## 2 Results
### Derivatives of 1018: Anti-Biofilm and Immunomodulatory Activity  
As peptide 1018 has demonstrated potent anti-biofilm activity against diverse bacterial species [22] and excellent immune modulatory activity [28; 29; 30], we attempted to investigate structure-activity relationships by designing peptides based on the amino acid sequence of 1018 but with various amino acid substitutions (Table 4), designed to test the effects of sequence inversion and especially altering the five amino acid hydrophobic stretch L3 IVAV7. Interestingly, inverting the sequence of 1018 in peptide HE1 maintained the anti-biofilm activity _vs. P. aeruginosa_ and only slightly reduced it against _B. cenocepacia_. In contrast, altering three amino acids including switching an R and V residue in the sequence substantially reduced activity, which was also true for the reversed sequence in peptide HE2. These initial screens also identified peptides HE4 and HE10 as two of the more active anti-biofilm peptides, and a scrambled shorter peptide HE12 as a virtually inactive peptide (Figure 2) that we subsequently used as a negative control. The three most effective 1018 derivatives were then further evaluated for anti-biofilm activity against _P. aeruginosa_ PA14 using the flow cell method (Figure 2). Peptides HE4 and HE10 demonstrated strong anti-biofilm activity comparable with that of 1018, while HE12 was substantially less active against _P. aeruginosa_ biofilms, both under inhibition and eradication conditions (Figure 2). We then considered whether these peptide derivatives had conserved the ability of peptide 1018 to inhibit biofilms formed by Gram-positive bacteria. Interestingly, HE4 and HE10 were notably more active against methicillin-resistant _Staphylococcus aureus_ (MRSA) compared to 1018 under static conditions, while HE12 showed less activity (Figure 3). These results demonstrate that the broad-spectrum activity displayed by 1018 [22] is generally conserved in these 1018 derivatives. \begin{table}
\begin{tabular}{c c c} \hline \hline \multicolumn{1}{c}{**Bacterial Strains**} & \multicolumn{2}{c}{**Proportion of Dead Cells (\%)**} \\ \cline{2-3}  & **Untreated** & **10 \(\mu\)g/mL 1018** \\ \hline _P. aeruginosa_ PA14 & \(7\pm 4\) & \(48\pm 17\)1  \\ _B. cenocepacia_ (IIIa) 4813 & \(6\pm 3\) & \(42\pm 13\)1  \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Proportion of dead biofilm cells in hydroxyapatite plates after treatment with sub-MIC levels of peptide 1018. Biofilms were grown in HA discs for 3 days and treated with peptide 1018 every 24 h. After scanning confocal microscopy the volume ratio of red fluorescence (due to the dead cell stain propidium iodide to green (all bacteria stain Syto-9) plus red fluorescence indicated the proportion of killed cells. Three independent assays were performed. Different superscript letters indicate statistically significant differences between groups (\(p<0.05\)). Antibiotics **2014**, 3  
\begin{table}
\begin{tabular}{l l l l l} \hline \hline \multirow{2}{*}{**Peptide**} & \multirow{2}{*}{**Sequence 1** **} & \multirow{2}{*}{**Modification**} & & \multicolumn{2}{c}{**Anti-Biofilm Activity**} \\  & & & **(\% Decrease in Biofilm)**} \\ \cline{5-5}  & & & _P. aeruginosa_ & _B.