## 3 Discussion  
The global spread of multidrug resistant strains is a major concern for many bacterial species including _P. aeruginosa_ as they are highly pathogenic compared to other Gram-negative bacteria and are able to survive and persist in various environments. Therefore, it is not surprising that _P. aeruginosa_ can be found alive in the hostile lung environment of CF patients, thus evading the host immune system, leading to morbidity and mortality in CF and immunocompromised patients [25]. Unfortunately, this opportunistic pathogen is resistant to common available antibiotics such as aminoglycos, quinolones, and \(\beta\)-lactams [17; 26]. In addition to known intrinsic and acquired resistance mechanisms, _Pseudomonas_ also uses an adaptive antibiotic resistance that includes virulence factors such as biofilm formation, motility, and the secretion of toxins and proteases [27]. In the lungs of infected patients, biofilm acts as a physical barrier formed by a robust extracellular matrix (ECM) encapsulating resilient communities of surface-adhered bacteria resistant to antibiotics; bacteria in biofilm are responsible for chronic recalcitrant infections of CF patients. Hence, there is a need to develop innovative approaches that are able to increase the sensitivity of a pathogen to the available therapies. On the other hand, the development of new antibiotics is very limited and time-consuming, and quite costly from an industrial point of view. In this scenario, to counteract _Pseudomonas_ infections, novel therapeutic strategies could also be used to implement the conventional ones. In this regard, it could be promising to target _P. aeruginosa_ virulence factors playing a relevant role in chronic infections, thus increasing bacterial susceptibility to known antimicrobials [27]. In this context, antimicrobial peptides (AMPs) are new potential candidates as anti-virulent compounds: they could be more useful as anti-biofilm molecules than conventional antibiotics, since their target is the bacterial membrane and their action leads to cell death, regardless of metabolic state [28]. Moreover, several AMPs (such as defensin, LL-37, IDR-1018, GL13K, NLF20, melittin, magainin II, ranalexin, T9W, cecropin P1, indolicidin, and nisin) have shown antimicrobial activity against _P. aeruginosa_ via either the reduction/inhibition of bacterial viability or biofilm formation [26; 27; 28; 29]. Most of these anti-pseudomonal peptides have been isolated from humans or animals [18]. In this study, we focused on a novel AMP derived from the so-called Chionodracine (Cnd) peptide, isolated from Antarctic iciefish _Chionodraco hamatus_, which represents an untapped reservoir of biodiversity [20]. Starting from the natural peptide sequence (22 amino acids), Cnd was modified by introducing charged residues; namely histidines and serines were all replaced by lysines, thus obtaining the mutant of interest named KHS-Cnd. We previously demonstrated that KHS-Cnd versus Cnd: (i) selectively disrupts bacterial versus mammalian membranes; (ii) displays a reduced cytotoxic activity against human primary cells and a low hemolytic activity; and (iii) shows a significantly high bactericidal activity against the ESKAPE pathogens [21; 22]. \begin{table}
\begin{tabular}{c c c c c} \hline  & **Untreated** & \multicolumn{2}{c}{**KHS-Cnd Treated**} \\ \hline  & **Adhesion** & **Invasion** & **Adhesion** & **Invasion** \\ \hline _P. aeruginosa_ 27P & 3.02 \(\times\) 10\({}^{5}\)\(\pm\) 0.21 \(\times\) 10\({}^{5}\) & 8.06 \(\times\) 10\({}^{3}\)\(\pm\) 0.08 \(\times\) 10\({}^{3}\) & 2.53 \(\times\) 10\({}^{4}\)\(\pm\) 0.45 \(\times\) 10\({}^{4}\) & 3.0 \(\times\) 10\({}^{3}\)\(\pm\) 0.2 \(\times\) 10\({}^{3}\) \\ \hline \end{tabular} Data represent the mean \(\pm\) SD of three independent experiments.  
\end{table} 
Table 2: The adhesion and invasion capabilities of _P. aeruginosa_ on A549 cells in the presence and absence of 40 μM KHS-Cnd. Given the above, we tested the anti-virulence effect of KHS-Cnd against _P.