## 2 Results
### In Vitro Directed Evolution and Genome Analysis  
The propensity of the peptide to select resistant isolates was evaluated by increasing the subinhibitory concentrations of the antimicrobial agent over 30 days. The increase in MIC is shown in Figure 8 for both Gram-positive and Gram-negative bacteria. The peptides exhibited a moderate propensity to select resistants. The MIC increments observed were comparable to those of daptomycin and polymyxin B, which are last-resort antibiotics with peptide composition and action on the cell wall and membrane, as suggested for Pln149-PEP20. The initial and final strains with reduced susceptibility to Pln149-PEP20 were sequenced to determine whether mutations related to the mode of action occurred. All mutations obtained for _S. aureus_ and _A. baumannii_ are listed in Tables 6 and 7, respectively. Figure 7: TEM for _A. baumannii_ ATCC 19606 treated with Pln149-PEP20 (MIC = 32 mg/L). (**a**) non-treated cells. Scale bar = 1 \(\upmu\)m (**b**) non-treated cell in detail, with homogeneous intracellular content. Scale bar = 200 nm (**c**) 0.5 \(\times\) MIC treated cells, showing some cell debris and condensation of cytoplasmic content. Scale bar = 1 \(\upmu\)m (**d**) 0.5 \(\times\) MIC treated cells, showing breakage of cell wall and membrane, besides condensation of cytoplasmic content in the peripheral area of cells. Scale bar = 500 nm (**e**) 1 \(\times\) MIC treated cells, showing peripheral cytoplasmic condensation. Scale bar = 1 \(\upmu\)m (**f**) 1 \(\times\) MIC treated cells, showing peripheral cytoplasmic condensation and diffuse membranes. Scale bar = 500 nm (**g**) 4 \(\times\) MIC treated cells, showing a heterogenous intracellular content distribution and bleb formation, typical of membrane disturbing treatments. Scale bar = 1 \(\upmu\)m (**h**) 4 \(\times\) MIC treated cell, with completely disturbed cell wall and membranes, surrounded by bleb. Scale bar = 200 nm. _Antibiotics_**2023, 12**, 391 \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Replicate** & \
\begin{tabular}{c} **Sequencing** \\ **Coverage * \\ \end{tabular} & **N50** & **Mutation** & **Change** & **Altered Protein** \\ \hline \multirow{6}{*}{A} & \multirow{6}{*}{153\(\times\)} & \multirow{6}{*}{19,562} & G \textgreater{} A & Ala111 Thr & Hypothetical protein \\ \cline{3-6}  & & & G \textgreater{} A & Ala111 Thr & Hypothetical protein \\ \cline{3-6}  & & & & &
\begin{tabular}{c} Bifunctional synthetase \\ (P)ppGpp/guanosine-3\({}^{\prime}\)-5\({}^{\prime}\)- \\ bis(diphosphate)-3\({}^{\prime}\)- \\ pyrophosph hydrolase \\ \end{tabular} \\ \cline{3-6}  & & & A \textgreater{} T & Gln152 His & DUF1672 domain protein \\ \cline{3-6}  & & & T \textgreater{} A & Ser194 Thr & FAD-containing oxidoreductase \\ \hline \multirow{2}{*}{B} & \multirow{2}{*}{173\(\times\)} & \multirow{2}{*}{14,266} & 394\_395 Ins ** & Frameshift mutation & Fibronectin binding precursor (fnbA) \\ \cline{3-6}  & & & C \textgreater{} A & Leu171 Ile & Major facilitor superfamily \\ \cline{3-6}  & & & 5\_6 Ins *** & Frameshift mutation & Major facilitor superfamily \\ \hline \multirow{2}{*}{C} & \multirow{2}{*}{138\(\times\)} & \multirow{2}{*}{12,146} & T \textgreater{} C & Leu893 Ser & Clumping factor A (CIfA) \\ \cline{3-6}  & & & A \textgreater{} T & Gln152 His & DUF1672 domain protein \\ \hline \hline \end{tabular}
\end{table} 
Table 6: Comparison between the initial and final strains of the _S. aureus_ ATCC 25923 in vitro selection with Pln149-PEP20. Figure 8: Relative MIC increase for each 30-day exposure, including exposure with Pln149-PEP20, each replicate represented in a shade of gray. (**a**) _S. aureus_ ATCC 25923 (**b**) _A. buumanmii_ ATCC 19606. There was no mutation in common between all biological replicas in _S. aureus_, but the DUF1672 domain protein appeared in two of the three final strains, which makes it a strong candidate for future investigation as a target for Pln149-PEP20. All mutations in _A.