*7.4. C14-NleRR-NH2 and C14-WRR-NH2*

Two lipopeptides (C14-NleRR-NH2 and C14-WRR-NH2) were studied to assess the antifungal activity against azole-resistant *Aspergillus fumigatus*. Both lipopeptides showed antifungal activity, with MICs ranging from 8 to 16 mg/L, and a dose-dependent effect was confirmed by both time-kill curves and XTT assays. Moreover, microscopy showed that hyphae growth was hampered at concentrations equal to or higher than MICs. Our results showed that both C14-NleRR-NH2 and C14-WRR-NH2 are effective against the resistant isolates tested, and this further prompts the research of lipopeptides as alternatives in antifungal therapy [95].

Finally, our experience also includes studies [96,97] evaluating the in vitro efficacy of AMPs on *Pneumocystis carinii* taken from patients with pneumonia. Cecropin P1, magainin II, indolicidin, and ranalexin alone and in combination with macrolides and dihydrofolate reductase inhibitors (DHFRs) were investigated. The four peptides suppressed the growth of *P. carinii* with no change in combination, with the exception of ranalexin, which showed synergistic activity with the macrolide [96]. Furthermore, an in vitro study on cell monolayers revealed that cecropin P1 and magainin II may be effective in inhibiting *P. carinii* growth at non-toxic cell monolayer concentrations [97].

#### **8. Conclusions**

In conclusion, our experience shows that AMPs can be a key option for treating infections sustained by multiresistant microorganisms and overcoming resistance mechanisms against currently used antibiotics or antifungals. The combination of AMPs allows antibiotics and antifungals that are no longer effective due to resistance to exploit the synergistic

effect by restoring their efficacy. This will be crucial in the near future, considering the growing spread of antibiotic resistance. In conclusion, our experience shows that AMPs can be a key option for treating infections sustained by multiresistant microorganisms and overcoming resistance mechanisms against currently used antibiotics or antifungals. The combination of AMPs allows antibiotics and antifungals that are no longer effective due to resistance to exploit the synergistic effect by restoring their efficacy. Our research has shown that the peptides allow the penetration of antibiotic molecules inside the bacterial bodies, which would otherwise be primarily ineffective against those bacterial species, thus allowing an antibiotic action in some ways "unexpected" as in the case of some betalactams, macrolides, or tetracyclines when combined with peptides for the treatment of Gram-negative microorganism infections.

This will be crucial in the near future, considering the growing resistance.

Moreover, our studies and other more recent ones have highlighted the possibility of coating some devices with peptides (we did it by hand by immersing the device, e.g., Dacron, in a peptide solution), and now other researchers do it with covalent bonds. This possibility can be exploited, for example, for orthopedic prostheses, long-lasting catheters, etc. A current limitation is the lack of data on human patients, the high cost of some AMPs, and their safety (which is improving thanks to cytotoxicity studies on cell monolayers). In addition, the problem of the frequent peptides' short half-life must be considered. This issue will have to be addressed in the future by seeking solutions similar to those obtained with glycopeptides such as dalbavancin and oritavancin, glycolipopeptides with prolonged half-lives (250–350 h), allowing once-weekly (dalbavancin) administration or a unique single-dose regimen (oritavancin).

**Author Contributions:** Conceptualization, O.S. and O.C.; methodology, G.R.; validation, A.G. and A.O.; data curation, E.M. and L.B.; writing—original draft preparation, D.G., A.M., and M.C.; writing review and editing, G.R.; supervision, O.S.; All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The animal study protocol was approved by the Institutional Animal Care Committee of the Ministery of Health and by the Animal Research Ethics Committee of IRCCS-INRCA (Istituto di Ricovero e Cura a Carattere Scientifico–Istituto nazionale di Riposo e Cura per Anziani) 767/2016 Pr 28/07/2016.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
