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Antimicrobial Agents and Chemotherapy, April 2004, p. 1168-1174, Vol. 48, No. 4
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.4.1168-1174.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Dynamics and Spatial Distribution of ß-Lactamase Expression in Pseudomonas aeruginosa Biofilms

Niels Bagge,1* Morten Hentzer,2 Jens Bo Andersen,2 Oana Ciofu,1 Michael Givskov,2 and Niels Høiby1

Department of Clinical Microbiology, Rigshospitalet, and Department of Bacteriology, Institute for Medical Microbiology and Immunology, Panum Institute, University of Copenhagen, Copenhagen,1 Center for Microbial Interactions, BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark2

Received 25 June 2003/ Returned for modification 25 September 2003/ Accepted 30 December 2003

The development of resistance to ß-lactam antibiotics is a problem in the treatment of chronic Pseudomonas aeruginosa infection in the lungs of patients with cystic fibrosis. The main resistance mechanism is high-level expression of the chromosomally encoded AmpC ß-lactamase of P. aeruginosa cells growing in biofilms. Several genes have been shown to influence the level of ampC expression, but little is known about the regulation of ampC expression in P. aeruginosa biofilms. To study the expression of ampC in P. aeruginosa biofilms, we constructed a reporter that consisted of the fusion of the ampC promoter to gfp(ASV) encoding an unstable version of the green fluorescent protein. In vitro biofilms of P. aeruginosa were exposed to the ß-lactam antibiotics imipenem and ceftazidime. Sub-MICs of imipenem significantly induced the monitor system of the biofilm bacteria in the peripheries of the microcolonies, but the centers of the microcolonies remained uninduced. However, the centers of the microcolonies were physiologically active, as shown by experiments with another monitor construction consisting of an arabinose-inducible promoter fused to gfp(ASV). The whole biofilm was induced in the presence of increased imipenem concentrations. Ceftazidime induced the monitor system of the biofilm bacteria as well, but only bacteria in the peripheries of the microcolonies were induced in the presence of even very high concentrations. The experiments illustrate for the first time the dynamic and spatial distributions of ß-lactamase induction in P. aeruginosa cells growing in biofilms. Thus, our experiments show that P. aeruginosa cells growing in biofilms constitute a heterogeneous population unit which may create different antibiotic-selective environments for the bacteria in the biofilm.

* Corresponding author. Mailing address: Department of Bacteriology, Institute for Medical Microbiology and Immunology, Panum Institute, University of Copenhagen, Copenhagen 2200, Denmark. Phone: 45 3532 7890. Fax: 45 3532 7693. E-mail: nbagge2000{at}yahoo.dk.

Antimicrobial Agents and Chemotherapy, April 2004, p. 1168-1174, Vol. 48, No. 4
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.4.1168-1174.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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