Genetic characterization of tigecycline resistance in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes.
OBJECTIVE
The intrinsically encoded ramA gene has been linked to tigecycline resistance through the up-regulation of efflux pump AcrAB in Enterobacter cloacae. The molecular basis for increased ramA expression in E. cloacae and Enterobacter aerogenes, as well as the role of AraC regulator rarA, has not yet been shown. To ascertain the intrinsic molecular mechanism(s) involved in tigecycline resistance in Enterobacter spp., we analysed the expression levels of ramA and rarA and corresponding efflux pump genes acrAB and oqxAB in Enterobacter spp. clinical isolates.


METHODS
The expression levels of ramA, rarA, oqxA and acrA were tested by quantitative real-time RT-PCR. The ramR open reading frames of the ramA-overexpressing strains were sequenced; strains harbouring mutations were transformed with wild-type ramR to study altered ramA expression and tigecycline susceptibility.


RESULTS
Tigecycline resistance was mediated primarily by increased ramA expression in E. cloacae and E. aerogenes. Only the ramA-overexpressing E. cloacae isolates showed increased rarA and oqxA expression. Upon complementation with wild-type ramR, all Enterobacter spp. containing ramR mutations exhibited decreased ramA and acrA expression and increased tigecycline susceptibility. Exceptions were one E. cloacae strain and one E. aerogenes strain, where a decrease in ramA levels was not accompanied by lower acrA expression.


CONCLUSIONS
Increased ramA expression due to ramR deregulation is the primary mediator of tigecycline resistance in clinical isolates of E. cloacae and E. aerogenes. However, some ramA-overexpressing isolates do not show changes in ramR, suggesting alternate pathways of ramA regulation; the rarA regulator and the oqxAB efflux pump may also play a role in tigecycline resistance in E. cloacae.
Centre for Infection and Immunity, Queen's University Belfast, Health Sciences Building, 97 Lisburn Road, Belfast BT9 7AE, UK.
Mark
M
Veleba
Shyamasree
S
De Majumdar
Michael
M
Hornsey
Neil
N
Woodford
Thamarai
T
Schneiders
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D021902:Enterobacter aerogenes / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D016972:Enterobacter cloacae / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D004756:Enterobacteriaceae Infections / Q000382:microbiology*; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006801:Humans; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000235:genetics; D008911:Minocycline / Q000031:analogs & derivatives* / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D000078304:Tigecycline; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D000900:Anti-Bacterial Agents; D026901:Membrane Transport Proteins; D014157:Transcription Factors; D000078304:Tigecycline; D008911:Minocycline