## 3 Results and Discussion
### Antimicrobial Activity of AMP-ELR and AMP-SELP Films
aureus_ to grow as planktonic aggregates [72], thus reducing the direct contact with the films. This observation has been previously reported in a similar direct contact assay using CM4-A200 films [12] and fiber mats [13]. Indeed, scanning electron microscopy micrographs clearly demonstrated the ability of _S. aureus_ to form aggregates reducing the contact of the overall microbial community with the film surface [12]. Similarly, in the present study, the antimicrobial action of the films was mediated by direct contact, in which cells must be available to contact with the film surface. If cells are able to form aggregates, only those in contact with the film surface are subjected to an antimicrobial effect, explaining the high variability of the assay. Still, analysis of the mean values demonstrates that, overall, the AMP-ELR films demonstrate a better antibacterial performance than AMP-SELP films. In addition to the structural backbone (ERL or SELP), the antimicrobial activity was also influenced by the type of AMP. Comparing the effect of the functional module in the antibacterial activity of the AMP-SELP films against _E. coli_, best results were obtained with BMAP18 with average % kill values greater than 60%, whereas the lowest antimicrobial performance was observed for Hep-SELP with % kill values lower than 20% (Table 2). The apparent low antimicrobial performance of Hep-SELP films may be due to the secondary structure of hepcidin. Unlike Synoeca, CM4 and BMAP18, which show a linear \(\alpha\)-helix secondary structure in membrane-mimicking environments [48, 73, 74], hepcidin forms a less flexible \(\beta\)-hairpin structure, with a central core cross-linked by four disulfide bonds [75] that might intertwine with SELP \(\beta\)-sheets, compromising the peptide interaction with bacteria membranes. Regarding the assays performed with _E. coli_ (Figure 3a), results revealed that, for the confidence interval considered, all polymer films (except for Hep-SELP films prepared \begin{table}
\begin{tabular}{c c c c} \hline
**Sample** & **\% Kill (_E. coli_) & **\% Kill (_S. aureus_)** & **Reference** \\ \hline
**Hep-A200 (a)** & 69.9 \(\pm\) 10.2 & 60.8 \(\pm\) 23.5 & This work \\
**Hep-A200 (b)** & 87.7 \(\pm\) 8.3 & 76.7 \(\pm\) 13.7 & This work \\
**Syn-A200 (a)** & 85.9 \(\pm\) 9.0 & 75.4 \(\pm\) 25.4 & This work \\
**Syn-A200 (b)** & 95.9 \(\pm\) 6.2 & 85.6 \(\pm\) 8.2 & This work \\
**CM4-A200 (b)** & 86.5 \(\pm\) 0.7 & 69.7 \(\pm\) 4.5 & [12] \\
**BMAP18-A200 (b)** & 100.0 & 98.8 \(\pm\) 2.0 & [8] \\
**Hep-SELP (a)** & 11.4 \(\pm\) 5.5 & 33.9 \(\pm\) 41.9 & This work \\
**Hep-SELP (b)** & 17.7 \(\pm\) 6.1 & 65.9 \(\pm\) 30.8 & This work \\
**Syn-SELP (a)** & 28.9 \(\pm\) 4.9 & 29.3 \(\pm\) 44.7 & This work \\
**Syn-SELP (b)** & 16.9 \(\pm\) 5.8 & 30.3 \(\pm\) 62.2 & This work \\
**CM4-SELP (a)** & 28.1 \(\pm\) 6.8 & 10.7 \(\pm\) 59.6 & This work \\
**CM4-SELP (b)** & 34.8 \(\pm\) 8.9 & 30.1 \(\pm\) 4.8 & This work \\
**BMAP18-SELP (a)** & 73.7 \(\pm\) 4.2 & 41.5 \(\pm\) 35.3 & This work \\
**BMAP18-SELP (b)** & 60.4 \(\pm\) 14.4 & 48.4 \(\pm\) 24.5 & This work \\ \hline \end{tabular}
\end{table} 
Table 2: Percentage lethality (% kill) of the different protein polymer films after 2 h incubation at 37 \({}^{\circ}\)C with _Escherichia coli_ and _Staphylococcus aureus_. Films were produced by solvent casting using (a) water and (b) formic acid as solvents. Experiments were performed in triplicate in independent days with three replicates each. using water as solvent) are statistically significant, demonstrating the antimicrobial effect of these films. The antimicrobial activity observed for the fusion proteins is attributed to the functional module (AMPs) and not to the structural module (A200 or SELP). Previously, we have demonstrated that CM4-A200 displays potent antimicrobial activity against _P. aeruginosa_, reaching lethality percentage values of ca. 97% [12].