Introduction 
Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections.
It is the third-leading cause of nosocomial infections and is the predominant pathogen associated with morbidity and mortality of CF patients [1],[2].
The biofilm-forming ability of P. aeruginosa, and indeed other bacteria, is thought to contribute to their ability to thrive in hostile host environments and result in chronic infection [3],[4].
Biofilms are multicellular surface-associated microbial communities encased in an extracellular matrix which display a characteristic structure and increased resistance to antimicrobial compounds and environmental stresses.
P. aeruginosa biofilms are up to 1000-fold more antibiotic tolerant than planktonic cells, to single and combination antibiotics [5]-[7].
As acute CF exacerbations caused by P. aeruginosa are often treated with combination antibiotic therapy [8]-[10], the increased resistance of biofilms to combination antibiotics is of direct clinical relevance.
Eighty five percent of P. aeruginosa strains isolated from the lungs of CF patients with advanced stages of disease have a distinctive mucoid colony morphology [11].
This mucoid phenotype is a result of overproduction of the alginate exopolysaccharide (EPS) [1],[12].
Alginate production has been shown to inhibit phagocytic killing of Pseudomonas, to protect from antibiotic exposure [13],[14], and is associated with poor prognosis for the infected patients [15],[16].
The direct observation of P. aeruginosa microcolonies encased in an alginate matrix in microscopy studies of CF bronchial samples [17], along with a large body of additional in vitro and in vivo data [7], [18]-[21] suggests that P. aeruginosa forms biofilms in the lungs of CF patients.
The mechanisms of biofilm-associated antibiotic resistance are distinct from the well studied intrinsic resistance mechanisms such as drug efflux, drug inactivation, membrane permeability and target site alterations.
Although the basis of biofilm-associated antibiotic resistance is not fully understood, it is likely that multiple mechanisms operate simultaneously in biofilms to contribute to antibiotic resistance.
Cells in a biofilm may be protected from antibiotic exposure due to the restricted penetration of antibiotics through the biofilm matrix [19].
However, while the biofilm matrix may limit diffusion initially for certain antibiotics such as beta-lactams and aminoglycosides [14],[22], the penetration of fluoroquinolones occurs immediately and without delay [23]-[25].
The rate of diffusion through the matrix is presumably dependent on binding of the antibiotic molecules to the EPS matrix.
Once the matrix becomes saturated, diffusion and antimicrobial activity of the drug will resume [26].
It is the general consensus that reduced diffusion through the biofilm matrix only provides a short-term protective effect and does not play a significant role during long-term antibiotic exposure [26].
Other resistance mechanisms include the presence of subpopulations of multidrug tolerant persister cells [27]-[29], drug indifference of slow-growing, nutrient-limited cells [30], and unique resistance mechanisms specifically associated with biofilms [31],[32].
Despite the fact that biofilms are recognized as the predominant mode of bacterial growth in nature and are responsible for the majority of refractory bacterial infections [19], little is known regarding the mechanisms of biofilm-specific antibiotic resistance.
Furthering our understanding of the mechanisms underlying biofilm-associated antibiotic resistance will significantly improve the treatment options available to patients with chronic bacterial infections.
Signal transduction systems have been documented to be involved in the regulation of biofilm formation in multiple bacterial species including P. aeruginosa, S. aureus, E. coli and V. fischeri [33]-[38].
These two component systems (TCS) are comprised of an membrane-anchored histidine kinase sensor and a cytoplasmic response regulator.
After detecting specific environmental signals, a signal transduction cascade is initiated that results in phosphorylation of the response regulator, which activates or represses the necessary target genes.
A number of regulatory systems that influence biofilm formation have been described.
These include, but are not limited to, the global virulence factor regulator GacA, mutation of which results in a 10-fold decrease in biofilm formation and failure to form microcolony structures [33].
Additionally, the hybrid sensor kinases, LadS and RetS appear to work upstream of GacA to possibly control the switch to a biofilm lifestyle [34],[35].
Mutations in algR, a response regulator protein required for synthesis of alginate, which is a major component of the matrix of biofilms in the cystic fibrosis lung [1] results in a P. aeruginosa strain that has decreased type IV pili-dependent motility and biofilm formation [39].
The three-component system SadARS which regulates the formation of mature microcolonies [40] and PvrR, a response regulator involved in the switch from planktonic to antibiotic-resistant biofilm cells in P. aeruginosa are additional examples of regulators of biofilm formation [41].
During the course of an infection, one of the first lines of defense encountered by colonizing bacteria is the production of cationic antimicrobial peptides (CAPs) by a variety of host cells including neutrophils, platelets and epithelia.
CAPs are short, amphipathic peptides that bind to and disrupt both the outer and cytoplasmic membranes resulting in cell death.
The broad-spectrum antimicrobial activity of CAPs against Gram-negative and Gram-positive bacteria accounts for their role as an essential component of the innate immune response of humans, animals and insects.
Cationic peptides, which have antimicrobial and immunomodulatory activities, are being developed as a promising new class of therapeutically relevant drugs [42].
In P. aeruginosa, resistance to CAPs is inducible by the PhoPQ and PmrAB TCSs, both of which are activated independently in response to limiting Mg2+ [43]-[46].
Under conditions of limiting magnesium, PhoP and PmrA bind to the promoter of the CAP resistance operon PA3552-PA3559 (arnBCADTEF-ugd) and induce its expression [45]-[47].
These genes encode an LPS modification pathway required for the addition of aminoarabinose to lipid A, which reduces the OM permeability to CAPs [48].
The PhoPQ and PmrAB regulatory systems are well studied in planktonic cultures and have been shown to induce modest resistance to CAPs (8-fold) under low Mg2+ conditions [45].
However, while the PA3552-PA3559 operon has been reported to be expressed in biofilms cultivated in flowcells, and is required for survival in response to colistin treatment [49], little else is known regarding these systems and the role they may play in biofilm-associated antibiotic resistance.
The extracellular matrix of P. aeruginosa biofilms includes extracellular DNA [50],[51], multiple bacterial exopolysaccharides and host proteins [4],[52].
Extracellular DNA, which is a matrix component of both Gram-positive and Gram-negative bacterial biofilms [51],[53], functions to maintain the 3D biofilm architecture by acting as a cell-cell interconnecting compound [50].
Genomic DNA has been shown to localize to the biofilm surface, surrounding the mushroom-shaped microcolonies [51].
DNA in the biofilm matrix is likely released by dead bacteria or immune cells.
It has been reported that prophage-mediated cell death is an important mechanism in the differentiation and dispersal of biofilms [54],[55].
Additional sources of DNA in biofilms may include the quorum sensing regulated release of DNA [51] and/or DNA contained within outer membrane vesicles (OMV) that bleb and are released from the OM of living P. aeruginosa cells [56],[57].
Furthermore, while a specific mechanism of DNA release has not been reported for P. aeruginosa it is possible that such a method may exist, similar to the autolysin-mediated DNA release observed in Staphylococcus epidermidis biofilms [53].
In this study we sought to examine if the presence of DNA in biofilms may contribute to biofilm-specific antibiotic resistance.
Here we identify a novel cation chelating property of DNA, which has several important consequences for biofilm physiology and antibiotic resistance in biofilms.
