## 3 Discussion
aeruginosa_ clinical isolates from CF patients. We first analyzed the anti-biofilm activity of KHS-Cnd in the pre-adhesion phase of biofilm growth and on the mature biofilm. Notably, anti-biofilm peptides are biochemically equivalent to AMPs: they share the same amino acid length (12-50), amphipathic properties in terms of basic residues (2-9 residues of K or R), and the amount of hydrophobic residues (about 50% on total) [29]. Different AMPs have been demonstrated to prevent biofilm attachment, starting from the observation of their antimicrobial activity on planktonic bacteria [30], although some do not act on mature and well-structured biofilm. We, herein, demonstrated that KHS-Cnd impaired biofilm development without affecting bacterial viability up to the tested concentration of 40 uM (and 20 uM for 32P strain). Similarly, among the AMPs tested against different clinical isolates of _P. aeruginosa_ from CF patients, the ability of the IDR-1018 peptide to prevent biofilm formation has been reported, despite its weak activity against planktonic bacterial forms [31]. Regarding biofilm inhibition by KHS-Cnd, it affected the biofilm development of five out of six tested strains with different efficacy, while only 40P strain biofilm formation was strongly promoted by KHS-Cnd; notably this strain was the lower biofilm producer compared to the others (Table 1). Since biofilm development is a multifactorial event, this result is not entirely unexpected. Particularly interesting is, in contrast, the disaggregation induced by KHS-Cnd on the mature biofilm, since it is notoriously very hard to disrupt an established biofilm. The IDR-1018 peptide also displayed disaggregating activity on the mature biofilm produced by _Pseudomonas_ clinical isolates, but the underlying molecular mechanisms still remain to be clarified [31]. In general, it has been hypothesized that AMPs' mechanism of action targets bacterial species-specific properties such as motility, cell adhesion, and quorum sensing [29]. In particular, for the anti-biofilm broad spectrum activity of IDR-1018, the authors evidenced that this peptide induces the degradation of the second messenger (p)ppGpp nucleotides, which play an important role as signals in biofilm formation and maintenance [32]. Several published studies report AMPs' ability to interfere with various stages of biofilm formation because they prevent initial bacterial adhesion to surfaces or destroy mature biofilms by detachment and/or killing of biofilm-embedded bacteria. Most AMPs are more effective in inhibiting the early phases of biofilm development rather than in eradicating the mature biofilm. This is probably due to the multiple interactions that AMPs may establish with the main components of the extracellular matrix (DNA, polysaccharides, and proteins) that surround and protect bacteria in the mature biofilm [33]. Moreover, we observed a disruption of the mature biofilm in four out of six bacterial strains (Figure 1, panel B). The disrupting activity was even evidenced on the 40 P strain despite the peptide not showing an inhibitory activity during the pre-adhesion period of the same strain. Notably, there was no effect on the 23 P strain, whose biofilm at 48 h was clearly lower compared to those produced by other strains. We can speculate that the effect of KHS-Cnd seemed to be more marked for stronger biofilm producers. Concerning the 23 P strain, the biofilm measured at 48 h was much thinner than that observed at 24 h. This phenomenon is not surprising, since biofilm development is the result of a dynamic process characterized by a fluctuation in biofilm amplification and the cell detachment phases, as already reported [34]. Biofilm development is a multi-stage process that requires different actors playing in temporally distinct moments in the different phases.