Investigating the potential use of an ionic liquid (1-Butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide) as an anti-fungal treatment against the amphibian chytrid fungus, Batrachochytrium dendrobatidis

Abstract

The disease chytridiomycosis, caused by the pathogenic chytrid fungus, Batrachochytrium dendrobatidis (Bd), has contributed to global amphibian declines. Bd infects the keratinized epidermal tissue in amphibians and causes hyperkeratosis and excessive skin shedding. In individuals of susceptible species, the regulatory function of the amphibian's skin is disrupted resulting in an electrolyte depletion, osmotic imbalance, and eventually death. Safe and effective treatments for chytridiomycosis are urgently needed to control chytrid fungal infections and stabilize populations of endangered amphibian species in captivity and in the wild. Currently, the most widely used anti-Bd treatment is itraconazole. Preparations of itraconazole formulated for amphibian use has proved effective, but treatment involves short baths over seven to ten days, a process which is logistically challenging, stressful, and causes long-term health effects. Here, we explore a novel anti-fungal therapeutic using a single application of the ionic liquid, 1-Butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide (BMP-NTf2), for the treatment of chytridiomycosis. BMP-NTf2 was found be effective at killing Bd in vitro at low concentrations (1:1000 dilution). We tested BMP-NTf2 in vivo on two amphibian species, one that is relatively tolerant of chytridiomycosis (Pseudacris regilla) and one that is highly susceptible (Dendrobates tinctorius). A toxicity trial revealed a surprising interaction between Bd infection status and the impact of BMP-NTf2 on D. tinctorius survival. Uninfected D. tinctorius tolerated BMP-NTf2 (mean ± SE; 96.01 ± 9.00 μl/g), such that only 1 out of 30 frogs died following treatment (at a dose of 156.95 μL/g), whereas, a lower dose (mean ± SE; 97.45 ± 3.52 μL/g) was not tolerated by Bd-infected D. tinctorius, where 15 of 23 frogs died shortly upon BMP-NTf2 application. Those that tolerated the BMP-NTf2 application did not exhibit Bd clearance. Thus, BMP-NTf2 application, under the conditions tested here, is not a suitable option for clearing Bd infection in D. tinctorius. However, different results were obtained for P. regilla. Two topical applications of BMP-NTf2 on Bd-infected P. regilla (using a lower BMP-NTf2 dose than on D. tinctorius, mean ± SE; 9.42 ± 1.43 μL/g) reduced Bd growth, although the effect was lower than that obtained by daily doses of itracanozole (50% frogs exhibited complete clearance on day 16 vs. 100% for itracanozole). Our findings suggest that BMP-NTf2 has the potential to treat Bd infection, however the effect depends on several parameters. Further optimization of dose and schedule are needed before BMP-NTf2 can be considered as a safe and effective alternative to more conventional antifungal agents, such as itraconazole.

Fig 1. The chemical structure of BMP-NTf2.

Fig 2. Identifying Bd populations in flow cytometry.

Top: Zoospore and zoosporangia populations can be distinguished by their differing forward and side scatter, an indicator of cell size and internal granularity, respectively. Flow cytometry also reveals qualitative information about cell viability in a culture since dead cells exhibit higher side scatter. In the leftmost panel, cells from a live culture show one zoospore grouping. In the middle panel (a mixed culture of live and dead cells) there are two distinct zoospore groups, one at higher side scatter. The right panel, a heat-killed culture, exhibits one zoospore group, again at higher side scatter than the live culture. While the zoosporangia do not resolve into two different groups, there is a significant upward shift in side scatter for cultures with dead cells. The ungated population at low forward scatter signal is debris. Bottom: Zoospore and zoosporangia via light microscopy from samples sorted via flow cytometry. Microscope magnification 10х. In addition, images were enlarged 1430х (zoospores) or 530х (sporangia) to show detail.

Fig 3. BMP-NTf2 treatment of Bd cultures reduces viability of both zoospores and zoosporangia.

A single 30-minute treatment of BMP-NTf2 resulted in a loss of Bd viability in vitro. A) Flow cytometry plots of FDA/PI-stained cultures treated with differing concentrations of BMP-NTf2 show a reduction in live cells and increase in dead cells for both zoospore (blue circled) and zoosporangia (purple circled) populations. B) Quantification of live and dead populations in the stained samples via gating showed that zoospores were susceptible to BMP-NTf2 in a dose-dependent manner. The 1:1000 and 1:10 doses were significantly different from one another (p < 0.05). C) BMP-NTf2 also reduced the zoosporangia viability; however, a dose-dependent trend was not observed. Mean ± SE for N = 6. **p < 0.01, ****p < 0.0001.

Fig 4. The relationship between the amount of BMP-NTf2 applied to amphibian skin versus the amount of BMP-NTf2 recovered in water samples (μL).

Following BMP-NTf2 application, frogs were placed in plastic condiment containers with 25 mL of water for one hour to determine the amount of BMP-NTf2 that did not adhere to the skin. The thick line represents the predicted values from a linear model, and the points are samples collected.

Fig 5

Bd infection intensity over time for (A) Dendrobates tinctorius and (B) Pseudacris regilla. The time series of individual Bd infection intensity over time for three treatment groups in each of two species. Blue represents Bd + BMP-NTf2 group, black indicates the control Bd only group, and yellow are Bd + itraconazole group. Dashed vertical lines represent day of treatments, and solid lines represent model predictions from linear mixed effects models.