## 3 Discussion
In the early stages, bacterial cells adhere to a surface in a reversible way by cell appendages such as flagella and type IV pili and, successively, they switch to irreversible attachment thanks to the involvement of specific proteins [35; 36]. Subsequently, the attached bacteria form microcolonies that spread into a more structured architecture and develop three-dimensional mushroom-like structures that are the hallmark of biofilm maturation [35]. Therefore, it is not surprising that in different phases, the same molecule can act to impair or enhance the biofilm development,as observed for the 40P strain. We can speculate that KHS-Cnd could have an effect, in the case of the 40P strain, on structures critical for the mature biofilm rather than in its pre-adhesion stage. Regardless, the obtained data did not show a homogeneous response of the tested strains to KHS-Cnd. This latter could be attributed to the high genotypic and phenotypic variability of _P. aeruginosa_ strains induced by the environmental conditions in the airway infections of CF patients [37]. Bacterial adaptation and evolution involve, indeed, variations that are also in the composition of extracellular matrix structure (e.g., qualitative and quantitative differences in polysaccharide content); the different structures exposed by the extracellular matrix during biofilm evolution can likely act as a target of the AMPs. VP-SEM analysis provided interesting information on the action of KHS-Cnd on the PA14 reference and 27P clinical strains. The morphological analysis highlighted that the effect of KHS-Cnd treatment on the biofilm formed by the PA14 strain, and moderately by the 27P strain, is not only related to a reduction in the biofilm amount, but also to a different compactness of the matrix. Bacteria were still mostly immersed in the matrix, but the matrix seemed more porous, without dramatic changes in the ECM filaments. Concerning protease activity, another virulence factor that we took into consideration, our results showed a strain-dependent effect of KHS-Cnd in terms of increased and/or decreased secretion in three out of six tested isolates. In the literature, little information is available on the protease secretion by _P. aeruginosa_ isolates from CF patients, whose pathogenicity is undoubtedly influenced by this virulence factor [38]. It has been suggested that the production of proteases could be a direct and rapid bacterial defense mechanism to counteract the AMPs [39]. Despite this, the 31P strain displayed a significant increase, of about 60%, in protease secretion, showing an inhibition in the pre-adhesion biofilm of 37%. KHS-Cnd was also able to reduce the adhesion to a biotic substrate such as pulmonary cell lines (one order of magnitude). Consequently, the invasion of host cells by _P. aeruginosa_ was also significantly impaired after treatment (the reduction was higher than 50%). Disrupting a biofilm without killing cells raises concerns about the further dissemination of a pathogen. A feasible solution to this risk could be the administration of AMPs in synergy with conventional antibiotics to overcome this effect [40]. With regard to the possible selection of resistant strains due to selective pressure exerted by AMPs on bacteria, it is conceivable that AMP exposure may lead to a lower number of resistant mutants compared with small-molecule antibiotics [41]. Indeed, if the peptide acts by weakening bacterial virulence and pathogenicity without affecting its viability, the evolutionary driving-force toward the development of resistance is certainly less strong.