Genomewide screening for novel genetic variations associated with ciprofloxacin resistance in Bacillus anthracis

Abstract

Fluoroquinolone (FQ) resistance of Bacillus anthracis is a serious concern in the fields of biodefense and bioterrorism since FQs are very effective antibiotics and are recommended as first-line treatment against this lethal bacterium. In this study, we obtained 2 strains of B. anthracis showing resistance or intermediate resistance to ciprofloxacin (CIP) by a stepwise selection procedure with increasing CIP concentrations. Fifteen genetic variations were identified between the parental and CIP-resistant strains by next-generation sequencing. Nonsynonymous mutations in the quinolone resistance-determining region (QRDR) of type II DNA topoisomerase were identified in the resistant strain but not in the intermediate-resistant strain. The GBAA0834 (TetR-type transcriptional regulator) locus was also revealed to be a novel "mutation hot spot" that leads to the increased expression of multidrug efflux systems for CIP resistance. As an initial step of CIP resistance in B. anthracis, such disruptive mutations of GBAA0834 appear to be more easily acquired than those in an essential gene, such as that encoding type II DNA topoisomerase. Such an intermediate-resistant phenotype could increase a cell population under CIP-selective pressure and might promote the emergence of highly resistant isolates. Our findings reveal, in addition to QRDR, crucial genetic targets for the investigation of intermediate resistance of B. anthracis to FQs.

FIG. 1.

(A) Schematic gene organization around the GBAA0834 locus. Vertical arrowheads indicate positions of the mutations (black, deletion mutation; white, insertion mutation; gray, SNV). The detailed sequence is shown in Fig. S4 in the supplemental material. Numbers indicate the genomic position of the Ames 0581 strain. Relative transcriptional expression around GBAA0834 in BA103 (B) and BA104 (C) derivatives. (D) Testing of susceptibility to CIP on Muller-Hinton agar containing 10 mg/liter reserpine, which acts by blocking the multidrug efflux protein. Dimethyl sulfoxide was used as the control solvent (−). Arrows indicate MICs for CIP.