Production of the cryptic EefABC efflux pump in Enterobacter aerogenes chloramphenicol-resistant mutants

Objectives: AcrAB-ToIC is the major tripartite multidrug efflux pump in Enterobacter aerogenes while EefABC is a cryptic efflux system. This study was conducted to identify and characterize E. aerogenes mutants producing the EefABC efflux pump. Methods: Four spontaneous chloramphenicol-resistant (CMR) mutants were isolated. The expression level of the eefABC promoter and the production of the EefA and B proteins were analysed in the mutants. Antibiotic susceptibilities were compared for wild-type and mutant strains. Efflux activity was investigated using an efflux pump inhibitor. Results: The activation of the eefABC promoter was detected in four CMR mutants. These mutants showed increased resistance to erythromycin and ticarcillin, but not to fluoroquinolones, ketolides and detergents. Two additional efflux proteins were detected in the mutants. The CMR mutants bear no mutation in hns, which encodes a repressor of eefABC. No alteration of porin expression, a phenotype observed in marA or ramA multidrug-resistant mutants, was detected in the mutants. Conclusions: These observations suggest that eefABC activation can occur in vitro independently of the H-NS, MarA or RamA global regulators.