## 2 Results
### Mechanism of Action, Scanning Electron Microscopy, and Resistance Induction  
The membrane depolarization assay, which utilizes the cationic fluorescent dye DiSC\({}_{3}\)(5) to evaluate the ability of peptides to damage bacterial membranes causing ion leakage, of HRZN-15 was assessed against AB5075 upon exposure to 50 \(\mathrm{\SIUnitSymbolMicro g}\)/mL (Figure 2A). Although the MIC is 4 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, we tested for the mechanism of action at 50 \(\mathrm{\SIUnitSymbolMicro g}\)/mL following our previous mechanistic studies [26; 27; 16; 28; 29]. HRZN-15 depolarized the bacterial membrane in a significant manner (\(p<0.01\)) compared to the untreated bacteria. LL-37 and polymyxin B also showed a significant membrane depolarization effect. We previously demonstrated peptide depolarizing effect against bacteria [14; 15; 16; 17; 18; 27]. The data for the kinetics of membrane depolarization are found in Figure S5. Figure 2: Mechanism of action, visualization of bacteria under SEM, and resistance induction of HRZN-15 against _A. baumannii_ AB5075. (**A**) Membrane depolarization (DiSC\({}_{3}\)(5)) and (**B**) permeabilization (EtfBr) of AB5075 upon exposure to 50 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of peptide or antibiotics after 20 min of incubation. SEM images of (**C**) untreated and (**D**) HRZN-15-treated AB5075. (**E**) In vitro resistance induction of AB5075 upon exposure to subinhibitory concentrations of HRZN-15 and colistir for 15 days. The figure shows an MIC fold change in HRZN-15 and colistir on peptide or antibiotic-exposed bacteria. “Passage #” indicates the passage number at which the MIC was determined. Statistical significance is indicated relative to the untreated samples by asterisks (\(\text{\textasciibles*}p<0.01\), \(\text{\textasciibles*}p<0.001\), \(\text{\textasciibles*}p<0.0001\)). The loss of membrane integrity was assessed using ethidium bromide, a DNA intercalating agent, upon peptide exposure, as we have previously reported [14; 15; 16; 17; 26; 27; 28]. When large-scale disruptions in the membrane occur, the ethidium bromide can enter the bacterial cells and stain the DNA. AB5075 was challenged with 50 \(\upmu\)g/mL of HRZN-15, LL-37, or polymysxin B, and fluorescence intensity was measured. Our results show that HRZN-15 permeabilized _A. baumannni_ bacterial membrane (Figure 2B) as relative fluorescence units increased compared to an untreated control containing only bacteria and ethidium bromide (\(p<0.001\)). The control antimicrobials LL-37 and polymysxin B also disrupted bacterial membranes, as we have shown previously [14; 15; 16; 17; 26; 27; 28]. The kinetics of membrane disruption revealed the rapid action of HRZN-15 on bacteria, as reflected at the first time point, time 0 (Figure S9), while LL-37 and polymysxin B affected the bacterial membrane much later. To understand the ability of the peptide to induce changes in the bacteria, a scanning electron microscope (SEM) was used to visualize the bacteria with or without peptide treatment. _A. baumannni_ was treated with 50 \(\upmu\)g/mL of HRZN-15 for 20 min. Untreated bacteria (Figure 2C) showed irregular morphology that might be attributable to shrinkage due to freeze drying (lyophilization). Conversely, HRZN-15-treated bacteria (Figure 2D) showed significant alterations in bacterial morphology compared to the control with course structures protruding from the bacteria, suggesting cell damage. One of the advantages of membrane lytic peptides is the difficulty for bacteria to acquire resistance due to the quick killing and fundamental nature of the membrane. We evaluated the induction of resistance of HRZN-15 and colistin upon exposure to subinhibitory levels. AB5075 acquired resistance rapidly after the fifth day of colistin exposure with increased MIC from 0.5 to 64 \(\upmu\)g/mL (128 MIC fold) (Figure 2E). AB5075 resistance to colistin continued to increase, reaching 512\(\times\) MIC compared to the initial growth-inhibitory concentration (Figure 2E), which was consistent with published results [50].