## 3 Discussion
Pln149-PEP20 seemed to maintain an action on bacterial growth to the same degree as polymyxin B. The similarity of PAE and time-kill results between the peptide and polymyxin B suggests that Pln149-PEP20 could have similar pharmacodynamic properties to those reported for polymyxin B. Both polymyxin B and Pln149-PEP20 can be classified as concentration-dependent, meaning they are more effective if they reach a concentration higher than the MIC. However, the time at which the concentration is maintained is less important than the concentration level [16]. As AMPs are reported to be versatile in their action spectrum, we also investigated the activity of the proposed molecule against cancer cells. Although no activity against HepG2 was observed, assays that include other cells must be performed to exclude anticancer action. In some cancer cells, the flip-flop phenomenon occurs, in which there is an exchange between phospholipids from the inner and outer leaflets of the eukaryotic cytoplasmic membrane. Consequently, changes occur in the phospholipid constitution, membrane fluidity, and surface charge. These changes can attract AMPs, as observed in the bacterial membrane [28]. In addition, other plantaricins also had activity against cancer cells, encouraging investigation for the newly designed peptide [19]. The assay against eukaryotic cells also showed that Pln149-PEP20 selectivity was lower than the hemolysis assay initially suggested. Ideally, an IS \(\geq\) 10 is expected to be considered a biologically effective antibiotic [29]. The hydrophobic surface of this molecule may interact with both cell membranes [10]. Beyond that, the outer membrane could be an obstacle to the peptide, explaining the higher MICs against Gram-negative bacteria. The synergism of commercial antibiotics was assessed to obtain a better selectivity index for these Gram-negative bacteria. The concomitant therapeutic use of antibiotics has been an alternative to overcome the problem of antimicrobial resistance [3]. Synergism with vancomycin was further explored because, besides having the lowest FIC index, this may indicate that Pln149-PEP20 acts on the outer membrane. vancomycin is a glycopeptide that inhibits cell wall synthesis by binding to the D-ala-D-ala portion of the cell wall precursor, preventing the action of transeptidases and peptidoglycan polymerases and thus inhibiting the cross-linking of peptidoglycans that make up the cell wall [30].ancomycin could have a broad spectrum of activity [31]; however, the Gram-negative outer membrane hinders the antibiotic from permeating the periplasmic space. Therefore, the synergism observed was likely due to the outer membrane destabilization caused by Pln149-PEP20, which allowed vancomycin to act on the cell wall. Of all strains tested, only three did not show Pln149-PEP20 synergism with vancomycin. The lack of synergism in _P. aeruginosa_ can be explained by the low permeability of its outer membrane [32]. For the other two strains, both are polymyxin-resistant, which could have led to lower Pln149-PEP20 activity. The polymyxin resistance mechanism in these strains is due to an insertion element in the \(mgrB\) gene. Therefore, these strains have an increased surface charge due to the addition of 4-amino-4-deoxy-L-arabinose to lipid A. Despite also being resistant to polymyxins, ACI50 harbors mutations in the \(pmrCAB\) system [20], resulting in a lower charge difference than that caused by the insertion element disruption of the \(mgrB\) gene in _K. pneumoniae_ isolates [18,33]. That could explain why _A. baumannii_ ACI50 has a lower FIC index than the other polimixin-resistant strains. After characterizing the peptide's action, we tried to elucidate how it acts. The investigation of membrane depolarization allowed for a better understanding of peptide interactions. Although the 0.5 \(\times\) MIC concentration did not cause relevant depolarization in _S. aureus_, the time-kill results showed that this concentration could reduce the bacterial population.