There is evidence that the microbiome of salamanders protects them from this wildlife disease. Previous studies on amphibian infectious disease conducted by the Lewis lab at Fordham University’s Calder Center have shown that the eastern redback salamander, Plethodon cinereus, which resists infection by the chytrid fungus, possesses cutaneous bacteria that inhibit growth of the fungus (Higashino & Lewis 2016). In this study, DNA was extracted from pure cultures and sequenced with 16s rRNA primers that produced a roughly 250 base pair product. 39 species were identified.
Figure 1-Figure 1: The zone of inhibition produced by plating zoospores and streaking antifungal bacteria as well.
Figure 2-Table 1: The leftmost column is the isolate number that corresponds to individual DNA samples used. The inside left column shows what the initial identifications of each isolate were after amplifying with 16s rRNA primers that produced a ~250bp product. The inside right column is the new identification of each isolate based on the 1000bp product. The “group” column indicates how the isolates were grouped in the PCR reaction and for alignment analysis.
Figure 3-Figure 2: The PCR results using degenerate primers for the chitinase and reductase genes.
Figure 4-Figure 3: Top: The amino acid alignment for a region of the chitinase gene. Blue letters indicate the mismatch of an amino acid based on the reference sequence of S. rhizophila. Bottom: the amino acid alignment for a region of the reductase gene. Blue letters indicate the mismatch of an amino acid based on the reference sequence of S. rhizophila.
Evidence suggests that the microbiome of salamanders protects them from wildlife disease caused by cutaneous fungal growth. A previous study identified 39 species that constitute the microbiome of salamanders in the Greater New York area using 16s rRNA primers that generated a ~250bp product. Of those species, somee exhibited antifungal ability as indicated by a zone of inhibition on a plate of fungal zoospores. The mechanism of Stenotrophomonas rhizophila’s antifungal ability was recently characterized as due to production of antifungal metabolites and use of a chitinase to degrade fungal cell walls. The two genes of interest were thus a chitinase and aldo/keto reductase. Degenerate primers were designed based on S. rhizophila sequences aligned with other organisms’ gene sequences. PCR showed amplification of these genes in S. rhizophila, as expected, but also in Bacillus weidmannii, Serratia liquefaciens, and Serratia fonticola. The 16s rRNA primers used as a control generated a ~1000bp product that was sufficient to recharacterize Bacillus, Pseudomonas, Serratia, and Acinetobacter as other species than originally thought. One of the S. rhizophila isolates was determined to be another Stenotrophomonas species, S. maltophila based on the chitinase and reductase sequences. This indicated that the region amplified was identical in both of these species.
I would like to thank Elle Barnes for allowing me to work on this portion of her dissertation and for all of her guidance throughout my time on this project. I would also like to thank Tony Evans and Faaria Fasih-Ahmad for all their help and dedication to seeing my project come to fruition. Lastly, I would like to thank Dr. Berish Rubin for his guidance and support in completing the project. The three of them were instrumental in making it the best it could be.
|This document was last modified 05/15/2018.|
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