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 λ 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.