## 2 Results and Discussion
### The Anti-Biofilm Activity of P13#1  
Many CAMPs show the ability to inhibit biofilm formation, and the most effective are even able to eradicate preformed biofilms [67; 68; 69]. Accordingly, we studied the ability of P13#1 to eradicate a preformed biofilm of _P. aeruginosa_ PAO1. In this case LL-37, the sole human cathelicidin, was used as a positive control because its antibiofilm activity is well known [69]. Overnight bacterial cultures were diluted to 1 \(\times\) 10\({}^{8}\) colony-forming units/mL (CFU/mL) in BM2 medium, and the bacterial biofilms were formed for 24 h at 37 \({}^{\circ}\)C in 96-well plates. The eradication of the mature biofilms was evaluated by incubating the biofilms with increasing concentrations (0-30 \(\upmu\)M) of LL-37 or P13#1 for 4 h. The biofilm mass and cell viability were measured using crystal violet and XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium) reduction assays, respectively, and the minimum biofilm eradication concentration (MBEC) was determined by counting the viable bacterial cells inside a biofilm's structure upon the disruption of the biofilm with 1% Triton X-100 (Figure 3).  
LL-37 induced both a reduction in the biofilm's mass and a strong reduction in cell viability at concentrations \(\geq\) 12.5 \(\upmu\)M (Figure 3A). These data were confirmed by deter  
Figure 3: The antibiofilm activity of LL-37 (**A**) and P13#1 (**B**) on _P. aeruginosa_ PAO1 and the antibiofilm activity of P13#1 on _P. aeruginosa_ RP73 (**C**). Blue circles—the amount of biofilm-bound crystal violet, expressed as a percentage with respect to the untreated wells. Red circles—XTT reduction, expressed as percentage with respect to the untreated wells. (**D**) Percentage of the CFU remaining after the treatment.  
mining the percentage of the CFU remaining after the treatment. At 25 \(\upmu\)M, LL-37 caused a reduction of slightly more than 99%. However, we were not able to measure an MBEC value, which is usually defined as the concentration of an antibiofilm agent which reduces the CFU by 99.9% or more. Differing from LL-37, P13#1 did not significantly reduce the biofilm's mass (Figure 3B) but caused a larger reduction in the cell viability, beginning from the lowest concentration assayed (1.56 \(\upmu\)M), and a larger reduction in the CFU. Indeed, at concentrations of P13#1 \(\geq\) 20 \(\upmu\)M, no colony was observed, thus allowing for an MBEC = 20 \(\upmu\)M to be defined. Therefore, it can be concluded that LL-37 kills the cells embedded into the biofilm to some extent and partially solubilizes the biofilm matrix, whereas P13#1 is not able to disrupt the biofilm's architecture; however, it is more efficient at killing the embedded cells.  
We also analyzed the antibiofilm effects of P13#1 on the clinical stain _P. aeruginosa_ RP73, which is a very efficient biofilm-former (Figure 3C). The obtained results were similar to those observed on PAO1; however, it was not possible to define an MBEC value since even at the highest concentrations tested, we still found about 0.2-0.3% of the CFU. It could be speculated that the _P. aeruginosa_ RP73 biofilm contains a subpopulation of cells which were not accessible or not sensitive to the peptoid. This hypothesis would be consistent with the origin of _P. aeruginosa_ RP73, a late clinical strain of _P. aeruginosa_ adapted to cause chronic infections.