Recently, there are increasing concerns regarding the role of snake fungal disease (SFD) in population declines of snake species. SFD is a severe ulcerative dermatitis caused by infection with Ophidiomyces ophiodiicola. This fungus was originally described in a diseased captive black rat snake in 2009 from Georgia and has since been documented in at least 15 Eastern and Midwestern states and one Canadian province. The vertebrate cutaneous microbiome has been documented to serve as the first line of pathogen defense in many different species, however, very little is known about symbiotic microbial associations in snakes. It is postulated that the reptilian skin microbiome may affect the pathogenicity of Oo by outcompeting the pathogen for space, producing antifungal metabolites, and/or stabilizing the microbial community and potentially increasing innate defensive immunity. Identification of these beneficial bacterial species, known as probiotics, and understanding the effects of microbial community dynamics on pathogen defense may aid in future conservation management plans. The objectives of this project were to 1) isolate the resident skin microflora from three snakes representing three different species in Tennessee, 2) genotype all isolated bacteria and fungi, and 3) determine if the resident bacteria possess antifungal activity when challenged against Oo.
A timber rattlesnake, black racer, and black king snake were captured in Tennessee with signs suggestive of SFD. At necropsy, lesions consistent with SFD were detected and Oo infection was confirmed with quantitative PCR in the black racer. Infection with Oo was not detected in the timber rattlesnake or king snake. Using standard microbiological practice, we obtained 23 bacterial and 12 fungal isolates from all three snakes. Isolated microbes were DNA extracted, PCR amplified, Sanger sequenced, and compared to the UNITE (fungi) or Greengenes (bacteria) data bases for genotyping. All three snake species possessed their own unique microbiome with no overlapping associations. Two fungal taxa belonged to the Fusarium solani complex and genus Trichophyton, potentially accounting for the dermatophytic lesions on the timber rattlesnake and king snake, respectively. Bacterial isolates were challenged using in vitro assays against Oo and zones of fungal inhibition recorded. Three bacterial strains belonging to the genera Myroides, Morganella, and Aeromonas, all isolated from the timber rattlesnake, inhibited Oo growth. The remaining 20 bacterial isolates did not show any activity against Oo. Results highlight the importance of the snake microbiome in pathogen defense. Future research will be directed at developing antifungal treatments using these probiotic species.