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

Bacteria Isolated from Bats Inhibit theGrowth of Pseudogymnoascus destructans,the Causative Agent of White-NoseSyndrome Joseph R. Hoyt*, Tina L. Cheng, Kate E. Langwig, Mallory M. Hee, Winifred F. Frick, A.
Marm Kilpatrick Department of Ecology and Evolutionary Biology, University of California Santa Cruz, Santa Cruz, California,United States of America * [email protected] Emerging infectious diseases are a key threat to wildlife. Several fungal skin pathogens Citation: Hoyt JR, Cheng TL, Langwig KE, Hee MM, have recently emerged and caused widespread mortality in several vertebrate groups, in- Frick WF, Kilpatrick AM (2015) Bacteria Isolated from cluding amphibians, bats, rattlesnakes and humans. White-nose syndrome, caused by the Bats Inhibit the Growth of Pseudogymnoascus fungal skin pathogen Pseudogymnoascus destructans, threatens several hibernating bat destructans, the Causative Agent of White-Nose species with extinction and there are few effective treatment strategies. The skin micro- Syndrome. PLoS ONE 10(4): e0121329. doi:10.1371/journal.pone.0121329 biome 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.
Academic Editor: R. Mark Brigham, University ofRegina, CANADA 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 Received: August 26, 2014 Pseudomonas) to quantify the effect of these bacteria on the growth of P. destructans. All Accepted: January 30, 2015 six Pseudomonas isolates significantly inhibited growth of P. destructans compared to non- Published: April 8, 2015 inhibitory control bacteria, and two isolates performed significantly better than others in sup- Copyright: 2015 Hoyt et al. This is an open pressing P. destructans growth for at least 35 days. In both challenge experiments, the ex- access article distributed under the terms of the tent 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 unrestricted use, distribution, and reproduction in anymedium, provided the original author and source are 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 syn- Data Availability Statement: All relevant data are drome. In addition, the presence of these bacteria may influence disease outcomes among within the paper and its Supporting Information files.
individuals, populations, and species.
Funding: This work was supported by United StatesFish and Wildlife Service (F12AP01081) to WFF;National Science Foundation (DEB-1115895) to AMK;and Bat Conservation International StudentScholarship to JRH. The funders had no role in study design, data collection and analysis, decision topublish, or preparation of the manuscript.
Emerging infectious diseases can have devastating impacts on wildlife, and they currently Competing Interests: The authors have declared threaten many species with extinction ]. With an increase in anthropogenic disturbance that no competing interests exist.
and rise in global trade and transportation, the threat posed by wildlife disease is likely to PLOS ONE DOI:10.1371/journal.pone.0121329 Bacteria Isolated from Bats Inhibits Pseudogymnoascus destructans increase [Wildlife diseases can be exceedingly challenging to manage because free ranginganimals are difficult to treat with drugs or vaccines, and many strategies require constanthuman intervention[]. For example, the re-establishment of Black-footed ferrets into their na-tive range required vaccination of adults and young born each year for both plague and caninedistemper ]. New approaches that do not require continued intervention are needed to re-duce the impacts of these devastating diseases ].
Several recently emerged wildlife pathogens infect host dermal tissue, and interactions with host skin microbiota could play an important role in disease severity. Vertebrate skin is an eco-system composed of different habitats which harbor diverse assemblages of microorganisms]. Previously, studies of skin microbiota primarily examined the pathogenic roles of skin mi-crobes, with little attention to the beneficial function that many microorganisms may provideHowever, beneficial bacteria on skin can provide vital functions to their hosts, includingprocessing of skin proteins, freeing fatty acids to reduce invasion of transient microorganisms,and inhibition of pathogenic microorganisms []. Some bacteria, termed probiotics, or benefi-cial bacteria have been developed to reduce the impact of a broad range of diseases.
