Confocal microscopy.

Overnight cultures of P. aeruginosa were centrifuged at 5,000 × g for 5 min, washed with LB medium, and adjusted to an OD₆₀₀ of 0.1. The bacterial suspension was added to a 35-cm² plate and incubated at 37°C for 24 h. The formation of biofilm was then evaluated using the crystal violet staining technique (38). Bacterial cell viability within biofilms was also assessed using fluorescent markers. At 24 h, P. aeruginosa biofilms were washed three times with PBS and rinsed in 10 mM sodium phosphate buffer, 100 mM NaCl, pH 7.4, containing 1:1 Syto9/propidium iodide, DAPI, or FITC-WGA (both at 10 μg/μl) and incubated in the dark for 15 min. Afterwards, the stained biofilms were gently washed with PBS in order to remove the excess fluorescent dye. Syto9/propidium iodide staining allowed us to monitor the bacterial viability within the biofilm as the Syto9 dye is able to permeate the cell membrane and stain the nucleic acid of healthy cells; whereas, when the cell membrane is damaged, propidium iodide can diffuse into the cell and displace Syto9. On the other hand, DAPI/WGA staining allows us to monitor the antimicrobial action on the biofilm at both, exopolysaccharide (EPS) and cellular levels. The lectin WGA-FITC conjugated is able to interact with the β-N-acetylglucosamine bonds present on the EPS molecules, allowing the observation of the AMPs action on the EPS. At the same time, DAPI is able to stain the bacterial DNA, showing the antimicrobial effect on the bacteria that form the biofilm. Then biofilms were incubated with 10 μM protein/peptides for 4 h at 37°C. Changes were imaged using a laser scanning confocal microscope (Olympus FluoView 1000 equipped with a UPlansApo 60× lens in 1.4 oil immersion objective, United Kingdom).

For quantitative analysis of live/dead bacterial cells, biofilms were grown as above in a plate coverslip system. The grown biofilms were then washed three times with PBS. Then, each well was treated with 10 μM protein/peptide resuspended in 10 mM sodium phosphate buffer pH 7.5, 0.1 M NaCl and incubated at 37°C for 4 h. After incubation, the wells were washed three times with PBS to remove planktonic bacteria. Then, the biofilms were prestained using the SYTO9/propidium iodide 1:1 mixture provided by the Live/Dead staining kit (Molecular Probes, Eugene, OR, USA) and incubated in the dark at 37°C for 15 min. Three washes were performed to remove excess dye, and then the biofilm was imaged using a laser scanning confocal microscope (Olympus FluoView 1000, equipped with an UPlansApo 40× lens with a 1.4 numerical aperture oil immersion objective. Mortality was calculated as the mean area of 10 randomly selected fields and quantified using ImageJ software (40). Unspecific background was subtracted, and particles with a diameter larger than 0.8 μm were analyzed. Biofilm three-dimensional reconstruction and estimation of the biofilm depth were performed with the IMARIS Bitplane imaging software (Oxford Instruments, Zurich, Switzerland).