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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2006, p. 2064–2069 Vol. 72, No. 3 0099-2240/06/$08.00 0 doi:10.1128/AEM.72.3.2064–2069.2006

Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Chelator-Induced Dispersal and Killing of Pseudomonas aeruginosa Cells in a Biofilm†

Ehud Banin,1 Keith M. Brady,2 and E. Peter Greenberg1*

Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195-7242,1 and Department

of Microbiology, Roy and Lucille Carver College of Medicine, University of Iowa, Iowa City, Iowa 522422 Received 2 November 2005/Accepted 20 December 2005

Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Bacteria in biofilms are more resistant to the immune system and to antibiotics than their free-living planktonic counterparts. Thus, biofilm-related infections are persistent and often show recurrent symptoms. The metal chelator EDTA is known to have activity against biofilms of gram-positive bacteria such as Staphylococcus aureus. EDTA can also kill planktonic cells of Proteobacteria like Pseudomonas aeruginosa. In this study we demonstrate that EDTA is a potent P. aeruginosa biofilm disrupter. In Tris buffer, EDTA treatment of P. aeruginosa biofilms results in 1,000-fold greater killing than treatment with the P. aeruginosa antibiotic gentamicin. Furthermore, a combination of EDTA and gentamicin results in complete killing of biofilm cells. P. aeruginosa biofilms can form structured mushroom-like entities when grown under flow on a glass surface. Time lapse confocal scanning laser microscopy shows that EDTA causes a dispersal of P. aeruginosa cells from biofilms and killing of biofilm cells within the mushroom-like structures. An examination of the influence of several divalent cations on the antibiofilm activity of EDTA indicates that magnesium, calcium, and iron protect P. aeruginosa biofilms against EDTA treatment. Our results are consistent with a mechanism whereby EDTA causes detachment and killing of biofilm cells.

Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Bacterial biofilms are abundant in the environment and are involved in several human bacterial infections (reviewed in references 11, 14, and 31). Of medical importance, biofilms can withstand host im- mune responses (19–21) and are much more resistant to anti- biotic treatments than their nonattached, individual, free-living (planktonic) counterparts (28, 36). For these reasons, biofilm infections are persistent, and individuals often show recurring symptoms following antibiotic therapy. One of the best-studied models for biofilm formation is the bacterium Pseudomonas aeruginosa (reviewed in references 27 and 30), which causes many types of infections, including biofilm-associated chronic lung infections in cystic fibrosis patients, acute ulcerative ker- atitis in users of extended-wear soft contact lenses, and bacter- emia in severe-burn victims.

The metal chelator EDTA has been shown to cause lysis, loss of viability, and increased sensitivity of planktonic Proteo- bacteria to a variety of antibacterial agents (reference 13; re- viewed in references 25, 29, and 40). This has led to the use of EDTA as a preservative in many products. Little is known about the influence of EDTA on biofilms of Proteobacteria. Raad et al. (32, 33) have shown that EDTA combined with minocycline is an effective treatment for microorganisms embedded in biofilms on catheter surfaces. Their studies focused on Staphylococcus epider- midis, Staphylococcus aureus, and Candida albicans; however, they also reported two cases of P. aeruginosa-infected catheters where

* Corresponding author. Mailing address: Box 357242, Department of Microbiology, University of Washington School of Medicine, Seattle, WA 98195-7242. Phone: (206) 616-2881. Fax: (206) 616-2938. E-mail: epgreen @u.washington.edu.

† Supplemental material for this article may be found at http://aem .asm.org/.

the EDTA-minocycline treatment caused a large decrease in the number of viable biofilm cells (32). Recently, Kite et al. (23) reported that tetrasodium EDTA could be used to eradicate biofilms on catheters. Ayres et al. (3) have examined the effects of permeabilizing agents on antibacterial activity against a P. aeruginosa biofilm grown on a metal disk. Their results further suggest increased anti-P. aeruginosa biofilm activity for several antibiotics when combined with EDTA (3).

We have further characterized the activity of EDTA against P. aeruginosa biofilms. We show that EDTA treatment of Pseudomonas biofilms results in 1,000-fold greater killing than treatment with gentamicin, an antibiotic commonly used to treat P. aeruginosa infections. Furthermore, a combination of EDTA and gentamicin can result in eradication of P. aeruginosa in our model biofilms. We present evidence that, in addition to killing, EDTA causes a rapid dispersion of P. aeruginosa cells from biofilms. Our data suggest that magnesium, calcium, and iron are involved in P. aeruginosa biofilm maintenance.

MATERIALS AND METHODS

Bacterial strains and culture conditions. We used P. aeruginosa PAO1 (17). For the flow cell experiments, we used PAO1 containing pMRP9-1. The strain constitutively expresses green fluorescent protein (GFP) when carrying this plas- mid (12). Both flow cell and disk reactor biofilms were grown in 1% tryptic soy broth (TSB) (Becton Dickinson, Sparks, MD). All cultures were incubated at 37°C unless otherwise indicated.

Disk reactor biofilm experiments. The rotating disk reactor was similar to that described previously (16). Reactors were inoculated with stationary-phase cul- tures (1%, vol/vol). After overnight growth, a flow of fresh medium was initiated (dilution rate, 0.7 h 1). After 24 h in a flow of medium, the polycarbonate chips with attached biofilm bacteria were removed from the spinning disk and washed three times in phosphate-buffered saline (PBS). We assessed the resistance of biofilm cells to EDTA or antibiotics as follows. Washed biofilms were incubated in either 1 ml of PBS (pH 7.4) or 20 mM Tris buffer (pH 7.4). EDTA (0.1 to 50 mM), gentamicin (1, 10, and 50 g/ml), or a combination of the two was added as indicated. The chips were incubated for 1 or 24 h in 24-well tissue culture plates

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