title: Identification of ciprofloxacin resistance by SimpleProbe, High Resolution Melt and Pyrosequencing nucleic acid analysis in biothreat agents: Bacillus anthracis, Yersinia pestis and Francisella tularensis.
abstract: The potential for genetic modification of biological warfare agents makes rapid identification of antibiotic resistant strains critical for the implementation of suitable infection control measures. The fluorinated quinolone, ciprofloxacin, is an antibiotic effective for treating bacterial infections by inhibiting the enzyme activity of the DNA type II topoisomerases DNA gyrase and topoisomerase IV. The genes that encode the subunits of DNA gyrase (gyrA and gyrB) and topo IV (par C and parE) contain hotspots within an area known as the quinolone resistance-determining region (QRDR). Base pair changes within this region give rise to mutations that cause resistance to the antibiotic by altering amino acids within the enzymes. Ciprofloxacin-resistant (cipro(r)) strains of Bacillus anthracis, Yersinia pestis, and Francisella tularensis with one or more known mutations within the QRDR of gyrA, gyrB, parC, and parE genes were tested with SimpleProbe and High Resolution Melt (HRM) dye chemistries and Pyrosequencing genetic analysis to evaluate the ability to rapidly detect ciprofloxacin-induced mutations. While SimpleProbe and Pyrosequencing successfully identified all known mutants, the HRM assay identified all but those resulting from G<-->C or A<-->T substitutions.
authors.affiliation: Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA.
authors.forename: Bonnie M
authors.initials: BM
authors.lastname: Loveless
authors.forename: Anastasiya
authors.initials: A
authors.lastname: Yermakova
authors.forename: Deanna R
authors.initials: DR
authors.lastname: Christensen
authors.forename: John P
authors.initials: JP
authors.lastname: Kondig
authors.forename: Henry S
authors.initials: HS
authors.lastname: Heine
authors.forename: Leonard P
authors.initials: LP
authors.lastname: Wasieloski
authors.forename: David A
authors.initials: DA
authors.lastname: Kulesh
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology; D001408:Bacillus anthracis / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002939:Ciprofloxacin / Q000494:pharmacology*; D027081:DNA Gyrase / Q000235:genetics / Q000378:metabolism; D027101:DNA Topoisomerase IV / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D005604:Francisella tularensis / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D009154:Mutation; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D015010:Yersinia pestis / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism
chemical_list: D000890:Anti-Infective Agents; D001426:Bacterial Proteins; D002939:Ciprofloxacin; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase