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.
Microorganisms represent a large portion of global biodiversity and are responsible for many important ecological functions, yet the majority of microbes have not been cultured (1-3). It is estimated that only 0.001% to 15% of microbes can be cultured by use of commonly used techniques and media (2, 4), although some recent attempts at culturing the uncultured have been successful. For example, a prevalent and abundant human gut bacterium with potential biomedical importance was recently cultured by a gene-targeted approach (5). Still, only a third of known bacterial phyla have cultured representatives (6). Recent advances in culture-independent rRNA-based molecular approaches have allowed for a greater understanding of bacterial diversity (1,7,8), but in most cases, the only data we have about uncultured microbes are the DNA sequences obtained directly from the environment. Next-generation sequencing in some cases has led to the discovery of entire groups, such as the TM7 candidate division, often found in terrestrial, aquatic, and clinical habitats (9). This group is phylogenetically distinct and widespread yet has not been isolated in pure culture (9-12). There are also bacteri...