Probiotics that can establish on hosts have the potential to provide a long-lasting solution for managing disease and, unlike chemical fungicides, may be able to coevolve with a pathogenProbiotics are regularly used in the biological control of disease in both aquaculture andagriculture, but have yet to be widely implemented in controlling wildlife disease, possibly be-cause of perceived risks and lack of demonstrated success [–]. Risks associated with aug-menting micro-organisms on a host, which can either be ineffective or accidentally harmful,can be minimized by using bacteria that naturally occur in the hosts' environment []. Resis-tant or tolerant species that are phylogenetically closely related to a heavily impacted speciesmay host bacteria that could serve as probiotics, and these bacteria may be more likely to beable to colonizing the target species' skin Here, we assess whether bacteria naturally occurring on bats can reduce the growth of Pseu- dogymnoascus destructans, the pathogen that causes white-nose syndrome (WNS)[]. White-nose syndrome first emerged in Howe's Cave, New York, in 2006, and spread quickly, causingprecipitous declines in hibernating bats throughout Eastern North America,]. Four species(Myotis septentrionalis, Myotis sodalis, Myotis lucifugus, and Perimyotis subflavus) have suf-fered >90% declines in regional populations and one species, M. septentrionalis, is on a trajec-tory towards extinction ,]. Myotis septentrionalis has recently been proposed by the U.S.
Fish and Wildlife Service for listing under the Endangered Species Act and has been listedunder the Canadian Species at Risk Act as Endangered.
Pseudogymnoascus destructans infects the dermal tissue of bats and grows optimally be- tween 10–14°C similar to the temperature of hibernating bats. Pseudogymnoascus destruc-tans infection may disrupt bats' physiological processes including heat and water loss andelectrolyte balance and typically results in increased arousal frequency by hibernatingbats [Increased arousal frequency may prematurely deplete bats' fat stores resulting indeath approximately 70–120 days after infection, based on laboratory infection ,].
Individuals able to survive through hibernation until spring appear to clear infection and fullyrecover [However, these bats become re-infected the following fall, in part becausePseudogymnoascus destructans is capable of persisting for long periods of time in the absenceof bats [ Currently, there are few management options that can reduce mortality in affected regions ]. Preliminary investigations of treatments to reduce mortality using antifungal drugs causedhigher mortality in the treated groups than control groups, possibly due to toxicity ]. Otherproposed chemical treatment options have avoided the toxic effects of the direct application ofchemicals, but have yet to be validated in situ [Thus, treatment options are urgently needed, PLOS ONE DOI:10.1371/journal.pone.0121329 Bacteria Isolated from Bats Inhibits Pseudogymnoascus destructans and a probiotic may be an effective way to reduce WNS impacts if it could at least partially in-hibit P. destructans growth and delay mortality long enough for bats to survive hibernation.
We cultured bacteria isolated from the skin of four species of hibernating bats from eastern North America to determine whether naturally occurring bacterial species might exist withinthe skin microbiome of bats that could inhibit growth of P. destructans. We then quantified theanti-fungal efficacy of these bacteria across a range of concentrations in twochallenge experiments.
We conducted sampling for cutaneous bacteria on hibernating bats at two hibernacula in NewYork and two in Virginia (exact locations of study sites are not provided to protect sensitivewildlife habitat). We rubbed sterile polyester swabs dipped in sterile water back and forth fivetimes along each bat's forearm and muzzle. Swabs were frozen in 20% glycerol stock for laterculturing. We collected swabs from ten individuals from each of four species Eptesicus fuscus,Myotis leibii, M. lucifugus, and M. sodalis.
Epidermal swab sample collection protocols for this study were approved by the University of California, Santa Cruz IACUC (protocol # FrickW1106). Sample collection was permittedby authorized state biologists from the New York Department of Environmental Conservationand Virginia Department of Game and Inland Fisheries. Handling and sampling of endangeredspecies (Myotis sodalis) was conducted under the appropriate state and federal permits.
Each swab was plated on two types of general media, Reasoner's 2A agar (R2A) and sabour- aud dextrose agar (SDA), and plates were incubated at 9°C for three weeks. We classified bacte-ria on each plate by morphotype, using color, growth form, and gram staining techniques. Weisolated one colony from each sample by morphotype (to reduce repeat sampling of the sameisolate) using a sterile inoculating loop and re-plated each isolate on R2A media and grew themfor 2–5 days at 9°C. Each isolate was cryo-banked by sampling from each of these colonies witha sterile inoculating loop, placing the sample in 30% glycerol, and freezing it at -80C forlater use.
