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Antimicrobial Agents and Chemotherapy, June 2001, p. 1761-1770, Vol. 45, No. 6
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.6.1761-1770.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Multidrug Efflux Pumps: Expression Patterns and Contribution to Antibiotic Resistance in Pseudomonas aeruginosa Biofilms

Teresa R. De Kievit,1 Michael D. Parkins,2 Richard J. Gillis,1 Ramakrishnan Srikumar,3 Howard Ceri,2 Keith Poole,3 Barbara H. Iglewski,1 and Douglas G. Storey2,*

Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14642,1 and Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4,2 and Department of Microbiology and Immunology, Queen's University, Kingston, Ontario K7L 3N6,3 Canada

Received 27 November 2000/Returned for modification 18 December 2000/Accepted 5 March 2001

Pseudomonas aeruginosa biofilms are intrinsically resistant to antimicrobial chemotherapies. At present, very little is known about the physiological changes that occur during the transition from the planktonic to biofilm mode of growth. The resistance of P. aeruginosa biofilms to numerous antimicrobial agents that are substrates subject to active efflux from planktonic cells suggests that efflux pumps may substantially contribute to the innate resistance of biofilms. In this study, we investigated the expression of genes associated with two multidrug resistance (MDR) efflux pumps, MexAB-OprM and MexCD-OprJ, throughout the course of biofilm development. Using fusions to gfp, we were able to analyze spatial and temporal expression of mexA and mexC in the developing biofilm. Remarkably, expression of mexAB-oprM and mexCD-oprJ was not upregulated but rather decreased over time in the developing biofilm. Northern blot analysis confirmed that these pumps were not hyperexpressed in the biofilm. Furthermore, spatial differences in mexAB-oprM and mexCD-oprJ expression were observed, with maximal activity occurring at the biofilm substratum. Using a series of MDR mutants, we assessed the contribution of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY efflux pumps to P. aeruginosa biofilm resistance. These analyses led to the surprising discovery that the four characterized efflux pumps do not play a role in the antibiotic-resistant phenotype of P. aeruginosa biofilms.

* Corresponding author. Mailing address: Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, AB T2N 1N4, Canada. Phone: (403) 220-5274. Fax: (403) 289-9311. E-mail: storey{at}acs.ucalgary.ca.

Antimicrobial Agents and Chemotherapy, June 2001, p. 1761-1770, Vol. 45, No. 6
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.6.1761-1770.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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