*6.1. Protegrin-1*

*Acinetobacter baumannii*, in our experience, is a Gram-negative pathogen with a high risk of developing multiple antibiotic resistances, particularly in the hospital setting and in immunocompromised patients. Although it has been shown that the combination of colistin with daptomycin or teicoplanin can make antibacterial therapy effective in a mouse model [64], colistin may not be sufficient in cases of *A. baumannii* MDR. In an in-vitro model of cultures of *A. baumannii* MDR, also resistant to colistin, taken from surgical wounds, the minimum inhibitory concentration (MIC), 2 mcg/mL, and minimum bactericidal concentration (MBC), 8 mcg/mL, of Protegrin-1 (PG-1) were assessed. PG-1 is an 18-amino-acid beta-hairpin AMP belonging to the cathelicidin family, with excellent bactericidal action in monotherapy and excellent synergy with colistin. No resistance to PG-1 developed, but there was also no effect on biofilm. However, PG-1 is proposed as an interesting future perspective in gram-negative MDR infections [65].

#### *6.2. Pexiganan*

Another interesting AMP is pexiganan, a 22-amino acid synthetic magainin-based lysine-rich peptide that showed effective action against *A. baumannii* in a mouse model of sepsis. Both the groups treated with pexiganan (1 mg/kg intraperitoneal) and colistin (1 mg/kg intraperitoneal) showed good antibacterial efficacy, but the lowest bacterial count occurred in the pexiganan plus colistin combination group, which also achieved the highest survival rate (90%) [66]. This AMP may also help overcome MDR phenomena involving last-line antibiotics such as colistin.

In addition, pexiganan was shown to be synergistic with tigecycline in a mouse model infected with *Pseudomonas aeruginosa*, making a normally ineffective antibiotic effective against Gram-negative bacteria [67]. This offers new perspectives, considering the possible use of antibiotics that would not normally be effective against Gram-negative bacteria. In another study on a mouse model with urethral stents, the effect of pexiganan and imipenem at sub-MIC concentrations on the biofilm produced by *P. aeruginosa*, a slime producer was evaluated [68]. Studying biofilm production in vitro, the group of mice treated with pexiganan and imipenem showed a marked reduction in adhesion and biofilm expression compared to untreated controls (average reductions of 34 ± 8% and 27 ± 4%, respectively), highlighting a role for this AMP in the management of infections sustained by *P. aeruginosa* capable of producing biofilm.

### *6.3. Alpha-Helical AMPs*

Magainin II and cecropin A, alpha-helical AMPs, were used in vitro and in vivo in a mouse model against *P. aeruginosa* MDR [69]. Magainin II and cecropin A showed a strong antimicrobial action, achieving a significant reduction in plasma endotoxin (≤0.015 ± 0.0 EU/mL and ≤0.015 ± 0.0 EU/mL, respectively) and TNF-alpha concentrations (0.38 ± 0.02 ng/mL and 0.44 ± 0.03 ng/mL, respectively) compared to control (38.40 ± 2.89 EU/mL and 145.16 ± 18.32 ng/mL) and rifampicin-treated groups (29.45 ± 3.43 EU/mL and 98.0 ± 8.7 ng/mL). The latter, in monotherapy, showed no action against *P. aeruginosa*, as in other studies [70] while the combination of magainin II and cecropin A was proved significantly effective in reducing bacterial counts and mortality. This study highlights how the combination of AMPs and antibiotics that are normally ineffective against *P. aeruginosa* may be a novel solution for new therapeutic needs.

### *6.4. Tachyplesin III*

Tachyplesin III, a potent disulphide-linked peptide, showed synergistic action in vitro with beta-lactams and colistin against *P. aeruginosa* MDR [71]. In a study by Cirioni et al. [72], the activity and in vivo efficacy of Tachyplesin III, colistin, and imipenem against a multiresistant *P. aeruginosa* strain, were investigated in a murine model of sepsis. Bacteremia levels were significantly lower in the combination therapy groups (1.1 × 101 ± 0.1 × 101 CFU/mL, Tachyplesin III and imipenem), (4.6 × 101 ± 0.5 × 101 CFU/mL colistin and imipenem) than in the single agent groups (control 5.8 × <sup>10</sup><sup>7</sup> ± 0.8 × 107 CFU/mL, Tachyplesin III 3.6 × <sup>10</sup><sup>3</sup> ± 0.6 × 103 CFU/mL), in particular Tachyplesin III with imipenem had the highest efficacy in terms of bacterial lethality, quantitative blood cultures, and plasma levels of lipopolysaccharide, tumour necrosis factor alpha, and interleukin-6. Once again, combination therapy with AMPs and traditional antibiotics proved to be a very useful option. Additionally, in a study with piperacillin/tazobactam (TZP), the authors [73] reported how mice treated with Tachyplesin III in combination with TZP demonstrated the greatest efficacy compared to monotherapy, implying that a urethral stent coated with Tachyplesin III can reduce *P. aeruginosa* bacterial growth by 1,000-fold.

Finally, the effects of Tachyplesin III and clarithromycin were studied in a mouse model of *Escherichia coli* sepsis. It was seen that Tachyplesin III (1 mg/kg intraperitoneally) alone resulted in greater antimicrobial action and a significant reduction in endotoxin and TNF-alpha plasma concentrations compared to the control and clarithromycin (50 mg/kg intraperitoneally) alone groups. The latter showed no antimicrobial activity but resulted in the reduction of endotoxins and TNF-alpha plasma concentrations. The combination group of Tachyplesin III and clarithromycin was seen to be the most effective in all parameters analysed [74].