We determined whether isolates could inhibit the growth of P. destructans using a challengeprotocol adapted from the National Committee for Clinical Laboratory Standards. All cultur-ing was done on SDA. []. A suspension containing 1.7 x 107 P. destructans conidia/ml (quan-tified using a hemocytometer) was prepared by flooding a 3 week-old culture of P. destructansgrown on SDA with 20 ml of 1X phosphate buffered saline with tween20 (PBST20). Colonieswere submersed for 5 minutes, and then gently rubbed with a sterile inoculating loop to freethe conidia. The supernatant was drawn off and placed into a 50ml falcon tube and vortexed tohomogenize the suspension. Each 90mm plate was inoculated with 200ul of the P. destructanssuspension and allowed to air dry for 10 minutes. We added bacteria on the plate already inoc-ulated with P. destructans from a growing culture using pinpoint inoculation at three equallyspaced points on top of the dried P. destructans suspension. Bacteria cultures were grown fromfrozen stock 24 hours earlier on SDA. Plates were placed into incubators at 9–10°C, which iswithin the range that bats hibernate [], and growth was monitored every other day for 14days and on day 14 any bacteria that produced a zone of inhibition (a visible reduction of P.
destructans growth surrounding the bacterial colony) were included in subsequent challengeexperiments described below In addition to bacterial isolates from bats, a Pseudomonas fluores-cens isolate PfA506 commonly used in biocontrol of agricultural fungal pathogens was included PLOS ONE DOI:10.1371/journal.pone.0121329 Bacteria Isolated from Bats Inhibits Pseudogymnoascus destructans as a positive control and two types of negative controls, 1) a sham inoculation with 20%sterile glycerol stock, and 2) two bacteria isolated from bats in the genera Chryseobacteriumand Sphingomonas (both gram-negative rod shaped bacteria) that are not known to produceanti-fungal compounds We identified bacterial isolates used in the following inhibition assays using PCR amplificationand DNA sequencing. DNA for PCR was obtained by suspending a small amount of a bacterialcolony in 100 μl of sterile deionized water (SDW) and lysing the cells at 95°C (10 min). Univer-sal bacterial 16S rRNA gene primers (16S_F (5 - ACC GCG ATA ATA CGT CCC GAT CG—3 )and 16S_R(5 - TGC GGA CGT GAA GTG CTA G -3 )) were used to amplify the 1.5 kb 16SrRNA gene fragment The following was added to each PCR template: 1 μl of crude lysateDNA template, 1.5 μl of each 0.6 μM forward and reverse primer, and 5 μl of Taq 5X MM(NEB) at 1X concentration, which contains 1.5 mM MgCl2, 2 mM dNTPs, and PCR buffer. Re-action volumes were made up to 25 μl with SDW. The reaction conditions involved an initialdenaturation at 95°C for 3 minutes, followed by 35 cycles of denaturation at 95°C for 15 sec,primer annealing for 15 seconds at 49°C, and extension for 90 seconds at 42°C. The 16S rRNAgene sequences were compared with known sequences in the EMBL database using MEGABLAST (BLASTN 2.1.1, to identify the most similar sequence alignment. Pseudomonasfluorescens isolate PfA506 was used to assure proper alignment of sequences.
Two separate inhibition assays were performed. In the first inhibition assay, we determined theability of each bacterial isolate to grow on lawns of different starting concentrations of P.
destructans, and to produce a zone of inhibition in which P. destructans growth was either de-layed, halted adjacent to the bacteria colony. In the second inhibition assay, we measured thegrowth of P. destructans on a lawn of different starting concentrations of each bacterial isolate.
In the first inhibition assay, the growth of each bacterial isolate was quantified in media in- oculated with four P. destructans concentrations estimated using hemocytometry (107, 106,105, and 104 conidia/ml). After the P. destructans suspension was dry and conidia fixed to theplate, we used a pipette to inoculate the plates with 0.1 μl of a 108 cfu/ml suspension of a givenbacterial isolate at three evenly spaced points on the plate. The bacterial suspension was pre-pared by suspending whole colonies in 30% glycerol, and using an inoculating loop to suspendthe colony. Each treatment was replicated nine times, and cultures were grown at 9°C for 37days. Zones of inhibition were quantified by measuring the distance from the edge of the bacte-rial colony to the edge of the visible P. destructans growth every other day We also micro-scopically examined the zones of inhibition on the final day of the experiment (day 43) tocharacterize the effects of bacteria on the growth of P. destructans.
In the second inhibition assay we determined the ability of each bacterial isolate to prevent growth of P. destructans across a series of six bacterial concentrations. Each bacterial isolatewas plated from cryobanked glycerol stock onto a Petri dish with SDA media and allowed toincubate for two days at 9°C before being added to a 30% sterile glycerol suspension. The con-centration of each isolate was standardized by making serial ten-fold dilutions of the culturingstock and then counting the number of colony forming units (cfu) per ml. The bacteria glycerolsuspension was frozen at -20°C while calculating the concentration. Each stock was standard-ized to the same concentration of 7.5x107 cfu/ml using 30% glycerol. We plated 50 μl of eachbacterial glycerol dilution on SDA in 60 mm Petri dishes. We used three replicates per treat-ment for bacterial concentrations 106, 102, and 101 cfu/ml, and five replicates for PLOS ONE DOI:10.1371/journal.pone.0121329 Bacteria Isolated from Bats Inhibits Pseudogymnoascus destructans concentrations 105, 104, and 103 cfu/ml. For the control plate, we added 50 μl of sterile 30%glycerol solution to the plates and then inoculated with P. destructans using a pinpoint inocula-tion. The diameter of the P. destructans colony was measured for a total of 42 days. Measure-ments were made every other day for the first 14 days, and then once every seven daysthereafter until the end of the experiment.
We used cell-free supernatant plated on a lawn of P. destructans to determine if anti-fungal compounds were being produced by the bacteria in the initial culture. We used cultures offresh bacteria grown in isolation, and co-cultured with P. destructans, in lysogeny broth. Cul-tures were then centrifuged for 30 minutes and the supernatant was drawn off. We then inocu-lated three plates with 50 μL of the supernatant on a lawn of P. destructans using the methodsdescribed above.
Bacteria motility experiments were conducted to assess whether the Pseudomonas isolates frombats preferentially move towards P. destructans. A 0.3% agar SDA media was prepared and asterile inoculating loop was dipped into a 7.5x107 cfu/ml of bacterial suspension and thenstabbed 5 cm into the soft agar for all nine bacterial isolates. To determine whether or not thebacteria preferentially moved towards P. destructans, we repeated the same methods describedabove, but included a small colony of P. destructans that was stabbed into the media on oneside of the tube. The tubes were incubated for 1 week at 10°C, and then stabs were visually in-spected for signs of whether bacteria moved away from the initial stab or moved towards the P.
destructans stab.
We fit linear mixed effects models (function glmer in package lme4 [in R v. 3.02 ]) withday as a categorical random effect and bacteria type and concentration as fixed effects to exam-ine the influence of bacteria type and serial dilution on the zone of inhibition (first inhibitionassay, ) and diameter of fungal colony (second inhibition assay, ). We fit five apriori models including additive and interactive effects ) and compared models usingAkaike's Information Criterion (AIC) [].
We isolated a total of 133 bacterial morphotypes from the 40 bats we swabbed. Four isolatesfrom E. fuscus (from 3 bats) and two isolates from two individual M. lucifugus inhibited P.
destructans growth in standard challenge assays These six bacteria were selected forfurther testing in the two inhibition assays. All were identified as members of the genus Pseudo-monas, with five of the six isolates most closely related to the Pseudomonas fluorescens groupand the other isolate (PA6) being most closely related to Pseudomonas abietaniphila(HF952541) In the first inhibition assay, bacterial colonies initially expanded quickly and then plateaued insize, with growth continuing for longer at lower initial concentrations of P. destructans in themedia ., ). Some bacterial isolates formed much larger colonies than others,with PF1, PF2, and PF4 forming the largest colonies (.). The size of bacterial colonies in-creased with decreasing initial concentrations of P. destructans ., PLOS ONE DOI:10.1371/journal.pone.0121329 Bacteria Isolated from Bats Inhibits Pseudogymnoascus destructans Bacteria isolated from Myotis lucifugus and Eptesicus fuscus used in challenge experiments.
Collection County Sphingomonas sp.
Pseudomonas sp.
Pseudomonas sp.
Pseudomonas sp.
Pseudomonas sp.
Pseudomonas sp.
