Anti-biofilm Properties

The biofilm eradication assay was carried out by crystal violet staining as described previously (Sass and Bierbaum, 2007). MDR P. aeruginosa strain 8328 was cultured in the wells of a polystyrene 96-well plate (BD Falcon) at 37°C for 48 h to allow biofilm formation. The planktonic cells in the culture were discarded and the plate washed with phosphate-buffered saline three times. LysPA26 (100 μg in 200 μl) was added to the plate wells, incubated at 37°C for 2 h, and then washed twice with phosphate-buffered saline. Crystal violet (100 μl 1%) was added and incubated for 30 min; subsequently, 33% acetic acid was added to dissolve the stain, and the optical density was measured at OD₆₀₀. The elimination activity of LysPA26 was also measured by enumerating the reduction of the residual live biofilm cells (Fu et al., 2010). Briefly, after discarding planktonic cells, the biofilm cells were treated with LysPA26 for 2 h. The contents of the treated biofilm-containing wells were mixed fully with a pipetting device, followed by sonication at 42 kHz in a water bath sonicator to make the biofilm cells become planktonic cells. For each experiment, samples were analyzed in triplicate.