DNA induces resistance to CAPs and aminoglycosides 
To determine if DNA-induced expression of PA3552-PA3559 resulted in increased resistance to antimicrobials, antibiotic susceptibility testing of P. aeruginosa biofilms grown in the presence and absence of extracellular DNA was performed. Biofilms were cultivated on 96-well format, polystyrene pegs submerged in BM2 defined media, with or without sub-inhibitory concentrations of extracellular DNA (0.75% (w/v)), and challenged with antibiotics. After antibiotic challenge, this assay allows for determination of both the minimum inhibitory concentration (MIC) of planktonic cultures and the minimum biofilm eradication concentration (MBEC). Consistent with previous results reporting on the antibiotic resistance phenotype of bacterial biofilms [6],[19], the MBEC values of biofilms cultivated in magnesium-replete conditions and treated with CAPs (polymyxin B, colistin) or aminoglycosides (gentamycin, tobramycin) were up to 64-fold higher than the MIC values of planktonic cultures (Table 1). The MBEC values of biofilms supplemented with extracellular DNA were 8 and 64-fold more CAP and aminoglycoside resistant than biofilms without exogenous DNA, respectively (Table 1). DNA-enriched biofilms were dramatically more resistant than planktonic cultures, up to 256-fold, and this resistance phenotype to CAPs and aminoglycosides was also observed in planktonic cultures supplemented with DNA. The simple addition of sub-inhibitory DNA amounts to planktonic cultures closely simulated the resistance-inducing effects of DNA in a biofilm (Table 1). The MIC values for polymyxin B and gentamicin are equal to 1 microg/ml and 2 microg/ml, respectively, using the standard microbroth dilution method for antimicrobial susceptibility testing (National Committee on Clinical Laboratory Standards (NCCLS) protocol) (data not shown). Thus, depending on the method used to determine the MIC (CBD or NCCLS protocol), DNA-enriched biofilms can be up to 2560-fold more polymyxin B resistant and up to 640-fold more aminoglycoside resistant than planktonic cultures. DNA-enriched biofilms did not show an increased tolerance to ceftazidime (beta-lactam) or ciprofloxacin (fluoroquinolone) (data not shown). Since extracellular DNA is a natural matrix component of PAO1 biofilms (Fig 5A and 5B), DNA-induced antibiotic resistance is likely to be a phenomenon unique to biofilms or other DNA rich environments. The presence of DNA in peg-cultivated biofilms (Fig 5A), grown in the absence of exogenous DNA, likely contributes to the increased antibiotic resistance generally observed in biofilms (Table 1). We have shown previously that the PA3552-PA3559 operon is required for resistance to cationic antimicrobial peptides in planktonic cultures grown in limiting magnesium conditions [47]. To determine if DNA-induced resistance requires these genes in biofilms, the resistance phenotype of the PA3553::lux mutant was determined. PA3553::lux had no significant DNA-induced CAP resistance in biofilm or planktonic cultures, confirming that these genes are essential for CAP resistance in the presence of extracellular DNA (Table 1). The PA3553 mutant also displayed decreased DNA-induced resistance to aminoglycosides compared to PAO1. The differences observed between CAP and aminoglycoside resistance in PA3553::lux suggests that DNA-induced resistance to aminoglycosides is not limited to PA3553 induction. The biofilms formed by the PA3553::lux mutant were unaltered compared to PAO1 biofilms under these conditions, ensuring that the difference observed was not due to an altered biofilm phenotype (Fig 6B). The CAP resistance phenotype of biofilms grown in limiting magnesium (20 microM) was similar to biofilms grown in DNA, confirming that DNA imposes a magnesium limitation stress (Table 2). Biofilms that were exposed to DNA during either the cultivation or challenge stages only, showed similar resistance profiles to biofilms grown and challenged in magnesium-replete conditions (Tables 1-2). Therefore, the DNA-induced resistance of biofilms requires both the cultivation and challenge under cation-limiting conditions. These latter two observations rule out the possibility that negatively charged DNA simply interacts with cationic antimicrobial peptides and prevents their access to bacterial cells.