P. fluorescens A506 Zones of inhibition could not be visualized until P. destructans growth was visible on days 9–11 (). At this time, zones of inhibition already differed significantly among bacterialisolates and initial concentrations (, S2 Fig., and ). Two bacterial isolates, PF1and PF2, generated larger zones of inhibition across most initial concentrations of P. destruc-tans by the end of the experiment Three isolates (PF1, PF2, and PF7) establishedtwo zones, one where growth of P. destructans was suspended immediately upon germination), and another outside of this zone where growth was arrested, but only after the myceli-al mat had begun to develop (Zones of inhibition on the last day of the experiment(day 37) increased with increasing initial concentrations of P. destructans for the Pseudomonasisolates showing the strongest inhibition (; PF1, PF2, and PF4; all concentration slopeswere significantly negative, all p-values <0.03). For the other four Pseudomonas isolates, thezone of inhibition was either variable across concentrations (; PF3, PF5, PA6) or increasedwith 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 inhibitionThe two control bacteria (Chryseobacterium sp. and Sphingomonas sp.) and the sham-inoculated control produced no zones of inhibition In the second inhibition experiment, P. destructans grew optimally in the absence of bacte- ria, and on media with low initial concentrations of the control bacteria By the end ofthe experiment, the size of P. destructans colonies differed between bacterial isolates and initialconcentrations and the effect of bacterial isolate varied among initial concentrations (and Tables). At the highest initial concentration (106 cells/ml), all bacteria (including the two con-trol bacteria) formed lawns and all reduced growth of P. destructans. As the starting concentra-tion of the bacteria lawn decreased, fewer isolates significantly reduced the growth of P.
destructans. At the three highest initial bacterial concentrations (106–104 cfu/ml), only isolatesPF1, PF2, and PF5 completely suppressed P. destructans growth for the duration of the experi-ment (day 42; At the three lowest initial concentrations of the bacteria, where therewere relatively few colonies, two Pseudomonas isolates, PF1 and PF2 performed significantlybetter than other isolates in reducing P. destructans growth and prevented P. destructans fromgrowing for the full duration of the experiment (, ). In both experiments, isolatesPF1 and PF2 produced the maximum reduction of mycelial growth across all concentrations,regardless of the way the isolates and P. destructans were co-cultured.
Cell-free supernatant drawn from liquid bacterial cultures had no effects on the growth of P.
destructans. Pseudogymnoascus destructans grew uniformly across all plates, regardless ofwhether the supernatant added to the plates was from bacteria co-cultured with P. destructansor cultured alone.
PLOS ONE DOI:10.1371/journal.pone.0121329

Bacteria Isolated from Bats Inhibits Pseudogymnoascus destructans 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 growthat 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 awidely 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 gramstaining techniques to help better visualize conidia (purple) and hyphae (pink) (scale bars, 10 μm). Within the first zone, indicated by the dark ringsurrounding the yellow bacteria colony (PF2), the bacteria either arrested or delayed conidia growth, (g) which can be seen by the small hyphael extensionfrom the conidia. Outside of this first zone, the growth of P. destructans was much more extensive (f), producing a mycelial network before its growthwas arrested.
All seven Pseudomonas isolates exhibited signs of motility but no chemotaxis towards P.
destructans colonies was observed. Using microscopy, two of the Pseudomonas isolates, PF1and PF2, were observed dispersing along P. destructans hyphae. The two control bacteriashowed no signs of motility ).
PLOS ONE DOI:10.1371/journal.pone.0121329

Bacteria Isolated from Bats Inhibits Pseudogymnoascus destructans First inhibition assay measuring the width of the zone of inhibition produced by bacteria on a lawn of P. destructans. The zones of inhibitionproduced by bacterial isolates when inoculated on a plate with four concentrations of P. destructans. Lines denoted by the same letter do not differsignificantly on the last day of the experiment ). CHR and SPH are isolates in the genus Chryseobacterium and Sphingomonas that are not knownto produce antifungal compounds. The control is an inoculation of 30% glycerol stock. PF1-5,7 and PA6 are all isolates in the genus Pseudomonas.
Second inhibition assay measuring the diameter of P. destructans colonies grown on a lawn of bacteria. Pseudogymnoascus destructans wasplated with nine bacterial isolates at six different concentrations (highest to lowest, left to right). Lines denoted by the same letter did not differ significantly onthe last day of the experiment. CHR and SPH are isolates in the genus Chryseobacterium and Sphingomonas that are not known to produce antifungalcompounds. The Control is a sham inoculation of 30% glycerol stock. PF1-7 and PA6 are all isolates in the genus Pseudomonas.
PLOS ONE DOI:10.1371/journal.pone.0121329 Bacteria Isolated from Bats Inhibits Pseudogymnoascus destructans As the threat of emerging infectious disease grows with increased global travel and trade [],new ways of managing wildlife disease must be considered ]. Traditionally, fungal pathogenshave been managed using chemical fungicides but toxicity effects on non-target organ-isms, and application challenges makes it difficult for broad-scale use on wildlife fungal patho-gens The results from our two sets of experiments demonstrate that in vivo, bacteriacultured from bats can inhibit the growth of P. destructans. Our results suggest that augmenta-tion prior to P. destructans exposure might reduce colonization, whereas bacterial augmenta-tion after exposure could displace P. destructans. Our results also suggest that a key challengefor successful treatment is applying bacteria such that they will persist on bat skin at highenough concentrations to limit P. destructans growth below levels that cause lethal disease.
