title: Emergence of AcrAB-mediated tigecycline resistance in a clinical isolate of Enterobacter cloacae during ciprofloxacin treatment.
abstract: Tigecycline resistance remains rare amongst Enterobacteriaceae in the UK, as elsewhere, but has been associated with upregulation of the AcrAB efflux system. Using isolates of an Enterobacter cloacae strain that developed tigecycline resistance in vivo during ciprofloxacin therapy as well as laboratory-selected mutants, we investigated the role of this pump and the global regulator RamA in tigecycline resistance. Laboratory mutants were selected from a susceptible clinical isolate in vitro by exposure to increasing concentrations of tigecycline. Expression of the acrAB operon and the ramA gene was monitored by real-time reverse-transcription polymerase chain reaction (RT-PCR). Overexpression of ramA was achieved using the pBAD expression vector, whilst insertional inactivation of acrB with a gentamicin resistance cassette was achieved with the bacteriophage lambda Red recombination system. Increased tigecycline minimum inhibitory concentrations in the clinical isolate and a laboratory mutant were associated with increases in acrAB and ramA transcripts. Induction of increased ramA expression resulted in increased acrAB expression, whilst insertional inactivation of acrB restored full susceptibility to tigecycline. Treatment with ciprofloxacin, a substrate of AcrAB in E. cloacae, possibly selected for cross-resistance to tigecycline as a result of RamA-mediated AcrAB upregulation.
authors.affiliation: Antibiotic Resistance Monitoring and Reference Laboratory, Health Protection Agency Centre for Infections, London NW9 5EQ, UK. michael.hornsey@hpa.org.uk
authors.forename: Michael
authors.initials: M
authors.lastname: Hornsey
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Ellington
authors.forename: Michel
authors.initials: M
authors.lastname: Doumith
authors.forename: Geoff
authors.initials: G
authors.lastname: Scott
authors.forename: David M
authors.initials: DM
authors.lastname: Livermore
authors.forename: Neil
authors.initials: N
authors.lastname: Woodford
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology* / Q000627:therapeutic use*; D001426:Bacterial Proteins / Q000235:genetics; D015373:Bacterial Typing Techniques; D010582:Bacteriophage lambda / Q000235:genetics; D002939:Ciprofloxacin / Q000627:therapeutic use*; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial; D016521:Electrophoresis, Gel, Pulsed-Field; D016972:Enterobacter cloacae / Q000187:drug effects* / Q000302:isolation & purification; D004756:Enterobacteriaceae Infections / Q000188:drug therapy / Q000382:microbiology*; D020869:Gene Expression Profiling; D006801:Humans; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics; D008826:Microbial Sensitivity Tests; D008911:Minocycline / Q000031:analogs & derivatives* / Q000494:pharmacology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D000078304:Tigecycline; D006113:United Kingdom
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D026901:Membrane Transport Proteins; D002939:Ciprofloxacin; D000078304:Tigecycline; D008911:Minocycline