Culture Media and Individual Hosts Affect the Recovery of Culturable Bacterial Diversity from Amphibian Skin

Daniel Medina, Jenifer B. Walke, Zachary Gajewski, Matthew H. Becker, Meredith C. Swartwout, Lisa K. Belden

One current challenge in microbial ecology is elucidating the functional roles of the large diversity of free-living and host-associated bacteria identified by culture-independent molecular methods. Importantly, the characterization of this immense bacterial diversity will likely require merging data from culture-independent approaches with work on bacterial isolates in culture. Amphibian skin bacterial communities have become a recent focus of work in host-associated microbial systems due to the potential role of these skin bacteria in host defense against the pathogenic fungus Batrachochytrium dendrobatidis (Bd), which is associated with global amphibian population declines and extinctions. As there is evidence that some skin bacteria may inhibit growth of Bd and prevent infection in some cases, there is interest in using these bacteria as probiotic therapy for conservation of at-risk amphibians. In this study, we used skin swabs from American toads (Anaxyrus americanus) to: (1) assess the diversity and community structure of culturable amphibian skin bacteria grown on high and low nutrient culture media, (2) determine which culture media recover the highest proportion of the total skin bacterial community of individual toads relative to culture-independent data, and (3) assess whether the plated communities from the distinct media types vary in their ability to inhibit Bd growth in in-vitro assays. Overall, we found that culture media with low nutrient concentrations facilitated the growth of more diverse bacterial taxa and grew distinct communities relative to media with higher nutrient concentrations. Use of low nutrient media also resulted in culturing proportionally more of the bacterial diversity on individual toads relative to the overall community defined using culture-independent methods. However, while there were differences in diversity among media types, the variation among individual hosts was greater than variation among media types, suggesting that swabbing more individuals in a population is the best way to maximize culture collections, regardless of media type. Lastly, the function of the plated communities against Bd did not vary across culture media type or between high and low nutrient media. These results inform current efforts for developing a probiotic-based approach for amphibian conservation and help to ensure that culture collections are capturing the majority of the important diversity in these systems.