Role of TolC in Klebsiella oxytoca resistance to antibiotics

Objectives: The Gram-negative human pathogen Klebsiella oxytoca is often resistant to several antibiotics such as fluoroquinolones, erythromycin, tetracycline, chloramphenicol and others. The aim of this study was to look at the mechanisms leading to this resistance and particularly the role of TolC and efflux mechanisms in determining resistance. Methods: Ciprofloxacin accumulation was measured spectrofluorometrically. Growth inhibition assays were performed in the presence or absence of carbonyl cyanide m-chlorophenylhydrazone (10 mg/L, final concentration). The genome of K. oxytoca was analysed for the existence of loci encoding tolC by PCR using primers for the Enterobacter aerogenes tolC gene and subsequently sequenced. A plasmid named pUC18TolC was constructed and inserted into Escherichia coli C600tolC,Tn5, and the function of TolC was analysed. The structure modelling was performed using the Modeller program. Results: The existence of the AcrAB efflux mechanism was demonstrated in the species, and a TolC-like protein, a channel-forming protein at the external membrane that allows the extrusion of antibiotics by the AcrAB efflux pump, was cloned, sequenced and a model proposed. Conclusions: K. oxytoca express a functional TolC that lacks a fragment of six amino acids characteristic of the external loops of TolC in E. coli. This makes this species resistant to a few colicins.