## 2 Results
### Microscopic Imaging of Anti-Biofilm Activity  
The ability of peptide 1018 to inhibit biofilms was further investigated using hydroxyapatite (HA) discs as a biologically-relevant biofilm substratum [34]. _P. aeruginosa_ PA14 (Figure 1A) and _B. cenocepacia_ 4813 (Figure 1B) were resuspended in brain-heart infusion broth (BHI) and grown for 3 days on HA disks. Subsequently the biofilm coated HA disks were treated or not with 10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of peptide. Fresh BHI medium and peptide were added every 24 h for a total of 3 days, and the effect of the peptide was evaluated using confocal microscopy and scanning electron microscopy. Samples for confocal microscopy were rinsed in 0.85% physiological saline for 2 min to remove the culture broth. After live/dead cell staining, five random areas of the biofilm on each disk were examined. Treatment with peptide 1018 significantly increased the number of dead biofilm cells to around 50% for both pathogens (Table 3, Figure 1). Interestingly, peptide-induced cell death was observed in _B. cenocepacia_ biofilms grown on hydroxyapatite discs, which differed from previous results obtained using the flow cell methodology in which no major increase in cell death was recorded [20]. Additional experiments using scanning electron microscopy further supported the notion that the peptide killed biofilm cells, as cell death was apparent from the substantial amount of extracellular debris (presumably from compromised cells) accumulated within the biofilm (Figure 1C). Also, biofilm cells exhibited disrupted morphologies and were smaller in size in the treated samples (Figure 1C).  
\begin{table}
\begin{tabular}{l l l} \hline \hline
**Strains** & **Biofilm** & **Inhibition by** \\ **(Colonial Morphology or Genomovar)** & **Formation 1 ** & **10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL 1018** \\ \hline _B. cenocepacia_ C5424 (IIIa) & \(0.75\pm 0.18\) & 59\% \\ _B. cenocepacia_ CEP0055 (IIIb) & \(0.33\pm 0.11\) & 58\% \\ _B. cenocepacia_ CEP509 (IIIb) & \(0.42\pm 0.08\) & 61\% \\ _B. stabilis_ C6061 (IV) & \(0.39\pm 0.07\) & 53\% \\ _B. dolosa_ CEP0021 (VI) & \(0.41\pm 0.06\) & 55\% \\ _B. ambifaria_ CEP0996 (VII) & \(0.29\pm 0.10\) & 62\% \\ \hline \hline \end{tabular}
\end{table}
Table 2: _Cont._Antibiotics_**2014**, 3  
**Figure 1.** Sub-MIC concentrations of anti-biofilm peptide 1018 triggered biofilm cell death in the hydroxyapatite biofilm model. (A and B) Biofilms were stained using SYTO-9 to stain live cells green and propidium iodide (PI) to stain dead cells red. Merged images showing bacteria stained with both SYTO-9 and PI are also shown, and a yellow or red merged color was observed for dead cells. Samples were then examined using confocal laser scanning microscopy (CLSM). Each panel shows reconstructions from the top in the large panel and sides in the right and bottom panels (xy, yz, and xz dimensions). Three-dimensional reconstructions of biofilms are shown on the far right panel (3D image). The scale bar represents 100 \(\upmu\)m in length. (**A**) _P. aeruginosa_ PA14 untreated and treated with 10 \(\upmu\)g/mL of peptide; (**B**) _B. cenocepacia_ (IIIa) 4813 untreated and treated with peptide. Two independent experiments were performed for each condition; (**C**) SEM images showing the effect of peptide treatment on biofilm cells. _P. aeruginosa_ and _B. cenocepacia_ biofilms were developed on hydroxyapatite (HA) discs for 3 days and treated three times (every 24 h) with 10 \(\upmu\)g/mL of peptide 1018. Biofilms were observed using SEM at a magnification of 20,000\(\times\) operating at 5 kV. Untreated biofilms grown using the HA biofilm model were typically 30-40 \(\upmu\)m thick, while biofilm thickness was reduced to 10-20 \(\upmu\)m in peptide-treated samples.