Ciprofloxacin selects for RNA polymerase mutations with pleiotropic antibiotic resistance effects.
Resistance to the fluoroquinolone drug ciprofloxacin is commonly linked to mutations that alter the drug target or increase drug efflux via the major AcrAB-TolC transporter. Very little is known about other mutations that might also reduce susceptibility to ciprofloxacin. We discovered that an Escherichia coli strain experimentally evolved for resistance to ciprofloxacin had acquired a mutation in rpoB, the gene coding for the β-subunit of RNA polymerase. The aim of this work was to determine whether this mutation, and other mutations in rpoB, contribute to ciprofloxacin resistance and, if so, by which mechanism.



Independent lineages of E. coli were evolved in the presence of ciprofloxacin and clones from endpoint cultures were screened for mutations in rpoB. Ciprofloxacin-selected rpoB mutations were identified and characterized in terms of effects on susceptibility and mode of action.



Mutations in rpoB were selected at a high frequency in 3 out of 10 evolved lineages, in each case arising after the occurrence of mutations affecting topoisomerases and drug efflux. All ciprofloxacin-selected rpoB mutations had a high fitness cost in the absence of drug, but conferred a competitive advantage in the presence of ciprofloxacin. RNA sequencing and quantitative RT-PCR analysis showed that expression of mdtK, encoding a multidrug efflux transporter, was significantly increased by the ciprofloxacin-selected rpoB mutations. The susceptibility phenotype was shown to depend on the presence of an active mdtK and a mutant rpoB allele.



These data identify mutations in RNA polymerase as novel contributors to the evolution of resistance to ciprofloxacin and show that the phenotype is mediated by increased MdtK-dependent drug efflux.
Department of Medical Biochemistry and Microbiology, Box 582 Biomedical Center, Uppsala University, Uppsala, Sweden.
Franziska
F
Pietsch
Department of Medical Biochemistry and Microbiology, Box 582 Biomedical Center, Uppsala University, Uppsala, Sweden.
Jessica M
JM
Bergman
Department of Medical Biochemistry and Microbiology, Box 582 Biomedical Center, Uppsala University, Uppsala, Sweden.
Gerrit
G
Brandis
Department of Medical Biochemistry and Microbiology, Box 582 Biomedical Center, Uppsala University, Uppsala, Sweden.
Linda L
LL
Marcusson
Department of Medical Biochemistry and Microbiology, Box 582 Biomedical Center, Uppsala University, Uppsala, Sweden.
Anna
A
Zorzet
Department of Medical Biochemistry and Microbiology, Box 582 Biomedical Center, Uppsala University, Uppsala, Sweden.
Douglas L
DL
Huseby
Department of Medical Biochemistry and Microbiology, Box 582 Biomedical Center, Uppsala University, Uppsala, Sweden diarmaid.hughes@imbim.uu.se.
Diarmaid
D
Hughes
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002939:Ciprofloxacin / Q000494:pharmacology*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D020869:Gene Expression Profiling; D009154:Mutation; D060888:Real-Time Polymerase Chain Reaction; D012641:Selection, Genetic; D017423:Sequence Analysis, RNA
D000900:Anti-Bacterial Agents; D002939:Ciprofloxacin; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit