An Update on the Therapeutic Potential of Antimicrobial Peptides against Acinetobacter baumannii Infections

The rise in antibiotic-resistant strains of clinically important pathogens is a major threat to global health. The World Health Organization (WHO) has recognized the urgent need to develop alternative treatments to address the growing list of priority pathogens. Antimicrobial peptides (AMPs) rank among the suggested options with proven activity and high potential to be developed into effective drugs. Many AMPs are naturally produced by living organisms protecting the host against pathogens as a part of their innate immunity. Mechanisms associated with AMP actions include cell membrane disruption, cell wall weakening, protein synthesis inhibition, and interference in nucleic acid dynamics, inducing apoptosis and necrosis. Acinetobacter baumannii is a critical pathogen, as severe clinical implications have developed from isolates resistant to current antibiotic treatments and conventional control procedures, such as UV light, disinfectants, and drying. Here, we review the natural AMPs representing primary candidates for new anti-A. baumannii drugs in post-antibiotic-era and present computational tools to develop the next generation of AMPs with greater microbicidal activity and reduced toxicity.


Introduction
The rise of antibiotic resistance is a major aid to global mortality statistics and represents a challenge for societies, including healthcare providers, governmental agencies, and the pharmaceutical industry.The inability to develop new antibiotics to interfere with drug-resistant pathogens suggests the world is heading toward a post-antibiotic era [1,2].For bacteria, three types of antimicrobial resistance have been described: intrinsic, acquired, and adaptive; the last is known as resistance due to changes in bacterial phenotype [3][4][5][6][7][8][9][10][11].The main mechanisms of antimicrobial resistance are target modification or mutation, efflux pumps, permeability reduction, hydrolysis or enzymatic inactivation, metabolic enhancement or auxotrophy, community cooperative resistance, target protective protein (TPPs), changes in cell morphology, and self-repair systems (Table 1).While many mechanisms lead to resistance, the exposure of microbes to inadequate doses of antimicrobial drugs can trigger their evolution, contributing to the selection of antimicrobial resistance [12,13].
According to Magiorakos et al. (2012), a multidrug-resistant (MDR) strain shows resistance to at least one antimicrobial in more than three classes of antimicrobials; an

Target modification or mutation
Mutation or modification of bacterial site will interfere with target matching, thus affecting the effect of antibiotics Modifying PBPs in MRSA, production of β-lactamases or carbapenemases in genus Klebsiella; fluoroquinolone-resistant S. aureus Mycobacterium tuberculosis resistance to rifampicin is mainly caused by the mutation of the rpoB gene and vancomycin-resistant Enterococcus (VRE) [29] Reduced permeability Deletion or damage of Omps is a source of bacterial resistance Loss of porin D2 from outer cell wall in imipenem-resistant P. aeruginosa [30] Inactivating enzymes Inactivating enzymes produced by bacteria, such as antibiotic hydrolases or similar enzymes, can hydrolyze or modify antibiotics inside the cell, rendering their inactivation before reaching the target site Production of penicillin-inactivating β-lactamase by penicillin-resistant S. aureus, Haemophilus influenzae, and Escherichia coli bacteria, gentamicin-resistant enterococci via enzymatic inactivation of aminoglycosides and carbapenem-producing Enterobacteriaceae [31] Efflux pumps Pumping of harmful molecules out of the bacterial cell Increased efflux of tetracycline, macrolides, clindamycin, or fluoroquinolones in S. aureus [32] Metabolic enhancement or auxotrophy

Core genome mutations change metabolic pathways and induce antibiotic resistance
The genome of clinically pathogenic E. coli [33,34] Community cooperative resistance Most bacteria coexist in communities, collectively resisting antibiotic effects; bacterial biofilms are efficiently protective of biofilm-forming bacterial species P. aeruginosa, S. aureus, S maltophilia, and other bacteria [35] Pharmaceuticals 2023, 16, 1281 3 of 34 Target protective proteins (TPPs) Bacterial synthetic protein protects antibiotic targets from antibiotics, eliminating their bacteriostatic effects Clinically isolated S.aureus and other staphylococcus resistance to fusidic acid due to the level acquisition of genes encoding the FusB-type protein [36] Cell morphology changes Modulating the body's relative area via absorption efficiency changes can lead to the dilution of antibiotics entering the bacterial cell Cells of the commonly used model organism Caulobacter crescentus [37] Self-repair systems The multiple antibiotic resistance operon of enteric bacteria manipulates DNA repair and outer membrane integrity, enhancing antibiotic resistance E. coli multiple antibiotic resistance (mar) loci was recognized as a determinant for cross-resistance to tetracyclines, quinolones, and β-lactams [38] AR, antibiotic resistance; Ref., reference; MLSB = macrolide, lincoside, streptogramin; PB, penicillin-binding.
Colistin is, currently, the main therapeutic option against resistant strains of A. baumannii.Unfortunately, since its reintroduction, reports on A. baumannii colistin resistance mechanisms have been reported, including the complete loss of LPS, modifications of the LPS target or plasmid-encoded MCR genes, and colistin efflux from the cell [39].
The World Health Organization (WHO) recently highlighted the resistance of A. baumannii to carbapenems (CRAb) [40,41], which classifies the species as a "priority for research and development of new antibiotic treatments."CRAb is a "critical" pathogen [42].Antimicrobial peptides (AMPs) have a high potential for use in the research and development of anti-Acinetobacter drugs [43,44].
In this review, from January 2000 to April 2023, an extensive literature search was carried out at PubMed to update current knowledge about the activity of antimicrobial peptides (AMPs), combining keywords related to Acinetobacter baumannii and antimicrobial peptides (Figure 1) and finding several AMPs capable of acting against MDR A. baumannii.According to our search criteria, no previous publication on this topic was found.

Community cooperative resistance
Most bacteria coexist in communities, collectively resisting antibiotic effects; bacterial biofilms are efficiently protective of biofilm-forming bacterial species P. aeruginosa, S. aureus, S maltophilia, and other bacteria [35] Target protective proteins (TPPs) Bacterial synthetic protein protects antibiotic targets from antibiotics, eliminating their bacteriostatic effects Clinically isolated S.aureus and other staphylococcus resistance to fusidic acid due to the level acquisition of genes encoding the FusB-type protein [36] Cell morphology changes Modulating the body's relative area via absorption efficiency changes can lead to the dilution of antibiotics entering the bacterial cell Cells of the commonly used model organism Caulobacter crescentus [37] Self-repair systems The multiple antibiotic resistance operon of enteric bacteria manipulates DNA repair and outer membrane integrity, enhancing antibiotic resistance E. coli multiple antibiotic resistance (mar) loci was recognized as a determinant for crossresistance to tetracyclines, quinolones, and βlactams [38] AR, antibiotic resistance; Ref., reference; MLSB = macrolide, lincoside, streptogramin; PB, penicillinbinding.
Colistin is, currently, the main therapeutic option against resistant strains of A. baumannii.Unfortunately, since its reintroduction, reports on A. baumannii colistin resistance mechanisms have been reported, including the complete loss of LPS, modifications of the LPS target or plasmid-encoded MCR genes, and colistin efflux from the cell [39].
The World Health Organization (WHO) recently highlighted the resistance of A. baumannii to carbapenems (CRAb) [40,41], which classifies the species as a "priority for research and development of new antibiotic treatments."CRAb is a "critical" pathogen [42].Antimicrobial peptides (AMPs) have a high potential for use in the research and development of anti-Acinetobacter drugs [43,44].
In this review, from January 2000 to April 2023, an extensive literature search was carried out at PubMed to update current knowledge about the activity of antimicrobial peptides (AMPs), combining keywords related to Acinetobacter baumannii and antimicrobial peptides (Figure 1) and finding several AMPs capable of acting against MDR A. baumannii.According to our search criteria, no previous publication on this topic was found.

Antimicrobial Peptides
Antimicrobial peptides, also known as host defense peptides, are produced naturally by living organisms as a part of their innate immune system against pathogens.AMPs are amphipathic molecules of varying molecular weights containing 11-50 amino acids with an overall positive electric charge [45,46], classified as α-helical, β-sheet, or extended peptides [47][48][49].AMPs are essential in regulating immune processes such as inflammation, activating and recruiting immune system cells [45].In addition, they can inhibit protein and nucleic acid synthesis, occasionally leading to apoptosis and necrosis [50,51].
AMP activities begin on cell membranes through electrostatic interactions.As polycationic peptides, their multiple positive amino acids drive electrostatic interactions with lipid membranes that are also influenced by hydrophobic interactions (Figure 2, Table 2).Due to inherent differences between bacterial and mammalian cell surfaces, there would be preferences when AMPs associate with a cell surface, leading to an accumulation at the surface and self-assembly reaching a particular concentration [52,53].At this stage, several models have been proposed to describe the mechanism of action (MOA) of AMPs.

Antimicrobial Peptides
Antimicrobial peptides, also known as host defense peptides, are produced naturally by living organisms as a part of their innate immune system against pathogens.AMPs are amphipathic molecules of varying molecular weights containing 11-50 amino acids with an overall positive electric charge [45,46], classified as α-helical, β-sheet, or extended peptides [47][48][49].AMPs are essential in regulating immune processes such as inflammation, activating and recruiting immune system cells [45].In addition, they can inhibit protein and nucleic acid synthesis, occasionally leading to apoptosis and necrosis [50,51].
AMP activities begin on cell membranes through electrostatic interactions.As polycationic peptides, their multiple positive amino acids drive electrostatic interactions with lipid membranes that are also influenced by hydrophobic interactions (Figure 2, Table 2).Due to inherent differences between bacterial and mammalian cell surfaces, there would be preferences when AMPs associate with a cell surface, leading to an accumulation at the surface and self-assembly reaching a particular concentration [52,53].At this stage, several models have been proposed to describe the mechanism of action (MOA) of AMPs.

