###### Abstract  
In our previous work, we showed that short antimicrobial hexapeptides (AMPs) containing three Trp and three Arg residues had a potent antib biofilm activity against a pathogenic Gram-positive _Staphylococcus aureus_ MRSA strain. However, the activity of these hexapeptides against a Gram-negative _Pseudomonas aeruginosa_ PA01 strain was relatively poor. Herein, we tested the longer 13-residue synthetic AMP tritrpticin-NH\({}_{2}\) (Tritrp) and several of its analogs as potential antib Biofilm agents that can prevent biofilm formation (MBIC) and/or cause biofilm dissolution (MBEC) for two _P. aeruginosa_ PA01 strains, one of which expressed the GFP protein. Tritrp, a porcine cathelcidin, is currently the only known naturally occurring cationic AMP that has three Trp in sequence (WWW), a feature that was found to be important in our previous study. Our results show that several Tritrp analogs were effective. In particular, analogs with Pro substitutions that had altered peptide backbone structures compared to the naturally occurring amphipathic two-turn structure showed more potent MBIC and MBEC antib Biofilm activities. Selectivity of the peptides towards _P. aeruginosa_ could be improved by introducing the non-proteinogenic amino acid 2,3-diaminopropionic acid, rather than Arg or Lys, as the positively charged residues. Using \({}^{1}\)H NMR spectroscopy, we also reinvestigated the role of the two Pro residues in cis-trans isomerism of the peptide in aqueous solution. Overall, our results show that the WWW motif embedded in longer cationic AMPs has considerable potential to combat biofilm formation in pathogenic Gram-negative strains. Keywords:antibiofilm peptides; antimicrobial peptides; biofilms; diaminopropionic acid; _Pseudomonas aeruginosa_; tryptophan _Mathematics of the Universe of  
naturally in many organisms and have been found to attack bacteria in multiple ways, such as bacterial membrane disruption or destabilization, pore formation, or through targeting essential intracellular proteins, RNA/DNA synthesis, or by interfering in metabolic or regulatory pathways [9; 10]. Numerous synthetic peptide analogs with improved killing characteristics and greater stability have now been tested against various disease-causing bacteria. Renewed interest in the clinical use of AMPs arose when it was found that they could also interfere in the formation of bacterial biofilms or cause the dissolution of preformed biofilms [11]. Nowadays, it is believed that such sessile communities of microbes, rather than planktonic bacteria, are responsible for more than two-thirds of all human infections [12]. Bacterial biofilms typically form on surfaces and they are usually made up of groups of cells that are encased in a matrix that is built-up of bacterially secreted polysaccharides, extracellular DNA, proteins and vesicles [13]. The matrix provides a protected environment that is difficult to penetrate for mammalian immune systems or for antibiotics. Consequently, much higher concentrations of antibiotics are required to treat bacteria that are living in biofilms compared to those existing in the free-living state [14; 15]. Furthermore, biofilms also like to form on biomedical devices such as catheters and prosthetic joints, creating significant problems for clinicians [16]. In addition to bacteria, biofilms can also form for pathogenic fungi, such as _Candida albicans_ (e.g., [17]). As such, the need has arisen for agents that can prevent the formation of biofilms of pathogenic microorganisms, or that can help in the breakdown of existing preformed biofilms. As a result, antibiotic film peptides are receiving considerable attention from researchers [14; 18; 19; 20], although such peptides have not yet reached clinical application. Pathogenic Gram-negative and Gram-positive strains, such as _Pseudomonas aeruginosa_ and _Staphylococcus aureus_, often form biofilms and these two organisms have been studied frequently as representatives for the multidrug-resistant ESKAPE strains.