant isolates of the Sterne and Russian Anthrax Vaccine STi strains were obtained following serial passage in the presence of increasing concentrations of four different fluoroquinolones. The quinolone-resistance-determining regions of the type II topoisomerase genes from the resistant strains were amplified by PCR and characterized by DNA sequence analysis. The MICs in the presence and absence of reserpine were determined using broth microdilution as a means of detecting active efflux.


RESULTS
Single and double amino acid substitutions in the GyrA (Ser-85-Leu; Glu-89-Arg/Gly/Lys) and GrlA (Ser-81-Tyr; Val-96-Ala; Asn-70-Lys) were most common. A single amino acid substitution in GyrB (Asp-430-Asn) was also identified. Efflux only applied to isolates selected for by either levofloxacin or ofloxacin.


CONCLUSIONS
Specific amino acid substitutions in the type II topoisomerase enzymes significantly contributed to the development of high-level fluoroquinolone resistance in B. anthracis. However, notable differences between the strains and the drugs tested were identified including the role of efflux and the numbers and types of mutations identified.
Bast
Darrin J
DJ
Toronto Centre for Antimicrobial Research and Evaluation (ToCARE), Room 1483, Department of Microbiology, Mount Sinai Hospital, 600 University Avenue, Toronto, Canada. dbast@mtsinai.on.ca
Athamna
Abed
A
Duncan
Carla L
CL
de Azavedo
Joyce C S
JC
Low
Donald E
DE
Rahav
Galia
G
Farrell
David
D
Rubinstein
Ethan
E
D019943:Amino Acid Substitution; D000890:Anti-Infective Agents / Q000494:pharmacology*; D001408:Bacillus anthracis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D017931:DNA Primers; D004250:DNA Topoisomerases, Type II / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D024841:Fluoroquinolones / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D009154:Mutation / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000890:Anti-Infective Agents; D017931:DNA Primers; D004269:DNA, Bacterial; D024841:Fluoroquinolones; D004250:DNA Topoisomerases, Type II