## 2 Results
### Synergism Assays  
Pln149-PEP20 did not exhibit any synergism or antagonism when tested against _S. aureus_ (ATCC 25923). Only indifferent interactions were observed, which allowed us to infer that Pln149-PEP20 does not compete directly with the targets of any of these antibiotics. For _A. baumannii_ ATCC 19606, Pln149-PEP20 showed synergism with ciprofloxacin, polymyxin B, ampicillin, and vancomycin. The fractional inhibitory concentration (FIC) values are presented in Table 4. No antagonism was observed.  
Synergism with vancomycin was investigated further. Although this antibiotic is used primarily for treating Gram-positive infections, investigating this synergism may help clarify the mechanism of action of Pln149-PEP20. In addition, potentiating molecules  
\begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Antibiotics**} & \multirow{2}{*}{**Antibiotics MIC (mg/L)**} & \multicolumn{2}{c}{**Combination (mg/L)**} \\ \cline{3-4}  & & **MIC\({}_{\text{ANTIBIOIC}}\)** & **MIC\({}_{\text{Pln149-PEP20}}\)** & **FIC Index** \\ \hline Ciprofloxacinacin & 1 & 0.06 & 8 & 2 \\ Tobramycin & 4 & 4 & 16 & 1.5 \\ Polymyxin B & 1 & 0.25 & 2 & 0.312 \\ Vancouver & \textgreater{}64 & 2 & 4 & 0.133 \\ Ampicillin & \textgreater{}64 & 16 & 4 & 0.375 \\ \hline \hline \end{tabular}
\end{table}
Table 4: Synergism for Pln149-PEP20 in _A. baumannii_ ATCC 19606 (MIC = 32 mg/L).  
\begin{table}
\begin{tabular}{c c c c} \hline \hline \multicolumn{1}{c}{**Bacterial Strains**} & \multicolumn{2}{c}{**PAE of the Treatments (\(\mathbf{h\pm s.d.}\))**} \\ \hline  & & Pln149-PEP20 0.5 \(\times\) MIC & Pln149-PEP20 1 \(\times\) MIC \\ _S. aureus_ ATCC 25923 (MIC = 8 mg/L) & \(\mathbf{4.0\pm 1.0}\) & \(\mathbf{5.5\pm 2.0}\) \\ _S. aureus_ SA43 [17] (MIC = 8 mg/L) & \(\mathbf{4.0\pm 0.5}\) & \(\mathbf{5.0\pm 1.0}\) \\ _A. baumannii_ ATCC 19606 (MIC = 32 mg/L) & \(\mathbf{3.5\pm 0.5}\) & \(\mathbf{4.5\pm 0.5}\) \\ _A. baumannii_ ACI50 [18] (MIC = 64 mg/L) & N.O. & \(\mathbf{2.5\pm 0.5}\) \\ \hline \hline \end{tabular}  
N.O.: not observed; s.d.: standard deviation.  
\end{table}
Table 2: Post-antibiotic effect of Pln149-PEP20.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline \multicolumn{1}{c}{**CIM\({}_{\text{50}}\) (mg/L)**} & **SI (THP-1)** & **SI (HFF-1)** \\ \hline Gram-positives & 8 & 8 & 7 \\ Gram-negatives & 32 & 2 & 2 \\ \hline \hline \end{tabular}
\end{table}
Table 3: Selectivity Index (SI) for Pln149-PEP20.  
that can increase the spectrum of antibiotics already well established in clinical use is an important strategy to combat antimicrobial resistance.  
Synergism with vancomycin was observed in other species (Table 5), such as _E. coli_ and _K. pneumoniae_, except for polymyxin B-resistant _K. pneumoniae_ AMKP4 and AMKP10.  
A synergism assay with vancomycin was performed in the presence of excess magnesium to assess whether these ions directly influence the action of the peptide on the outer membrane. This assay is represented as a heat map in Figure 3. The presence of exogenous Mg\({}^{+2}\) prevented synergism with vancomycin and prevented the action of Pln149-PEP20 itself. The presence of this cation in excess indeed prevents Pln149-PEP20 from establishing interactions that are fundamental for its activity. Bacterial membranes have divalent cations, such as Mg\({}^{+2}\) and Ca\({}^{+2}\), that interact and stabilize the membrane on its outer facet. Cationic AMPs, such as Pln149-PEP20, compete with these ions for electrostatic interactions, displacing them and establishing initial interactions with negative components [21].