Estudo das bactérias cultiváveis da pele de Adelophryne maranguapensis (ANURA: ELEUTHERODACTYLIDAE)

Abstract

Adelophryne maranguapensis is a critically endangered bromeliad frog endemic to the Serra de Maranguape, Ceará. Although there are studies on its natural history, little is known about its symbiotic microorganisms. Skin bacteria are essential in amphibian defense against pathogens, especially against the threat of the fungus Batrachochytrium dendrobatidis (Bd), which causes chytridiomycosis—a disease that has contributed to the decline of several amphibian populations globally. To expand knowledge about the skin microbiota of A. maranguapensis, this study performed the first cultivation of symbiotic bacteria of the species, followed by molecular identification of the strains. Three individuals were collected from two bromeliad species. After capture, the individuals were washed with distilled water, and their microbiota was collected with sterile swabs applied to their entire body surface. The swabs were stored in microtubes with TSB and glycerol (1:1) for transport and preservation. In the laboratory, 20µL of each sample were inoculated onto plates with R2A medium, maintained at 24–26°C for 14 days. Colonies with different morphologies were selected and picked to obtain pure cultures. Genomic DNA was extracted by thermolysis, and the 16S rRNA gene was amplified by PCR and sequenced using the Sanger method. The sequences were edited in CodonCode Aligner 12.0.3 and compared to GenBank using the BLAST algorithm. The three samples presented 150, 375, and 425 CFU/mL, totaling 34 morphotypes. DNA was successfully amplified in 13 samples, but one did not generate a usable sequence, resulting in 12 sequenced strains, with coverage ranging from 769 to 1,107 bp. Half of the strains belong to the Microbacteriaceae family, common in soil, plants, and animals, and already associated with biotechnological potential, including activity against Bd. The genera Jatrophihabitans and Mycobacterium were also identified. The first is abundant in the skin of anurans and on the leaves of V. cearensis, while the second, associated with diseases in herpetofauna, may pose a threat to the species. Other isolated genera include Kocuria, Paenibacillus, and Bacillus, all with records of anti-Bd activity. Furthermore, Bacillus and Paenibacillus are used in the agricultural and medical sectors. The results indicate that the skin of A. maranguapensis harbors cultivable bacteria that may play an important role in protecting these animals against disease. The literature highlights not only the biotechnological potential of these strains, but also their potential role in host health, through the production of antimicrobials, including metabolites with anti-Bd activity.