## 3 Discussion  
Several studies have shown that some AMPs possess antibiofilm properties, while others are devoid of such activities [11; 14]. There are also examples of naturally occurring bioactive peptides, such as the atrial natriuretic peptide, that have no antimicrobial activities against planktonic cells, but that can interfere in bacterial biofilm formation (e.g., [65]). While the chemical features that make a potent AMP are now quite well understood (cationicity, hydrophobicity, amphipathic membrane-bound structure), there are currently no well-established design features for developing antibiofilm peptides. This is probably related to the fact that widely different mechanisms of action have been proposed that can achieve antibiofilm action [14; 66; 67]. Moreover, cells directly dispersed from biofilms  
Figure 8: Two-dimensional ROESY spectrum of 1M tritrpticin-NH\({}_{2}\) (Tritrp-Arg) were collected in aqueous solution at 37 \({}^{\circ}\)C. The imino-region of the spectrum is shown, and the observed exchange interactions are depicted below over the 1D spectrum. A, B and C refer to the three main peaks observed. seem to have unique properties when compared to normal planktonic cells, providing further complexity [68]. Hence, the screening of AMPs for antibiofilm properties, such as performed in this study, is currently the best way to uncover new antibiofilm peptides. For example, through a screening approach, we recently identified that short Trp- and Arg-rich hexapeptides are very potent at eliminating biofilms from an MRSA strain of _S. aureus_[23]. These peptides with only six amino acids residues were much shorter than the twelve to thirty residues that are normally cited as being required to form an antibiofilm peptide [14; 20]. While theoretical QSAR studies [69] have had some success in uncovering some new 12-residue peptides, this approach could not identify the chemical or structural features that characterize a potent antibiofilm peptide. Interestingly, one of the most potent QSAR-predicted peptides was quite rich in Trp and Arg residues [69]. However, it lacked the Pro residues and the proximal Trp residues that characterize indolicidin and tritrpticin. In studies with several peptides, it has been observed that some AMPs can display antibiofilm activities at concentrations that are lower than their MIC values, which represents the concentration of an AMP required to kill planktonic cells (see, for example, [11; 70]). Indeed, in their early studies, [PERSON] et al. demonstrated that LL37 and indolicidin both displayed such antibiofilm activities, while several other known AMPs did not show any activity, revealing that such effects are highly peptide-specific. Also, in our current work, we see such concentration effects, for example, comparison of the MIC and MBIC's curves for Tritrp-P9A in Figures 1 and 4 show that the latter curve is shifted to lower peptide concentrations. Likewise, in Figure 2, the shape of the curve for the MBEC suggests that more than half of the cells are already removed at a concentration of 4 uMol/L, which is below the MIC value. However, removing the remaining cells requires substantially higher peptide concentrations, suggesting that not all biofilm-entrapped bacteria in our MBEC assays are equally accessible to the AMPs. In previous studies, it has been shown that the Trp-rich AMPs indolicidin and puroin-dolineA can display antibiofilm activities against several pathogenic bacteria [11; 42]. In view of their comparable Trp-rich amino acid sequences, both these peptides are related to Tritrp, and hence we expected that the latter would display antibiofilm properties as well. Indeed, our assay results (summarized in Tables 2 and 5) support this presumption, and they show that of these three Trp-rich peptides that are of equal length, Tritrp has the most promising antibiofilm activities.