Linking culture-dependent and -independent characterizations of amphibian skin microbial communities: Important insights into the use of probiotics in amphibian conservation

Background/Question/Methods

Cutaneous microbial symbionts of amphibians may inhibit the fungal pathogen, Batrachochytrium dendrobatidis (Bd), and there is great interest in using these symbiotic bacteria as probiotics for the conservation of amphibians threatened by Bd. However, in other systems, it is estimated that only 0.001-15% of microbes can actually be cultured with commonly used techniques and media. This study examined the portion of the culture-independent microbial community that is culturable with R2A low nutrient agar, and which taxonomic groups are over- or under-represented by our current culturing methods. To address these questions, we performed culture-dependent and culture-independent molecular characterizations of the bacteria associated with 64 individuals from four amphibian species in Virginia: bullfrogs (Rana catesbeiana), eastern newts (Notophthalmus viridescens), spring peepers (Pseudacris crucifer), and American toads (Bufo americanus). Using the culture sequences as a reference database, we clustered the whole community Illumina sequences at 97% similarity. We examined the proportion and taxonomy of the amphibians’ microbial communities that were cultured. 

Results/Conclusions

For any amphibian species, our culturing approach captured between 0.6-1.1% of the skin microbial community, which falls within the range observed in other environments. Based on the culture-independent analysis, the four amphibian species we examined harbored OTUs from 32 bacterial phyla, while only four of these phyla (Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes) were represented in our cultures. However, these four phyla were dominant on amphibian skin, representing a combined 94.5% of the total community in our culture-independent dataset. Although only a small portion of amphibian skin microbes are captured with our current culturing approach, the dominant phyla are represented in our culture collections. Clearly, the culture-independent approach provides a more complete characterization of the microbiota, but culturing is still critically important to investigate function, in our case, the inhibitory activity of the microbes against Bd. The fact that at least some members of the dominant bacterial taxonomic groups are represented by culturing is encouraging, as these dominant groups are likely to play an important role in defense against Bd. Future research should focus on optimizing culturing conditions to target the less dominant phyla, as well as uncultured taxa within the dominant phyla, to increase the pool of potential probiotic bacteria.