*2.3.8 XT-7*

XT-7 was first isolated from norepinephrine-stimulated skin secretions of *Xenopus tropicalis* [183]. The activity anti-*Acinetobacte*of this peptide was first reported against A. baumannii Euroclone I NM8 strain (MIC = 22.2 μg/ml) [111]. Later, the amino acid substitution of lysine at position 4 [G4K] increased the therapeutic index [115] principally. Subsequent studies were based on this new analog that inhibited sensitive and drug-resistant *A. baumannii* strains at concentrations of 4–32 and 4–64 μg/ml, respectively [104].

#### *2.3.9 Buforins*

Buforin II is a potent antimicrobial peptide derived from Burforin I, isolated from the stomach tissue of the Asian toad *Bufo gargarizans* [184]. It causes bacterial death by crossing the membrane, binding to intracellular targets, including DNA and RNA, and inhibiting cellular functions [116]. This peptide has a potent anti-*Acinetobacter* activity since it can hinder the growth of both sensitive and resistant isolates of *A. baumannii* at concentrations of 0.25–39 μg/ml [87, 98]. Buforin II alone or in combination with an antibiotic showed highly potent on *A. baumannii* sepsis treatment in a rat model [104].

### **2.4 Melittin**

Melittin is a cationic amphipathic α-helical AMP isolated from the venom (approximately 50% of the dry weight) of the European honeybee (*Apis mellifera*) [185] with numerous reported properties such as antifungal [186], antiparasitic [187], antibacterial [185], antiviral, and anticancer properties [188]. The primary mechanism of melittin action is the membrane lysis through pore formation (a carpet-like mechanism) [189]. This potent anti-*Acinetobacter* peptide inhibits MDR and XDR clinical isolates at 0.125–2 μg/ml concentration [118, 119]. A study demonstrated that topical administration of melittin at concentrations of 16 and 32 μg/ mL in mice killed 93.3% and 100% of an XDR *A. baumannii* on a third-degree burned area, respectively [118]. No toxicity was observed on the injured or healthy derma and circulating red blood cells in the examined mice. Recently, a study that evaluated the melittin against Brazilian clinical strains revealed that most strains were susceptible, except for one pan drug-resistant strain [190].

#### **2.5 Cecropins**

Cecropins, the lytic peptides, were initially isolated from the hemolymph of the giant silk moth, *Hyalophora cecropia*, and possess antibacterial and anticancer activity *in vitro* [191, 192]. The primary antimicrobial mechanism of cecropins is membrane lysis [193]. Cecropin A is a cationic amphipathic α-helical AMP that can induce apoptosis by oxidative stress in addition to attacking the membrane [194]. This peptide has potent antimicrobial activity against *A. baumannii*, inhibiting MDR clinical isolates at 0.5–32 μg/ml [99]. Vila-Farres et al. reported that this peptide inhibited the growth of sensitive and colistin-resistant strains of *A. baumannii* at 32 and 256 μg/ ml, respectively [86]. A pilot study that evaluated the viability of *Caenorhabditis elegans* infected by *A. baumannii* in the presence of 68 insect-derived AMPs identified 15 cecropin or cecropin-like peptides that prolonged the survival of worms infected with *A. baumannii* [121]. Interestingly, the direct investigation of the anti-*Acinetobacter* effect also showed that these 15 AMPs could inhibit the growth of *A. baumannii* at 4.5 to over 20 μg/ml concentrations. BR003 cecropin A, isolated from *Aedes aegypti*, is the most active member of this group. This peptide inhibited sensitive and MDR *A. baumannii* strains at 4.5 μg/ml [100]. Musca domestica cecropin (Mdc) isolated from the *larvae* of a housefly inhibits both standard (ATCC 19606) and MDR strains of *A. baumannii* at 4 μg/ml with high speed (half an hour) [122]. Cecropin P1, an AMP isolated from *Ascaris suum* of pig intestine, showed high activity against colistin-sensitive *A. baumannii* with MIC at 1.6 μg/ml. In contrast, there was less activity against the colistin-resistant strains with MIC >25 μg/ml [86].

Other peptides that showed great activity against susceptible MDR and extensively drug-resistant (XDR) *A. baumannii* strains were Cecropin-4, an α-helical

synthetic AMP [124], and CAMEL, a hybrid AMP consisting of cecropin from *H. cecropia* and melittin from *Apis melífera* [102]. In addition, AMPs with activity against biofilms have been observed in cecropins identified in *M. domestica* [124], myxinidin isolated from *Myxine glutinosa* [104], and in the naturally occurring AMP complex isolated from the maggots of blowfly *Calliphora vicina* (Diptera, *Calliphoridae*) named FLIP7 (Fly Larvae Immune Peptides 7) [126].

#### **2.6 Mastoparan**

Mastoparan is a small cationic amphipathic α-helical AMP isolated from the hornet venom of *Vespula lewisii* [195, 196] with a robust anti-*Acinetobacter* activity. However, the anti-*acinetobacter* solid activity, the high hemolytic activity, and toxic effects affected highly therapeutic applications [197]. Mastoparan inhibited the growth of a sensitive wild-type *A. baumannii* ATCC 19606 and a colistin-resistant *A. baumannii* ATCC 19606 mutant at 4 and 1 μg/ml, respectively. This study also used 14 colistin-susceptible *A. baumannii* clinical isolates and 13 pan-resistant *A. baumannii* strains isolated in a hospital outbreak [198] and reported the MIC of 1–16 and 2–8 μg/ ml for sensitive and colistin-resistant isolates, respectively [86]. Mastoparan-AF (MP-AF), isolated from the hornet venom of *Vespa affinis*, also showed effective antimicrobial activity with MICs ranging from 2 to 16 μg/ml against MDR *A. baumannii* isolates [129]. Analogs of mastoparan were made to increase the stability of the peptide in serum. These analogs had an equal inhibitory effect with mastoparan against XDR *A. baumannii* strains (4 μg/ml); in addition, it showed stability in the presence of human serum for more than 24 h [86].

### **2.7 Histatins**

Histatins belong to a distinct family of at least 12 low-molecular weight, histidine-rich cationic, salivary gland peptides with antimicrobial effect through the plasma membrane disruption [199]. Histatin-8, known as hemagglutinationinhibiting peptide [200], was the only member of this group that showed antimicrobial activity against *A. baumannii*, inhibiting the growth of both sensitive standard strains colistin-resistant mutant *A. baumannii* ATCC 19606 at 32 μg/ml [86].

#### **2.8 Dermcidins**

Dermcidin is an anionic AMP encoded by the DCD gene in humans essentially produced in eccrine sweat glands, secreted into a sweat, and further transported to the skin's epidermal surface [130, 201]. It has two parts; N-terminal peptide promotes neural cell survival under severe oxidative stress conditions called DCD-1 L [130]. DCD-1 L, a C-terminal peptide with the net electric charge of 2, is the only anionic anti-*Acinetobacter* natural AMP found in the literature that shows partial helicity in solution [130, 182]. Interestingly, in exposure to this AMP, the PDR *A. baumannii* isolates are twice more susceptible as XDR isolates and the standard strain (ATCC 19606) (MIC = 8 μg/ ml) [131].

### **2.9 Tachyplesin III**

Tachyplesin III, isolated from the hemolymph of the Southeast Asian horseshoe crabs *Tachypleus gigas* and *Carcinoscorpius rotundicauda*, consists of 17 amino acids with two disulfide bridges and is a representative antimicrobial peptide with a cyclic β-sheet structure. However, its potential toxicity hampers its use in mammalian cells [202]. Nevertheless, Tachyplesin III could inhibit the

XDR *A. baumannii* strains (8–16 μg/ml) and at 2 MIC, eliminating the XDR *A. baumannii* strains [203].

### **2.10 Computationally designed antimicrobial peptide**

The biosynthesis of AMPs can be a starting point for obtaining AMPS with functions similar to natural ones, being an attractive therapeutic option for preventing and controlling infections. In this sense, bioinformatics and computer science have been widely used in various aspects in many studies of *A. baumannii*, such as design evaluation of AMPs [136, 204–208], which includes two general principles that increased antimicrobial activity and reduced toxicity against eukaryotic cells [209, 210]. As an example of synthetic AMPs, we have stapled AMP [137] and PNA (RXR) 4XB, an antisense nucleic acid peptide compound [138] with intense bactericidal activity. The synthetic RR is a small α-helical AMP with fast bactericidal activity capable of retaining the antimicrobial property at physiological concentrations of NaCl and MgCl2 [132]. The anti-*A. baumannii* effect of RR against sensitive and MDR strains inhibits the growth at 25–99 μg/ml concentration. Two new analogs of this peptide were introduced with much stronger anti-*A. baumannii* properties than RR, and the AMPs RR2 and RR4 inhibit the growth of sensitive and drug-resistant strains (3–6 μg/ml) [211]. The peptide DP7 inhibits the growth of antibiotic-resistant *A. baumannii* strains at 4–16 μg/ml concentration, and the synergistic effects were showed after simultaneous treatment of some drug-resistant *A. baumannii* isolates with DP7 and antibiotics such as amoxicillin, azithromycin, and vancomycin [133]. Zhang et al. showed that DP7 invades the microbial cell through various pathways after sequencing the transcriptome of the bacteria exposed to this peptide [134]. Omega76 is a cationic AMP with an α-helical structure, causing death in *A. baumannii* through membrane disruption. This peptide was designed based on the maximum common subgraph of helices and further introduced as an appropriate alternative for colistin due to its high anti-*A. baumannii* activity against carbapenem and tigecycline-resistant isolates (MBC = 2–8 μg/ml) and lack of toxicity in the mouse model [135].

#### **3. Resistance to AMPS**

Although AMPs have a low likelihood to select for resistance, similar to the conventional antibiotics, another challenge is represented by the numerous reports describing the development of resistance mechanisms against some AMPs, including proteolytic degradation or sequestration by secreted proteins, impedance by exopolymers, and biofilm matrix molecules, circumvention of attraction by cell surface/membrane alteration, and export by efflux pumps [212–216]. The development of resistance to colistin by *A. baumannii* following long-term clinical application was observed [217, 218]. In *A. baumannii* stable colistin resistance was also observed following direct plating with the complete loss of LPS production due to the inactivation of one of three genes involved in lipid A biosynthesis (*lpxA, lpxD, or lpxC*). Resistance to colistin is an important clinical issue, considering that colistin is a last-resort drug used to treat MDR nosocomial pathogens [218–220]. Several mechanisms have been reported responsible for resistance to AMPs, including expression of efflux pumps, increased secretion of proteolytic enzymes, and surface charge modification to avoid membrane-peptide electrostatic interactions [213, 221, 222].

For delivering the AMPs, several nanocarriers were developed, which may help avoid the low bioavailability, proteolysis, or susceptibility and toxicity associated

with APMs [223, 224]. Changes in the molecular structure, modifications of biochemical characterization, and combination with common antibiotics have been reported to reduce AMP resistance [214]. The aprotinin is the first inhibitor identified to inhibit AMP resistance in multiple pathogens [225].
