## 3 Discussion  
As multidrug-resistant wound infections continue to be a danger to both civilians and military personnel, there is an urgent need to discover new antimicrobials to target these bacteria, especially MDR _A. baumannii_. AMPs represent a new class of antimicrobials that have rapid killing activity, different mechanisms of action than antibiotics, and a low propensity for resistance acquisition [80]. AMPs with activity against _A. baumannii_ have been developed through rational design and other similar approaches [64; 81], and we seek to expand the number of active peptides against this organism. Computational approaches to designing new antimicrobial peptides are emerging as a useful and productive approach to generating novel peptide candidates with desired or useful properties [39]. We implemented a novel computational analysis method of our input dataset, using DFT developed by [PERSON] and [PERSON], 2012 [48], followed by a positional analysis protocol developed by us [40], \"DFT plus PA\". We designed our novel peptides (HRZN) by analyzing a dataset of collected peptide sequences with activity against any strain of _A. baumannii_, as this was the peptide property on which we wanted to focus our design efforts. Additional design parameters included designing a helical peptide with a moderate net cationic charge and a favorable hydrophobic index. We produced a small set of new computationally designed HRZN peptides, which were all previously unknown or unidentified and were tested against MDR _A. baumannii_ for their antibacterial and antibiofilm activities. Our aggressive approach of testing only against MDR _A. baumannii_ sets a high bar for the performance of the peptides, but also quickly eliminates any inactive peptides from consideration for our final desired clinical application, which is the treatment of complex wounds infected with MDR _A. baumannii_. HRZN peptides demonstrated robust antibacterial activity against 3 MDR _A. baumannii_ strains, with HRZN-15 exhibiting the most potent activity (MIC 4 ug/mL). In a previous study where we designed new peptides (PHNX), MIC values were higher [40], illustrating the advantage of using this highly curated dataset for analysis. We also added amide groups at the C-terminus of each peptide, as they have been shown to increase the propensity to form \(\alpha\)-helix as well as increase bacterial membrane disruption [82; 83]. The most active peptide, HRZN-15, showed rapid killing activity in a dose- and time-dependent manner in the MIC range. The peptide exhibits complete killing against two different MDR _A. baumannii_ strains within 3 h at MIC levels and within 1 h at higher peptide concentration. Unlike conventional antibiotics, antimicrobial peptides tend to act directly on bacterial membranes [84]. An analysis of the mechanism of action revealed that HRZN-15 mainly interacts with bacterial membranes. The peptide disrupted the membrane causing depolarization and permeabilization effects. Therefore, HRZN-15 is likely to perturb the membrane, causing cell damage and leading to leakage of intracellular constituents, as evident by SEM images. A similar mode of action was previously demonstrated in LL-37, indicating its potential to interact with bacterial membranes [85]. Another extensively studied AMP, magainin 2, was found to induce membrane permeabilization by forming pores on bacterial membranes [86]. The HRZN-15 peptide also exhibited significant antibiofilm activity against MDR _A. baumannii_ biofilm. The antibiofilm activity correlated with the MIC; thus, the effect is likely due to killing the bacteria in the biofilm rather than inhibiting biofilm production itself. This is a strong positive characteristic of this peptide that can kill bacteria when they are within the biofilm. HRZN-15 was even able to eradicate pre-formed 24 h biofilm from MDR _A. baumannii_ biofilm, unlike the other two antibiofilm peptides tested in this study.