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Bacteria isolated from bats inhibit the growth of Pseudogymnoascus destructans, the causative agent of white-nose syndrome.

Hoyt JR, Cheng TL, Langwig KE, Hee MM, Frick WF, Kilpatrick AM - PLoS ONE (2015)

Bottom Line: White-nose syndrome, caused by the fungal skin pathogen Pseudogymnoascus destructans, threatens several hibernating bat species with extinction and there are few effective treatment strategies.The skin microbiome is increasingly understood to play a large role in determining disease outcome.In both challenge experiments, the extent of suppression of P. destructans growth was dependent on the initial concentration of P. destructans and the initial concentration of the bacterial isolate.

View Article: PubMed Central - PubMed

Affiliation: Department of Ecology and Evolutionary Biology, University of California Santa Cruz, Santa Cruz, California, United States of America.

ABSTRACT
Emerging infectious diseases are a key threat to wildlife. Several fungal skin pathogens have recently emerged and caused widespread mortality in several vertebrate groups, including amphibians, bats, rattlesnakes and humans. White-nose syndrome, caused by the fungal skin pathogen Pseudogymnoascus destructans, threatens several hibernating bat species with extinction and there are few effective treatment strategies. The skin microbiome is increasingly understood to play a large role in determining disease outcome. We isolated bacteria from the skin of four bat species, and co-cultured these isolates with P. destructans to identify bacteria that might inhibit or kill P. destructans. We then conducted two reciprocal challenge experiments in vitro with six bacterial isolates (all in the genus Pseudomonas) to quantify the effect of these bacteria on the growth of P. destructans. All six Pseudomonas isolates significantly inhibited growth of P. destructans compared to non-inhibitory control bacteria, and two isolates performed significantly better than others in suppressing P. destructans growth for at least 35 days. In both challenge experiments, the extent of suppression of P. destructans growth was dependent on the initial concentration of P. destructans and the initial concentration of the bacterial isolate. These results show that bacteria found naturally occurring on bats can inhibit the growth of P. destructans in vitro and should be studied further as a possible probiotic to protect bats from white-nose syndrome. In addition, the presence of these bacteria may influence disease outcomes among individuals, populations, and species.

No MeSH data available.


Related in: MedlinePlus

Challenge plates showing the inhibition of Pseudogymnoascus destructans.Bacteria were plated with an initial starting concentration of 104 cfu/ml (PF2). The plate (a) shows no visible P. destructans growth on day 43, compared to the (b) control plate showing uninhibited P. destructans colony growth at day 43. (d) The zones of inhibition produced by one of the top performing P. fluorescens isolates (PF2) compared to the sham inoculated control (c) and a widely used strain of P. fluorescens, (e; PF7: PfA506). There are two distinct zones of inhibition produced by the top performing strain (as shown in panel d) indicated by the grey solid circle and the dashed grey circle. Microscopic images of the inner and outer zones are shown in panels (f) and (g). We used gram staining techniques to help better visualize conidia (purple) and hyphae (pink) (scale bars, 10 μm). Within the first zone, indicated by the dark ring surrounding the yellow bacteria colony (PF2), the bacteria either arrested or delayed conidia growth, (g) which can be seen by the small hyphael extension from the conidia. Outside of this first zone, the growth of P. destructans was much more extensive (f), producing a mycelial network before its growth was arrested.
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pone.0121329.g001: Challenge plates showing the inhibition of Pseudogymnoascus destructans.Bacteria were plated with an initial starting concentration of 104 cfu/ml (PF2). The plate (a) shows no visible P. destructans growth on day 43, compared to the (b) control plate showing uninhibited P. destructans colony growth at day 43. (d) The zones of inhibition produced by one of the top performing P. fluorescens isolates (PF2) compared to the sham inoculated control (c) and a widely used strain of P. fluorescens, (e; PF7: PfA506). There are two distinct zones of inhibition produced by the top performing strain (as shown in panel d) indicated by the grey solid circle and the dashed grey circle. Microscopic images of the inner and outer zones are shown in panels (f) and (g). We used gram staining techniques to help better visualize conidia (purple) and hyphae (pink) (scale bars, 10 μm). Within the first zone, indicated by the dark ring surrounding the yellow bacteria colony (PF2), the bacteria either arrested or delayed conidia growth, (g) which can be seen by the small hyphael extension from the conidia. Outside of this first zone, the growth of P. destructans was much more extensive (f), producing a mycelial network before its growth was arrested.

Mentions: Zones of inhibition could not be visualized until P. destructans growth was visible on days 9–11 (Fig. 1). At this time, zones of inhibition already differed significantly among bacterial isolates and initial concentrations (Fig. 2, S2 Fig., and S5 Table). Two bacterial isolates, PF1 and PF2, generated larger zones of inhibition across most initial concentrations of P. destructans by the end of the experiment (S2 Table). Three isolates (PF1, PF2, and PF7) established two zones, one where growth of P. destructans was suspended immediately upon germination (Fig. 1g), and another outside of this zone where growth was arrested, but only after the mycelial mat had begun to develop (Fig. 1f). Zones of inhibition on the last day of the experiment (day 37) increased with increasing initial concentrations of P. destructans for the Pseudomonas isolates showing the strongest inhibition (Fig. 2; PF1, PF2, and PF4; all concentration slopes were significantly negative, all p-values <0.03). For the other four Pseudomonas isolates, the zone of inhibition was either variable across concentrations (Fig. 2; PF3, PF5, PA6) or increased with decreasing initial P. destructans concentration (PF7; concentration coef. 1.72 ± 0.64, p = 0.008). Two isolates, PF1 and PF2, out-performed the reference P. fluorescens strains (PF7; PfA506) at all initial concentrations with at least a two-fold difference in zone of inhibition (Fig. 2). The two control bacteria (Chryseobacterium sp. and Sphingomonas sp.) and the sham-inoculated control produced no zones of inhibition (Fig. 2).


Bacteria isolated from bats inhibit the growth of Pseudogymnoascus destructans, the causative agent of white-nose syndrome.

Hoyt JR, Cheng TL, Langwig KE, Hee MM, Frick WF, Kilpatrick AM - PLoS ONE (2015)

Challenge plates showing the inhibition of Pseudogymnoascus destructans.Bacteria were plated with an initial starting concentration of 104 cfu/ml (PF2). The plate (a) shows no visible P. destructans growth on day 43, compared to the (b) control plate showing uninhibited P. destructans colony growth at day 43. (d) The zones of inhibition produced by one of the top performing P. fluorescens isolates (PF2) compared to the sham inoculated control (c) and a widely used strain of P. fluorescens, (e; PF7: PfA506). There are two distinct zones of inhibition produced by the top performing strain (as shown in panel d) indicated by the grey solid circle and the dashed grey circle. Microscopic images of the inner and outer zones are shown in panels (f) and (g). We used gram staining techniques to help better visualize conidia (purple) and hyphae (pink) (scale bars, 10 μm). Within the first zone, indicated by the dark ring surrounding the yellow bacteria colony (PF2), the bacteria either arrested or delayed conidia growth, (g) which can be seen by the small hyphael extension from the conidia. Outside of this first zone, the growth of P. destructans was much more extensive (f), producing a mycelial network before its growth was arrested.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390377&req=5

