## 2 Results
### Effect of KHS-Cnd on Biofilm Formation
In this case, bacterial strains were grown in BHI medium in microtiter plates for 24 h at 37 \({}^{\circ}\)C; then, the growth medium was replaced with BHI containing or not containing KHS-Cnd (40 \(\mu\)M for all strains except for 32P, where we used 20 \(\mu\)M). The microtiter plates were then incubated for a further 24 h at 37 \({}^{\circ}\)C. Surprisingly, after the addition of the peptide, in four out of the six tested strains, biofilm reduction was observed, ranging from about 20 to 33% (Figure 1, panel B). Nevertheless, it is worth noting that the biofilm measured after 48 h of incubation was more abundant and doubtless more structured and difficult to eradicate. Interestingly, in the 40P clinical isolate, KHS-Cnd was also able to disaggregate about 25% of the preformed biofilm. These results suggest that KHS-Cnd action is not restricted to the initial bacterial attachment on the abiotic surface, but is also effective on mature biofilm. Ultrastructural Morphology of P. aeruginosa PA14 and 27P Strains Following the Treatment with KHS-Cnd  
Ultrastructural morphology of _P. aeruginosa_ biofilm was observed using a variable pressure scanning electron microscope (VP-SEM). This analysis was performed on the PA14 reference strain and the 27P clinical strain treated or not-treated with KHS-Cnd. 27P was chosen because it was a good biofilm former and KHS-Cnd strongly disaggregated its mature biofilm. The observation using VP-SEM of the untreated _P. aeruginosa_ PA14 samples (Figure 2A,B) showed that the sample was characterized by the presence of an abundant extracellular matrix (ECM), whose surface appeared irregular, being smooth and compact (A, asterisk) in some areas while in others, it appeared spongy and rough (A, star). At higher magnification (B), the ECM area with the spongy appearance was appreciable in detail. The three-dimensional network structure was well visible and included larger meshes in some areas (arrow) and smaller ones in others (dotted oval). Network meshes were lined by ECM trabeculae that interconnected one another, developing a regularly branching trabecular system, and the overlapping of the meshes created a labyrinthic system of narrow channels. The treatment with KHS-Cnd induced relevant ultrastructural modifications, as illustrated in Figure 2C,D. The picture at low magnification (C) showed a spongy appearance of the sample, almost on all of its surface (C, Star); compact and smooth areas were rare (C, asterisk). The image at higher magnification (D) clarified that the spongy areas contained trabeculae formed by aggregates of fine granular filaments, showing a \"pearl necklace\" aspect (arrow). Comparing Figure 2B of the control sample with Figure 2D of the treated sample, the disaggregating effect of KHS-Cnd was evident. Its action was exerted homogeneously on the sample surface and was not too aggressive. Filaments forming the trabeculae appeared compactly assembled in the control sample, and, after the treatment, were arranged more loosely, increasing the overall sample porosity. The low aggressiveness of the treatment was also revealed by the observation that, even if we had an increase in porosity, this aspect was not dramatically changed: ECM filaments maintained their fine and regularly granulated aspect, and no filaments had curly free endings, which means that filament disruption was observed. To further quantitatively analyze the effect of KHS-Cnd on the _P. aeruginosa_ PA14 ECM ultrastructure, we used the Hitachi Map 3D Software (v.8.2, Digital surf, Besancon, France). The three-dimensional reconstruction of the samples and the circular area extracted are magnified and shown in false colors in Figure 3A-D for a representative experiment. High elevated areas corresponding to the ECM trabeculae lining the meshes are represented in green. The deep channels of the labyrinthic system are represented in blue.