*WINNER* Improved taxonomic identification techniques for cutaneous microbiota of salamanders

Abstract

Improved taxonomic identification techniques for cutaneous microbiota of salamanders

C. Grant McCormick, Aubree J. Hill, and Dr. John H. Gunderson

Tennessee Technological University, Department of Biology

Batrachochytrium salamandrivorans (Bsal) and B. dendrobatidis (Bd) are fungal pathogens causing major conservation concern for amphibian populations in North America. Resistance against Bd infection has been observed in salamanders of the southeastern United States, and is likely due to their cutaneous microbiota. We hypothesize that Bd inhibitory bacterial symbionts of salamanders can be used to develop probiotic treatments for Bd and Bsal infections. We collected 110 bacterial isolates from cutaneous swab samples of wild Smoky Mountains salamanders, including multiple Bd inhibitors. However, previous attempts to taxonomically identify bacteria through traditional PCR-assisted gene sequencing techniques were largely unsuccessful. In fact, our results indicate that roughly 30% of the isolates were misidentified when only one primer pair was used. Our objective was to re-sequence the isolates’ 16s rRNA gene in a more complete way using four pairs of internal sequencing primers. Our improved PCR protocol yielded high-quality, publishable gene sequences for all isolates. Now that isolates have been accurately identified, we may begin development of a probiotic treatment containing antifungal bacteria to treat disease caused by Bd and Bsal.