pone.0121329.g001: Challenge plates showing the inhibition of Pseudogymnoascus destructans.Bacteria were plated with an initial starting concentration of 104 cfu/ml (PF2). The plate (a) shows no visible P. destructans growth on day 43, compared to the (b) control plate showing uninhibited P. destructans colony growth at day 43. (d) The zones of inhibition produced by one of the top performing P. fluorescens isolates (PF2) compared to the sham inoculated control (c) and a widely used strain of P. fluorescens, (e; PF7: PfA506). There are two distinct zones of inhibition produced by the top performing strain (as shown in panel d) indicated by the grey solid circle and the dashed grey circle. Microscopic images of the inner and outer zones are shown in panels (f) and (g). We used gram staining techniques to help better visualize conidia (purple) and hyphae (pink) (scale bars, 10 μm). Within the first zone, indicated by the dark ring surrounding the yellow bacteria colony (PF2), the bacteria either arrested or delayed conidia growth, (g) which can be seen by the small hyphael extension from the conidia. Outside of this first zone, the growth of P. destructans was much more extensive (f), producing a mycelial network before its growth was arrested.
Mentions: Zones of inhibition could not be visualized until P. destructans growth was visible on days 9–11 (Fig. 1). At this time, zones of inhibition already differed significantly among bacterial isolates and initial concentrations (Fig. 2, S2 Fig., and S5 Table). Two bacterial isolates, PF1 and PF2, generated larger zones of inhibition across most initial concentrations of P. destructans by the end of the experiment (S2 Table). Three isolates (PF1, PF2, and PF7) established two zones, one where growth of P. destructans was suspended immediately upon germination (Fig. 1g), and another outside of this zone where growth was arrested, but only after the mycelial mat had begun to develop (Fig. 1f). Zones of inhibition on the last day of the experiment (day 37) increased with increasing initial concentrations of P. destructans for the Pseudomonas isolates showing the strongest inhibition (Fig. 2; PF1, PF2, and PF4; all concentration slopes were significantly negative, all p-values <0.03). For the other four Pseudomonas isolates, the zone of inhibition was either variable across concentrations (Fig. 2; PF3, PF5, PA6) or increased with decreasing initial P. destructans concentration (PF7; concentration coef. 1.72 ± 0.64, p = 0.008). Two isolates, PF1 and PF2, out-performed the reference P. fluorescens strains (PF7; PfA506) at all initial concentrations with at least a two-fold difference in zone of inhibition (Fig. 2). The two control bacteria (Chryseobacterium sp. and Sphingomonas sp.) and the sham-inoculated control produced no zones of inhibition (Fig. 2).

Bottom Line: White-nose syndrome, caused by the fungal skin pathogen Pseudogymnoascus destructans, threatens several hibernating bat species with extinction and there are few effective treatment strategies.The skin microbiome is increasingly understood to play a large role in determining disease outcome.In both challenge experiments, the extent of suppression of P. destructans growth was dependent on the initial concentration of P. destructans and the initial concentration of the bacterial isolate.

View Article: PubMed Central - PubMed

Affiliation: Department of Ecology and Evolutionary Biology, University of California Santa Cruz, Santa Cruz, California, United States of America.

ABSTRACT
Emerging infectious diseases are a key threat to wildlife. Several fungal skin pathogens have recently emerged and caused widespread mortality in several vertebrate groups, including amphibians, bats, rattlesnakes and humans. White-nose syndrome, caused by the fungal skin pathogen Pseudogymnoascus destructans, threatens several hibernating bat species with extinction and there are few effective treatment strategies. The skin microbiome is increasingly understood to play a large role in determining disease outcome. We isolated bacteria from the skin of four bat species, and co-cultured these isolates with P. destructans to identify bacteria that might inhibit or kill P. destructans. We then conducted two reciprocal challenge experiments in vitro with six bacterial isolates (all in the genus Pseudomonas) to quantify the effect of these bacteria on the growth of P. destructans. All six Pseudomonas isolates significantly inhibited growth of P. destructans compared to non-inhibitory control bacteria, and two isolates performed significantly better than others in suppressing P. destructans growth for at least 35 days. In both challenge experiments, the extent of suppression of P. destructans growth was dependent on the initial concentration of P. destructans and the initial concentration of the bacterial isolate. These results show that bacteria found naturally occurring on bats can inhibit the growth of P. destructans in vitro and should be studied further as a possible probiotic to protect bats from white-nose syndrome. In addition, the presence of these bacteria may influence disease outcomes among individuals, populations, and species.

No MeSH data available.


Related in: MedlinePlus