Metabolites from Microbes Isolated from the Skin of the Panamanian Rocket Frog Colostethus panamansis (Anura: Dendrobatidae).

The Panamanian rocket frog Colostethus panamansis (family Dendrobatidae) has been affected by chytridiomycosis, a deadly disease caused by the fungus Batrachochytrium dendrobatidis (Bd). While there are still uninfected frogs, we set out to isolate microbes from anatomically distinct regions in an effort to create a cultivable resource within Panama for potential drug/agricultural/ecological applications that perhaps could also be used as part of a strategy to protect frogs from infections. To understand if there are specific anatomies that should be explored in future applications of this resource, we mapped skin-associated bacteria of C. panamansis and their metabolite production potential by mass spectrometry on a 3D model. Our results indicate that five bacterial families (Enterobacteriaceae, Comamonadaceae, Aeromonadaceae, Staphylococcaceae and Pseudomonadaceae) dominate the cultivable microbes from the skin of C. panamansis. The combination of microbial classification and molecular analysis in relation to the anti-Bd inhibitory databases reveals the resource has future potential for amphibian conservation.

Viscosin-like lipopeptides from frog skin bacteria inhibit Aspergillus fumigatus and Batrachochytrium dendrobatidis detected by imaging mass spectrometry and molecular networking.

Amphibian populations worldwide have declined and in some cases become extinct due to chytridiomycosis, a pandemic disease caused by the fungus Batrachochytrium dendrobatidis; however, some species have survived these fungal epidemics. Previous studies have suggested that the resistance of these species is due to the presence of cutaneous bacteria producing antifungal metabolites. As our understanding of these metabolites is still limited, we assessed the potential of such compounds against human-relevant fungi such as Aspergillus. In this work we isolated 201 bacterial strains from fifteen samples belonging to seven frog species collected in the highlands of Panama and tested them against Aspergillus fumigatus. Among the 29 bacterial isolates that exhibited antifungal activity, Pseudomonas cichorii showed the greatest inhibition. To visualize the distribution of compounds and identify them in the inhibition zone produced by P. cichorii, we employed MALDI imaging mass spectrometry (MALDI IMS) and MS/MS molecular networking. We identified viscosin and massetolides A, F, G and H in the inhibition zone. Furthermore, viscosin was isolated and evaluated in vitro against A. fumigatus and B. dendrobatidis showing MIC values of 62.50 µg/mL and 31.25 µg/mL, respectively. This is the first report of cyclic depsipeptides with antifungal activity isolated from frog cutaneous bacteria.