Mechanism of AMP Mode of Action Reference
Direct killing: Membrane target Electrostatic interactions and hydrophobic interactions (peptide and bacterial cell surface), membrane rupture-bound types of machinery and bacterial lysis-bilayer disruption [54][55][56][57][58][59] Direct killing: Non-membrane target Action on the bacterial cell wall, activation of autolysin, intracellular targets: inhibition of protein/nucleic acid synthesis, disruption of enzymatic activities and bacterial lysis [54][55][56][57]60] Immune modulation Chemotaxis, activation of immunocytes, microbial killing; antiendotoxin activity, suppression of toll-like receptors (TLRs) and/or cytokine-mediated production of proinflammatory cytokines and preventing excessive and harmful proinflammatory responses, controls the inflammation [57,61,62] Multiple modes of action have been proposed for AMPs during interactions with bacterial cell surfaces, commonly known as transmembrane pore and non-pore models (Figure 3).The pore model presents differentiated forms such as barrel-stave and toroidal,

Mechanism of AMP Mode of Action Reference
Direct killing: Membrane target Electrostatic interactions and hydrophobic interactions (peptide and bacterial cell surface), membrane rupture-bound types of machinery and bacterial lysis-bilayer disruption [54][55][56][57][58][59] Direct killing: Non-membrane target Action on the bacterial cell wall, activation of autolysin, intracellular targets: inhibition of protein/nucleic acid synthesis, disruption of enzymatic activities and bacterial lysis [54][55][56][57]60] Immune modulation Chemotaxis, activation of immunocytes, microbial killing; anti-endotoxin activity, suppression of toll-like receptors (TLRs) and/or cytokine-mediated production of proinflammatory cytokines and preventing excessive and harmful proinflammatory responses, controls the inflammation [57,61,62] Multiple modes of action have been proposed for AMPs during interactions with bacterial cell surfaces, commonly known as transmembrane pore and non-pore models (Figure 3).The pore model presents differentiated forms such as barrel-stave and toroidal, reflecting the bilayer's net arrangement.The barrel-stave shape preserves bilayer organization and begins as AMPs are parallel to the surface before perpendicularly inserting into the lipid bilayer [63].The amphipathic structure of α and/or β sheet peptides permits lateral peptide-peptide interactions between hydrophilic amino acids to form the lumen, as well as the hydrophobic regions' interaction with bilayer lipids [64,65], such as organizing and resembling a protein ion channel (Figure 3A).A minimum length of 22 residues in an α-helical structure or eight residues in a β sheet is needed to span a lipid bilayer.Only a subset of known AMPs, such as alamethicin [66], pardaxin [67,68], and protegrins [63], have been shown to form barrel-stave channels.The carpet model describes AMPs that do not insert into the lipid bilayer to form pores [70,[74][75][76]; peptides adsorb to the cell surface (Figure 3C).Upon reaching a threshold concentration, membrane integrity is compromised by a detergent-like effect that leads to the formation of micelles (Figure 3D).As the results in the carpet model are not dependent on specific amino acid compositions, lengths, or interactions, they can describe the MOA of several AMPs at high concentrations due to their amphiphilic nature, such as cecropin [77], indolicidin [78], aurein 1.2 [76], and LL-37 [75].It has been suggested that the carpet-like mechanism is a prerequisite step for the toroidal pore model [71].Other models have been proposed, including interfacial activity, electroporation, and Shai- Toroidal pores also result from the perpendicular insertion of AMPs into the lipid bilayer but do not display lateral peptide-peptide interactions [66].Rather, peptides disrupt the hydrophobic/hydrophilic arrangement of the bilayer and induce a local curvature in the lipid bilayer (Figure 3B).Pores are formed from a dynamic interaction between the inserted peptides and phospholipid head groups, creating a transient lipid-peptide supramolecule.
The carpet model describes AMPs that do not insert into the lipid bilayer to form pores [70,[74][75][76]; peptides adsorb to the cell surface (Figure 3C).Upon reaching a threshold concentration, membrane integrity is compromised by a detergent-like effect that leads to the formation of micelles (Figure 3D).As the results in the carpet model are not dependent on specific amino acid compositions, lengths, or interactions, they can describe the MOA of several AMPs at high concentrations due to their amphiphilic nature, such as cecropin [77], indolicidin [78], aurein 1.2 [76], and LL-37 [75].It has been suggested that the carpet-like mechanism is a prerequisite step for the toroidal pore model [71].Other models have been proposed, including interfacial activity, electroporation, and Shai-Huang-Matsazuki models [71].However, in most cases, the studies used the results from model membrane systems.Only a few AMPs have been studied in whole bacterial cells to define their MOAs [79,80], suggesting that the results from model membranes describing potential MOAs may need to explain their actions against the full pathogen.
Many AMPs are currently being studied to describe their therapeutic efficacy against A. baumannii strains.We have curated the online antimicrobial peptide database, APD3, to list the many examples of AMPs under study (Tables 3 and 4).These include both peptides produced by living organisms and novel peptides inspired by their activities.AH, α-helical; BS, β-sheet; NA, unavailable; *, result in µM; aa, amino acid; >, bigger then; <, less than; NF, not found; NPs, nanoparticles; Ref., reference.

