title: Antibiotic combination therapy can select for broad-spectrum multidrug resistance in Pseudomonas aeruginosa.
abstract: Combination therapy with several antibiotics is one strategy that has been applied in order to limit the spread of antimicrobial resistance. We compared the de novo evolution of resistance during combination therapy with the β-lactam ceftazidime and the fluoroquinolone ciprofloxacin with the resistance evolved after single-drug exposure. Combination therapy selected for mutants that displayed broad-spectrum resistance, and a major resistance mechanism was mutational inactivation of the repressor gene mexR that regulates the multidrug efflux operon mexAB-oprM. Deregulation of this operon led to a broad-spectrum resistance phenotype that decreased susceptibility to the combination of drugs applied during selection as well as to unrelated antibiotic classes. Mutants isolated after single-drug exposure displayed narrow-spectrum resistance and carried mutations in the MexCD-OprJ efflux pump regulator gene nfxB conferring ciprofloxacin resistance, or in the gene encoding the non-essential penicillin-binding protein DacB conferring ceftazidime resistance. Reconstruction of resistance mutations by allelic replacement and in vitro fitness assays revealed that in contrast to single antibiotic use, combination therapy consistently selected for mutants with enhanced fitness expressing broad-spectrum resistance mechanisms.
authors.affiliation: Department of Veterinary Disease Biology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Martin
authors.initials: M
authors.lastname: Vestergaard
authors.affiliation: Department of Veterinary Disease Biology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Paulander
authors.affiliation: Center for Genomic Medicine, Rigshospitalet, Copenhagen, Denmark.
authors.forename: Rasmus L
authors.initials: RL
authors.lastname: Marvig
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark.
authors.forename: Julie
authors.initials: J
authors.lastname: Clasen
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Copenhagen, Denmark.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Jochumsen
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Copenhagen, Denmark.
authors.forename: Søren
authors.initials: S
authors.lastname: Molin
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Copenhagen, Denmark.
authors.forename: Lars
authors.initials: L
authors.lastname: Jelsbak
authors.affiliation: Department of Veterinary Disease Biology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Hanne
authors.initials: H
authors.lastname: Ingmer
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark. Electronic address: afol@vet.dtu.dk.
authors.forename: Anders
authors.initials: A
authors.lastname: Folkesson
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics; D002442:Ceftazidime / Q000378:metabolism; D002939:Ciprofloxacin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D004338:Drug Combinations; D024901:Drug Resistance, Multiple, Bacterial; D004359:Drug Therapy, Combination; D006801:Humans; D008962:Models, Theoretical; D009154:Mutation; D046915:Penicillin-Binding Proteins / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000187:drug effects*; D012097:Repressor Proteins / Q000235:genetics; D012641:Selection, Genetic; D014157:Transcription Factors / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D004338:Drug Combinations; C423620:MexR protein, Pseudomonas aeruginosa; C077901:NfxB protein, Pseudomonas aeruginosa; D046915:Penicillin-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D002939:Ciprofloxacin; D002442:Ceftazidime
keywords: Antibiotics; Combination therapy; Drug efflux; Fluoroquinolones; Multidrug resistance; β-Lactams