Since no unwanted harmful action at the lung level was pointed out when all the selected AMPs were administered at 0.1 mg/kg, their in vivo efficacy in treating P. aeruginosa-induced respiratory infection was then examined using a mouse model of acute lung infection. The peptides were intra-tracheally administered at 2 hours after bacterial challenge and the number of colony forming units (CFUs) in lung, BAL, and spleen was determined at 6 hours after bacterial instillation. As indicated in Fig. 4a, the total lung burden as shown in number of CFU (lung homogenate + BAL) was significantly reduced (90% reduction) after AMP treatment, compared to the number found in control animals that were infected but received only the vehicle PBS (red bars). Overall, Esc(1–21)-1c revealed to be the peptide with the best in vivo antimicrobial efficacy, probably due to its longer biostability29. In addition, the significant lowering in the number of bacteria in the spleen of Esc(1–21)-1c-treated mice compared to that of control animals or those treated with Esc(1–21) or LL-37 (Fig. 4a) indicated a substantial reduction in the systemic dissemination of bacterial cells and spread of infection. In parallel, the total number of leukocytes in the infected mouse lungs after AMPs treatment was investigated. As reported in Fig. 4b, in line with the diminished bacterial burden provoked by all peptides, a lower number of immune cells, especially neutrophils, was counted in comparison with PBS-treated infected animals, 4 hours after peptide instillation (e.g. 6 hours from infection).Figure 4Comparison among Esc(1–21), Esc(1–21)-1c and LL-37 on the number of viable P. aeruginosa cells (CFU). CFU in mouse lungs (Total = lung homogenate + BAL) and spleens (panel a) as well as on the number of inflammatory cells in the BAL (panel b) of pulmonary-infected mice at 4 hours after peptide administration at 0.1 mg/kg were enumerated. Animals were intra-tracheally infected with 3 million PAO1 cells. The peptides were administered intra-tracheally, at 2 μg (0.1 mg/kg) at 2 hours after bacterial infection. The number of viable Pseudomonas cells in the lung and spleen as well as the number of inflammatory cells in the BAL were evaluated 6 hours after infection, as described in the Experimental section. Results are mean ± SEM from three independent experiments; n = 4–6 mice for each treatment group. Following one-way analysis of variance (ANOVA), posthoc comparisons were made using the Dunnett’s multiple comparison test when the P-value was significant (p < 0.05). *p < 0.05, **p < 0.01, ***p < 0.001 for peptide-treated animals versus PBS-treated mice.