Cathelicidins
Cathelicidins are a group of more than 30 cationic AMPs (CAMPs) identified from the immune system of several vertebrates.Their structure comprises two domains involved in antimicrobial activity [214,215].Cathelicidins have shown good activity compared to broad-spectrum carbapenems (imipenem and meropenem), antibiotics of choice to treat MDR A. baumannii (MIC = 16-32 mg/L) [216].
LL-37-based synthetic peptides showed potent microbicidal activity against ESKAPE pathogens (P.aeruginosa, A. baumannii, and S. aureus) without selecting for resistance.They could eliminate persistent cells and biofilms at micromolar concentrations [164].SAAP-148 is an α-helical AMP that is able to inhibit A. baumannii MDR growth and prevent biofilm formation at a concentration of 6 µg/mL.An ex vivo human skin infection model and an in vivo murine skin infection model eliminated acute and biofilm-related infections at concentrations of ~5% [164].Its antibiofilm activity improved when incorporated into nanoparticles of Poly(lactic-co-glycolic) (PLGA) that gradually increased over time, suggesting a sustained local release of the peptide based on the dose-effect in vitro profile [105].
P10, a synthetic derivative of LL-37, is cationic, showing stronger activity than LL-37 [155,222].The de novo pepD2, also LL-37-based, was designed as a trigonal distribution of positive charges in its helical structure.It displayed an 8 µg/mL MIC against the A. baumannii ATCC-type strain.WLBU2 (also called PLG0206) is an engineered cationic amphipathic α-helix, derived from LL-37 peptide, that can be inserted into bacterial membranes, leading to cell death as well as potent antibacterial effects against the biofilms of MDR A. baumannii and K. pneumoniae [168].MIC values for WLBU2 were reported to be 1.5-3.2µM for an XDR A. baumannii [223], 7.484 µM for clinical isolates [92], and 7.943 µM for K. pneumoniae.

Snake
A large number of cathelicidins have been identified from snakes.Cathelicidin-BF (Cath-BF) was isolated from the venous glands of a banded krait (Bungatus fasciatus) [224].It is one of the best-known cathelicidins, presenting an α-helical structure.Two mechanisms are attributed to its antimicrobial activity: disrupting bacteria membranes and directly pointing intracellular targets [224].It has been proven to be highly active against the drug-resistant clinical isolates of A. baumannii and can inhibit growth at 12.8 µg/mL [105].ZY4, a disulfide bridge, stabilized the cyclic peptide derivative of Cath-BF and displayed activity against clinical MDR isolates of A. baumannii, with MIC values ranging between 4.6 and 9.4 µg/mL.ZY4 killed bacteria via membrane permeabilization and inhibited biofilm formation [169].With a half-life of 1.8 h in vivo, ZY4 displayed good stability and a low tendency to induce resistance.NA-CATH has an N-terminal α-helical structure with an unstructured C-terminal [85].Identified from the Chinese cobra (Naja atra) [225], it can inhibit the growth of drug-resistant strains of A. baumannii at 10 µg/mL [225].Its antimicrobial MOA appears to occur through membrane deformation and the formation of transient pores [226].OH-CATH was identified from the king cobra (Ophiophagus hannah) [122].Its analog, DOH-CATH30, exhibits microbicidal activity against MDR A. baumannii (1.56 to 12.5 µg/mL MIC).

Alligator
The antibacterial activity of American alligator (Alligator mississippiensis) serum can be attributed to the presence of CAMPs, and several have been identified [227].AM-CATH36 inhibited the growth of both drug-resistant and susceptible A. baumannii at 2.5 µg/mL, while its two fragments, AM-CATH28 and AM-CATH21, inhibited at 10 µg/mL [85].All three appear to permeabilize bacterial membranes.MDR clinical isolates seemed more susceptible to the fragments than the full-length peptide.The recently identified As-CATH8 displayed in vitro activity profiles similar to the last-resort vancomycin and polymyxin B antibiotics.In a murine abscess model of high-density bacterial infections, As-CATH8 showed good activity against A. baumannii (MIC = 0.6 µg/mL) and S. aureus [86].