The bacteria we isolated from bats, Pseudomonas spp., is ubiquitous in the environment and is well known to have anti-fungal properties The group of bacteria that these isolates weremost closely related to, Pseudomonas fluorescens, has previously been detected on several mam-mals (including bats), as well as amphibians, fish, and plants [–]. Members of the P. fluor-escens group are known to produce a suite of antifungal compounds that can inhibit manyplant fungal pathogens [as well as the amphibian fungal pathogens, Batrachochytrium den-drobatidis Some strains in the P. fluorescens group are also capable of producing mycolys-ing enzymes that can colonize the mycelia and conidia of fungi rendering them no longerviable All of our Pseudomonas spp. isolates were motile, which might allow them to usethe mycelial networks of fungal colonies to aid in dispersal and colonization []. All of theseattributes make P. fluorescens ideal as a proposed candidate to be tested as a biological controlagent for reducing infection intensity and increasing survival of bats exposed to P. destructans.
Whether these antifungal bacteria that naturally occur on bat skin could partially explain differences in mortality from WNS among populations and species is currently unknown. Theisolates with strongest inhibitory properties were cultured from E. fuscus, which has lower mor-tality from WNS compared to other species [However, we also isolated two strains of P.
fluorescens (PF3 and PF4) that showed moderate P. destructans inhibition from M. lucifugus, aspecies that has suffered severe mortality from ]. Further research is needed to deter-mine the relative abundance, distribution, and inhibitory ability of P. fluorescens on wild batsand whether presence and abundance of P. fluorescens influences disease severity.
The next steps in developing a probiotic for WNS should include testing, in vivo, one or more of the P. fluorescens strains that we isolated against P. destructans using a bat species thatsuffers high disease mortality from WNS, such as M. lucifugus, M. septentrionalis, or Perimyotissubflavus ]. Studies with live hibernating bats will determine whether interactions observedin vitro have functional significance in disease outcomes for bat species currently threatenedby WNS.
Bacterial colony size during first inhibition assay. Colony size of nine bacterial iso-lates grown on plates inoculated with four different concentrations of Pseudogymnoascusdestructans with fungal concentrations decreasing from left to right. CHR and SPH are isolatesin the genus Chryseobacterium and Sphingomonas that are not known to produce antifungalcompounds. The Control is a sham inoculation of 30% glycerol stock. PF1-7 and PA6 are bac-terial isolates in the genus Pseudomonas.
(TIF) PLOS ONE DOI:10.1371/journal.pone.0121329 Bacteria Isolated from Bats Inhibits Pseudogymnoascus destructans BLAST results of 16S rRNA sequence from the National Center of Biological In-formation database for the six bacterial isolated in this study from bats (Myotis lucifugusand Eptesicus fuscus).
(DOCX) AIC values for the first inhibition assay measuring the zone of inhibition pro-duced by bacteria on a lawn of P. destructans.
(DOCX) AIC values for the second inhibition assay measuring how different bacterial iso-lates influenced the diameter of a P. destructans colony.
(DOCX) AIC values for the first inhibition assay measuring the diameter of bacteria colo-nies on different concentrations of P. destructans.
(DOCX) Coefficients for linear models of the influence of nine bacterial treatments and acontrol on the radius of the zones of inhibition of P. destructans produced by bacteria atfour different initial concentrations of P. destructans on day 37 for the data shown in(DOCX) Coefficients for linear models of the influence of nine bacterial isolates on the di-ameter of P. destructans colonies for each bacterial concentration on day 43 for the datashown in .
(DOCX) We thank P. Peng, K. Gambel and M. McCree for lab assistance, W. Stone from the NYS DECfor help with collecting preliminary data. We thank G. Gilbert, M. Brigham, and an anonymousreviewer for helpful comments on the manuscript.
Author Contributions Conceived and designed the experiments: JRH AMK TLC WFF KEL. Performed the experi-ments: JRH TLC MHH. Analyzed the data: JRH AMK KEL WFF. Wrote the paper: JRH AMKWFF KEL.
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PLOS ONE DOI:10.1371/journal.pone.0121329

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