## 3 Discussion
By itself, the latter phospholipid does not form stable bilayers, and this could contribute to the bacterial membrane instability and to antibiofilm action [84; 90]. The WWW motif of the hexapeptides was shown to be important to this action, and since Tritrp also carries this motif, it is possible that some membrane unmixing occurs here as well although the cytoplasmic membrane composition in Gram-negative strains is somewhat different than in Gram-positive strains [84]. Moreover, as discussed above, since longer antibiofilm peptides are often active as antibioofilm agents at concentration below the MIC, it is tempting to speculate that the enhanced lipid flip-flop seen in the recent studies [88; 89] also plays a role in the antibiofilm action against _P. aeruginosa_. Indeed, it has been shown that Tritrp, like indolicidin, can also cause lipid flip-flop for model bacterial membranes [37]. Finally, as Tritrp, indolicidin and puroinodine A can enter bacterial cells, where they have been shown to disturb macromolecular DNA, RNA and protein synthesis, these peptides could also bind to intracellular regulatory nucleotides, such as (p)ppGpp. Another well-studied antibiofilm peptide (IDR-1018), can bind to (p)ppGpp, and has been shown to also give rise to antibiofilm dispersal activities at low peptide concentrations [91]. While this peptide may exert other effects in the cell as well [92], (p)ppGpp is known to play an important role in bacterial antimicrobial resistance [93]. Antibiofilm peptides have also been shown to bind to the cyclic-di-GMP regulatory nucleotide that is known to play a role in biofilm formation [94; 95]. Nonetheless, all these intracellular actions require the AMP to first cross the bacterial cytoplasmic membrane, and hence membrane interaction of the peptide with bacteria in biofilms must play a pivotal role in such activities. Finally, some researchers have suggested that these peptides seem to behave like 'dirty drugs' that lack specificity and can act on various targets simultaneously [10]. As such, it seems likely that several of these different mechanisms could all play together, perhaps sequentially, and it is therefore difficult at this time to pinpoint a unique antibiofilm target and a specific mode of action. Based on the results reported herein, taken together with our previous study [23], we can conclude that Trp- and Arg-rich hexamer peptides appear to be the best way forward to try to address biofilm formation by Gram-positive organisms, while abbreviated-Lys and Pro-substituted versions of Tritrp offer the best path forward towards tackling Gram-negative bacteria. In combatting biofilms comprised of mixed bacterial communities that contain both Gram-negative and Gram-positive strains, which occur quite frequently [96], the Tritrp peptides seem to offer the most optimal choice, as these are equally effective (see Table 4) at killing Gram-positive _S. aureus_ strains when compared to the hexepeptides. Be that as it may, this screening study is only a first step towards the development of antibofilm agents that may be of clinical use. While the biofilm matrices studied in our work would certainly contain extracellular DNA that originates from the bacteria [97; 98], biofilm matrices found in human hosts also contain DNA from the host which seems to provide a coating that surrounds the bacterial biofilm [99]. It is well known that many cationic AMPs have the propensity to bind to DNA (e.g., [100]). Hence, such a human host DNA coat around the biofilm could prevent an AMP from successfully penetrating such biofilms, which would render them inactive. Clearly, future studies will have to focus on measuring the antibofilm efficacy of AMPs in wounds and skin infections or in animal models of infectious diseases [15], to provide better insights into their potential clinical effectiveness.