Wallaby
WAM-1 is a cathelicidin in marsupial milk that is isolated from the Tammar wallaby (Macropus eugenii) mammary gland [167,228].It inhibited biofilm formation in clinical isolates and dispersed the 24-h-old biofilms of tested isolates, including MDR strains [89].In comparison to LL-37, WAM-1 shows several desirable properties.WAM-1 in vitro activity was 12 to 80 times more effective than LL-37 at eliminating the clinical isolates of A. baumannii, and its activity as a peptide is not reduced in the presence of total serum or high NaCl concentrations.Although its MOA is unknown, it does not lead to hemolysis and has the potential for in vivo applications [89].

Hoofed Animals
Domesticated animals have yielded several cathelicidins.Bovine neutrophils cytoplasmic granules contain indolicidin, a short tryptophan-rich cationic peptide that displaces divalent cations on the surface of cell membranes, forms pores, and can inhibit DNAprocessing enzymes [90,[229][230][231][232].Indolicidin showed potent anti-A.baumannii activity (4-32 µg/mL MIC) on susceptible clinical isolates and 16 µg/mL against colistin-resistant strains [106].In an in vitro combination with antimicrobial agents, indolicidin MIC was tested against 12 MDR clinical isolates and was reported to be between 2 and 64 µg/mL [90].Bactenecin is a cyclic, arginine-rich cationic AMP isolated from cows, sheep, and goats with a type I β-turn structure and a disulfide bond between cysteines at positions 3 and 11 [90,133].Bactenecin can make cell membranes more permeable and inhibit RNA and protein synthesis: 16 and 64 µg/mL MIC against susceptible and colistin-resistant A. baumannii, respectively [79,106].Other studies of cathelicidins include bovine BMAP-27, sheep SMAP29, and goat minibactenecins [152], which have been shown to inhibit the growth of clinical MDR A. baumannii [93].

Defensins
Animals, plants, and fungi produce an ancient class of AMPs called defensins that contain six to eight conserved cysteine residues.Their MOA includes binding cell membranes, forming pores, and, consequently, killing pathogens [233].Defensins have been categorized into α, β, and θ-defensins subfamilies [234].

Insect Defensins
The insect defensin, CL-defensin, can partially permeabilize A. baumannii and, different from others, is predicted to have an N-terminal loop, an α-helix segment, and an antiparallel β-sheet according to circular dichroism spectroscopy [109].

Alyteserins
Alyteserin-1c and Alyteserin-2a are two cationic AMPs that show that anti-A.baumannii activity is released from the skin secretions of midwife toads (Alytes obstetricans) following norepinephrine stimulation [83,165,251].Alyteserin-1c inhibited MDR A. baumannii growth and caused death between 11.3 and 22.6 µg/mL, showing low hemolytic activity [83].The substitution E4K further reduced the effects on red blood cells while improving growth inhibition of colistin-resistant and XDR A. baumannii isolates [120].Structural changes of Alyteserin-2a also resulted in an analog with 4-8-fold greater antimicrobial activity and less hemolytic effects [95].

Caerulein Precursor Fragment
Also isolated from the volcano-clawed frog, the caerulein precursor fragment (CPF-AM1) is a cationic AMP that binds bacterial LPS [97,120].CPF-AM1 inhibits the growth of susceptible and colistin-resistant isolates, showing minimal fibroblast toxicity and hemolytic activity [136].CPF-B1 was isolated from a Marsabit clawed frog (Xenopus borealis), displaying anti-A.baumannii activity at concentrations between 11.4 and 22.8 µg/mL and low hemolysis [113].From the Peracca clawed frog (Xenopus clivii), CPF-C1 is another member of the caerulein family of peptides with proven activity against A. baumannii, including inhibitory activity as low as 5 µg/mL concentration [112].

Hymenochirins
Hymenochirin-1B was isolated from a Zaire Dwarf clawed frog (Hymenochirus boettgeri) and is the first member of the hymenochirins class of AMPs of their host defense system [252,253].Hymenochirin-1B is an α-helical cationic peptide able to inhibit the growth of MDR A. baumannii at 19.1 µg/mL MIC [123].In addition to its antimicrobial activity, it displays anticancer and immunomodulatory properties.Hymenochirin-1B, generated by E6K and D9K substitutions, showed a nearly 4-fold increase in activity against MDR and XDR A. baumannii isolates and reduced toxicity to human erythrocytes [123].Hymenochirin-1Pa was isolated from Merlin's dwarf gray frog (Pseudhymenochirus merlini) and was able to inhibit the growth of XDR A. baumannii between 7.5 and 15 µg/mL; however, it showed moderate hemolytic activity [136,253].

Buforins
The stomach of an Asian toad (Bufo gargarizans) yielded Burfoin I [255].Its derivative, Buforin II, is a potent antimicrobial peptide that kills bacteria by crossing the membrane to bind intracellular targets, including DNA and RNA, inhibiting cellular activities [102].Buforin II can hinder the growth of susceptible and resistant Acinetobacter isolates between 0.25 and 39 µg/mL [100,101].By itself, or in combination with antibiotic treatments, Buforin II demonstrated good potential when tested in an A. baumannii rat sepsis model [96].

