CHARACTERIZATION OF THE BACTERIAL MICROFLORA ON THE SKIN OF BOREAL TOADS, Anaxyrus (Bufo) boreas boreas, AND COLUMBIA SPOTTED FROGS, Rana luteiventris, IN GRAND TETON NATIONAL PARK, WYOMING USA

A.M. HILLIS¹*, S.T. PRANGE², M. ADOMAKO³, L.M. CHRISTENSEN⁴, S. ST-HILAIRE⁵, P.P. SHERIDAN^6
1Department of Biological Sciences, Idaho State University, 921 South 8th Ave., Pocatello, ID 83209-8007, USA.
²Department of Biological Sciences, Idaho State University, 921 South 8th Ave., Pocatello, ID 83209-8007, USA.
³Department of Biological Sciences, Idaho State University, 921 South 8th Ave., Pocatello, ID 83209-8007, USA.
⁴Department of Biological Sciences, Idaho State University, 921 South 8th Ave., Pocatello, ID 83209-8007, USA.
⁵Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, 550 University Ave. Charlottetown, PEI Canada C1A 4P3.
⁶Department of Biological Sciences, Idaho State University, 921 South 8th Ave., Pocatello, ID 83209-8007, USA.
* Corresponding Author : collaman@isu.edu

Received : 23-12-2015     Accepted : 09-03-2015     Published : 06-04-2015
Volume : 7     Issue : 1       Pages : 588 - 597
Int J Microbiol Res 7.1 (2015):588-597

Keywords : bacterial microflora, skin of boreal toads, phylogenetic lineages, AMMI analysis, phylogenetic diversity of microorganisms
Academic Editor : Jesus Ortega
Conflict of Interest : None declared
Acknowledgements/Funding : This research was supported USGS Park-Oriented Biological Support (#77-NRMS) and the Office of Research at Idaho State University. We thank Peter J. Murphy and Sara Bruer for assistance in the collection of skin samples.
Author Contribution : SP carried out cultivation of the bacterial colonies, the molecular genetic studies, the TRFLP study, the sequence alignment, the phylogenetic analysis, and drafted the manuscript. SP, AH, and LC conducted the TRFLP analysis and interpretation. AH and LC

Background: Cutaneous bacteria inhabiting the skin of boreal toads (Anaxyrus (Bufo) boreas boreas) and columbia spotted frogs (Rana luteiventris) from Grand Teton National Park were isolated and identified using their 16S SSU rRNA gene sequence. We also used a culture-independent method, Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis of the 16S SSU rRNA gene sequence, to characterize and compare the bacterial microbiota of these two amphibian species within and between different collection sites in Grand Teton National Park, Wyoming, USA. Results: Bacterial isolates belonged to 5 major phylogenetic lineages: the Actinobacteria, the Bacteroidetes/Chlorobi Group, and the Alpha-, Beta-, and Gamma-Proteobacterial lineages. TRFLP analyses showed a high species richness between sites and between amphibian species, as well as a significant amount of diversity. All three measures of diversity used (Margalef Species Richness, the Shannon Index, and the Simpson Index) were higher for frog samples than toad samples, but varied between sites. Additive Main effects and Multiplicative Interaction (AMMI) analysis of the TRFLP results showed more variability in the 3’ fragments than in the 5’ fragments of the 16S SSU rRNA gene sequences amplified from metagenomic DNA extracted from amphibian skin surface samples. Furthermore, within the 3’ fragments one site was shown to be significantly different than the other four sites by AMMI analysis. Conclusions: This study illustrated the extensive phylogenetic diversity of microorganisms present on the skin of frogs and toads present in GTNP. The identification of some of the bacterial isolates present as belonging to lineages known to produce antifungal or antibiotic compounds (thereby enabling microbial antagonism) forms the basis for a plausible hypothesis for the disease resistance of amphibians to Batrachochytrium dendrobatidis in GTNP.