JAC Advance Access originally published online on February 24, 2009
Journal of Antimicrobial Chemotherapy 2009 63(4):668-674; doi:10.1093/jac/dkp027
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Original research |
Role of TolC in Klebsiella oxytoca resistance to antibiotics
1 Laboratory of Microbiology, Department of Pathology and Experimental Therapeutics, Medical and Dental Schools, University of Barcelona, Feixa Llarga s/n Hospitalet, 08907 Barcelona, Spain 2 Department of Microbiology and Parasitology, University of Santiago de Compostela, Santiago de Compostela, Spain 3 Life Sciences Department, Barcelona Supercomputing Center, Barcelona, Spain
Received 29 September 2008; returned 12 November 2008; revised 15 January 2009; accepted 17 January 2009
* Corresponding author. Tel: +34-934024265; Fax: +34-934024249; E-mail: mvinyas{at}ub.edu
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.
Keywords: antimicrobial agents , Klebsiella spp. , antibiotic accumulation , efflux , Enterobacteriaceae , modelling
Present address: IACA Department, University of Vic, Carrer de la Laura 13, 08500 Vic, Spain.