Hylin a1
Hylin a1 is an α-helical cationic amphipathic AMP that was isolated from the skin secretion of a white spotted tree frog (Hypsiboas albopunctatus) [257].Its antimicrobial activity has been attributed to its action on bacterial membranes.However, it also displays a strong hemolytic activity.Two analogs, Hylin a1-11K and Hylin a1-15K, showed good antimicrobial activity against carbapenem-resistant A. baumannii clinical isolates at 1-2 µM without changes in hemolytic activity [82].

Fish Piscins
Fish possess a strong innate immune system as a first-line defense against various pathogens [258].Several antimicrobial components can be found within the epidermal mucus, including AMPs, lysozyme, proteases, and lectins [259].Piscidins are cationic AMPs expressed by fish mast cells [260], which comprise a family of structurally related mature peptides between 21 and 44 residues.They are made of an amphipathic α-helical structure, which suggests that piscins have bactericidal activities against microorganisms [261].The piscidin AMP family includes pleurocidin, moronecidin, chrysophsin, dicentracin, epinecidin-1, and myxinidin [262].
Pleurocidin is an amphipathic α-helical cationic peptide found in the gills, gut, and on the skin of winter flounder (Pseudopleuronectes americanus) [263], which is genetically similar to piscidin [264].It displays broad-spectrum activity against pathogenic bacteria and fungi such as K. pneumoniae, S. aureus, P. aeruginosa, and the opportunistic oral pathogen C. albicans [263,265].Against MDR A. baumannii isolates, pleurocidin inhibits growth between 8 and 32 µg/mL [93].Its MOA appears to be caused by membrane disruption due to its binding [266]; however, it shows lower hemolysis when compared to other natural peptides using in vitro toxicity studies [267].

Melittin
The cationic amphipathic α-helical AMP melittin was isolated from European honeybee (Apis mellifera) venom, comprising nearly half its dry weight [278].Numerous melittin properties have been reported, including antibacterial [278], antiparasitic [279], and antifungal [280], along with anticancer and antiviral properties [281].Its primary MOA is a carpet-like interaction with membranes, leading to pore formation and lysis [282].Melittin displays potent antimicrobial activity against clinical MDR and XDR Acinetobacter at concentrations as low as 0.125 µg/mL [149,150].In a mouse model of third-degree burns, the topical application of melittin at 16 µg/mL eliminated 93.3% of an XDR A. baumannii [149].Importantly, the injured derma and surrounding tissue, including red blood cells, showed no toxicity.Brazilian clinical studies confirmed melittin activity against most Acinetobacter strains except for one PDR [283].
The fusion of cecropin A to endolysin ST01 has been shown to have increased bactericidal activity against ESKAPE pathogens, with A. baumannii (ATCC 17978) being eliminated at a concentration of 0.25 [289].Another hybrid of cecropin with melittin, CAMEL, rapidly kills A. baumannii [88].OMN6 is a 40-amino acid synthetic cyclic peptide based on cecropin A that displays increased stability and a significant decrease in proteolytic degradation and low cytotoxicity against eukaryotic cells.This peptide exerts a rapid bactericidal effect causing a selective bacterial membrane disruption [195], which is effective in A. baumannii laboratory (MIC = 8 µg/mL) and clinical isolates (MIC = 4-8 µg/mL), suggesting a low likelihood for resistance development [195].

Mastoparan
Mastoparan was isolated from hornet (Vespula lewisii) venom [236].While it displays good activity against wild-type, colistin-resistant, and PDR clinical A. baumannii [106,290], it also shows high hemolytic activity, which would prevent its therapeutic application [291].Action against clinical MDR A. baumannii (2-16 µg/mL MIC) was observed for mastoparan-AF isolated from Vespa affinis [148].Improvements in serum stability (24 h) have been achieved for mastoparan analogs, resulting in the growth inhibition of XDR clinical isolates [106].Higher therapeutic efficiency against MDR clinical isolates has been acquired by conjugating mastoparan to chitin, resulting in nanoconstructs (Afreenish hassan).Improvements in hemolytic toxicity have not been reported.

Dermcidin
The dcd gene in humans encodes dermcidin, a two-region anionic AMP produced and secreted by eccrine sweat glands and transported to the skin surface [116,292].The Nterminal peptide is involved with neuronal cell survival in response to oxidative stress [116]; meanwhile, the C-terminal fragment shows anti-Acinetobacter activity [293].With a net charge of -2, DCD-1L can interact with negatively charged bacterial phospholipids.Clinical PDR A. baumannii shows a two-fold increase in susceptibility compared to XDR isolates and the standard ATCC 19606 strain [117].DCD-1L can also inhibit bacterial attachment and biofilm formation, which could affect infection initiation [117].

