###### Abstract
However, the morphology and overall appearance of the biofilms made by these two organisms is quite distinct, and several studies have shown that they are sensitive to different concentrations of antib Biofilm peptides [21]. Moreover, the process of biofilm formation and the efficacy of the subsequent attack by antib biofilm peptides further depends on the culture medium in which the cells are grown [21]. In two previous studies [22; 23], we tested two different groups of antimicrobial peptides for their potency as antibiofilm agents acting on pathogenic _P. aeruginosa_ PA01 and on _S. aureus_ MRSA strains. Under the experimental conditions employed in those studies it was found that biofilms formed by the Gram-positive _S. aureus_ strain were extremely sensitive to very low concentrations of Trp- and Arg-rich AMPs. In particular, hexappeptides made up of three Trp and three Arg residues could disrupt the _S. aureus_ biofilms or prevent their formation, while being mostly ineffective against _P. aeruginosa_. Hexappeptides containing the three Trp in sequence (e.g., RRWWWR-NH\({}_{2}\)) proved to be the most effective and at higher concentrations, these could also act on _P. aeruginosa_[23]. These results prompted us to consider using longer Trp- and Arg-rich peptides as potential antibiofilm agents against the latter Gram-negative strain. For this purpose, we selected the 13-residue porcine cathelicidin peptide tritrpicin, which naturally contains the WWW sequence at its center and has an additional four Arg residues and a positively charged N-terminal end distributed around it (VRRFPWWWPFLRR). Tritrpicin was first described in 1996 [24] and has since been shown to have broad antimicrobial, antifungal as well as anticancer activities [25]. In an animal model study, its potential was shown for helping combat sepsis [26]. Tritrp is part of a group of potent, yet relatively short Trp- and Arg-rich AMPs; their shorter length makes them more economical to produce and hence they are an interesting target for further study [27; 28]. Numerous variants of Tritrp, with natural and unnatural amino acid substitutions, have already been studied [29; 30; 31; 32; 33; 34]. In most studies, it has been found that Tritrp acts as an antimicrobial on planktonic cells by perturbing bacterial membranes, although additional intracellular modes of action have not been excluded [31; 34]. In this contribution, we used three distinct antibiofilm assays to study an amidated version of the Tritrp peptide and several analogs with selected amino acid substitutions. To our knowledge, this is the first time that the antibiofilm activity of Tritrp and its analogs have been surveyed. All the peptides used in this study were carboxy-amidated because the removal of the C-terminal negative charge improves the antibacterial activity, as is commonly seen for many AMPs [35]. The antimicrobial (MIC) and hemolytic activities of the majority of these peptides have been reported before, as well as their structural characterization by NMR spectroscopy [30; 36; 37]. The amino acid sequences of the Tritrp analogs used in this study, as well as those of the related Trp-rich bovine cathelicidin peptide indolicidin and the plant puorioIndicine A peptide which were also analyzed in this work, are indicated in Table 1[38; 39; 40; 41; 42]. In membrane-mimicking environments, Tritrp normally adopts a well-defined amphipubic two turn structure, where the two Pro residues are involved in the formation of the two turns in the peptide backbone [36]. Structural NMR and spectroscopic studies have shown that substitution of both Pro residues by Ala in Tritrp results in the creation of a mostly alpha helical backbone structure ([37; 43], see also [44]). In a similar fashion, substitution of Pro residues by Ala in the related Trp-rich AMP indolicidin also led from a turn structure to a more alpha-helical structure [45; 46]. Our results show that several Tritrp analogs possess good antibiofilm activities against _P. aeruginosa_.