Most of the Dominant Members of Amphibian Skin Bacterial Communities Can Be Readily Cultured

Abstract

Currently, it is estimated that only 0.001% to 15% of bacteria in any given system can be cultured by use of commonly used techniques and media, yet culturing is critically important for investigations of bacterial function. Despite this situation, few studies have attempted to link culture-dependent and culture-independent data for a single system to better understand which members of the microbial community are readily cultured. In amphibians, some cutaneous bacterial symbionts can inhibit establishment and growth of the fungal pathogen Batrachochytrium dendrobatidis, and thus there is great interest in using these symbionts as probiotics for the conservation of amphibians threatened by B. dendrobatidis. The present study examined the portion of the culture-independent bacterial community (based on Illumina amplicon sequencing of the 16S rRNA gene) that was cultured with R2A low-nutrient agar and whether the cultured bacteria represented rare or dominant members of the community in the following four amphibian species: bullfrogs (Lithobates catesbeianus), eastern newts (Notophthalmus viridescens), spring peepers (Pseudacris crucifer), and American toads (Anaxyrus americanus). To determine which percentage of the community was cultured, we clustered Illumina sequences at 97% similarity, using the culture sequences as a reference database. For each amphibian species, we cultured, on average, 0.59% to 1.12% of each individual's bacterial community. However, the average percentage of bacteria that were culturable for each amphibian species was higher, with averages ranging from 2.81% to 7.47%. Furthermore, most of the dominant operational taxonomic units (OTUs), families, and phyla were represented in our cultures. These results open up new research avenues for understanding the functional roles of these dominant bacteria in host health.

FIG 1

Amphibian skin bacterial OTU richness (number of OTUs) and phylogenetic diversity values based on culture-independent (a and b) and culture-dependent (c and d) characterizations. Dashes represent the mean diversity for each amphibian species. Correlations between culture-dependent and -independent measures of OTU richness (e) and phylogenetic diversity (f) are shown for all amphibians combined. Lines in panels e and f represent GLM and LM predictions, respectively.

FIG 2

NMDS ordinations of Sorensen similarity (a and b) and unweighted UniFrac distance (c and d) matrices for culture-independent (a and c) and culture-dependent (b and d) microbial communities associated with four amphibian species.

FIG 3

Mean percentages of total OTUs “individually matched” and “species matched” to cultured OTUs for each amphibian species. An OTU was considered individually matched if it was present in the Illumina data for the same individual from which it was cultured, and it was considered species matched if it was cultured from a different individual of the same amphibian species.

FIG 4

16S rRNA gene tree of culture-independent OTUs, with the mean relative abundance of OTUs for each amphibian species (blue = bullfrogs, teal = newts, red = spring peepers, and green = toads). Only OTUs with mean relative abundances of ≥0.01% across all amphibian species were included in this analysis. Cultured OTUs are indicated with black bars, and bacterial phyla are indicated with colored branches. The phylum Proteobacteria was divided into classes (Alpha-, Beta-, Delta-, and Gammaproteobacteria).

FIG 5

Relative abundances of culture-independent bacterial phyla associated with bullfrogs (a), newts (b), spring peepers (c), and toads (d). The complete bar represents the total mean relative abundance of each phylum, while the black portion of the bar represents the cultured portion of each phylum. The numbers in parentheses show the number of “species-matched” cultured OTUs (in bold) out of the total number of OTUs in the phylum for each amphibian species. Only phyla with mean relative abundances of >0.5% for each species are shown. Error bars represent standard errors.