Tachyplesin III
The hemolymph of Southeast Asian horseshoe crabs (Tachypleu gigas and Carcinoscorpius rotundicauda) contains tachyplesin III and 17 amino acids AMP.As opposed to an αhelical structure, this peptide presents a cyclic β-sheet with two disulfide bridges.Against an XDR clinical A. baumannii, tachyplesin III had 8-16 µg/mL MIC and could fully eliminate the bacteria at twice the MIC concentration [294].However, it also displays high toxicity against mammalian cells, preventing therapeutic applications [294].

Spider Peptides
Several AMPs have been isolated from spider venom.Lycosin-I is a 23-amino acid peptide from a Chinese wolf spider (Lycosa singoriensis) venom, resulting in 8-32 µg/mL MIC against MDR A. baumannii [140,295].Ant spider venom (Lachesana tarabaevi) and latarcins 2a also displayed potent antimicrobial activity against clinical MDR A. baumannii (8-64 µg/mL) [93].Like Lycosin-I, LS-AMP-E1, and LS-AMP-F1, those from burrowing wolf spiders (Lycosa sinensis) had different inhibitory activity against other clinical drugresistant bacteria and could effectively inhibit the formation of biofilms with no obvious hemolytic effects.Among ESKAPE pathogens, LS-AMP-F1 was the most effective against A. baumannii, with the lowest being 3.1 µM MIC [140].LyeTx I was isolated from a wolf spider from Brazil (Lycosa erythrognatha) and showed inhibitory activity against several MDR bacteria.However, it also showed hemolytic and cytotoxic effects.Conjugating a derivative, LyeTx I-b, to PEG could eliminate these contradictory effects while maintaining MIC values against A. baumannii, such as antibiofilm formation, and did not induce resistance [186].

Scorpion
Many AMPs have been identified from scorpion venom, displaying antimicrobial activity against A. baumannii, such as Hp1404, ctriporin, and Im5 [94].Notably, these peptides also show harmful effects, such as hemolysis, requiring sequence alterations to fix.Hp1404 was isolated from the venomous gland of a giant forest scorpion (Heterometrus petersii) and is an amphipathic α-helical peptide that exhibits antimicrobial activity against methicillin-resistant S. aureus along with cytotoxicity.Many Hp1404 analogs showed lower cytotoxic activity against MDR A. baumannii [134].BmKn2 is another naturally occurring cationic α-helical AMP in the Chinese scorpion (Mesobuthus martensii Karsch), showing antimicrobial and strong hemolytic activity.It only shows activity against Grampositive bacteria, such as S. aureus.Its mutant BmKn2-7 has lower hemolytic activity and presents a broad antimicrobial spectrum [296].Another analog, BmKn2-7K, is non-toxic at antimicrobial dosages and exhibits potent antimicrobial activity via a membrane-lytic mechanism against antibiotic-resistant ESKAPE pathogens.For MDR A. baumannii, BmKn2-7K and BmKn2-7R (MIC = 2.5-5 µg/mL) showed potent and improved antimicrobial activity compared to BmKn2-7 (MIC = 5-10 µg/mL) [95].

Hybrid Peptides
The combination of different AMPs offers a rational approach to developing nonnatural AMPs.PapMA peptide consists of 18 amino acids, combining the first eight amino acids from papiliocin, a 37-residue AMP purified from the larvae of a swallowtail butterfly (Papilio xuthus) with resides 4-12 of magainin 2, and a 23-residue AMP purified from African clawed frog (Xenopus laevis) skin.A proline hinge joined the two fragments.While PapMA showed high antimicrobial activity, it was cytotoxic to mammalian cells [298].The hybrid peptides P7A3 and A3P7 that combined cathelicidin (P7) and aurein (A3) were obtained using the flipping technique [299].The serial truncation of the C-terminal led to an optimal candidate, AP19, that was stable against proteolytic enzymes via a D-amino acid substitution (D-AP19).The final peptide rapidly killed A. baumannii ATCC 19606 (MIC = 7.81 µg/mL) via membrane disruption and showed a low tendency to induce bacterial resistance.It also exhibited potent antibacterial activity against MDR and XDR A. baumannii (MIC = 3.91-15.63)[176].BP214 is a cationic amphipathic all-D decapeptide developed from a short cecropin A-melittin hybrid peptide BP100 [300], which showed excellent activity against colistin-resistant A. baumannii and modest hemolytic properties [301].

Resistance to AMPS
Resistance to AMPs can be acquired through their degradation, sequestration, and impedance by exopolymers or biofilm matrix molecules, as well as through the alteration of membrane lipid composition and exporting mechanisms [53,[302][303][304][305][306][307] (Table 5).Following its long-term clinical use, colistin resistance has been documented for A. baumannii [308,309].Resistance was also observed after inactivating one of the genes involved in LPS biosynthesis.As colistin is a last-resource drug to treat MDR nosocomial pathogens, resistance is an important clinical issue [309][310][311].Several nanocarriers have been developed to overcome low bioavailability, proteolysis, and toxicity associated with AMPs [312,313].Changes in molecular structure, biochemical modifications, and their combination with common antibiotics have been reported to minimize AMP resistance [303].

