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

Abstract

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 immune responses (19)(20)(21) and are much more resistant to antibiotic 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 keratitis in users of extended-wear soft contact lenses, and bacteremia in severe-burn victims.The metal chelator EDTA has been shown to cause lysis, loss of viability, and increased sensitivity of planktonic Proteobacteria to a variety of antibacterial agents (reference 13; reviewed 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 epidermidis, Staphylococcus aureus, and Candida albicans; however, they also reported two cases of...

The assessment of the antibacterial and antifungal activities of aspirin, EDTA and aspirin-EDTA combination and their effectiveness as antibiofilm agents

“…Several recent publications have indicated that EDTA alone and in combination is an effective antibiofilm agent with a spectrum covering both Gram-positive and Gramnegative bacteria as well as C. albicans (Raad et al 2003(Raad et al , 2007Kite et al 2004;Percival et al 2005;Banin et al 2006;Sherertz et al 2006). Its antibiofilm effect was suggested to be because of its capability to sequester the divalent ions essential to the extracellular polymeric matrix structure of biofilms (Percival et al 2005).…”

“…Nevertheless, both agents were reported to possess a weak and broad-spectrum antimicrobial activity towards some planktonic and biofilm cultures (Root et al 1988;Gil et al 1994;Nicolau et al 1995;Raad et al 2003;Alem and Douglas 2004;Stepanovic et al 2004;Percival et al 2005;Banin et al 2006;Yakandawala et al 2007).…”

The potential of desferrioxamine-gallium as an anti- Pseudomonas therapeutic agent

“…All incubations for spinning disk experiments were at 37°C. The spinning disk biofilms were then exposed to the indicated agents in water for 24 h without shear (31). To estimate the number of remaining attached biofilm cells, we placed the discs in 1 ml PBS and dispersed the cells by using a tissue homogenizer (Brinkman Homogenizer).…”

“…To determine the affect of an agent on mature biofilms the biofilm was allowed to grow for six days before addition of the test compound. To discriminate live and dead cells we used propidium iodide (PI, 30 M, Sigma Chemical Co.) as described elsewhere (31). We also used a spinning disk reactor as a third method to study biofilms (32).…”

Selection of hyperadherent mutants in Pseudomonas putida biofilms

“…Genetic determinants that play a role in biofilm formation have been unravelled in different bacterial species, and environmental and cellular signals that influence this process have also been described. These include iron availability, quorum sensing, and the intracellular secondary messenger cyclic di-GMP (Banin et al, 2006;Patriquin et al, 2008;Ueda & Wood, 2009;Coenye, 2010). Changes in the levels of cyclic di-GMP correlate with phenotypic changes associated with virulence, motility, colony morphology, production of exopolysaccharides, and the transition between planktonic and sessile lifestyles (Hengge, 2009;Römling & Simm, 2009, and references therein).…”