title: Genomic characterization of ciprofloxacin resistance in a laboratory-derived mutant and a clinical isolate of Streptococcus pneumoniae.
abstract: The broad-spectrum fluoroquinolone ciprofloxacin is a bactericidal antibiotic targeting DNA topoisomerase IV and DNA gyrase encoded by the parC and gyrA genes. Resistance to ciprofloxacin in Streptococcus pneumoniae mainly occurs through the acquisition of mutations in the quinolone resistance-determining region (QRDR) of the ParC and GyrA targets. A role in low-level ciprofloxacin resistance has also been attributed to efflux systems. To look into ciprofloxacin resistance at a genome-wide scale and to discover additional mutations implicated in resistance, we performed whole-genome sequencing of an S. pneumoniae isolate selected for resistance to ciprofloxacin in vitro (128 μg/ml) and of a clinical isolate displaying low-level ciprofloxacin resistance (2 μg/ml). Gene disruption and DNA transformation experiments with PCR fragments harboring the mutations identified in the in vitro S. pneumoniae mutant revealed that resistance is mainly due to QRDR mutations in parC and gyrA and to the overexpression of the ABC transporters PatA and PatB. In contrast, no QRDR mutations were identified in the genome of the S. pneumoniae clinical isolate with low-level resistance to ciprofloxacin. Assays performed in the presence of the efflux pump inhibitor reserpine suggested that resistance is likely mediated by efflux. Interestingly, the genome sequence of this clinical isolate also revealed mutations in the coding region of patA and patB that we implicated in resistance. Finally, a mutation in the NAD(P)H-dependent glycerol-3-phosphate dehydrogenase identified in the S. pneumoniae clinical strain was shown to protect against ciprofloxacin-mediated reactive oxygen species.
authors.affiliation: Centre de recherche en Infectiologie du Centre de recherche du CHUL and Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada.
authors.forename: Andréanne
authors.initials: A
authors.lastname: Lupien
authors.forename: Dewan S
authors.initials: DS
authors.lastname: Billal
authors.forename: Fereshteh
authors.initials: F
authors.lastname: Fani
authors.forename: Hafid
authors.initials: H
authors.lastname: Soualhine
authors.forename: George G
authors.initials: GG
authors.lastname: Zhanel
authors.forename: Philippe
authors.initials: P
authors.lastname: Leprohon
authors.forename: Marc
authors.initials: M
authors.lastname: Ouellette
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002939:Ciprofloxacin / Q000494:pharmacology*; D016680:Genome, Bacterial / Q000235:genetics; D008826:Microbial Sensitivity Tests; D009154:Mutation; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013296:Streptococcus pneumoniae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D017382:Reactive Oxygen Species; D002939:Ciprofloxacin