Fig. 4. Anti-infective activity of modern and archaic EPs in pre-clinical animal models.

(a) Schematic of the skin abscess mouse model used to assess the anti-infective activity of the modern and archaic EPs with activity against A. baumannii cells. (b) Peptides were tested at their MIC in a single dose one hour after the establishment of the infection. Each group consisted of six mice (n = 6) and the bacterial loads used to infect each mouse derived from a different inoculum. (c) To rule out toxic effects of the peptides, mouse weight was monitored throughout the whole extent of the experiment. (d) Schematic of the neutropenic thigh infection mouse model in which bacteria is injected intramuscularly in the right thigh and modern and archaic EPs were administered intraperitoneally to assess their systemic anti-infective activity. Mice were euthanized six and eight days after the beginning of the experiment, i.e., two- and four-days post infection. Each group consisted of four mice (n = 4) and the bacterial loads used to infect each mouse derived from a different inoculum. (e) All EPs, except TKN1-SSI17, showed bacteriostatic activity inhibiting proliferation of bacteria. Peptides with bacteriostatic activity were able to maintain their effect during the entire experiment (eight days), except for A7E2T1-SPR39 that was effective for six days. (f) Mouse weight was monitored throughout the duration of the neutropenic thigh infection model (8 days total) to rule out potential toxic effects of cyclophosphamide injections, bacterial load, and the EPs. The antibiotic polymyxin B was used as positive control in both models. Statistical significance in b and e (day 6) was determined using one-way ANOVA, and in e (day 8) using Kruskal-Wallis test because of the non-normal distribution and unequal variance across groups; p values are shown for each of the groups, all groups were compared to the untreated control group; features on the violin plots represent median and upper and lower quartiles. Data in c and f are the mean plus and minus the standard deviation. Figure created with BioRender.com and the PyMOL Molecular Graphics System, Version 2.1 Schrödinger, LLC.