## 3 Discussion  
Currently, there are no clinically approved antimicrobial agents that target bacterial biofilms. Previously, we described that the mechanism of action of broad-spectrum anti-biofilm peptide 1018 involved binding and degradation of the second messenger nucleotides (p)ppGpp, which are involved in biofilm development [22]. In this report, we extended these studies on anti-biofilm peptide 1018 to examine whether this peptide inhibited biofilms formed by clinical strains isolated from CF patients. In addition, we examined if the dual properties of immunomodulatory and anti-biofilm activity were conserved in 1018 derivatives, since both of these activities have been reported to be independent of antimicrobial activity _vs._ planktonic cells [17, 21, 30]. Figure 4: Immunomodulatory activity and cytotoxicity of peptide 1018 and its derivatives HE1, HE4, HE10, HE12. (**A**) Human peripheral blood mononuclear cells were subjected to 20 \(\upmu\)g/mL and 100 \(\upmu\)g/mL of HE1, HE4, HE10, HE12, and 1018 for 24 h. Monocyte chemotactic protein-1 (MCP-1) production was assessed using sandwich enzyme linked immunosorbent assay (ELISA). The level of MCP-1 induction was similar for HE1, HE4, HE10, and 1018 at 100 \(\upmu\)g/mL; (**B**) LPS (20 ng/mL) was co-administered with 20 \(\upmu\)g/mL of each of the aforementioned peptides for 24 h. The tumor necrosis factor-alpha (TNF-\(\upalpha\)) production in response to both stimulants was assessed using enzyme linked immunosorbent assay (ELISA) and compared to lipopolysaccharide (LPS) alone. (The peptides themselves did not induce any significant TNF-\(\upalpha\) production). In both A and B, statistical significance was assessed using one-way ANOVA (**, \(p<0.01\); ***, \(p<0.001\)); (**C**) Peptide 1018 and its derivatives were not cytotoxic. Lactate dehydrogenase release from peripheral blood mononuclear cells (PBMCs) was measured to assess the toxicity of 20 \(\upmu\)g/mL and 100 \(\upmu\)g/mL of 1018 and its derivatives. It was demonstrated that 10 ug/mL of 1018 inhibited biofilm formation by most strains screened (Table 1), which was generally well below the MIC _vs._ planktonic cells (Table 1). Indeed, consistent with previous studies [21], all _Burkholderia_ genomovars were found to be highly resistant to peptides 1018, LL-37, and 1037, as well as to the cationic antibiotic polymyxin B (Table 1). Against _P. aeruginosa_ clinical strains, the peptides showed modest to very weak antimicrobial activity in the order 1018 \(>\) LL-37 \(>\) 1037. Biofilm formation by most _P. aeruginosa_ isolates (Table 2) was substantially inhibited by 10 ug/mL of 1018, except strain 7632C, for which biofilm formation was only inhibited by 19% (Table 2). Biofilm formation by _P. aeruginosa_ mucoid strain 4608 was affected the most by peptide action (67% inhibition). Peptide 1018 also inhibited biofilm formation by at least 52% in all _Burkholderia_ CF isolates tested, with _B. multivorans_ D2661 being the most susceptible strain (75% inhibition) (Table 2). Additional experiments using a hydroxyapatite disc biofilm model revealed that the peptide (10 ug/mL) triggered cell death in biofilms formed by both _P. aeruginosa_ PA14 and _B. cenocepacia_ (IIIa) 4813 (Figure 1 and Table 3), although interestingly, 1018-induced death of _B. cenocepacia_ biofilms had not been previously observed using the flow cell biofilm assay [22]. However, _B. cenocepacia_ cell death, as observed here, might reflect the different model systems. Killing was further confirmed using scanning electron microscopy (Figure 1C). Thus, the biofilm inhibitory activity of peptide 1018 (Table 2) might be due in part to the ability of 1018 to induce biofilm cell death (Figures 1 and 2; Table 3). Peptide 1018 has two independent activities, as an immunomodulator [28; 29; 30] and as an antibiofilm agent [22]. To understand if these properties were evident in related peptides we designed a series of 1018 derivatives. The best of these showed similar (HE4) or reduced (HE10) anti-biofilm activity against _P.