Amphibians skin features an extraordinary exocrine system with several granular and mucous glands, located at strategic places in the individuals, that are able to secrete highly concentrated poisons and other defense related substances in each species (9–11). Being part of protection strategies, the granular glands are specialized in producing diverse compounds such as steroids, alkaloids and peptides (4,9,12). From these secretions the first AMP molecules were isolated more than 45 years ago, to date more than 500 structures have been reported for having biological activity, besides these substances can be modified by interactions with the microbiota found in the skin, working synergically (13).
The peptides contained in these secretions are marked by intrinsic characteristics to each family that secretes them, for instance AMP is found mainly in Bufonidae, Hylidae, Leptodactylidae, Phyllomedusidae y Ranidaefamilies (12). The present research began evaluating the antibacterial activity of raw secretions of 16 species belonging to eleven families, the selection was done through a Kirby-Bauer antibiogram with fresh collected samples, where a greater antibacterial activity was observed in six species, belonging to six families: Bufonidae, Hylidae, Leptodactylidae, Phyllomedusidae, Dendrobatidae y Microhylidae; which were then selected for the microdilution test, in which thet also were filtered for sterilization purposes.
The microdilution test helped identify that some families such as Leptodactylidae and Bufonidae, showed a marked antibiotic activity and this diminished after filtration, which suggests that this is a critical step in handling the samples and that it is also necessary to check what microorganisms are present in these secretions that power the antibacterial activity of the raw secretions.
The sample with greater biological activity, was that taken from the Phyllomedusidae family, this species have not been investigated previously, the antibacterial effect was similar to those of control antibiotics, an extended spectrum beta lactam: ampicillin and a quinolone: ciprofloxacin, but this bactericidal activity was achieved at lower protein concentrations (fig 1) when compared to other secretions and to those normally used for the reference antibiotics, this can be explained by antimicrobial peptides described formerly in other organisms belonging to this family (14). These peptides also showed activity against the same microorganisms used in this research but in other investigations, where they have also been active against resistant strains of S. aureus and E. coli (15).
The Phyllomedusidae species is distributed from tropical Mexico to Argentina, of these, two families of AMP with great antibacterial activity have been identified, dermaseptin, which has been widely studied (12,14) and, recently, medusin is described (16), this new family consists of three peptides and each one has demonstrated the ability to inhibit the growth of gram positive bacteria (S. aureus) and gram negative bacteria (E. coli) besides yeast (C. albicans),, these microorganisms were also sensitive to the tests carried out by (14).
Other researches (17) describe at least seven AMP families in secretions of the Phyllomedusidae family, taking into account the results of the raw extract assay, it is reasonable to corelate the antibiotic activity to the presence of peptides, and as can be seen in figures 1 and 2, the higher the protein concentration the higher the antibiotic activity, at 47 and 94 g/ mL concentration.
Finally it is worth noting that the concentration of proteins obtained using the new method “under stress”, frogs in bags, it is considerably higher than that reported in the literature, even using the conventional methods such as intravenous administration of norepinephrine; (18) reported that after using this novel method, the animals were returned to the aquarium. Other methods (19) sacrificed the individual for skin removal and later proceeded to extract the secretion through solvents, altogether these methods managed to obtained proteins in a concentration range in between 0,1- 1000 µg/mL. Whereas using the frog in bag method obtained a protein concentration range of 380–1380 µg/mL, demonstrating that this new technique to obtain secretions from anurans is effective and its negative impact for the subjects is minimal, avoiding the sacrifice step, which might also contribute to preserve the species and make research sustainable, although further investigations are needed to clarify the mechanism of action and the time or concentration action on bacteria.