Conclusions
Among ESKAPE pathogens, A. baumannii is of major concern for nosocomial and community-acquired infections.Due to its high ability to acquire resistance and biofilm formation, there has been an alarming loss of antibiotic efficacy and a rise in MDR isolates worldwide.The shortage of new antibiotic treatments shows the need to transition to a "post-antibiotic era" by developing new alternative therapeutical approaches.AMPs have emerged as excellent candidates due to the broadness of natural peptides found as part of innate immune systems, demonstrating activity against many A. baumannii, including clinical MDR and XDR isolates.While many AMPs display undesirable effects, such as hemolysis and host toxicity, studies have demonstrated the ability to modify their sequences to improve performance.AMPs isolated from natural sources have attracted significant interest in recent years as promising pharmacological substitutes for conventional antibiotics; moreover, extensive research has been undertaken on the discovery, production, and optimization of peptide drugs.Future advances in bioinformatics and studies on peptide sequence/structure/function could be able to develop synthetic AMPs to address major health concerns.Our review of AMPs highlighted common characteristics, such as cationic, α-helical structure, interactions with bacterial membranes, bilipid pore formation, and intracellular component targeting.Many possibilities for performance improvement combined with traditional treatments and their use as bioconjugates encourage future applications.Peptide drugs currently represent a significant proportion of the pharmaceutical market.Considering their therapeutic potential, market prospects, and economic values, antimicrobial peptides are expected to attract investments and research efforts, achieving success in the medium to long term.In addition to their antimicrobial properties, many AMPs have demonstrated other beneficial activities such as anticancer, antioxidant, wound healing, and angiogenesis that further support additional research.

Figure 1 .
Figure 1.Number of articles selected according to the year of publication.Figure 1. Number of articles selected according to the year of publication.

Figure 1 .
Figure 1.Number of articles selected according to the year of publication.Figure 1. Number of articles selected according to the year of publication.

Figure 2 .
Figure 2. Antimicrobial peptide (AMP) mechanisms on bacterial cells: (A) AMPs directly affect bacterial membrane and intracellular targets and disrupt lipid receptors and membrane-bound machinery.(B) AMPs indirectly trigger the activation and chemoattraction of immune cells.

Figure 2 .
Figure 2. Antimicrobial peptide (AMP) mechanisms on bacterial cells: (A) AMPs directly affect bacterial membrane and intracellular targets and disrupt lipid receptors and membrane-bound machinery.(B) AMPs indirectly trigger the activation and chemoattraction of immune cells.

Pharmaceuticals 2023 , 39 Figure 3 .
Figure 3. AMP mechanisms of action on bacterial membranes: (A) In the barrel-staved model, the accumulation of AMPs inserted into the membrane bilayer forms a pore.(B) In the carpet model, AMPs accumulate on the surface until a critical concentration displays detergent behavior to form micelles. (C) Accumulated AMPs inserted in vertical and bent orientations form a pore in the toroidal pore model.(D) Positively charged AMPs interact with negatively charged cell membranes adsorbing, leading to electroporation.(E) AMP interaction can interfere with membrane thickening, making the membrane more fragile.(F) Non-lytic membrane depolarization.(G) AMP oxidizes membrane lipids, leading to reactive oxygen species and increased lysis and permeability.(H) AMP generation of the non-bilayer intermediate that interacts with the membrane.

Figure 3 .
Figure 3. AMP mechanisms of action on bacterial membranes: (A) In the barrel-staved model, the accumulation of AMPs inserted into the membrane bilayer forms a pore.(B) In the carpet model, AMPs accumulate on the surface until a critical concentration displays detergent behavior to form micelles. (C) Accumulated AMPs inserted in vertical and bent orientations form a pore in the toroidal pore model.(D) Positively charged AMPs interact with negatively charged cell membranes adsorbing, leading to electroporation.(E) AMP interaction can interfere with membrane thickening, making the membrane more fragile.(F) Non-lytic membrane depolarization.(G) AMP oxidizes membrane lipids, leading to reactive oxygen species and increased lysis and permeability.(H) AMP generation of the non-bilayer intermediate that interacts with the membrane.

Table 1 .
Bacterial resistance mechanisms against antibiotics.

Table 2 .
General mechanism of AMP actions: direct killing by inhibiting membranes, bacterial lysis, and immune modulation.

Table 2 .
General mechanism of AMP actions: direct killing by inhibiting membranes, bacterial lysis, and immune modulation.

Table 3 .
AMPs produced by living organisms demonstrating anti-A.baumannii activity.

Table 4 .
Synthetic AMPs point out anti-A.baumannii activity.

Table 5 .
Gram-negative bacterial resistance mechanisms against AMP.