Avaliação da inibição do fungo Batrachochytrium dendrobatidis por bactérias isoladas de ninhos de espuma de anuros

Abstract

The Batrachochytrium dendrobatidis fungus is the pathogen that causes chytridiomycosis, a highly virulent disease that resulted, until now, in the decline of hundreds of amphibian species, many to extinction. Facing this threat, conservation strategies of the affected species are essential. Like the skin of amphibians, anuran foam nests, a reproductive strategy used by some species to place their eggs, contain an associated microbiome, little known in terms of function and composition. Thus, there is potential in these structures to isolate bacteria capable of inhibiting the Bd fungus from the moment the eggs hatch. Therefore, this work aims to evaluate the Batrachochytrium dendrobatidis inhibition potential of Adenomera hylaedactyla, Leptodactylus vastus and Physalaemus cuvieri foam nests isolates, as well as their environmental safety, through hemolytic activity tests. The anti-Bd diffusion assay was carried out in solid medium, by preparing the cultures of the isolates on T1% agar plates containing Bd, in which the isolates to be evaluated were inoculated. Then, the anti-Bd assay was carried out in liquid medium, for which co-cultures of Bd zoospores and bacterium cultures were prepared, which were then filtrated to obtain cell-free supernatant (CFS). The CFSs were added in 96-well plates containing Bd cultures, to analyze whether fungal inhibition has occurred. To evaluate the hemolytic activity, cultures of each isolate were inoculated in blood agar medium. On solid medium, 54 out of 183 strains showed anti-Bd activity. However, when testing 50 of these strains in liquid medium, results ranging from facilitation to high inhibition of Bd growth were obtained. Also, 111 strains (60.56%) showed no hemolytic activity, a promising result since the hemolytic activity is a potential virulence factor. Five isolates showed inhibitory activity in both experiments while showing no hemolytic activity, namely Serratia sp. A7, B8 (non-identified genus), Microbacterium sp. L6, Chryseobacterium sp. L15 e Pseudomonas sp. P137. Thus, foam nests contain bacteria that can be successfully used as probiotics in the treatment of chytridiomycosis, and further studies are needed to prove their safety and efficiency.