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UFlorida Project Design

Through our research on Chytridiomycosis, we learned several frog species existed which had natural resistance to infection as a result of symbiotic bacteria living on their skin. These symbiotic bacteria produce metabolites that kill or inhibit the growth of the chytrid fungus. Many strategies for combating Chytridiomycosis focus on inoculating amphibians with these symbiotic bacteria. However, these bacteria are not widespread among amphibian species. Our team decided to modify the more ubiquitous E. coli to produce a metabolite from the symbiotic bacteria as a potential treatment for Chytridiomycosis.

Initially we planned on using violacein, a metabolite of J. lividum. J. lividum is the most widely recognized bacteria to inhibit Bd growth and several studies indicate that violacein is the primary reason why the bacteria can confer resistance against Chytridiomycosis. However; violacein has broad antibacterial and antifungal properties and there was concern that it would be harmful to introduce it into to ecosystems in which it was not naturally present.

Tryptophol is a secondary metabolite produced by a Bacillus sp. and Chitinophaga arvensicola in co-culture. It was selected for this project because it showed the greatest inhibition of Bd out of 45 metabolites produced by a Bacillus sp., Janithobacterium lividum., a Pseudomonas sp., and Chitinophaga arvensicola. These species were cultured both independently and in combination (Loudon et al. 2014).

Our proposed method of application of the modified E. coli would be via soil bioaugmentation in regions of affected amphibian populations. Bioaugmentation with J. lividum has been shown to be an effective method of inoculating amphibians against Bd (Muletz et al. 2012). We believe it would be a useful application of our modified E. coli as well. Tryptophol is an antifungal well suited to this strategy, especially as it does not have disadvantageous antifungal activity on mycorrhizae (Barroso et al. 1986) which we believe will minimize the effects of the bacteria on local flora. Our aim is to reduce disturbance on the commensal bacterial relationships and avoid stimulation of any opportunistic pathogens.

We tested our bacteria to determine if tryptophol was being produced using HPLC and we plated tryptophol against a fungus in the Chytridiomycota family to verify tryptophol’s effectiveness against Bd.

Sources

Barroso, J., Neves, H. C., & Pais, M. S. (1986). Production Of Indole-3-E Thanol And Indole-3-A Cetic Acid By The Mycorrhizal Fungus Of Ophrys Lutea (Orchid Aceae). New Phytologist, 103(4), 745-749. doi:10.1111/j.1469-8137.1986.tb00849.x

Loudon, A. H., Holland, J. A., Umile, T. P., Burzynski, E. A., Minbiole, K. P., & Harris, R. N. (2014). Interactions between amphibians symbiotic bacteria cause the production of emergent anti-fungal metabolites. Frontiers in Microbiology, 5. doi:10.3389/fmicb.2014.00441

Muletz, C. R., Myers, J. M., Domangue, R. J., Herrick, J. B., & Harris, R. N. (2012). Soil bioaugmentation with amphibian cutaneous bacteria protects amphibian hosts from infection by Batrachochytrium dendrobatidis. Biological Conservation, 152, 119-126. doi:10.1016/j.biocon.2012.03.022